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Gas Exchange and Acclimation of Radish in Reduced Pressure Environments

Permanent Link: http://ufdc.ufl.edu/UFE0041065/00001

Material Information

Title: Gas Exchange and Acclimation of Radish in Reduced Pressure Environments
Physical Description: 1 online resource (162 p.)
Language: english
Creator: Gohil, Hemantkumar
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2010

Subjects

Subjects / Keywords: acclimation, hypobaria, model, radish, transpiration
Agricultural and Biological Engineering -- Dissertations, Academic -- UF
Genre: Agricultural and Biological Engineering thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: GAS EXCHANGE AND ACCLIMATION OF RADISH IN REDUCED PRESSURE ENVIRONMENTS By Hemant Laxmansinh Gohil August 2010 Chair: Melanie Correll Major: Agricultural and Biological Engineering Plants grown on long-term space missions will likely be grown in low pressure environments (i.e., hypobaria). In general, the fundamental growth and photosynthesis of plants grown in hypobaria are similar to plants grown at normal atmospheric conditions with some exceptions. For example, transpiration rates can be elevated in low pressure resulting in plant wilting if water is not readily available. Plants may also exhibit poor growth and germination if the partial pressures of critical gases are not maintained to certain levels (e.g., pO2 for seed germination and pCO2 for photosynthesis). Since the gas phase of the environment is so critical for successful growth of plants in both normal and low pressure environments, the effects of the gas phase composition, particularly CO2, on the gas exchange and growth of radish (Raphanus sativus var. Cherry Bomb II) in hypobaria were studied. Low pressure growth chambers were built that could monitor the environmental parameters for these studies. The fresh weight (FW), leaf area, dry weight (DW), CO2 assimilation rates (CA), dark respiration rates (DR), and transpiration rates from 26 day-old radish plants that were grown for an additional seven days at different total pressures (33, 66 or 101 kPa) and pCO2 (40 Pa, 100 Pa and 180 Pa) were measured. In general, the dry weight of plants was enhanced with CO2 enrichment and with decreased total pressure. In limited pCO2 (40 Pa), the transpiration for plants grown at 33 kPa was over twice that of controls (101 kPa total pressure with 40 Pa pCO2). Increasing the pCO2 from 40 Pa to either 100 or 180 Pa reduced the transpiration rates for plants grown in hypobaria and at normal atmospheric pressures. Plants grown at lower total pressures (33 and 66 kPa total pressure) and super-elevated pCO2 (180 Pa) had evidence of leaf damage. Taken together, radish growth can be enhanced and transpiration reduced in hypobaria by enriching the gas phase with CO2 although at high levels of CO2 leaf damage can occur. Since the diffusivities of gas increases as the atmospheric pressure drops, it is expected that transpiration and CO2 assimilation in plants would increase as plants grow in hypobaria. A mathematical relationship based on the principles of thermodynamics was developed for calculating the transpiration and photosynthesis for plants. Stomatal conductance is sensitive to total pressure. At 33 kPa total pressure, stomatal conductance increases with the boundary increasing by a factor of ?1.7, thus the boundary layer thickness conductance increases by 70%. Since the leaf conductance is a function of both stomatal conductance and the boundary layer conductance, the overall conductance will increase resulting in significantly higher levels of transpiration as the pressure drops. The conductance of gases is also regulated by stomatal aperture in an inverse relationship. Stomatal aperture is directly influenced by concentration of CO2 inside the leaf space. The higher CO2 concentration inside the leaf air space during low pressure treatments may result in stomata closing partially or fully which may reduce the excessive transpiration caused by increased diffusivity. Therefore, a reduced pressure environment with high CO2 may be an ideal scenario for minimizing transpiration and maximizing the plant biomass yield in BLSS. The application of this model to data for plants acclimated long term to hypobaria (33 kPa) and reference values (101kPa) suggest that for transpiration the model predicts transpiration for plants that are grown at normal pressure as well as reduced pressure. To understand the mechanisms of plant adaptation to hypobaria, plants were transferred to low pressure at different stages of growth (0, 2, 14, and 26 days after germination). The growth, CA, transpiration and stomatal density were compared between these plants. Plants exposed short term to hypobaria ( < 2 days in hypobaria) responded differently than those exposed to long-term hypobaria ( > 2 days). For example, plants that were grown entirely in hypobaria had smaller and thicker leaves compared to plants that were exposed for 2 days or less to hypobaria. These long-term treated plants also had higher CA (p < 0.1) and transpiration rates (p < 0.05) even though their overall growth (FW and DW) was not significantly affected by hypobaria. The stomatal density of plants grown long term in hypobaria was not significantly different than plants grown short term to hypobaria. Therefore, it appears that plants may respond to enhanced gas exchange in hypobaria by reducing their leaf area. Further studies on the mechanisms of plant adaptation are required to identify other biological or physiological mechanisms of plant acclimation to hypobaria. Some of the engineering constraints to grow plants in Martian plant growth facilities can be offset by growing plants at reduced atmospheric pressure. Characterization of hypobaria response at reduced pressure can provide data which can help in explaining some of the response which plants may encounter on Martian facility. These studies are important for understanding mechanism by which plant control water relations.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Hemantkumar Gohil.
Thesis: Thesis (Ph.D.)--University of Florida, 2010.
Local: Adviser: Correll, Melanie J.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2011-08-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2010
System ID: UFE0041065:00001

Permanent Link: http://ufdc.ufl.edu/UFE0041065/00001

Material Information

Title: Gas Exchange and Acclimation of Radish in Reduced Pressure Environments
Physical Description: 1 online resource (162 p.)
Language: english
Creator: Gohil, Hemantkumar
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2010

Subjects

Subjects / Keywords: acclimation, hypobaria, model, radish, transpiration
Agricultural and Biological Engineering -- Dissertations, Academic -- UF
Genre: Agricultural and Biological Engineering thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: GAS EXCHANGE AND ACCLIMATION OF RADISH IN REDUCED PRESSURE ENVIRONMENTS By Hemant Laxmansinh Gohil August 2010 Chair: Melanie Correll Major: Agricultural and Biological Engineering Plants grown on long-term space missions will likely be grown in low pressure environments (i.e., hypobaria). In general, the fundamental growth and photosynthesis of plants grown in hypobaria are similar to plants grown at normal atmospheric conditions with some exceptions. For example, transpiration rates can be elevated in low pressure resulting in plant wilting if water is not readily available. Plants may also exhibit poor growth and germination if the partial pressures of critical gases are not maintained to certain levels (e.g., pO2 for seed germination and pCO2 for photosynthesis). Since the gas phase of the environment is so critical for successful growth of plants in both normal and low pressure environments, the effects of the gas phase composition, particularly CO2, on the gas exchange and growth of radish (Raphanus sativus var. Cherry Bomb II) in hypobaria were studied. Low pressure growth chambers were built that could monitor the environmental parameters for these studies. The fresh weight (FW), leaf area, dry weight (DW), CO2 assimilation rates (CA), dark respiration rates (DR), and transpiration rates from 26 day-old radish plants that were grown for an additional seven days at different total pressures (33, 66 or 101 kPa) and pCO2 (40 Pa, 100 Pa and 180 Pa) were measured. In general, the dry weight of plants was enhanced with CO2 enrichment and with decreased total pressure. In limited pCO2 (40 Pa), the transpiration for plants grown at 33 kPa was over twice that of controls (101 kPa total pressure with 40 Pa pCO2). Increasing the pCO2 from 40 Pa to either 100 or 180 Pa reduced the transpiration rates for plants grown in hypobaria and at normal atmospheric pressures. Plants grown at lower total pressures (33 and 66 kPa total pressure) and super-elevated pCO2 (180 Pa) had evidence of leaf damage. Taken together, radish growth can be enhanced and transpiration reduced in hypobaria by enriching the gas phase with CO2 although at high levels of CO2 leaf damage can occur. Since the diffusivities of gas increases as the atmospheric pressure drops, it is expected that transpiration and CO2 assimilation in plants would increase as plants grow in hypobaria. A mathematical relationship based on the principles of thermodynamics was developed for calculating the transpiration and photosynthesis for plants. Stomatal conductance is sensitive to total pressure. At 33 kPa total pressure, stomatal conductance increases with the boundary increasing by a factor of ?1.7, thus the boundary layer thickness conductance increases by 70%. Since the leaf conductance is a function of both stomatal conductance and the boundary layer conductance, the overall conductance will increase resulting in significantly higher levels of transpiration as the pressure drops. The conductance of gases is also regulated by stomatal aperture in an inverse relationship. Stomatal aperture is directly influenced by concentration of CO2 inside the leaf space. The higher CO2 concentration inside the leaf air space during low pressure treatments may result in stomata closing partially or fully which may reduce the excessive transpiration caused by increased diffusivity. Therefore, a reduced pressure environment with high CO2 may be an ideal scenario for minimizing transpiration and maximizing the plant biomass yield in BLSS. The application of this model to data for plants acclimated long term to hypobaria (33 kPa) and reference values (101kPa) suggest that for transpiration the model predicts transpiration for plants that are grown at normal pressure as well as reduced pressure. To understand the mechanisms of plant adaptation to hypobaria, plants were transferred to low pressure at different stages of growth (0, 2, 14, and 26 days after germination). The growth, CA, transpiration and stomatal density were compared between these plants. Plants exposed short term to hypobaria ( < 2 days in hypobaria) responded differently than those exposed to long-term hypobaria ( > 2 days). For example, plants that were grown entirely in hypobaria had smaller and thicker leaves compared to plants that were exposed for 2 days or less to hypobaria. These long-term treated plants also had higher CA (p < 0.1) and transpiration rates (p < 0.05) even though their overall growth (FW and DW) was not significantly affected by hypobaria. The stomatal density of plants grown long term in hypobaria was not significantly different than plants grown short term to hypobaria. Therefore, it appears that plants may respond to enhanced gas exchange in hypobaria by reducing their leaf area. Further studies on the mechanisms of plant adaptation are required to identify other biological or physiological mechanisms of plant acclimation to hypobaria. Some of the engineering constraints to grow plants in Martian plant growth facilities can be offset by growing plants at reduced atmospheric pressure. Characterization of hypobaria response at reduced pressure can provide data which can help in explaining some of the response which plants may encounter on Martian facility. These studies are important for understanding mechanism by which plant control water relations.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Hemantkumar Gohil.
Thesis: Thesis (Ph.D.)--University of Florida, 2010.
Local: Adviser: Correll, Melanie J.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2011-08-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2010
System ID: UFE0041065:00001


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GAS EXCHANGE AND ACCLIMATION OF RADISH IN REDUCED PRESSURE
ENVIRONMENTS



















By

HEMANT LAXMANSINH GOHIL


A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA

2010


























Hemant L Gohil
































Dedicated in memory of


Kalpana Chawla









ACKNOWLEDGMENTS

I am thankful to the Agricultural and Biological Engineering Department at the

University of Florida for access to resources and facilities as well as providing me my

research assistantship. I am also thankful to NASA for funding my research studies.

I express my deepest gratitude to Dr. Melanie Correll who served as chair of my

committee. I would like to thank her for her guidance, support and patience during my

course work and research and I am thankful to her for helping me to improve in

scientific and technical writing. It was a great learning experience working with Dr.

Correll.

I am thankful to my committee members; Dr. Ray Bucklin for his friendly

guidance and enlightening discussions (academic and non academic); Dr. Thomas

Sinclair, for his keen interest in improving my fundamental understanding of gas

exchange in plants; Dr. Robert Ferl for broadening the perspective of my research and

Dr. John Sager for his valuable guidance whenever it was required.

Without the help of John Truett, a distinct colleague and a friend, the low

pressure growth chambers for my research would not have been so reliable. I am also

grateful to him for teaching me mechanical and electrical aspects of instrumentation. I

am also thankful to Alex Stimpson for his help in programming at the beginning of my

research. It was great fun working with Samantha, Stella and Derek. I am grateful to

Samantha for her help with experimentation and to Stella for seed germination studies.

I am thankful to our technical staff, especially Veronica Campbell, for meticulous

proofreading of my manuscripts and ordering research materials. I am grateful to Billy

Duckworth, Steve Feagle, Deniel Preston and Helena Niblack for technical assistance









whenever it was needed. I am also thankful to our departmental staff, Mary Hall, Robin

Snyder and Dawn Mendoza for their assistance with official and paperwork.

Throughout my research there were many friends and people who directly or

indirectly helped me, I am thankful to all of them. I am grateful to my parents who stood

by me whenever I made an important decision. My humble gratitude goes to shri Chinu

Bapji and Sadhaguru Jaggi Vasudev for their blessings and spiritual energy.

My deepest gratitude goes to my loving wife, Hiral whose perennial care and

courage motivated me through the ups and downs in research and in day to day life. I

also benefited a lot by discussing research problems with her. I feel extremely lucky to

have her endless support, without which, finishing Ph.D. would have been very

stressful.









TABLE OF CONTENTS



ACKNOWLEDGMENTS..................... .. ........... .......................................4

L IS T O F T A B L E S .............................................................................. ................... 9

L IS T O F F IG U R E S ............................................................................................................ 10

LIST O F PA RA M ETER S ................. ................... ................................................ 12

A B S T R A C T ....................................................................................................... ........... 13

CHAPTER

1 ATMOSPHERIC CONDITIONS USED FOR BIOLOGICAL SYSTEMS DURING
SPACE MISSIONS: IMPLICATIONS FOR HYPOBARIC PLANT BIOLOGY .......... 17

Advanced Life Support System s (A LS) ............................................... ................ 17
G as Phase for Biological Life S support ................................................. ................ 18
The International Space Station (ISS) ................................................. ................ 26
A advanced Plant Studies on the ISS ..................................................... ................ 27
H yp o b a ric P la nt B io lo g y .............................. ................... ........ ............... ................ 2 8
The Effects of Hypobaria on Seed Germination and Plant Growth .......................28
S u m m a ry .................................................................................................................. ... 3 2
S structure of the D issertation ................................................................. ................ 35

2 DESIGN AND CONSTRUCTION OF LOW PRESSURE GROWTH CHAMBERS
(L P G C S ) .................................................................................................... ....... .. 4 0

S system D description ............................................................................................. 43
G a s L e a k a g e T e s ts ..................................................................................................... 4 4
D ata A acquisition and C control System .................................................. ................ 45
S ensors and T heir C alibrations ............................................................ ................ 46
P pressure S ensor .................................................................................................. 46
R elative H um idity (R H ) S ensor...................................................... ................ 47
O xygen S ensor (0 2) ......................... .............................................................. 47
C arbon D ioxide (C O0 2) S ensor ....................................................... ................ 48
L o a d C e ll ........................................................................................................ 4 9
Light Sensor C alibration ...................................... .. ....... .......... ................... 50
T em perature S ensors ........................................ .. ..................... ............. 50
Sum m ary and Future Im provem ents ................................................... ................ 51

3 MODELING GAS DIFFUSIVITY, PHOTOSYNTHESIS AND TRANSPIRATION
U N D E R H Y P O BA R IA .. .................................................................... ................ 63

P lants in H ypobaria ............................................................................................. 64









Binary Diffusion of G cases .......................................................... ..... .. ...... .................. 65
The Effects of Background Gases on Overall Diffusivity..................................... 67
Diffusivity of CO 2 and H20 in Hypobaria.............................................. ................ 68
G as D iffusion in Leaves ........................................................................................ 70
L e af T ra nsp iratio n .......................................................... ................. ............. 7 1
Leaf P h otosynthesis ....................................................... ................ ............ 75
C o n c lu s io n ............................................................................................................... .. 7 5

4 THE INTERACTING EFFECTS OF CO2 AND HYPOBARIA ON GROWTH AND
TRANSPIRATION OF RADISH (RAPHANUS SATIVUS) .................................. 78

In tro d u c tio n ............................................................................................................. .. 7 9
M materials and M ethods ........................................................... ................ 82
P la n t M a te ria l ........................................................ .................. ... ............ 8 2
Growth Chambers and Environmental Control..............................................83
P la n t H a rv e s t ........................................................................................................ 8 4
S statistical A analysis ...................................................................................... 84
R e s u lts ...................................................................................................... .......... 8 4
P la n t G ro w th ......................................................................................................... 8 4
C O 2 A ssim ilation .......................................................................................... 85
T ranspiration R ates ...................................................................................... 86
D is c u s s io n ............................................................................................................... ... 8 7
S u m m a ry .................................................................................................................. ... 9 1

5 THE EFFECTS OF SHORT-TERM AND LONG-TERM ACCLIMATION OF
RA D IS H TO HY PO BA R IA ..................................... ....................... ................ 100

In tro d u c tio n ............................................................................................................ .. 1 0 0
M materials and M ethods ................................................................ .. ...... ........ ............... 102
Growth Chambers and Environmental Control..................... ................... 103
G as exchange R ates ............... ........... ........................................... 104
Plant Biomass and Leaf Area ...... .............. ...................... 104
Scanning Electron Microscopy ................................................................. 104
Leaf Stomatal Density and Stomatal Index...... ................... ................... 105
R e s u lts ............... ........ ..... ..... ... ..................... ....................................... 1 0 5
Seed Germination and Plant Growth in Hypobaria................. ................... 105
CO2 Assimilation Rates (CA) ...................................... 106
Transpiration R ates (T) ................................................. ..... ... ................. 107
Water Use Efficiency (WUE) ...... .................. ...................... 108
Stom ata D evelopm ent ..................................... .. ....... ........................... ... 108
D is c u s s io n ............................................................................................................. .. 1 0 9
S e e d G e rm in atio n ..................................................... ................... ............. 10 9
S eedling G row th in H ypobaria ...................................................................... 110
Gas Exchange, CO2 assimilation, and Transpiration in Hypobaria................112
Short-term Acclimation to Hypobaria...... .... ........................................ 114
Long-term Acclimation to Hypobaria ...... .... ........................................ 116
S u m m a ry ............................................................................................................... ... 1 1 9









6 TRANSPIRATION MODEL PERFORMANCE AT REDUCED PRESSURE....... 131

In tro d u c tio n ...............................................................................................................1 3 1
M ateria ls a nd M eth o d s .......................................... .. ...................................... 13 2
S sensitivity A analysis ............................................................................................. 132
R results and D discussion ...................................................................................... 133
C conclusion ............... ...................................................................... ...... 134

7 SUMMARY AND FUTURE WORK............................................. 142

APPENDIX: CR10 PROGRAM ......................................................... ................ 146

0 2 R ead ing .................................................................................. ........................ 146
0 2 C a lib ra tio n ........................................................................................................... 1 4 6
R H R e a d in g 1 .......................................................................................................... 1 4 7
R H C a lib ra tio n .......................................................................................................... 1 4 7
P pressure R leading ............................................................................................... 147
P ressu re C a lib ratio n ........................ ............................................ ................ 14 8
Pum p R elay C control .....................................................................................148
C O 2 R e a d in g ............................................................................................................ 1 4 9
L ig h t R e a d in g ........................................................................................................... 1 4 9
L ig h t C a lib ra tio n ....................................................................................................... 1 4 9
Therm ocouple R leading ......................................................................................149
P ro g ra m ............................................................... .................................................... 1 5 0

LIST O F R E FER EN C ES ...........................................................................................153

B IO G R A P H IC A L S K E T C H .............................................................................................. 16 2






















8









LIST OF TABLES


Table page

1-1 Total pressure and gas composition used to support life in various space
m is s io n s ............................................................................................................... .. 3 8

1-2 Hypobaria studies at various total pressures and gas composition .................. 39

4-1 Set points for the environm ental conditions .................................... ................ 98

4-2 The effect of pressure and (carbon dioxide) CO2 on plant growth (dry and
fresh weight), water content, CA and dark respiration (DR) and transpiration
rates of 26-day-old radish plants grown for six days at the given pressure
tre a tm e n t............................................................................................................. .. 9 9

5-1 Number of days plants exposure to normal pressure and hypobaria. ............. 127

5-2 Environmental conditions used for the experiments ................. ................ 128

5-3 Average fresh weight (FW), dry weight (DW) of shoots, roots, hypocotyls in
hypobaria treatments (A-D) and control treatment (E).................................. 129

5-4 Average specific leaf area, stomata density, stomata index and average
stomata pore length from leaf of hypobaria (33 kPa) and normal pressure
(1 0 1 k P a ) ......................................................................................................... 1 3 0

6-1 Parameters and reference values used in the model ................................. 139

6-2 Parameter description and reference values used for sensitivity analysis........ 140

6-3 Sensitivity analysis of the transpiration model at various conditions.. .............. 141









LIST OF FIGURES


Figure page

1-1 Theoretical depiction of a future Martian farm. Courtesy: nasa.gov.................. 36

1-2 Simplified equations showing human respiration (top) and plant
photosynthesis (bottom ) ...................................... .. ......... ... ..... ... .. ...... ........ 37

2-1 Low Pressure Growth Chamber (LPGC)......................................................... 53

2-2 Low Pressure Growth Chambers developed for this objective with gas tanks,
datalogger (C R 10) and PC interface .............................................. ................ 54

2-3 Calibration of the pressure sensor comparing pressure gauge reading
against m illivolts readings. ...... ............................................................ 55

2-4 Calibration of the relative humidity sensor against known saturated salt
s o lu tio n s .............................................................................................................. .. 5 6

2-5 Calibration curves of an oxygen sensor at different pressures (5A) and
curves of slope and intercept (5B) .................................................. ................ 57

2-6 Calibration curves for a CO2 sensor at different pressures (A) and curve of
slope and intercept (B) ..................................................................... 58

2-7 CO2 sensor reading against the equivalent syringe volume................................. 59

2-8 Calibration for a load cell used to measure the weight of the flask containing
p la n ts ............................................................ ..... ................................................ ... 6 0

2-9 Calibration curve for light sensor readings against handheld light meter ............ 61

2-10 Pressure (-33 kPa), 02 (-20 kPa) and temperature (-23 C) recordings for
five hour for chamber A (top panel), chamber B (middle panel) and chamber
C (b otto m p a ne l)............................................................................................. 62

3-1 The individual mass diffusivity of CO2 and H20 calculated from the binary
mass diffusivity using the empirical formula given by Fuller et al. (1966) ........... 77

4-1 Schematic of the low pressure growth chambers used for experiments. Each
chamber has 0.09 m3 total internal volume.. .................................................. 93

4-2 Radish (26 day-old) grown for six days in super elevated CO2 (180 Pa) at
101 (first row), 66 (second row), or 33 (third row) kPa total pressure.................. 94

4-3 Total leaf area of radish grown at various total pressures and CO2 levels ....... 95









4-4 C02 drawdown curves on the final day of the experiment of 26 day-old radish
grow n for six days at 33, 66, or 101 kPa......................................... ................ 96

4-5 Water loss due to transpiration of 26 day-old radish grown for 7 days for low
(40 Pa; A), high (100 Pa; B) or super-elevated CO2 (180 Pa; C) at 33, 66, or
101 kP a total pressure. ......................... ........................................................... 97

5-1 Germination of radish seedlings at 33 kPa total pressure and various p02.
Bars represents the mean STDEV (n=2) .............. ................... 121

5-2 Radish plants after four weeks of growth Treatment A (28 days at 33 kPa),
Treatment B (21 days at 33 kPa), Treatment C (14 days at 33 kPa), and
Treatment D (2 days at 33 kPa)...... ...... ......................... 122

5-3 Carbon dioxide assimilation (CA;A), Transpiration rates (B) and leaf area (C)
on the last day of the experiment. .............. ........................... 123

5-4 CA (A) and Transpiration rates (B) and Water Use Efficiency (WUE) (C) on
the day 7, 14, 21 and 28 of the experim ent...... ...................... ................... 124

5-5 Scanning electron microscopy (SEM) image from a youngest leaf of a plant
grown entirely in hypobaria (A; 33 kPa) or in normal pressure (B; 101 kPa)..... 125

5-6 The long-term and short-term responses of the shoot or root to water deficit,
low humidity and high temperature (adapted from Chaves et al., 2003) ........ 126

6-1 Predicted transpiration rates from 33 to 101 kPa.................... ................... 135

6-2 Predicted transpiration rate at various total pressures (33, 66, 101 kPa) and
various stomatal widths (0.0001, 0.0003 and 0.0004 cm). ............................... 136

6-3 Predicted transpiration rate at three different pressures (33, 66 and 101 kPa)
and two different vapor pressure deficit (VPD) levels (0.6 kPa and 0.9 kPa).... 137

6-4 Predicted transpiration rates at stomata numbers .................... ................ 138











LIST OF PARAMETERS


P pressure (kPa)
VPD vapor pressure deficit (kPa)
RH relative humidity (%)
pCO2 partial pressure of CO2 (Pa)
pO02 partial pressure of 02 (Pa)
L leak rate (% Vol/day)
t time interval (h)
Po initial pressure (kPa)
Pi end pressure (kPa)
Ca CO2 concentration outside leaf air space (Pa)
Ci CO2 concentration inside leaf air space (Pa)
CA CO2 assimilation rate (pmol m-2 S-1)
DR Dark respiration rate (pmol m-2 S-1)
T transpiration (mmol m2 s-1)
FW fresh weight (g)
DW dry weight (g)
WUE water use efficiency (mol CO2 mol H20-1)
k the Boltzmann constant, ergs/K
m molecular weight ( g mol-1)
CAB collision diameter, cm
a stomata length (cm)
b stomata width (cm)
d stomata depth (cm)
D diffusivity (cm2 S-1)
n number of stomata
hs stomatal conductance (cm s-1)
hb boundary layer conductance (cm s-1)
K thermal diffusivity (cm2 S-1)
L length (cm)
Kv kinetic viscosity (cm2 S-1)
u air speed (cm s-1)
Re Reynolds number
p viscosity ( g cm-1 s-1)
p density (g cm-3)
D diffusivity (cm2 S-1)
Sc Schmidt number










Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy

GAS EXCHANGE AND ACCLIMATION OF RADISH IN REDUCED PRESSURE
ENVIRONMENTS

By

Hemant Laxmansinh Gohil

August 2010

Chair: Melanie Correll
Major: Agricultural and Biological Engineering

Plants grown on long-term space missions will likely be grown in low pressure

environments (i.e., hypobaria). In general, the fundamental growth and photosynthesis

of plants grown in hypobaria are similar to plants grown at normal atmospheric

conditions with some exceptions. For example, transpiration rates can be elevated in

low pressure resulting in plant wilting if water is not readily available. Plants may also

exhibit poor growth and germination if the partial pressures of critical gases are not

maintained to certain levels (e.g., pO2 for seed germination and pCO2 for

photosynthesis). Since the gas phase of the environment is so critical for successful

growth of plants in both normal and low pressure environments, the effects of the gas

phase composition, particularly C02, on the gas exchange and growth of radish

(Raphanus sativus var. Cherry Bomb II) in hypobaria were studied. Low pressure

growth chambers were built that could monitor the environmental parameters for these

studies. The fresh weight (FW), leaf area, dry weight (DW), CO2 assimilation rates (CA),

dark respiration rates (DR), and transpiration rates from 26 day-old radish plants that

were grown for an additional seven days at different total pressures (33, 66 or 101 kPa)









and pCO2 (40 Pa, 100 Pa and 180 Pa) were measured. In general, the dry weight of

plants was enhanced with CO2 enrichment and with decreased total pressure. In limited

pCO2 (40 Pa), the transpiration for plants grown at 33 kPa was over twice that of

controls (101 kPa total pressure with 40 Pa pCO2). Increasing the pCO2 from 40 Pa to

either 100 or 180 Pa reduced the transpiration rates for plants grown in hypobaria and

at normal atmospheric pressures. Plants grown at lower total pressures (33 and 66 kPa

total pressure) and super-elevated pCO2 (180 Pa) had evidence of leaf damage. Taken

together, radish growth can be enhanced and transpiration reduced in hypobaria by

enriching the gas phase with CO2 although at high levels of CO2 leaf damage can occur.

Since the diffusivities of gas increases as the atmospheric pressure drops, it is

expected that transpiration and CO2 assimilation in plants would increase as plants grow

in hypobaria. A mathematical relationship based on the principles of thermodynamics

was developed for calculating the transpiration and photosynthesis for plants. Stomatal

conductance is sensitive to total pressure. At 33 kPa total pressure, stomatal

conductance increases with the boundary increasing by a factor of ~1.7, thus the

boundary layer thickness conductance increases by 70%. Since the leaf conductance is

a function of both stomatal conductance and the boundary layer conductance, the

overall conductance will increase resulting in significantly higher levels of transpiration

as the pressure drops. The conductance of gases is also regulated by stomatal aperture

in an inverse relationship. Stomatal aperture is directly influenced by concentration of

CO2 inside the leaf space. The higher CO2 concentration inside the leaf air space during

low pressure treatments may result in stomata closing partially or fully which may

reduce the excessive transpiration caused by increased diffusivity. Therefore, a reduced









pressure environment with high C02 may be an ideal scenario for minimizing

transpiration and maximizing the plant biomass yield in BLSS. The application of this

model to data for plants acclimated long term to hypobaria (33 kPa) and reference

values (101 kPa) suggest that for transpiration the model predicts transpiration for plants

that are grown at normal pressure fairly well, but for those grown at 33kPa the model

over predicts transpiration observed data by almost

To understand the mechanisms of plant adaptation to hypobaria, plants were

transferred to low pressure at different stages of growth (0, 2, 14, and 26 days after

germination). The growth, CA, transpiration and stomatal density were compared

between these plants. Plants exposed short term to hypobaria (< 2 days in hypobaria)

responded differently than those exposed to long-term hypobaria (>2 days). For

example, plants that were grown entirely in hypobaria had smaller and thicker leaves

compared to plants that were exposed for 2 days or less to hypobaria. These long-term

treated plants also had higher CA (p<0.1) and transpiration rates (p< 0.05) even though

their overall growth (FW and DW) was not significantly affected by hypobaria. The

stomatal density of plants grown long term in hypobaria was not significantly different

than plants grown short term to hypobaria. Therefore, it appears that plants may

respond to enhanced gas exchange in hypobaria by reducing their leaf area. Further

studies on the mechanisms of plant adaptation are required to identify other biological

or physiological mechanisms of plant acclimation to hypobaria.

Some of the engineering constraints to grow plants in Martian plant growth

facilities can be offset by growing plants at reduced atmospheric pressure.

Characterization of hypobaria response at reduced pressure can provide data which









can help in explaining some of the response which plants may encounter on Martian

facility. These studies are important for understanding mechanism by which plant

control water relations.










CHAPTER 1

ATMOSPHERIC CONDITIONS USED FOR BIOLOGICAL SYSTEMS DURING SPACE
MISSIONS: IMPLICATIONS FOR HYPOBARIC PLANT BIOLOGY

Advanced Life Support Systems (ALS)

Long-term, space exploration with humans will require an Advanced Life Support

system (ALS) that provides air, water, and food to explorers in a sustainable manner.

This system will consists of the latest technologies for atmosphere revitalization, water

supply, and food and fiber production as well as the recycling of these valuable

resources. These technologies may be based on physico-chemical (P-C) or biological

approaches. The P-C approach uses mechanical or chemical mechanisms to provide

an ALS whereas the biological approach, the Biological Life-Support-System (BLSS),

uses biological systems (e.g., plants, algae or microbes) to supply the requirements for

ALS. The efficiency of each approach has been compared and estimates of the time

until the system has reached sustainability have been modeled. This time to reach

sustainability is an important aspect for cost analysis and as an indicator to how long a

space colony can be supported without re-supply from Earth. Unfortunately, the results

of these studies are conflicting as to which method is more efficient for long-term space

missions. For example, Alan Drysdale (Boeing Corporation; Drysdale, 2001) suggests

that the BLSS may take three years for sustainability making it a viable option for ALS.

In contrast, Harry Jones (NASA- Ames: Jones, 2007) suggests that the BLSS could

take up to ten years to reach a sustainable state and therefore he supports a P-C

approach. Barry Fingers (Dynamac Corp., Fingers et al., 1996) suggests the ideal

scenario is the hybrid of both the approaches, where P-C approach is used for the initial

one to two years until the BLSS can become sustainable. The P-C approach has









already been used on the International Space Station (ISS) with much success but the

costs associated with transport of materials for P-C systems are considerably lower for

the station that is ~375 kilometers above earth than for distant planets. Arguments for

the P-C approach suggest that the advancements in water and air recovery and

biomass processing make the P-C more cost effective and reliable than BLSS.

Whereas, the supporters of the BLSS suggest that eventually a biological system for

material recycling will be required for long-duration missions and for their benefits to

explorers living with plants and having a regular supply of fresh food (Wheeler, 2004).

As one of the major components of developing a BLSS, the environment, including the

gas phase, temperature and pressure used to support the biological system must be

carefully monitored and controlled. This chapter discusses the environments (i.e., gas

phase composition and pressures) that have been used in the past to support life on

space missions and describes the effects of these types of environments on plants as

part of a BLSS.

Gas Phase for Biological Life Support

Here, a brief history of the environments that have been used for life support is

described with emphasis on those systems that have been used for humans and plants.

The first biological system to go up in space was a dog named Laika sent by the United

States of Soviet Russia (USSR) on Sputnik 2 on October 4, 1957. The pressure and

atmospheric composition of the vessel was controlled with an oxygen generator, CO2

and vapor scrubbing system with pressures and gas concentrations (CO2, N2 and 02)

maintained to levels of Earth sea level (Table 1-1). Unfortunately, the inadequate

temperature control system resulted in the heatstroke and the death of Laika within a

few hours (Malashenkov, 2002). After Laika, the USSR sent several dogs and cats in to









space of which some returned safely. Shortly thereafter (1958-62), during the Mercury

missions, the US sent monkeys into space with a total atmospheric pressures of -34

kPa at 100% oxygen. The advantage of using the low pressure in this case was that it

required less total gas to be transported from Earth to support life and thus increased

mission duration. In addition, the low pressure required a much lighter hull for the craft

since a smaller pressure difference between the inside of the craft and the vacuum of

space was maintained. High levels of 02, greater than 35 kPa, (Baker 1981), can be

toxic to humans and animals, thus 34 kPa of total pressure of 02 was chosen as the

highest pressure. This atmospheric composition and pressure was used for the

Mercury, Gemini, and Apollo space missions (Martin and McCormick, 1992).

The success of these first missions with animals led to more challenging human

missions by the USSR and the USA. On April 12, 1961, the first human, Yuri Gagarin,

was sent to space by the USSR in Vostok 1. This vessel had sea level atmospheric

conditions (100 kPa total pressure, 0.04 to 0.2 Pa CO2, and 21 kPa 02). The gas

composition and high pressure of the Vostok missions required strong materials for the

cabin construction and the necessity for prolonged de-nitrogenation before any extra-

vehicular operation since suits on these missions were much lower than cabin pressure.

As a benefit of this gas composition and pressure, the lower oxygen concentration of

the atmosphere prevented fire hazards and allowed for a more earthlike environment for

biological studies (Baker, 1981). The oxygen was provided by potassium superoxide,

CO2 and excess water was absorbed by lithium hydroxide. These atmospheric

conditions were used throughout the USSR program up until Mir (Baker, 1981). Shortly

after the USSR manned space mission on May 5, 1961, the US sent Alan Shepherd to









space on Mercury Freedom 3 in similar atmospheric conditions as were used for the

monkeys in the past Mercury missions (-34 kPa at 100% oxygen). A total of six manned

missions were performed during the Mercury period using this enriched oxygen, low-

pressure environment.

The first two-person mission and the first mission with the cosmonauts without a

spacesuit were in Voskhod 1 (Oct 1964). The Voskhod spacecraft were modified Vostok

vessels that had a parachute system removed to allow for a second cosmonaut. The

atmospheric conditions were similar to those of the Vostok missions (100 kPa, 0.04 to

0.2 Pa C02, and 21 kPa 02). Voskhod 2 (Mar 1965) was the first mission that included a

human spacewalk by Alexei Leonov. This required for extra vehicular activities the

cosmonaut had to reduce the pressure in his suit to either 40.6 kPa or 27.4 kPa total

pressures before exiting the vehicle which put the cosmonaut in danger of getting the

bends (Baker, 1981). There was some difficulty entering the airlock on the vessel due to

the rigidity of his pressurized suit relative to the airlock. The suit was designed to

provide 45 minutes of oxygen for breathing and cooling and allowed for venting of gas

and vapor to space.

After the success with upper orbit (Earth) flights, missions to the Moon became the

focus of space exploration. However, before the astronauts could land on the moon

several manned Gemini missions (1965-66) were used to survey the Moon and test the

capabilities of the rockets and for landing. As a part of Biostake series of experiments,

corn and mustard seeds were carried by astronaut Ed White in his space suit (at

25.5kPa, 100% oxygen; Grimwood et al., 1969) during the first US extravehicular

activity (EVA) on Gemini 4 (1967). Though seeds were germinated during subsequent









growth studies on Earth at ambient pressure, abnormality in the plants was observed

likely due to heavy ion radiation (Paul and Ferl, 2006). After Gemini, the Apollo missions

were developed to bring astronauts to the moon. Unfortunately, on January 27, 1967,

the Apollo 1 mission ended in disaster with a fire that killed the three astronauts on

board while on Earth (Edward White, Virgil Grissom, and Roger Chaffee). The fire was

caused by the high flammability of a pure oxygen environment inside the capsule (at

-100 kPa; Apollo 204 Review Board Report, 1967). This high pressure was required

during launch since the craft were not built to withstand high pressure differences

between the environments inside and outside of the vessel. A similar incident occurred

earlier in March 1961 in the USSR which claimed the life of Soviet cosmonaut trainee,

Valentin Bondarenko, when a fire started in the pure oxygen atmosphere in the

chamber he had been in, although this incident was concealed from the public for years

(Oberg, 2009). As a result of the Apollo fire, NASA set new criteria for gas phase

composition for space missions. These included that the gas phase would consist of

60% oxygen and 40% nitrogen at roughly sea-level pressure at launch, then the

pressure was lowered by releasing gas to maintain about a 40kPa difference between

the inside and outside of the vessel during ascent until the atmosphere reached

approximately 34kPa and 100% oxygen during the first 24 hours in space. The

astronauts were acclimated in space suits from the time of launch at 100% oxygen (34

kPa), approximately three hours before launch to prevent the bends during the

depressurization and ascent. Once the craft reached the set point atmospheric

conditions, the astronauts could remove their suits and move about the cabin. These

Apollo missions ran from 1967-72. Unfortunately, the hazards of oxygen flammability









were realized again during the Apollo-13 mission, when, on the return flight to Earth the

oxygen gas tank was damaged and ended up starting a fire. To address this, the Apollo

14 mission had several modifications to the gas tank systems. Interestingly, the Apollo

14 mission had one of the first long-term treatments of seed in space when astronaut

Stuart Roosa, the flight commander, carried 500 seeds in his personal belongings and

brought them back to Earth. The seeds were of several tree species including

sycamore, pines, and fir. After the 9-day mission, these seeds were given to several

educational institutes, including the University of Florida, the trees germinated from

these seeds appear to have no signs that the mission to the moon had negatively

affected their growth (Klein, 1981).

During the Biosatellite program (1966-1969) NASA sent the Bion series of

satellites with specimens of fruit fly, frog eggs, bacteria and wheat seedlings to study

the effects of weightlessness on living organism. Total pressure was maintained at

approximately 100 kPa and oxygen partial pressure was 21 kPa. In the Biosatellite II,

pepper plant experiment, a camera recorded the positions of plant leaves with respect

to time to study the effect of microgravity on plant movement (Thimann, 1968).

In order to conduct long-term research on living systems in space as well as to

establish a space laboratory for small animals, plants, microorganisms, and humans,

the USSR launched Salyut (1971-82) and the USA launched Skylab (1973-79). Salyut

was at approximately sea level pressure and atmosphere composition. Salyut carried

the Oasis, the first of its kind plant growth system with a cinematic recording system

(Neichitaleo and Mashinski, 1993). It was used to cultivate Brassica capitata, Linum

usitatissium and Allium porrum. The biological experiments also included variety of









species including spiders, worms, fishes, and frogs. Many of the experiments were

performed to study the effects of radiation and space environment on biological

systems. In contrast to Salyut, the Skylab platform was operated at 34 kPa total

pressure but the atmospheric composition was 70% oxygen and 30% nitrogen. Nitrogen

helped reduce the risk of pure oxygen while still maintaining the health of the Astronauts

from hypoxic conditions. One of the plant experiments (Skylab experiment ED-61/62)

studied phototropism of tomato in low gravity. This was the first time the seeds were

germinated in agar in space microgravity. These studies also compared various light

intensities that are required to produce photosynthesis for plants grown in space

(Cramer et al., 1984)

Interestingly, a combined Skylab-Salyut station was proposed by the US and the

USSR. But, as noted above, each station had different gas mixes and pressures. Since

each station would need to make modifications, it was proposed that a 55kPa total

pressure environment, slightly enriched in oxygen would be used to compromise and

minimize the modifications required for one station. However, this combined station

was never realized.

To replace Salyut, the USSR built Mir (1980 -98). The Mir, the Russian word for

world or peace, station had an atmosphere of 101 kPa total pressure and earth ambient

concentration of gases (21 kPa 02, 80 kPa N2 and 0.04-0.2 kPa CO2). Seed

germination experiments on board Mir were done with variety of small, closed or

ventilated/partially ventilated chambers, from simple a beaker containing moist soil

(Kosmos 1129; Parfeenov and Arbanova, 1981) to a sophisticated greenhouse, called

Svet, the Russian word for cosmos (Nechitailo and Mashinsky, 1993). During the early









experiments on seed germination, plants exhibited poor growth and stress (Kordyum et

al., 1985). This was possibly due to the lack of natural convection of air movement in

microgravity leading to formation of stagnant air layers around seedlings within the

closed chamber (Musgrave et al., 1988). Another attempt to germinate seeds and

complete the life cycle in space resulted in a failed experiment (Mashinsky et al, 1994).

However, a complete plant life cycle was performed with wheat grown in the 'Svet'

greenhouse which had a better control system for the gas phase (Svetlana et al., 2005).

However, after these plants were returned to Earth (STS-81, 1997), it appeared that the

wheat flowers had sterile seeds. Initially, microgravity was assumed to be the cause of

the seed sterility but when researchers grew a dwarf wheat variety at the same levels of

atmospheric composition that the super dwarf was exposed to in space, they found

higher concentration of ethylene on the station to be the cause (Salisbury 1995). Plants

tolerate up to 4-5 ppb concentration of ethylene, with anything higher resulting in

reduced plant growth. Salisbury et al. (1995) found that the ethylene in the growth

chamber was as high as 1-2 ppm, ~1000 times higher than the tolerable limit for plants.

Since ethylene produced by plants was indicated as the source of ethylene and since

convection is limited in microgravity, these researchers developed a dwarf variety of

wheat (Apogee) that is insensitive to higher concentrations of ethylene. The Apogee

wheat produced non-sterile flowers and viable seeds on board Mir and the ISS

suggesting that ethylene and not microgravity was the cause for flower sterility found in

previous missions (Levinskikh et al., 2000).

Due to the problems with high ethylene and CO2 concentrations in the cabin of

Mir, which were harmful to plant growth, it was decided that an independent,









controllable growth chamber was required to conduct specialized experiments which

would protect plants from exposure to cabin level CO2 and ethylene. Therefore, the Svet

greenhouse (1990-2000) was built by Russia and was the first automated plant growth

facility. The greenhouse was at 101 kPa total pressure and atmospheric composition

similar to Earth sea level except CO2 levels were up to 0.2 kPa. However, it had open

type air ventilation system that had air in contact with the Mir cabin atmosphere

(Svetlana et al., 2005). Improvements to the plant growth chambers were made to

SVET in the late 1990s which included the ability to monitor the plant growth in real time

by measurement of CO2 and H20 exchange rates, temperature, and relative humidity.

This allowed the measurements of photosynthesis and transpiration rates based on the

current plant CO2, vapor, temperature and relative humidity levels. However, the open

air system was not ideal for careful control of gas phase surrounding plants.

After the Biosatellite program was cancelled in 1968, it was not until 1982 during

the third space flight program (STS-3) that the first Plant Growth Unit (PGU) was

launched by the USA to perform seedling growth experiments (Cowles et al., 1984).

PGU served for 15 years for use on plant growth studies before Astroculture was

introduced. The Astroculture Growth Chamber (ASC-GC) was the first of its kind. It was

a completely automated growth chamber developed in the early 1990's to provide

support system for plant growth in closed environment. It was developed by the

Wisconsin Center for Space Robotics and Automation at the University of Wisconsin,

Madison. The CO2 was maintained in the range of 300 2000 ppm and the ethylene

concentration could be reduced to less than 50 ppb using a catalytic ethylene scrubber.

The main objective of ASC-GC was to perform short- and long-term plant experiments









in microgravity to study seed-to-seed cycle in space with automatic control for up to 30

days. The ASC-GC hardware was efficiently used for studies on Arabidopsis (STS-68).

In one study, the reproductive ability of pre-germinated Arabidopsis seedlings in various

gas compositions and ventilation regimes were studied. At a CO2 concentrations of 300-

2000 ppm Arabidopsis had sterile pollen and embryos, and at very high CO2

concentration (8000 ppm) plants exhibited early abortion of ovules and mature pollen,

and the release of pollen from anthers was restricted preventing fertilization (Kuang et

al., 1996). The Mir station was in operation for fifteen years until March 23, 2001, when

it was deliberately de-orbited, breaking apart during atmospheric re-entry over the South

Pacific Ocean.

The International Space Station (ISS)

The International Space Station (ISS) is a joint project of several space agencies

across the world led by NASA. The ISS construction began in 1998 and has had

continuous human presence since November 2000. The ISS platform runs at 101 kPa

total pressure with Earth sea level concentration of the gases. Advanced Astroculture

made its debut flight during the second ISS increment to study the effects of

microgravity on seed to seed development of Arabidopsis thaliana. The main objective

of this study was to grow a second generation of plants using the first generation of

seeds and harvesting the living plant tissues for gene expression analysis (Fourth and

Fifth increment; Link et al., 2003).

The completion of a complete life cycle by Brassica rapa L and wheat plants of

Apogee variety in the Svet greenhouse on board Mir indicated that plants can be grown

in consecutive generations in space (Levienskikh et al., 2000; Musgrave et al., 2002).

During March 2003 to April 2005, a Russian group led by Sychev studied five









consecutive generations of genetically engineered dwarf green peas in a greenhouse

called LADA in the Russian module of ISS. The LADA has mainly earthlike atmospheric

pressure and gas concentrations of CO2, N2 and 02; though CO2 concentration can

reach up to 0.1 kPa. They reported that pea plants grown over a complete ontogeny

cycle on board ISS were similar to ground controls in terms of plant development and

genetic characteristics (Sychev et al., 2007).

Advanced Plant Studies on the ISS

The ISS now has more sophisticated plant growth chambers such as the

European Modular Cultivation System (EMCS). The EMCS was launched in February

2007; as one of the European Space Agency (ESA) contributions to the ISS. It is the

part of European Columbus Lab section of the ISS. It has two centrifuge rotors, which

can provide different gravitational accelerations from 0.001 to 2g (Brinckmann, 2005).

The advantage of EMCS is that the hardware can be experiment specific. The

incubation chamber controls relative humidity down to 30 %, oxygen at 0-21 % and CO2

from 200- 2000 ppm. The EMCS also has an ethylene scrubber which prevents

ethylene accumulation. The EMCS was originally designed for plant experiments but

due to the recent budget cuts to the space program, it has also been used for other

organisms such as fruit flies (Brinckmann, 2005).

The US and the USSR have chosen different levels of gas phase composition and

total pressures to maintain environments for life support. In the future, it is likely that life

support systems will be maintained at lower pressures for the cost savings in

transporting valuable gases and reduction in the cost of materials to maintain structural

integrity of chambers used to house living systems. The effects of low pressure on

plant growth are described in the next section.









Hypobaric Plant Biology

Plants will be an integral part of BLSS for human space exploration since they can

provide a source of food, can revitalize air and water for human use, and offer

psychological benefits to space travelers during long-term missions away from Earth

(Figure 5-2). During many of these missions, plants will likely be grown at reduced

atmospheric pressure in environmentally controlled chambers (Paul and Ferl, 2006).

The low pressure environment will minimize the amount of atmospheric gases that need

to be transported from Earth and the low pressure will require less structural mass of

the chambers to withstand the low pressure/vacuum environments found in space or on

many planets or moons. The study of plants in low pressure is termed hypobaric plant

biology and, on Earth, it includes the study of plants at high altitudes. The existence of

flora and fauna at very high altitudes, up to 5000 m (total atmospheric pressure of 55

kPa) with warmer temperatures suggests that plants have existing mechanisms for

growth at low pressure at least down to approximately 1 of an atmosphere (Korner,

2004). Because plants will likely be grown in low pressure environments on the Moon or

Mars, a brief history of hypobaric plant biology as it relates to space biology is

described.

The Effects of Hypobaria on Seed Germination and Plant Growth

Successful crop growth in hypobaria depends on a variety of interacting factors

such as the relative gas composition (particularly 02 and CO2), absolute total pressure,

vapor pressure deficit (VPD) of the atmosphere, and the temperature, among other

factors. Many studies have been performed to identify the effects of low pressure on

germination, plant growth and development and many of these studies have recently

been reviewed (Paul and Ferl, 2006) but are described here briefly and summarized in









Table 1-2. In one of the first experiments on germination in hypobaria, rye seeds that

were grown in approximately 3 kPa total pressure with Mars level carbon dioxide partial

pressure (0.45.kPa) without oxygen had no seed germination (Siegel, 1963). However,

at 3kPa of pure oxygen the seeds germinated. Other studies also report seed

germination at partial pressure of oxygen of 5-8 kPa at various total pressures

(Musgrave et al., 1988; Schwartzkopf and Mancinelli; 1991). These studies indicate that

for many species the lower limit for germination in a pure oxygen environment is at

approximately 5kPa.

Although seed germination appears to be restricted to a small window of partial

pressure of oxygen, the results on the effects of these low pressures on plant growth

are conflicting. For example, tomato plants had reduced growth in 40 kPa compared to

control (93 kPa) in one study (Daunicht and Brinkjans, 1992), but showed enhanced

growth at 33kPa compared to control in another (Rule and Staby, 1981). Mungbean

growth was also enhanced in hypobaria at 22 kPa total pressure (Musgrave et al.,

1988). These differences in responses were attributed to the differences in composition

of the atmosphere with respect to CO2 and 02. Reduced growth may have been due to

the lower partial pressure of oxygen (pO2at ~8.4 kPa) which can induce hypoxia stress

in plants and result in poor growth (Musgrave et al., 1988). In general, for short-term

studies in hypobaria, plants have increased growth if enough oxygen and water are

supplied. This increased growth is partially attributed to enhanced photosynthetic rates

of the plants as a result of reduced pO02 or alternatively, due to the increases in diffusion

of CO2 (Iwabuchi et al., 1996; Corey et al., 1996, 2002; Goto et al., 1996; Richards et

al., 2006). Lower pO02 inhibits photorespiration since CO2 has less competition with 02









for the enzyme Ribulose Bisphosphate Carboxylase/Oxygenase (RUBISCO), the first

step in carbon fixation (Drake et al., 1997). The increased photosynthesis at low

pressure and low pO02 was decreased by injecting 02 into the environment suggesting

that indeed the ratio of CO2 to 02 was the cause of the increased growth in some

studies (Corey et al., 2002). Even under normal pressures (101 kPa), Arabidopsis had

increased photosynthetic rates when plants were grown in hypoxic conditions (2.1 kPa

ppO2; Richards et al., 2006). Further studies with Arabidopsis with greater range of

pressures showed less of a difference in carbon dioxide assimilation (CA) after 16 hours

in hypobaria relative to controls at 100 kPa compared to plants that were exposed to

hypobaria for only 1 hour, this suggests a possible adaptive response after 16h in

hypobaria (Richards et al., 2006). In rice, the negative effects of hypoxia on growth at

25kPa total pressure were alleviated by maintaining the pO02 at 10 kPa (Goto et al.,

2002). Therefore, the benefits of low pO02for increased photosynthesis in hypobaria

need to offset the negative effects of hypoxia on plant growth. Others suggest that the

enhanced diffusion of CO2 in hypobaria to the leaf allows for improved CO2 fixation and

this may account for the increased growth for plants grown in hypobaria (Goto et al.,

1995; Daunicht and Brinkjans, 1996; Massimino and Andre, 1999). In contrast, some

long-term experiments reported that plants had similar rates of photosynthesis in

hypobaria as those grown in normal atmospheric conditions and had no increase in

growth (Iwabuchi and Kurata, 1996; Spanarkel and Drew, 2002). This may be a result

of reduced stomata apertures in acclimated leaves during the long-term hypobaria

treatments thus reducing CO2 diffusion into the leaf (Iwabuchi and Kurata, 1996).









Further studies on the dynamics of C02 diffusion to the leaf and its effects on plant

growth are required to understand this response.

Other gases in the hypobaria environment also diffuse at a greater rate relative to

high pressures environments and these include ethylene and other plant volatiles.

Ethylene is known to cause a variety of responses in plants including enhanced leaf

senescence and reduced overall growth (Finlayson et al., 2004). To study the effects of

ethylene in hypobaria, He et al. (2007and 2009) exposed lettuce and wheat to 25 and

101 kPa total pressure at various oxygen concentrations and monitored ethylene. Their

results suggested that hypobaria does not affect the plant growth and that ethylene

concentration was the same in both the hypobaric conditions and in the normal pressure

controls. However, this low level of ethylene may be a result of the reduced oxygen

partial pressure since this can reduce ethylene synthesis in plants (Burg 2004).

The effects of hypobaria on gene regulation were studied in Arabidopsis plants

grown at 10 and 101 kPa total pressure and at 2 and 21 kPa pO02 (Paul et al., 2004).

This study identified genes that were regulated in low pressure but were not caused by

low levels of oxygen, i.e., not due to hypoxia. These genes that were specifically

regulated by low pressure included genes involved in desiccation-related pathways

such as dehydrins, ABA-related proteins, and cold-responsive (COR)-related proteins

(Paul et al., 2004). This suggests that plants do undergo stress in hypobaria, particularly

water stress. Future studies on the gene expression of plant grown in hypobaria are

required to identify the downstream pathways of this response and the effects of long

term acclimation on gene regulation.









Recent studies on plant growth for radish that were grown entirely in hypobaric

conditions (Levine et al., 2008; Wehkamp, 2009) suggest that plants reduce their

excessive water loss over time in hypobaria by reducing their transpiration and CO2

assimilation rates to levels similar to plants grown at ambient pressure. In spinach, the

transpiration rates were slightly higher during the short-term exposure (1 day) to

hypobaria but after 10 days in hypobaria, both the CO2 assimilation and transpiration

rates were similar to those of plants grown in ambient pressure (Iwabuchi and Kurata,

2003). However, it is still not clear how plants acclimate to long-term exposure. Ground-

based studies have also been performed to test the fruit and nutritional quality of radish

grown at low pressures (33, 66 kPa) and these studies found that the biochemical

composition of the radish was not significantly different from plants grown at ambient

levels for the compounds tested (Levine et al., 2008).

Summary

Maintaining life in space is a challenge. Throughout the history of the space

program, a careful balance of keeping the gas composition at pressures that allow life to

thrive at economical levels while minimizing the dangers of these atmospheres to life

has been a primary goal of engineers. As we advance to the next step of bringing life to

distant planets, further modifications in the atmospheric conditions to support life are

likely. Due to the cost savings for growing plants in reduced pressures, low pressure

growth chambers are likely to be used. Although the exact level of total atmospheric

pressure has not been set, it is suggested that plants will be grown at approximately

55kPa in a Moon or Mars greenhouse (personal communication, Ray Wheeler, NASA).

Studies on seed germination, CO2 enrichment, ethylene evolution, vegetative growth

and even gene expression profiles in low pressure on Earth and in space suggest that









plants can be grown at low pressures and be a viable food source for explorers. These

studies also give ample evidence that the fundamental biological processes of

photosynthesis and growth are essentially the same in low pressure and Earth sea level

pressures. The use of plants in a biological life support system (BLSS) seems to be a

promising approach, although it is far from mature. Since the inception of the concept

'salad machine', extensive research has been performed to study the plant response to

various environmental factors which have contributed to the development of the

hardware that is currently used in space for plant growth. However, there are several

technological and operational issues associated with ALS which will need to be

addressed before humans colonize space. For example, how does a low pressure

environment affect plants over several generations? The genetic studies suggest that

hypobaria may result in a metabolic drain on plants and therefore they may not be

physiologically healthy and over generations these may result in poor growth and

reproductive health. Comprehensive studies on the interacting effects of all

environmental parameters in hypobaria on plant growth and development has not been

performed. As we plan for the future to grow plants on the Moon or Mars, the proper

selection of pressure and environmental parameters is required to ensure a sustainable

plant growth system. The range of gas compositions that are likely to be selected will

depend on not only the species but also the stage of plant development since 02

requirements are different in the vegetative and reproductive periods (Wheeler 2004).

In addition, gas composition may be dictated by the available resources on each planet.

For example, CO2 may be used to pressurize plant growth chambers on Mars due to its









availability. The task of optimizing gas phase composition and pressures for plant

growth for space exploration will require many more studies.

During the last decade, our understanding of the effects of hypobaria on plant

growth from the molecular level to whole plant level has improved (Paul et al., 2004;

Wehkamp 2009). Studies have shown that seed germination, seedling growth and plant

development can occur in hypobaria when pO2, pCO2, and pH2Oare at levels that

support growth. Gas exchange studies on plants grown in low pressure suggest that

plants may have increased CO2 assimilation and transpiration but this may depend on

the duration of exposure to hypobaria (Rule and Staby, 1981; Andre and Massimino,

1992; Daunicht and Brinkjans, 1996; Corey et al., 1997; Iwabuchi and Kurata, 2003).

On Mars, CO2 is readily available and can be used to pressurize the chambers for plant

growth but the effects of very high CO2 concentration on plant growth in hypobaria are

not known. Also, only a few experiments have studied the effects of long-term

hypobaria on plant growth, thus the acclimation mechanisms that plants utilize to adapt

to hypobaria are unclear. Therefore, for this dissertation, the gas exchange rates of

radish in hypobaria in various levels of CO2 are studied and the effects of long-term

hypobaria on plant acclimation are explored. Radish was chosen as the model plant for

these studies since it is selected by NASA as one of the salad crops (Wheeler, 2001), it

has been used in several hypobaria studies (Wilkerson, 2005; Levine et al., 2008;

Wehkamp 2009), and the anatomy of radish lends itself to perform transpiration

studies. The radish roots and leaves are also edible and provide excellent nutritional

value to future space explorers (Levine et al., 2008). To study the gas exchange and

acclimation of radish in hypobaria, the following four objectives were addressed:









1. Design and build four, mid-size, low pressure growth chambers (LPGC) that can
monitor temperature, pressure, humidity, CO2, 02, light and plant weight.
2. Predict the effects of pressure on the diffusivity of H20 and CO2 and the
implication of these on plant growth.
3. Study the interacting effects of hypobaria and pCO2 on plant growth,
photosynthesis, and transpiration.
4. Compare growth, transpiration, photosynthesis and stomata between long- and
short-term acclimated plants in hypobaria.
Structure of the Dissertation

This dissertation is organized into topical research areas that address the above

objectives. Chapter 2 describes the construction and testing of low pressure growth

chambers (LPGCs) that can monitor environmental parameters important for plant

growth. Chapter 2 also describes the procedures for sensor calibration and data

management using data acquisition. The prediction of the effects of reduced pressure

on the diffusivity of H20 and CO2 and the implication of these on plant growth is

described in Chapter 3. Chapter 4 describes the interacting effects of total pressure (33,

66 and 101 kPa) and partial pressure of CO2 (0.04, 0.1 and 0.18 kPa) on growth,

photosynthesis and transpiration of radish plants. Chapter 5, describes studies on the

effects of long-term (1-4 weeks in hypobaria) and short-term (2 days in hypobaria)

acclimation to hypobaria on growth, photosynthesis and transpiration of radish plants. In

Chapter 6, the transpiration model described in Chapter 3 is applied to predict

transpiration rate at various reduced pressures, vapor pressure deficits (VPD) and

stomatal widths. Finally, in Chapter 7, the general conclusions and recommendations

for future studies are stated. The program for sensor and parameter control is included

in the appendices.








































Figure 1-1. Theoretical depiction of a future Martian farm. Courtesy: nasa.gov
































(Adapted from Wheeler et al., 2004)
Figure 1-2. Simplified equations showing human respiration (top) and plant
photosynthesis (bottom). The products of photosynthesis are oxygen (02),
which can be used by explorers and carbohydrate (CH20), which can be used
for food. Through transpiration, plants can be used to purify wastewater, i.e.,
the transpired water can be condensed as clean water.









Table 1-1. Total pressure and gas composition used to support life in various space


rr
Year
1957,
Sputnik

1961-63,
Vostok

1958-63,
Mercury
1964-65,
Voslhod

1965-66,
Gemini
1968,
Biosatellite

1967-72,
Apollo
1973-79,
Skylab


missions.
Atmospheric composition
Sea level gas
concentration and total
pressure (101 kPa)
Sea level gas
concentration and total
pressure (101 kPa)
Total pressure 34 kPa,
Pure 02
Sea level gas
concentration and total
pressure (101 kPa)
Total pressure 34 kPa,
Pure 02
Sea level gas
concentration and total
pressure (101 kPa)
Total pressure 34 kPa,
Pure 02
Total pressure 34 kPa, 70
% 02, 30 % N2


1971-83 Sea level gas
Salyut 1 concentration and total
pressure (101 kPa)
1980-98, Sea level gas
Mir-I concentration and total
pressure (101 kPa)
1998- Sea level gas
ISS concentration and total
pressure (101 kPa)


Comment
Dog (Laika) was the first to enter into space:
died of heat stroke (Maleshankov, 2002)

First human Yuri Gagarin entered into space
(Baker, 1981)

Monkeys and Chimpanzees (Martin and
McCormick, 1992)
Space walk by cosmonauts (Baker, 1981)


Corn (Baker, 1981)

First complete plant growth system by USA,
Gravitropism studies on pepper plants (Baker,
1981)
First Lunar Landing: seed material exposed to
lunar atmosphere (Klein, 1981)
First US Space Station with Astronauts (Klein,
1981)
Orchids for psychological benefit and peas,
Onion cultivated on board (Smolders, 1973)

Plant complete life cycle on several plant
species (Salisbury 1995)

Advanced plant level studies (eg. Gene
expression),(Brinckmann, 2005)










Table 1-2. Hypobaria studies at various total pressures and gas composition.

02 Pressure
Reference Crop CO2 (Pa) (kPa) kPa Comment


Seigel et al., 1963
Mansell et al., 1968
Rule and Staby, 1981
Musgrave et al., 1988
Goto et al., 1995
Corey et al.,1996
Daunicht, Brinkjans,1996
Iwabuchi et al., 1996
Corey et al.,1997a
Massimino and Andre 1999
Goto et al., 2002
Sparnakel and Drew, 2002
He et al., 2003
Iwabuchi and Kurata, 2003
Paul et al., 2004
Wilkerson, 2005
Paul and Ferl, 2006
Richards et al., 2006
He et al., 2007
Levine at al., 2008
He et al., 2009
Wehkamp, 2009
Rajapakse et al., 2009


rye
turnip
tomato
mungbean
spinach
lettuce
tomato
spinach
wheat
wheat
rice
lettuce
lettuce,Wheat
spinach
Arabidopsis
radish
Arabidopsis
Arabidopsis
lettuce
radish
lettuce
radish
lettuce


0.24 %
40
14,28,40
40
50, 100
40, 80
40
40
120
65, 100
50, 100
70
100
40
100
100
100
40, 100
100
100
100


0.1 %
21
3,7,10
2,5,8,21
2,8,15,21
10,21
40
21
14,21
8,18
21
21
6,12,21
20
2,21
21
2,21
5,10,15,21
6, 12, 21
21
12,21
2,6,12,21
6, 10,21


3, 10, 50, 100
50, 93
17,34,51
21-23
50, 75, 101
50, 101
40, 70, 100
25, 101
70
10, 20
50,70, 101
70, 100
30
25
10, 100
33, 100
25, 100
25,50,75,100
25
33, 66, 101
25, 100
10,33,66,101
25, 100


Seeds fail to germinate at 3 kPa total pressure
Transpiration rate increased at 50 kPa
Hypobaria increased CA and biomass
No adverse effects of 22 kPa on growth
No difference in growth at 50, 75 & 100 kPa
At 50 kPa Pn and biomass increased by 25 %
At 40 kPa tomato plant had reduced growth
Growth was same in hypobaria and 101 kPa
Pn rates slightly increased (15%) at 70 kPa
dry mass increased by 75 % at 10 kPa
Growth normal at 50 kPa, reduced at 34 kPa
Slightly enhanced growth at 70 kPa
Ethylene synthesis down by 65% at 30 kPa
At 25 kPa stomata pore size, aperture reduced
over expression of drought related genes
Transpiration enhances at reduced pressure
Expression of ADH gene under hypoxia
Pn rates and growth increased at hypobaria
Oxygen below 12 kPa reduced plant growth
Little effects of hypobaria on nutritional quality
Ethylene reduces growth under hypobaria
Long-term hypobaria reduced transpiration loss
Hypoxia and hypobaria increases phyto-chemical









CHAPTER 2

DESIGN AND CONSTRUCTION OF LOW PRESSURE GROWTH CHAMBERS
(LPGCS)

In order to grow plants at low pressures, a chamber capable of maintaining low

pressure for long periods and a system to control and monitor environmental

parameters such as temperature, light, relative humidity (RH) and gas composition is

required. The objective of this chapter is to describe the design and construction of four,

medium-scale; low pressure plant growth chambers (LPGCs) that were used for the

experiments that are described in Chapters 4 and 5 of this dissertation.

Maintaining a plant growth facility on Mars or the Moon at reduced atmospheric

pressure (hypobaria) compared to Earth sea level pressure will reduce the cost of

launching and transporting the components to build the facility since a smaller pressure

difference between the inside and the outside of the facility would require materials with

less structural mass. This low pressure facility would also need less gas to pressurize

the chamber thus reducing the amount of valuable gases that would be supplied from

Earth. These lighter structural materials will promote ease of construction but they must

also withstand the harsh conditions of a distant planet or moon. Such a LPGC should be

small enough to be efficient in modulating the thermal heat transfer and promote mixing

of the gases but minimize the re-supply of gases such as CO2 for maintaining plant

growth. In contrast, the chamber should be large enough to allow for plant growth

without hindrance to leaf and plant expansion. According to one estimate, the required

area to grow a crop to fulfill 100 % of the food requirements for one person is 50 m2

(Wheeler et al., 2001). Since building of such a large system is costly, the objective of

this project was to build smaller-scale, research chambers (0.09 m3) that could









accommodate three plants while being able to fit all three chambers into a larger

environmentally controlled incubator at UF (Environmental Growth Chambers, Chagrin

Falls, Ohio). Sensors in the LPGC were designed to monitor 02, CO2, relative humidity,

temperature, pressure, light and accommodate three load cells for monitoring

transpiration and plant growth.

Other LPGCs have been built in the past to study plant growth but unfortunately,

for most of these, there is little documentation or details as to the design and

construction of the systems (Purswell, 2002). Most of the LPGCs had limitations in

terms of the range of experiments that could perform either due to the small size or due

to other drawbacks in their construction. For example, Corey et al. (1996) used a

system which had very high leak rates thus the plant growth chamber could not be

operated below 70 kPa. Daunicht and Brinkjans (1996) used a mass flow controller to

regulate the air flow and gas composition and mechanical precision vacuum controller

to maintain low pressure; however specifics about the chamber were not given. In their

system, excess CO2 and ethylene was removed by constantly ventilating by bringing in

the outside air at normal pressure. Schwarzkopf et al. (1991) designed a LPGC which

could be operated down to 1 kPa with leak rates of 1 % chamber volume/h but the type

of sensors used to measure various parameters was not reported. Simpson and Young

(1998) devised a growth chamber to simulate the Martian atmosphere which could

operate down to ~0.133 kPa but the data collection system was not described. Recent

reviews on the design and construction of other LPGCs are described elsewhere (Mu

2005, Wilkerson 2005, Hublitz, 2006).









The low pressure chambers that were built for this project are based on the

designs of previous LPGCs by Wilkerson (2005) and Hublitz (2006) with minor

modifications. The largest of these systems used for hypobaria studies was constructed

to fit inside a large stainless steel vacuum chamber (1.2 m X 1.2 m X 1.0 m) that

simulated the Martian atmosphere of 0.6 kPa (Hublitz, 2006). The LPGC was

constructed with a polycarbonate hemispherical dome (1 m diameter) that served as the

top of a Mars greenhouse and an aluminum flat plate as a base. This chamber was

housed within the larger vacuum chamber. The primary purpose of this LPGC was to

study the heat and mass transfer in two of the main Martian conditions, i.e., reduced

pressure and extremely low temperature. An industrial freezer (~ 2m X 2m X 2 m)

brought the temperature of the vacuum chambers to as low as -22 C. Hublitz (2006)

and used mass flow controllers and solenoid valves to regulate the gases and pressure

and grew lettuce plants for approximately seven days in these chambers. A visual

interface (LabView software, National Instruments, TX) was used to collect the data and

control gas and pressure (Hublitz, 2006). However, localized temperature gradients

within the chamber and difficulties in collecting condensate resulted in large ice blocks

that accumulated in the lower portion of the chamber. To date, this is the only LPGC

that simulated the actual pressure conditions that would be used in a LPGC on Mars,

i.e., the chamber at higher pressures than the surrounding environment but still much

lower than Earth sea level.

Wilkerson (2005) used a simplified bell jar system which had an aluminum base

to house wiring, sensors, a cooling coil and a humidifier with a glass bell-jar top to

house the plants. This chamber was 22 cm in diameter and 38 cm high. These









chambers were mainly used to study plant growth and transpiration in low pressure.

Although they had a relatively small area for plant growth and were awkward to

instrument, the major advantage of these systems were the flexibility in their design with

many ports and the o-ring seal for the bell jar was leak proof. The top was also easy to

replaced before and after experiments. These chambers were used to grow radish at

various pressures for one day. The design of LPGCs described here are larger versions

(i.e., 6 times the volume) of the chambers of Wilkerson (2005).

System Description

The chambers designed for these studies (Figure 2-1) consist of two main

components, a transparent Plexiglas tube for the cylindrical wall of the cover (1.2 cm

thick, at 20 cm in diameter and 60 cm tall) that was topped with a circular (3 cm thick,

20 cm diameter) acrylic disk (Professional Plastics, Fullerton, CA). The top disc had a

groove cut out that had an O-ring (2.57 mm diameter) which provided a gas tight fit with

the tube. The top of an aluminum cube (25 cm X 25 cm X 25 cm) was welded to a

circular aluminum disk (46 cm diameter) that had a square (25 cm X 25 cm) cut out to

open the upper chamber to the lower cube. A circular groove (1.9 cm length) in the

aluminum disk housed a rubber gasket (Gainesville rubber & Co. Inc., FL) and provided

a gas-tight seal when the Plexiglas tube was placed on top and placed under low

pressure. The transparent cover makes it easy to monitor plant growth and allows for

light transmission. These chambers can be run at pressures as low as -2 kPa. The

chamber is large enough to contain three (7.4 cm X 7.4 cm) pots and all the various

sensors to monitor the environment. The aluminum base houses the cooling fan, wires

and circuitry for the many sensors. Four LPGCs (internal volume of 0.09 m3) were









assembled; one for testing and calibrating of the sensors and three for experimental

treatments (Figure 2-1). Six, feed-thru (type K) ports in the sides of LPGC allowed wires

to pass from the inside to the outside of the chamber (PFT2NPT-4CU, Omega Inc,

Stamford, CT). In the studies reported here, only three of the ports were utilized. The

chambers have sensors for temperature (DS-75, DS10 Dallas Semiconductors, Dallas,

TX), pressure (MPXH6115AC6U, ASCX15AN, Sensym ICT, Milpitas, CA), CO2 (T-6004,

OEM ultrasonic, 6004, Goleta, CA), 02 (MAX-250, Matec Co., Salt Lake, UT), relative

humidity (HH-4602-L-CPREF, Honeywell, Morristown, NJ), light (LI-190, Li-Cor, Lincoln,

NB) and three load cell (LPS-0.6 kg, Celtron Tech. Inc. Colvina, CA). A vacuum pump

(373 watts) was used to remove gases from the chambers to maintain the low pressure

environment.

Gas Leakage Tests

One of the objectives for the LPGC was to minimize the amount of gas leakage.

Leaks in the chambers result in the loss of gasses and difficulty in maintaining set points

for experimental procedures. The leak rate will also determine how often the vacuum

pump must be operated to keep pressures at the set levels. Leaks were prevented by

applying vacuum grease to the gaskets that was in the aluminum plate of the base, and

through the use of TeflonTM tape around all screw threads. The use of plastic tubing

instead of copper tubing that was previously used in other chambers (Wilkerson, 2005)

also improved this chamber to make an almost airtight system. These LPGCs are able

to hold a pressure as low as 1.3 kPa pressure with the leak rate of 0.03 kPa/h. This low

level of leakage required minimal use of the vacuum pump during the experimental

procedures (often only once a week). Solenoid valves (F-822-G-4 24VDC, Gulf Controls









Company LCC, Gainesville, FL) were fitted in the vacuum line for air-tight closing of the

system. The following formula was used to calculate the leak rate,


L = (Pi Po)* 100 1440 (2-1)
Pi*t
Where,
L = Leak rate (% Vol/day)
Pi = Initial pressure (kPa)
Po = Final Pressure (kPa)
t = Time interval (hr)
1 Day = 1440 minutes


Data Acquisition and Control System

Wilkerson et al. (2005) used Opto 22 interface hardware (SNAP ultimate brain,

Opto22, Temecula, CA) to monitor plant growth and collect data. Although the hardware

is very effective for monitoring and controlling sensors, it is bulky and is not easy to

modify. The present research used a CR10 data logging system (Campbell Scientific

Inc, Logan, USA). The CR10 is a centralized control system which requires fewer

components to interface with sensors, it is easy to use and scalable. The data logger is

a small computer combined with a sensitive voltmeter that records the voltages (raw

analog signals) from the sensors (Figure 2-2) and can be connected with a PC via a

cable interface. The PC used software PC208 (Campbell Scientific, Logan, Utah) to

convert the voltage data into sensor readings. For example, a 0 to 5 volt signal range

can represent either 0 -100 % relative humidity or 0 100 % oxygen. It provides the

input voltage of environmental parameters at small time interval (programmable) and

data can be saved and logged either on the data logger or on the PC. When an









excitation voltage is applied to a sensor, the sensor transmits a voltage signal to the

CR10 based on the environmental condition. The PC208W software (Campbell

Scientific Inc, Logan, Utah) converted voltage reading into a final reading that is

appropriate for the parameter being measure (e.g., relative humidity in %). The software

assisted with creating the program, monitoring real-time measurements, and retrieving

stored data.

The standard CR10X has 12 single ended inputs which can also be used as 6

differential inputs. It can measure DC voltage up to 2.5 volts. For each chamber, there

were two differential type of sensors (a load cell and light sensor) and four, single-ended

channels for C02, 02, RH and pressure sensors. The additional channels were not

required for the environmental parameters measured in the experiments presented in

this dissertation. A single-ended input measures the difference of a single conductor

relative to ground; whereas a differential input measures the voltage difference between

two conductors. The CR10 requires a nominal 12 volt DC power supply, which was

provided by a 12 volt battery. The battery was continuously charged using a 12 volt

battery charger.

Sensors and Their Calibrations

Pressure Sensor

The pressure sensors are small integrated circuit sensors. The pressure sensors

(ASCX15AN, Sensym ICT) were calibrated against a precision pressure gauge

(Digiquartz T60 series, Paroscientific Inc. Redmond, WA) following a method used by

Wilkerson (2005). They were calibrated for a range from 0 kPa to 101 kPa. All the

sensors were calibrated by linear regression analysis. The response of the sensor was

linear with respect to voltage and an example of this calibration for one of the four









sensors is shown in Figure 2-3. The program for controlling pressure 2 kPa via

solenoid valve is contained in Appendix A.

Relative Humidity Sensor

Relative humidity values are not dependent on total pressure, thus calibration at

normal pressure was conducted. Relative humidity sensors (HH-4602-L-CPREF,

Honeywell, Morristown, NJ) were calibrated in a closed container using saturated salts

(Greenspan, 1977). Five containers each had a 500 mL beaker containing saturated

salt solutions to maintain specific level of relative humidity at steady state and were

incubated for 24-48 h. They were saturated solutions of MgCI2 (32.8% RH), NaBr (57.6

% RH), NaCI (75.3 % RH), KBr (81.8% RH) and K2S04 (97.3% RH). One sensor at a

time was calibrated. The sensor was kept inside the container for a period of time until

the reading stabilized. Sensors were connected to the CR10 for recording the signals

and then the voltage reading of a sensor was recorded. The known relative humidity vs.

voltage reading was plotted for each chamber. A slope equation gives an offset values

and a multiplier values which were then input into the PC208 program. During the trials

all four RH sensors varied <1% of the range compared to each other. Figure 2-4 is an

example of one of the RH sensor calibration plots.

Oxygen Sensor

Oxygen concentration was measured by galvanic cell type oxygen sensors (MAX-

250, Maxtec, Salt lack city, UT). The Maxtec MAX-250 senses between 0 and 100%

oxygen. Due to the pressure differences that were used in this experiment, the factory

calibration data was not valid for the low pressure studies used here. The sensor was

calibrated following a method used by Wilkerson (2005) and Mu (2005). At the start of

the calibration, the chambers were purged twice using nitrogen gas to remove any









residual oxygen. The sensors were calibrated using different mixtures of oxygen and

nitrogen. For example, for a 10% oxygen concentration a chamber would be filled to 90

kPa with nitrogen, and an addition 10kPa of oxygen for a total pressure of 100 kPa. This

was repeated for other total pressure and oxygen levels and is shown in Figure 2-5A.

The slopes and intercepts were then plotted against pressure and regression curves

were performed using Excel (Microsoft, Seattle, WA 2003). The data from calibration of

one of the three oxygen sensor is this is shown in Figure 2-5B. These lines show the

slope and intercept as a function of pressure over time, and can be applied to the

sensor readings to determine the oxygen percent at any given signal and pressure

using equation 2-2. Where slope is defined by equation 2-3 and intercept is defined by

equation 2-4.

02 [%] = Slope (P)*mvolts + Intercept (P) (2-2)

Slope (P) = 367.15*P-1.1475 (2-3)

Intercept (P) = 0.0012*P2 -0.1581*P +3.4498 (2-4)


Carbon Dioxide Sensor

The C02 sensors used for these studies were the OEM-6004 module (T-6004,

OEM ultrasonic, 6004, Goleta, CA). These cost effective modules had a range of 0-

2000 ppm. Because these sensors are pressure sensitive, they had to be calibrated at

different pressures following a method used by Wilkerson (2005). At the start of

calibration, chambers were purged twice using nitrogen gas to get rid of any residual

C02. The sensor was calibrated using different mixtures of nitrogen (ranging 20 to 100

kPa) and carbon dioxide ranging 0.04 to 0.15 kPa C02. Since the carbon dioxide portion

was so small, it did not affect the total pressure. The C02 gas was added using known









amount of syringe volume (based on the total volume of chamber). Data at these

percentages were collected at different total pressures, ranging from 20 kPa to 100 kPa,

and plotted against the pressure in units of millivolts. These data were compared on the

basis of pressure and a linear regression of each pressure at different percentages was

found. These results are shown in Figure 2-6A. The slopes and intercepts were then

plotted against pressure and regression curves were performed using Excel (Microsoft,

Seattle, WA). The data from an example calibration of one of the three C02 sensor are

shown in Figure 2-6B. These lines show the slope and intercept as a function of

pressure over time, and can be applied to the sensor readings to determine the carbon

dioxide percentage at any given signal and pressure using equation 2-5. Where the

slope is defined by equation (2-6) and intercept is defined by equation (2-7). Later C02

sensor readings were compared against the known syringe volume of C02 gas in to

chamber (Figure 2-7)

C02 [%] = Slope (P)*mvolts + Intercept (P) (2-5)

Slope (P) = y = 225.51*P-1 .2942 (2-6)

Intercept (P) = y = -0.0845*P2 + 16.533*P 928.34 (2-7)

Load Cell

The voltage output from the load cells used in these experiments to record the

weight of the plants and the flasks did not depend on the total pressure of the

environment, thus, the load cells were calibrated at normal pressure following a method

used by Wilkerson (2005). A total of nine load cells (LPS-0.6 kg, Celtron Tech. Inc.

Colvina, CA)), three per each chamber, were used for the experimental chambers. Each

sensor was factory calibrated in terms of FSO (Full Scale Output), which is mV/V

output. Since the excitation voltage of CR10 was 5 volts, FSO was multiplied by 5. For









example, a load cell with FSO sensitivity of 1.0 mV/V has full scale = 5.1 mv. The

multiplier was calculated by dividing 600g (full scale weight) with FSO. For this example,

it was 600/5.1 = 115.88 grams. The offset value is a reading by the sensor when

nothing is on the load cell. Load cell readings were tested for accuracy with standard

weights (Figure 2-8).

Light Sensor Calibration

The light sensors (LI-190SA, Li-Cor Inc, Lincoln, NE) were insensitive to variation

in temperature, pressure and gas composition. Thus, the factory calibration was used

for the light sensor following a method used by Wilkerson (2005). Each sensor had

certified values of an offset and multiplier. Since the sensor output is in the millivolt

range, the signal was amplified in order to be read correctly. A LI-COR millivolt adapter

was used to amplify the mvolts into voltage data. The light readings were compared

against the reading by a hand held light meter (LI-250A, Li-Cor Inc, Lincoln, NE) from

the same company that had been calibrated at the factory. The plot of the hand held vs.

connected sensor is given in Figure 2-9.

Temperature Sensors

The temperature sensor (DS10 Dallas Semiconductors, Dallas, TX) is factory

calibrated and does not require further calibration even at very low pressures. The

temperature sensors were shielded with aluminum foil as they are sensitive to ambient

radiation. Leaf temperature was measured by fine gauge- type-K thermocouples

(OS36SM-K-140F, Omega; Stamford, CT). The performance of temperature sensor at

constant temperature in all three experimental chambers is given in Figure 2-10.









Summary and Future Improvements

Four, LPGCs were designed and met the specifications required. The chambers

designed for these studies are suited for studying physiological aspects of plant growth

and development in low pressures, including transpiration and the effects of the gas

phase on plant growth and development. The LPGSs were able to maintain pressure,

02 concentration, air temperature and relative humidity to given set points while

measuring the plant weight and CO2 uptake rates. Vacuum feed through ports

successfully prevented air leak in to the chambers.

There were a few limitations to this system. For example, nutrient and water

supply was not controlled. A circulating hydroponics system would be a better

alternative to the flask system that was used here. In addition, water that was

transpired, collected on the walls of the Plexiglas top and at the bottom of the metal

base for initial studies and this was improved with salt saturated solutions that absorbed

excess water. Future improvements would include a mechanism for collecting

condensate using a cooled coil or other mechanisms. Humidity control was not available

for values below 85%. Future improvements would include a humidity control system to

study the interacting effects of humidity and low pressure on plant growth. For these

studies, the the light levels were at 250 pmol m-2 s-1 using cool-white, florescent lamps

to avoid hazards in environmental chambers associated with other lamps. However, on

the surface of planets the light levels may be very high at or above 400-600 pmol m-2 s-1

so improvements to the lighting system may be required. Although the CR 10 data

logging system is easy to use, it requires battery power supply which needs to be

charged at regular intervals. The limited number of ports on the CR10 prevented the

addition of more sensors. A centralized data logging system will be more effective as it









requires less wiring and is less complex and could readily be adapted to connect to user

friendly software for programming such as LabView (National Instruments, Austin, TX)

and scale up of the instrumentation would be relatively easy. An efficient way to

maintain and control concentration of nitrogen, oxygen and carbon dioxide is required

and a mass flow controller which automatically controls the gas levels is needed.

Ethylene can be scrubbed by circulating a chamber air through stainless steel tube

containing potassium permanganate (He et al., 2009). Although, in the present study

temperature control within a chamber was not required, it will be necessary to have

temperature control for self-contained chambers that would be used on the Moon or

Mars.
















Low Pressure Plant Growth Chamber


All Dimensions in Inches
Base constructed of 6061 aluminum
Shell and Lid constructed of acrylic
All tolerances to within 0.1 in


,1/4" Silicone Gasket


Low Pressure Plant Growth Chamber
A) Bottom View (Aluminum Base)
B) Front View (With Plant)
C) Side View (With Plant)
D) Isometric View


Figure 2-1. Low Pressure Growth Chamber (LPGC)












































Figure 2-2. Low Pressure Growth Chambers developed for this objective with gas
tanks, datalogger (CR10) and PC interface.












54















y = 21.924x + 122.43
R2= 1 /


0 20 40 60 80
Digital reading (kPa)
Figure 2-3. Calibration of the pressure sensor comparing
against millivolts readings.


100

pressure gauge reading


2500


2000


1500



1000



500















4-

3.5 y = 0.0255x + 0.9798
R2 = 0.9974
3

2.5

2-

1.5

1

0.5

0
0 20 40 60 80 100 120


Figure 2-4. Calibration of the relative humidity sensor against known saturated salt
solutions.









30 A


y = 0.584x 2.8929


- 2.0423


O 40 kPa
o 60 kPa
A 80 kPa
o 101 kpa


y = 0.2471 x 1.6221


y = 0.1281 x- 1.046


10 20 30 40 50 6(
Oxygen %


y = 385.52x-1.1503
R2 = 0.9973


o Slope


o Intercept


y = 0.0023x2 0.3554)
R0 2 = 0.9826


0 20 40 60 80 100

Total pressure (kPa)
Figure 2-5. Calibration curves of an oxygen sensor at different pressures (5A) and
curves of slope and intercept (5B).













2000 1 A


1600


1200


800


400


0 500 1000 1500 2000

CO2 (ppm)


20 40 60
y = 225.51x-1.2942
R2 = 0.9982
R&


2500 3000


o Slope
* Intercept


y = -0.0845x2 + 16.533x 928.34
R2 = 0.9977


Total pressure (kPa)

Figure 2-6. Calibration curves for a C02 sensor at different pressures (A) and curve of
slope and intercept (B).


50

-50

-150

-250

-350

-450

-550

-650


- H -- H H -















6000


5000 --- 33 kPa
-- 66 kPa
4000 -A-101 kPa
E
0..
4 3000


2000 -


1000


0 -
0 0.05 0.1 0.15 0.2
C02 (kPa)
Figure 2-7. C02 sensor reading against the equivalent syringe volume













5 y = 0.0083x + 0.2995

2 4


0 3


0 2 o o Voltage Read
-J




0
0 100 200 300 400 500 600 700

Weight (g)


Figure 2-8. Calibration for a load cell used to measure the weight of the flask containing
plants.





















300


250 y = 28.271Ln(x) + 164.76
S0 R2 = 0.9965
200


150 -
S1 Light sensor calibration

0
) 100 -Log. (Light sensor
U) calibration)

1D 50


0 -
0 10 20 30 40 50 60 70 80

Handheld unit reading (pmol s"' m-z)


Figure 2-9. Calibration curve for light sensor readings against handheld light meter










A


AAB--- ArAsAuA-A-- OxygeA-A-- T ratu--r-e--







--Pressure -e- Oxygen Temperature
I I I II


50 B


(U
Q-


C)



x
0)


>.






O
0
n

U)
W


C/)
C/)
U)
0l
-I-

0
F--


0-- -0Q -0& -C --0 -Q --Q0 -Q -- C --Q -- --- --0 --0 --0 0 --0 --0 --0 0


0 1 2 3 4 5
Time (h)
Figure 2-10. Pressure (-33 kPa), 02 (-20 kPa) and temperature (-23 C) recordings for
five hour for chamber A (top panel), chamber B (middle panel) and chamber
C (bottom panel).


C


26

20

16

10

5

0
25
-2




2






25

20

15









CHAPTER 3

MODELING GAS DIFFUSIVITY, PHOTOSYNTHESIS AND TRANSPIRATION UNDER
HYPOBARIA

As an integral part of Bioregenerative Life Support System (BLSS) for long-term

space missions, plants will likely be grown at reduced pressure. At reduced pressures,

the diffusivity of gases increases. This will affect the rates at which CO2 is assimilated

and water is transpired through stomata. To understand the effects of reduced pressure

on plant growth, the diffusivities of CO2 and H20 at various total pressures (101, 66, 33,

22, 11 kPa) and CO2 concentrations (0.04, 0.1 and 0.18 kPa) were calculated. The

diffusivity is inversely proportional to total pressure and shows dramatic increase at

pressures below 33 kPa (1/3 atm.). A mathematical relationship based on the principles

of thermodynamics was developed for calculating the transpiration and photosynthesis

for plants. Stomatal conductance is sensitive to total pressure. At 33 kPa total pressure,

stomatal conductance increases with the boundary increasing by a factor of ~1.7, thus

the boundary layer thickness conductance increases by 70%. Since the leaf

conductance is a function of both stomatal conductance and the boundary layer the

overall conductance will increase resulting in significantly higher levels of transpiration

as the pressure drops. The conductance of gases is also regulated by stomatal aperture

in an inverse relationship. Stomatal aperture is directly influenced by concentration of

CO2 inside the leaf space. The higher CO2 concentration inside the leaf air space during

low pressure treatments may result in stomata closing partially or fully which may

reduce the excessive transpiration caused by increased diffusivity. Therefore, a reduced

pressure environment with high CO2 may be an ideal scenario for minimizing

transpiration and maximizing the plant biomass yield in BLSS.









Plants in Hypobaria

Long-term missions to Moon and Mars will require a well-studied and predictable

Advanced Life Support System (ALS) for space explorers. Among other components

such as like waste treatment and energy production, bio-regeneration (BLSS) through

the use of plants has been identified as an integral part of ALS. Plants will be utilized for

CO2 removal from the air that is respired from explorers, for oxygen regeneration and

water purification systems, and as a source of food for human use (NASA, 2003). There

is no atmospheric pressure on the Moon with very little of an atmosphere on Mars (less

than 1 kPa total pressure). Chambers used to grow plants will likely be maintained at

reduced total pressure (i.e., hypobaria). Operating the chamber at reduced pressure will

mitigate the negative effects caused by large pressure differences between the inside of

the chamber and the vacuum/low pressure environments of the Moon or Mars (Bucklin

et al., 2004). The reduced pressure will also reduce consumable and structural

components of ALS, thus launch cost. Another advantage of lower pressure would be

reduced leakage of valuable gases requiring less total gas to pressurize the chamber.

Richards et al. (2006) and Paul and Ferl (2006) reviewed the advantages of low

pressure environments for plant growth on space missions. One of the fundamental

differences in hypobaria compared to normal atmosphere is the diffusivity of gases

(Rygalov et al., 2004), which may influence rates of water vapor loss and carbon dioxide

(CO2) assimilation by plants. The effects of diffusivity on the plant transpiration at

normal atmosphere have been studied (Mott and Parkhurst, 1991) but there are very

few studies (Gale, 1972; Goto et al., 1996) on the effects of diffusivity of CO2 and water

on plant growth at hypobaria. This article reviews the physical relationships that









influence the diffusivity of H20 and CO02 in hypobaria, and the consequent implications

for plant growth.

Binary Diffusion of Gases

The general relationships defining gaseous diffusion in physical, chemical and

biological systems have been developed, although not applied for understanding gas

exchange by plants in hypobaria. Gas flux density, according to Fick's law is directly

proportional to the driving force, i.e. partial pressure gradient, and the conductance of

the gas in the gas mixture. Gas conductance is dependent on the mass diffusivity of the

binary gas system (DAB) as a function of temperature, pressure and the gas

composition. In a binary gas system, DAB is proportional to temperature and inversely

proportional to pressure. It is the sensitivity of DAB to pressure that is of special interest

in growing plants in hypobaria chambers. The earliest attempts to derive the

mathematical relationship to calculate binary gas diffusivity at reduced pressure used a

Stephan-Maxwell hard-sphere model (Hirshfelder et al., 1948; Bird et al., 1954). They

presented the mathematical expression of binary gas diffusion based on the kinetic

theory describing constant motion of atoms, ions and molecules at temperature above

absolute zero. The diffusion coefficient of the gases at reduced pressure can be

estimated from the intermolecular forces of the fluxes (Slattery and Bird, 1958). To

improve upon the calculations based on theoretical properties and get more accurate

gas phase co-efficient, Bird et al. (1960) simplified the derivation of Slattery and Bird

(1958) by using the Chapman-Enskog model (Equation 4-1; Chapman and Cowling,

1952), which defines the binary diffusion coefficient of gas A in gas B (DAB, cm2 S-1) in

terms of potential energy of interaction between a pair of molecules in the gas. They

presented the theoretical diffusion over the range of pressures from 10 to 100 kPa and








compared it with experimental data. Though, the predicted values were within 5% of the

calculated values, it required considerable calculations.

DAB 3 8kT 1 + 1 (3-1)
32no-A B \mA mB

where n = total concentration of both species, mol cm-3
T= temperature, K
k = the Boltzmann constant, ergs/K
mA,mB = molecular mass, g mol-1
oAB = collision diameter, separation between molecular centers of unlike pairs
upon collision, cm

In deriving Equation 3-1, the following assumptions were made,

The gases are non reactive.
The gases as a whole are assumed to be at rest but the molecular motion is taken into
account.
All molecules have velocities, representing the region of last collision.
Temperature profile of gas corresponds to the Fourier's law of heat conductance.
Thermal conductivity for polyatomic gases is calculated at low density.

Equation 3-1 has two main limitations because it is based on the hard-sphere

model (Fuller et al., 1966).

There is a difference between theory and experimental observations of the values of a
because the increased temperature softens the molecule which reduces the o.

Very few values of a are available in literature, and they are applicable for only a narrow
temperature range because of the temperature dependence of o.

Arnold's (1930) original suggestions to overcome these limitations were

implemented by Fuller et al. (1966) by,

Replacing the temperature dependence with Sutherland temperature

corrections to improve the temperature dependence.

Replacing a with cube root of sums of Le-Bas atomic volume parameter

which is an easily measured property of a diffusing substance.








An empirical analysis done by Fuller et al. (1966) described DAB by the following

general equation.
Tb1/2


A __mA mB (3-2)

pk[(VA)1 (vB )2Y]3

where c = an empirical constant
p = pressure, Pa
b = empirical temperature power dependence
Vi = special diffusion parameters to be summed over atoms of diffusing species
at normal boiling point, m3 kmol-1 here, Vair =20.1, VH20 = 12.7, Vco2 = 26.7
a,, a2, a3 = empirical exponents to the diffusion volumes.

Fuller et al. (1966) used results from about 300 experimental values to obtain the

empirical coefficients for various gas mixtures by using a non-linear least square

analysis. Optimization to obtain the smallest standard deviation resulted in the following

empirical relationship.

I _31/2
10- T175 -+ __l -
V P[(-- VA ) 3 (- V )13]2 (3-3)


The Effects of Background Gases on Overall Diffusivity

Generally, air is composed of multi-gas components and can be considered one

single gas if the concentration of individual gases does not change. However, in a

system where total pressure and the partial pressures of the individual gases are

varying, considering air as a single gas may not give correct values of overall diffusivity

of CO2 and H20. In complex biological systems such as alveolar regions of lungs,

closed plant/animal growth chambers, fermentation vessels and plant leaf stomata, the

air cannot be represented by binary gas constituents (Johnson, 1999). The partial








pressure of the background gases directly affects the overall diffusivity of a gas. For

example, diffusivity of oxygen in a mixture of nitrogen will not be the same as in a

mixture of helium, even though the total pressure and pO02 remains the same (Mott and

Parkhurst, 1991). Higher values of mass diffusivity mean larger amounts of mass

transfer.

Fuller et al. (1966) proposed a method to calculate the individual constituent mass

diffusivity (or overall diffusivity of particular gas) in a multi-component gas at different

mole fractions. The calculation of mass diffusivity of a constituent gas (for e.g. CO2 in air

composed of N2, C02, 02 and H20) requires first the calculation of binary diffusivity of

that gas (CO2) in all gas components (Equation 3-3).
1
DA = (3-4)
j=1 DAB

where, DA = mass diffusivity of a constituent A in multi gas (m2 S-1).
Pj1 = the mole fraction of gaseous component
DAB = Binary mass diffusivity of constituent A in a gas B (m2 s-1)
Eq. 5 can be applied to our case to define Dco2 in a chamber composed of CO2,
02, N2 and H20.

Dco = CO2 + IUH+2 + 1
D Dco co DCO H 2 Dco co- (3-5)

Diffusivity of CO2 and H20 in Hypobaria

The earlier studies on various aspects of plant growth under reduced pressure

have suggested that plant growth is possible down to 10 kPa (Musgrave et al., 1988).

Also, high partial pressures of CO2 Up to 200 Pa can be beneficial to plant growth.

Therefore, for clarity, the diffusivity of H20 and CO2 in binary gas systems was

calculated at various total pressures and pCO2 using equation (Equation 3-4). The









pH20 and temperature were held constant for all calculations at 2.64 kPa and 22 oC,

respectively.

As shown in Figure 3-1, the diffusivity of both H20 and CO2 is highly sensitive to

pressure. Diffusivity of the gases increases as pressure decreases, reflecting the

diminished interaction among gas molecules. The increase in diffusivity is not linear but

shows a dramatic increase at low pressures of below 20 kPa. Increasing the CO2

partial pressure in air does not significantly change the diffusivity from physiological

levels. For example, at 33 kPa total pressure, when CO2 is increased from 40 to 180

Pa, the mole fraction increases from 0.001 to 0.005 (mol kmol-1), which is much less

when compared to the sum of mole fraction of remaining gases (pO02 at 21 kPa = 0.606

mol kmol-1 and pN2 at 12 kPa = 0.313 mol kmol-1).

The ratio of the diffusivity of H20 to C02 is interesting because of the impact of the

exchange of gas by plants for these two gases. The diffusivity of H20 under 101 kPa

total pressure is generally about 1.6 times higher than CO2 due to two main reasons (i)

the difference in the molecular weight of water (18 g mol-1) and CO2 (44 g mol-1) and (ii)

the atomic volume of water (12.7 g mol-1) and CO2 (26.7 g mol-1). Since these two

factors are not sensitive to pressure, the ratio of diffusivity of the two gases is virtually

insensitive to pressure (inset Figure 3-1). At standard temperature and pressure, the

ratio of gaseous phase resistance of water and CO2 in air is 1.7 (Bieurhuizen and

Slayer, 1964a). The calculation of the ratio based on assumptions by Gale et al. (1972)

also agrees on value of the diffusive resistance around of 1.7 though their assumptions

for the diffusive resistance ratio equation did not include the low pressure.









Gas Diffusion in Leaves

The growth and health of plants is dependent on their ability to transpire H20 and

assimilate CO2. Both of these gas exchange processes by leaves is dependent on the

sensitivity of gas diffusivity. This can be shown by examining the basic equations

defining leaf gas flux density. The fundamental description of mass flux density for a leaf

is the Onsager expression for coupled non-equilibrium flow as a result of water vapor

and temperature gradients (Katchalsky and Curran, 1967).

J = L,A~, + LTAT (3-6)

where,J = Vapor flux density (mol cm-2 S-1)
Lv= Onsager coefficient of vapor
LVT= Onsager vapor-temp coupling coefficient
ApJv = vapor chemical potential difference across the diffusion path. (J mol-1)
AT= Temperature difference across the diffusion path (oK)

Since in most plant systems there is a small temperature gradient and a comparatively
small coupling coefficient, Equation 3-6 reduces to the familiar simplified expression of
flux density of individual gas species under steady state conditions. In Equation 3-7 the
Onsager coefficient is expressly defined by

J = mp, (3-7)


where m = mobility co-efficient, cm 2 mol J-1 s-1
pv= vapor density, mol cm -3
p = chemical potential of vapor, J mol-1
x = distance, cm

For the short distances of vapor flux between leaves and the bulk atmosphere, the

chemical potential is defined by the vapor pressure gradient (J cm-3). When the

atmosphere adjacent to the liquid surface is saturated with vapor (Pv*)


J m= (* -P) (3-8)
Ax









The mobility coefficient can be explicitly defined as a function of gas diffusivity.

m = D P (3-9)
P.

where, Pa = Pressure of air, J cm-3
Pa = density of air, mol cm-3
On the molar basis, the evaporation rate can be written simply as


E= D P- (p, *-Pa) (3-10)
Ax Pa

The D/Ax defines the conductance (cm s-1) for vapor flux from a surface at

saturated vapor pressure.

Leaf Transpiration

Transpiration is more complicated than simple diffusion because the surface for

evaporation is buried inside a leaf at the cell walls. Transpiration requires the diffusion

along the pathway from the cell walls through stomatal pores and then through the leaf

boundary layer. The conductance limitation to vapor in this pathway from inside leaves

is predominantly stomatal (Rand, 1977) at about 90 % or more of the limitation (Noble,

1990). Therefore, the conductance for transpiration must account for gas conductance

through stomata pores. The stomatal conductance (hs, cm s-1) is directly dependent on

gas diffusivity as well as pore aperture and pore depth (Parlange and Wagoner, 1970).

Equation 4-11 describes this relationship.

h, ;,r 1 b D) "
Ats = r-a-b-D-
d +- In
2 (3-11)

where a, b = length of major and minor axis of pore aperture, respectively, cm
d = pore depth, cm
D = diffusivity of water vapor (cm2 S-1)
n = stomata density (number cm-2)









The value 'b' (pore width) is dependent on conditions in the leaf and commonly

varies from 0 to 4*10-4 cm (Sinclair, 1980). Environmental factors such as light, CO2

concentration and vapor pressure deficit can have large influences on pore width.

Equation 3-11 also clearly demonstrates that stomata conductance is dependent

on gas diffusivity. As shown previously, the value of D is a function of a total pressure,

and hence total pressure will have direct influence on stomatal conductance. Under low

pressure situations Figure 3-1 shows that D is increased, which according to Equation

3-11 means stomata conductance is increased. For example, for plants grown at one

third of the standard earth atmospheric pressure stomatal conductance will increase by

the ratio of mass diffusivity at 0.3 atm to that of at 1 atm (Figure 3-1), thus increasing in

stomatal conductance by approximately a factor of 3. Thus, transpiration rate and CO2

assimilation rates would be substantially increased under hypobaria conditions.

The conductance of the boundary layer surrounding the leaf is also dependent on

gas diffusivity, although in a complex manner because of the convective movement of

air. The theoretical considerations used for the flat plate evaporative loss through

boundary layer can be applied for the plant leaf (Sinclair, 1980). The effects of boundary

layer on a flat plate of an infinite length (Gebhart, 1961) can be estimated by the

following equation.


hbl = 0.664 Re1/2 Sc1l3 (3-12)
L

where, hbl = vapor boundary layer conductance, cm s-1
K= fluid thermal diffusivity (k/ p*Cp), 0.215 cm2 s-1 at 20 C
Where,
k = thermal conductivity (W.m-.K1), Cp = specific heat (J kg-1 K1)
p= the fluid density (kg m-3)
Re = Reynolds number
Sc = Schmidt number









The fluid thermal diffusivity is a function of 1/p whereas the thermal conductivity

and specific heat will remain almost constant for constant temperature and low pressure

(p < 2 atm; Salazar, 2003). For example, at 33 kPa the K will be -3 times the value of K

at 101 kPa.

Re = Reynolds number (uL/Kv)

where L = length of flat surface, cm
u = air speed, cm s-1
Kv = kinematic viscosity at 0.15 cm2 s-1 at 20 oC.

The Re is inversely of related to kinematic viscosity (p/p). Thus, the Reynolds

number is significantly affected by pressure. The dynamic viscosity does not

significantly change with pressure. For example at 33 kPa, the Re will decrease by a

factor of 3 compared to normal atmospheric pressure since the density of gases is 1/3

of the normal pressure.

Sc = Schmidt number (pJ/pD)

where p = fluid viscosity (N s m-2)
D = mass diffusivity, m2 -1.

The Sc is a function of fluid density and diffusivity. At 33 kPa, the diffusivity of

gases increases by three times compared to normal atmospheric pressure but this

value is offset by the decrease in the density of air. The value of Sc at 101 kPa as well

as at 33 kPa (20 C) equals approximately 0.63 for water vapor. Overall, at 33 kPa the

value of hbI will be increased by a factor of ~1.7 compared to normal atmospheric

pressure (101 kPa).

Combining the constants at 20 C, the boundary layer conductance (hbl) for both

sides of leaf in a laminar flow can be predicted from Equation 3-13. Thus, the diffusion

coefficient also influences the boundary layer conductance through the Schmidt









number. However, the fact that the Schmidt number is taken to the cube root and the

boundary layer conductance offers little limitation to gas flux density, the following

simplified equation is generally appropriate (Sinclair, 1980).


hbi = 0.63 u
L (3-13)

where L is the length (cm) of the leaf
u is the wind velocity (cm2 s-1).

At 33 kPa, the co-efficient value will be 1.096, which is 1.74 times the co-efficient

for 101 kPa.

The combined conductance (stomatal and boundary layer) can be designated as h

(cm s-1).

hs hbl
h =-- (3-14)
hs + hbl

This equation suggests that the combined conductance will increase as pressure

decreases. For example for h at 33 kPa, the values would be 3 and about 1.7 times the

normal atmospheric pressure values, for hs and hbl, respectively. This is assuming

similar stomatal open geometries and air velocities between 33kPa and 101kPa.

Inserting the stomatal and boundary layer conductance into an equation in the

general form of Equation 3-15, the transpiration (T, units) can be defined by the

following equation (Sinclair, 1980).

T = h (PL Pv) (3-15)

where, PL = saturated vapor pressure at leaf temperature, Pa
Pv = vapor pressure outside the leaf air space, Pa

It should be noted that as transpiration rates increase in lower pressure, the

surface leaf temperature will drop due to latent heat of vaporization. This drop in









temperature will result in a lower vapor pressure deficit in low pressure compared to

higher pressures. This would decrease transpiration rate below the initial estimate.

Leaf Photosynthesis

Photosynthesis is a function of the partial pressure carbon dioxide gradient but the

direction of diffusion is opposite of that of transpiration. Similar to T, photosynthesis is a

function of the CO2 partial pressure (Pa) difference between the atmosphere and the

inside of the leaf, and total gas conductance (Equation 3-16; Sinclair 1980).

A = 44 h (Po2a P02) (3-16)

A is the photosynthetic rates and 44 is the molecular weight of CO2. The Pco2a and

Pco2i are the leaf outer and internal leaf partial pressures (Pa) of CO2, respectively. The

'h' is the total conductance for CO2 which was described for transpiration and would

increase as pressure drops. Stomatal conductance is a function of the stomata

aperture, which in turn is dependent on the diffusion coefficient of CO2 (Montieth and

Unsworth, 1990). Because the mass diffusivity is a function of a pressure and the

molecular weight, the effects of reduced pressures should be reflected in its effects on

plant growth (i.e., the CO2 assimilation and the transpiration rates). At reduced pressure

the diffusion limitation to CO2 and water transfer through stomata should be reduced

proportionately (Mott and Parkhurst, 1991). Thus, resulting in enhanced photosynthesis

and reduced transpiration.

Conclusion

There is a strong correlation between the total pressure and the diffusivity of C02

and H20 (Figure 3-1). At reduced pressure, the diffusivity of individual gases increases.

The increased diffusion should result in increased number of CO2 molecules transferred









through stomata thus increasing the plant biomass. Higher C02 levels in conjunction

with reduced pressure can enhance the plant growth. The CO2 assimilation via

photosynthesis (A) is also subject to the stomatal regulation which implies that there is a

threshold limit of CO2 beyond which stomatal aperture will tend to close preventing

further A and it is expected that this value be lower at lower pressures compared to

higher pressures. Similarly, due to increased diffusion more water molecules should

move out of the stomatal pore as less resistance for water in liquid phase to reach the

equilibrated gas phase water. Thus, transpiration rates will also increase. High CO2

partial pressure combined with reduced pressure can decrease transpiration through

stomatal closure. Further studies in low pressures with various gas compositions is

required to identify the threshold level of each gas to enhance the plant growth and

reduce transpiration rates without adverse effects.

This analysis of the sensitivity of the diffusivity of CO2 and H20 to pressure shows

that engineers designing ALS systems have two variables to consider. Transpiration

and photosynthesis are both sensitive to the partial pressure of each gas in the bulk

atmosphere around the plants and the total pressure of the atmosphere. Due to the

high sensitivity of the diffusion coefficients at hypobaria pressure, and hence gas flux,

plant growth and water loss can be influenced by the pressure of the growth chamber.

If water flux, for example, is a critical aspect of a water purification system, transpiration

rate of the plants can be increased or decreased by simply adjusting the plant chamber

pressure. Similarly, plant growth will be responsive to the pressure changes as

enhanced CO2 assimilation is predicted.









3.0 2
0 0 0 0
1.5
2.5 DH20
2.5 0\ 5
S \ +DCO2 1

2.0 i 0.5
o \
>1.5 0 30 60 90
S\ Total pressure (kPa)
1.0 -

S0.5 -^

0.0-
0 20 40 60 80 100
Total pressure (kPa)

Figure 3-1. The individual mass diffusivity of C02 and H20 calculated from the binary
mass diffusivity using the empirical formula given by Fuller et al. (1966).
Inset: The ratio of diffusivity of H20 over C02.










CHAPTER 4

THE INTERACTING EFFECTS OF CO2 AND HYPOBARIA ON GROWTH AND
TRANSPIRATION OF RADISH (RAPHANUS SATIVUS)

Plants grown on long-term space missions will likely be grown in low pressure

environments (i.e., hypobaria). However, transpiration rates can be elevated in low

pressure resulting in plant wilting or stress. It is possible to reduce transpiration by

increasing the partial pressure of CO2 (pCO2), but the effects of altering CO2 to super-

elevated levels on plant growth and transpiration in hypobaria are not known. Here, the

interacting effects of pCO2 and total atmospheric pressure on the growth and

transpiration of radish (Raphanus sativus var. Cherry Bomb II) were studied. This

material also appears in Gohil et al., (2010). The fresh weight (FW), leaf area, dry

weight (DW), CO2 assimilation rates (CA), dark respiration rates (DR), and transpiration

rates from 26 day-old radish plants that were grown for an additional seven days at

different total pressures (33, 66 or 101 kPa) and pCO2 (40 Pa, 100 Pa and 180 Pa)

were measured. In general, the dry weight of plants was enhanced with CO2 enrichment

and with decreased total pressure. In limited pCO2 (40 Pa), the transpiration for plants

grown at 33 kPa was over twice that of controls (101 kPa total pressure with 40 Pa

pCO2). Increasing the pCO2 from 40 Pa to either 100 or 180 Pa reduced the

transpirations rates for plants grown in hypobaria and at normal atmospheric pressures.

However, plants grown at lower total pressures (33 and 66 kPa total pressure) and

super-elevated pCO2 (180 Pa) had evidence of leaf damage. Radish growth can be

enhanced and transpiration reduced in hypobaria by enriching the gas phase with CO2

although at high levels of CO2 leaf damage can occur.









Introduction

Plants will be an integral part of an Advanced Life Support system for long-term,

human space exploration. Plants will be utilized for CO2 removal from the air that is

respired from explorers, they will act as oxygen regeneration and water purification

systems, and they will be a source of food and fiber for human use (NASA, 2002). On

planets with little to no atmospheres, the chambers that will be used to grow plants will

likely be maintained at reduced total pressure (i.e., hypobaria). The low pressure inside

the chamber will mitigate the negative effects caused by large pressure differences

between the inside of the chamber and the vacuum/low pressure environments of the

planet or moon (Bucklin et al., 2004). Maintaining the chambers at low pressure will also

reduce costs associated with transporting heavy materials used to build chambers that

could withstand large pressure differences and the lower pressure would reduce

leakage of valuable gases and require less total gas to pressurize the chamber. Since

transport costs associated with supplies are expected to be one of the major limitations

in long-term space missions, any mechanisms to reduce these costs should be

explored. For review on the advantages of low pressure environments for plant growth

on space missions see Richards et al., ( 2006) and Paul and Ferl (2006).

On Earth, many species of plants grow at high altitudes with low total pressure

(-55 kPa). This suggests that plants can adapt to hypobaria. Since pressures for

growing plants on the Moon or Mars will be even lower (i.e., -33 kPa, Paul and Ferl

2006) than that found on Earth, studies on the effects of these lower pressures on plant

growth have been performed. The results of these studies have shown that seed

germination (Musgrave et al., 1988), plant growth and development (Iwabuchi and









Kurata 1996; Goto et al., 2002; Spanarkel and Drew, 2002; Richards et al., 2006; He et

al., 2007), and fruit ripening (Burg and Burg, 1966) are possible in hypobaric conditions

for a broad range of species with species surviving in pressures as low as 10 kPa

provided there is enough pO02, pCO2, and pH20 (Massimino and Andre, 1999; Goto et

al., 2002). Although it is well known that plants can grow and develop in hypobaria, the

effects of low pressure on the photosynthesis, biomass accumulation, and transpiration

rates of plants are unclear. For example, some studies report increased biomass,

photosynthesis or transpiration in hypobaria (Rule and Staby, 1981; Andre and

Massimino, 1992; Daunicht and Brinkjans, 1996; Corey et al., 1997; Iwabuchi and

Kurata, 2003) while others report reduced growth or no change in either plant biomass,

photosynthesis or transpiration in hypobaric conditions (Goto et al., 1995; 1996;

Iwabuchi et al., 1996; Spanarkel and Drew, 2002; Iwabuchi and Kurata, 2003; Richards

et al., 2006; He et al., 2007). The differences in the experimental procedures between

these studies make it hard to interpret the effects of hypobaria on plant growth and

development. For example, the plant species, pCO2, p02, pH20, and the duration of the

experiments all varied between experiments and likely account for the differences in

plant responses to hypobaria. In order to develop decision support tools for growing

plants on the Moon or Mars, further studies on the effects of low pressure environment

on plant growth responses are required.

As total atmospheric pressure decreases, the diffusivity of water vapor and gases

increase (Gale, 1971). The enhanced transport of water from the leaf surface and CO2

toward the plant from the surrounding gas phase may account for the enhanced

evapotranspiration/transpiration and photosynthesis that was found in some studies for









plants grown in hypobaria (Iwabuchi et al., 1996; Goto et al., 1996; Daunicht and

Brinkjans, 1992, 1996; Corey et al., 1996, 2002; Wilkerson, 2005). Enhanced

transpiration in hypobaria can have significant negative effects on plants if the water is

not readily replaced. For radish, the increased evapotranspiration in hypobaria resulted

in severe wilting of radish within 30 minutes of transfer from high (101k Pa) to lower

pressure (25k Pa, Wilkerson, 2005). This water stress in hypobaria appears to cause

the differential expression of drought-related genes (Paul et al., 2004). These changes

in gene expression may trigger downstream responses in plants to induce stomata

closure to reduce water loss. For example, stomata apertures from spinach grown in

hypobaria long-term (10 days) were smaller with similar transpiration rates as plants

grown at normal atmospheric pressure (Iwabuchi and Kurata, 2003). Increasing the

pCO2 can also reduce stomata aperture and thus reduce transpiration rates even in

hypobaria. For example, a twenty percent decrease in transpiration rates for tomato

grown in hypobaria was reported when CO2 was increased from 40 to 100 Pa (Daunicht

and Brinkjans, 1996). The CO2 levels for Advanced Life Support systems may be at

super-elevated levels (-0.6 kPa or higher, Wheeler et al., 1999) and CO2 may be used

as a pressurizing gas for growing plants on Mars which would result in very high pCO2

(Wheeler, 2001). Unfortunately, the interacting effects of high levels of pCO2 and

pressure on plant growth, transpiration and photosynthesis are unclear. Therefore, the

objectives of the present study were to study the interacting effects of CO2 and pressure

on growth (dry weight, DW, and fresh weight, FW), CO2 assimilation (CA), and

transpiration rates of radish. These studies provide insight into the detrimental effects of









high levels of C02 on plant development at low pressures and identify methods for

reducing transpiration and improving growth in hypobaria with CO2 enrichment.

Materials and Methods

Plant Material

Seeds of radish (Raphanus sativus L. cv. Cherry Bomb) were germinated and

grown in pots containing soil (MetroMix 300, Sun Grow Horticulture, Bellevue, WA) with

nutrient solution of 1/6 strength MS (Murashige and Skoog, 1962) medium applied

three-times-a-week. Plants were grown for 20 days at 22C with a 12h photoperiod with

lighting at 150 [[mol m-2 s-1 from cool-white-florescent lambs in environmentally

controlled chambers at 85% relative humidity. After growth in pots, plants were rinsed in

ddH20 and transferred to 250 mL flasks containing ~300 mL of 1/6 strength MS

medium, pH adjusted to 6.8 with HCI (1 mM) or NaOH (1 M). The volume of flask was

large enough for the short-term studies that the level of nutrient solution at the end of

the experiment was well above the majority of roots. The roots were well-developed and

reached far into the flask at this growth stage. Plants were placed in the holes of the

stoppers and surrounded by putty (Plumbers Putty, P/N 43048, Ace Hardware, IL) to

minimize water loss during experiments. Plants were grown in flasks under similar

conditions of pots for 4 to 5 days before being transferred to experimental growth

chambers for acclimation. The fresh weight (FW) of plants was measured on the

acclimation date at 4.0 0.9 g. The nutrient level was then filled to ~300 mL prior to

acclimation. Plants were then acclimated in low-pressure chambers at normal

atmospheric pressure (101 kPa with 40, 100 or 180 Pa pCO2 and 21 kPa pO2 and 2.2

kPa pH20) for one day prior to experimental treatments. Plants were grown for an









additional seven days under pressure and gas treatments then removed for plant

harvest.

Growth Chambers and Environmental Control

The experiments were performed in three chambers (Figure 4-1) of total volume of

0.09 m3 with a fan (Delta Model, P/N BFB0512M, Silicon Valley Compucycle, San Jose,

CA). The chambers had sensors for temperature (DS10 Dallas Semiconductors, Dallas,

TX), pressure (P/N ASCX15AN, Sensym ICT, Milpitas, CA), CO2 (OEM ultrasonic,

6004, Goleta, CA), 02 (MAX-250, Maxtec Co., Salt Lake, UT), relative humidity (HH-

4602-L-CPREF, Honeywell, Morristown, NJ), light (LI-190, Li-Cor, Lincoln, NB) and

three balances for weight determination per chamber (LPS-0.6 kg, Celtron Tech. Inc.

Colvina, CA). Calibration of 02 and CO2 sensors was performed according to Richards

et al., (2006). CO2 and 02 levels were adjusted daily at 4 hours into the photoperiod.

CO2 assimilation studies were started at the same time in the photoperiod for all tests

and were repeated daily. After the acclimation period, the gas and pressure composition

were brought to set points daily (Table 4-1). Environmental conditions for the one day

acclimation in the growth chamber were 22 2 C, 12 hour photoperiod, at 250 [[mol m-
2 S-1 using cool-white, florescent lamps and 85% relative humidity. The pO2 was

maintained at -21 kPa with other environmental conditions described (Table 4-1).

Each chamber contained three individual plants. Pressure was maintained with a

vacuum pump (1/2 HP, JB Industries, Chicago, IL) and chambers had leakage rates of

~0.03% (chamber volume/h). Data collection and controls were performed using a

CR10 datalogger (Campbell Scientific, Logan, UT) that was connected to a PC.

Experiments were performed in triplicate.









Plant Harvest

After the experimental treatments in the chambers, plants were removed from the

chamber blotted dry and analyzed for fresh weight, leaf area, and dry weight. Dry weight

analysis was performed by placing plants on pre-weighed pans at 600C for 48h and

reweighing the contents. Leaf area was measured on day 7 with digital images using

Image Pro Plus software (ver. 6.0 Media Cybernetics, Bethesda, MD).

Statistical Analysis

For CO02 and pressure treatments, the analysis was treated as a two-factor

analysis of variance (ANOVA) using SAS (SAS institute NC, USA) and the mean of

three individual plants of independent experiments (here, n=3).

Results

Plant Growth

Plants grown with 40 or 100 Pa pCO2 appeared healthy at all total pressure

treatments (33, 66, or 101 kPa). However, at 180 Pa pCO2, leaves from plants grown at

33 and 66 kPa pressures were yellow, had red speckles and leaf tip burn, and appeared

to have reduced chlorophyll content. This "leaf damage" was not observed for plants

grown at 180 Pa pCO2 and 101 kPa pressure (Figure 4-2). Despite this apparent leaf

damage, plant dry weight was the greatest in the 180 Pa pCO2 treatments for all

pressures (33, 66, or 101 kPa, Table 4-2). The pressure and pCO2 both significantly

affected the dry weight (p < 0.01) but the interaction between these two parameters was

not significant (p > 0.05). The dry weight of plants was maximal at 66 kPa total pressure

and 180 Pa pCO2 with -0.65 g per plant and lowest at 101 kPa total pressure and 40 Pa

pCO2 at -0.42 g per plant (Table 4-2). For all pressure treatments, increases in pCO2

resulted in increases in dry weight (Table 4-2). Radish fresh weight and % water content








were not significantly affected by total pressure, pC02, or the interaction between the

two treatments (Table 4-2). Leaf area decreased as pC02 levels increased for plants

grown at 33 kPa total pressures (Figure 4-3). This reduction in leaf area with increasing

pC02 levels was also observed for plants grown at 66kPa and 101 kPa pressures but to

a much lesser extent (Figure 4-3). The reduction in leaf area may have been in part due

to the senescence of the hypocotyl leaves since this was accelerated in higher pC02.

Plants grown in low pC02 (40 Pa) and low pressure (33 kPa) had the largest leaf area

(-240 cm2 per plant) compared to other pC02 and pressure treatments (Figure 4-3).

C02 Assimilation

The C02 assimilation rates (CA) for plants remained approximately constant from

day 1 to day 7 for 40 and 100 Pa pC02 treatments for all pressures however, at 180 Pa

pC02, the CA decreased with each passing day (data not shown). This reduction in CA

was greater at 33 and 66 kPa than for plants grown at 101 kPa. The pressure and the

pC02 treatments as well as their interaction significantly affected CA with the greatest CA

at 100 Pa pC02 and 66 kPa at -12.42 [[mol m-2 s-1 and the lowest at 180 Pa pC02 and

101 kPa at -3.44 [[mol m-2 s-1 (Table 4-2). The C02 uptake rates at low pC02 (40 Pa)

and 33 kPa total pressure was greater than the uptake rates from plants grown at

similar pCO2 levels with 66 kPa or 101 kPa total pressure (Figure 4-4A). This

corresponded to a greater leaf area in the low pCO2, low pressure treatments compared

to plants from other pressure and C02 combinations (Figure 4-3). At 100 Pa pCO2,

plants grown at 66 kPa had the greatest C02 uptake (Figure 4-4B) which corresponded

to the maximal CA (based on leaf area) and the greatest dry weight of all experimental

treatments (Table 4-2). For plants grown at 101 kPa, the CA (based on leaf area) was









similar for all C02 treatments (Table 4-2) despite having an increased slope in C02

uptake as pCO2 levels increased (Figure 4-4). At super elevated levels of CO2 (180 Pa),

the CO2 uptake rate was the lowest for plants grown at 33 kPa total pressure compared

to plants from other treatment combinations (Figure 4-4C).

Dark respiration rates (DR) were significantly (p < 0.01) affected by total pressure

but not by the CO2 or interactions between the two (Table 4-2). In general, as total

pressure was reduced from 101 kPa to 33 kPa, the DR increased for plants grown at all

pCO2 levels. The exception to this was at 100 Pa pCO2 and 66 kPa total pressures

where the DR was maximal at -1.91 [mol m-2 s-1. This corresponded with plants with

the greatest dry weight (Table 4-2). In contrast, the lowest DR was found with plants

grown at 180 Pa pCO2 and 101 kPa total pressure at -0.6 [[mol m-2 s-1 that

corresponded to the lowest CA (Table 4-2).

Transpiration Rates

Plants grown at reduced pressures (33 or 66 kPa) had greater transpiration rates

than plants grown at 101 kPa, regardless of the CO2 treatment (Table 4-2). In fact,

plants grown at 33 kPa total and 40 Pa pCO2 had over twice the transpiration rate (-580

mL m-2 day-1) as plants grown at 101 kPa total and 40 Pa pCO2 (-210 mL m-2 day-1,

Table 4-2). Transpiration rates were reduced by increasing pCO2 from 40 to 100 Pa for

all pressure treatments but a further increase in pCO2 to 180 Pa only reduced

transpiration rates for plants grown at 101 kPa (Table 4-2, Figure 4-5). The cumulative

water loss per plant was greatest in low pCO2 (40 Pa) and low pressure (33 kPa) with

-50 mL of water lost per plant over the seven days in the chambers (1 day acclimation,

7 day treatment) compared to plants grown at 101 kPa total pressure with 180 Pa pCO2









which lost the least amount of water for any treatments over the experiment at -12 mL

per plant (Figure 4-5). Transpiration was minimal at night as indicated by the plateaus

on the curves of cumulative water loss for all pressure and pCO2 treatments (Figure 4-

5).

Discussion

Radish plants appeared healthy with no signs of water stress or leaf damage for

most of the pressure and pCO2 combinations that were used in these experiments. The

exception to this was for plants grown at lower pressures (66 or 33 kPa) and super-

elevated pCO2 (180 Pa, Figure 4-2). These plants had yellow leaves with red speckles,

and leaf tip damage. Previous studies with radish in hypobaria did not report any leaf

damage at similar pressures (33, 66, and 96 kPa) even after 21 days in treatments with

120 Pa pCO2 (Levine et al., 2008). The differences in leaf appearance between this and

the previous study may be that the 180 Pa pCO2 used here was at a high enough

concentration to induce leaf damage at low pressure. Low pressures enhance diffusion

rates of CO2 (Gale, 1971) therefore, the detrimental effects of elevated CO2 may occur

at lower concentrations in hypobaria. Since plants appeared healthy at 101 kPa and 180

Pa pCO2, it suggests that this level of pCO2 is not detrimental to growth at normal

atmospheric pressures. Previous studies at super-elevated levels of pCO2 (500 and

1000 Pa) reported leaf bleaching in potatoes, wheat and soybeans at normal

atmospheric pressure (Tisserat et al., 1997; Wheeler et al., 1999). In addition, high

pCO2 can cause accelerated leaf senescence (Usuda and Shimogawara, 1998). It is

also possible that ethylene buildup in the system could have caused the leaf damage in

our studies since ethylene was not scrubbed from the gas phase. Ethylene, which can









accumulate in closed systems with plants, can reduce chlorophyll levels in leaves and

promote leaf senescence (He et al., 2009). The much larger chambers that were used

in previous studies with radish in hypobaria (Levine et al., 2008) may have prevented

high concentrations of ethylene from accumulating near the plant thus preventing leaf

damage. Further studies with the removal of ethylene from the chambers may clarify the

roles of ethylene in inducing leaf damage at super-elevated pCO2 in hypobaria.

Despite the leaf damage in hypobaria with super-elevated pCO2, plants from these

treatments had enhanced dry weights compared to plants grown at lower pCO2 levels

(40 or 100 Pa pCO2, Table 4-2). Super-elevated pCO2 can greatly enhance starch

accumulation in leaves (Levine et al., 2009) which may account for the increased dry

weights that were observed. In general, CO2 enrichment (100 or 180 Pa) increased the

dry weight of plants for all pressure treatments compared to plants grown at limiting

pCO2 (40 Pa, Table 4-2). Many experiments have demonstrated that CO2 enrichment at

normal pressures causes significant increases in photosynthetic uptake of CO2 and

increases in biomass for a variety of plant species including radish (Usuda and

Shimogawara, 1998; Long et al., 2004).

Radish had an increase in biomass (dry weight) in hypobaria compared to plants

grown in normal atmospheric pressures (Table 4-2). While some studies report similar

increases in biomass from plants grown in hypobaria (Andre and Massimino 1992;

Corey et al., 1996; Spanarkel and Drew, 2002; Goto et al., 2002), others report no

change or a decrease in biomass (Daunicht and Brinkjans, 1992; Iwabuchi et al., 1994;

Goto et al., 1995; He et al., 2007, 2009). Not surprisingly, the photosynthetic rates

reported for plants grown hypobaria also conflicts, with some studies reporting









enhanced photosynthetic rates and others reporting no change (Iwabuchi et al., 1994;

Goto et al., 2002; Corey et al., 1997; Spanarkel and Drew, 2002; Iwabuchi and Kurata,

2003; Richards et al., 2006). For Arabidopsis, photosynthetic rates were increased with

decreasing pressure for limiting pCO2 (40 Pa) but were unchanged in response to

hypobaria at non-limiting pCO2 (70-100 Pa, Richards et al., 2006). In this case, the p02

was dropped as pressure was lowered thus resulting in lower pO02/pCO2 ratio. A lower

pO02/pCO2 ratio can result in enhanced photosynthesis. The pO02 was maintained in our

experiments at ambient levels (21 kPa) yet plants had enhanced photosynthetic rates in

hypobaria even in non-limiting CO2 (Table 4-2, Figure 4-4). A previous study with radish

also reported both an increase in shoot biomass and an increase in photosynthesis for

plants grown at low pressure (33 kPa) with normal atmospheric levels of pO02 (21 kPa;

Levine et al., 2008). Therefore, it appears that for radish the biomass and

photosynthetic rates are enhanced in hypobaria when the pO02 is maintained at normal

atmospheric levels.

The dark respiration rates (DR) were enhanced for radish in hypobaria (Table 4-2)

which correlates with results found for Arabidopsis and lettuce (Spanarkel and Drew

2002; Richards et al., 2006). In contrast, others report a lower DR for lettuce that was

grown longer-term in hypobaria (He et al., 2007). Since plants may adapt to low

pressure it may be possible that plants adapted and reduced their DR in response to

hypobaria.

Transpiration rates of radish were higher in hypobaria regardless of CO2 levels

compared to plants grown in normal atmospheric pressures (Table 4-2, Figure 4-5).

Enhanced evapotranspiration or transpiration rates have been reported in hypobaria for









a few species (Daunicht and Brinkjans, 1996; Iwabuchi and Kurata, 2003; Wilkerson,

2005; Richards et al., 2006; Levine et al., 2008). The increased diffusivity of gases in

low pressure can result in rapid water removal from leaves and promote wilting of plants

that are transferred from high pressure to low pressure environments (Rygalov et al.,

2004; Wilkerson, 2005). Drought related genes are differentially regulated in response

to hypobaria suggesting that plants do respond to the enhanced water loss in

hypobaria. Approximately 200 drought-stress-related genes were differentially regulated

in Arabidopsis in response to hypobaria and included genes involved in desiccation-

related pathways such as dehydrins and ABA-related proteins (Paul et al., 2004).

Similar to the results of photosynthetic rates in hypobaria, the evapotranspiration rates

in Arabidopsis studies were increased in hypobaria for low pCO2 treatments but were

similar to plants grown at normal atmospheric pressures when pCO2 levels were non-

limiting (Richards et al., 2006). For radish, enhanced transpiration rates in hypobaria (33

or 66 kPa) occurred even in non-limiting CO2 (Table 4-2, Figure 4-5). These differences

may be accounted for the different mechanisms of acclimation to hypobaria for different

species with Arabidopsis responding quicker to the hypobaria or the differences in the

measured parameter evapotranspiration versus transpiration. However, for spinach

grown in non-limiting CO2 (100 Pa) and short-term in hypobaria the transpiration rates

were enhanced compared to plants grown at normal atmospheric pressure but after ten

days acclimation in hypobaria plants had similar rates of transpiration as plants grown at

normal atmospheric pressure (Iwabuchi and Kurata, 2003). The stomata apertures from

the spinach that were acclimated long-term to hypobaria were smaller than those that

were grown at higher pressure. It may be that the radish had not yet acclimated to









hypobaria even after seven days (Table 4-2, Figure 4-5). Transpiration rates were

reduced by increasing pCO2 from 40 to 100 Pa for all pressures (Table 4-2, Figure 4-5).

However, an increase in pCO2 from 100 to 180 Pa only reduced transpiration for plants

grown in normal atmospheric pressure (101 kPa) and not for plants grown in hypobaria

(33 or 66 kPa; Figure 4-5). Many studies have shown that at normal atmospheric

pressure, stomata closure occurs in plants as pCO2 is increased from 40 to 100 Pa but

above 100 Pa there is little further decrease in the stomata aperture (Jarvis 1976;

Stanghellini and Bunce, 1993; Assmann, 1999; Wheeler et al., 1999). This may explain

why a further increase in pCO2 from 100 to 180 Pa did not reduce transpiration for

plants grown in hypobaria. Taken together, it appears that plants acclimate to hypobaria

and the enhanced transpiration by closing stomata when CO2 is not limited. However, in

limited CO2, stomata remain open in hypobaria provided that water is non-limiting.

Further studies on the development and response of stomata in hypobaria may lead to

identifying the mechanisms of adaptation for plants to the low pressure environment.

Summary

Radish plants grown in hypobaria had increased biomass (DW), C02 assimilation,

dark respiration (DR), and transpiration compared to plants grown in ambient pressures.

Transpiration was reduced and growth enhanced by enrichment with CO2 for all

pressure treatments. Plant transpiration rates remained constant over seven days of

hypobaria treatments suggesting that plants did not acclimate to hypobaria by reducing

stomata aperture during this period. Very high pCO2 (180 Pa) when combined with

hypobaria (33 or 66 kPa) induced leaf damage. The leaf damage was not found at lower

pCO2 (100 Pa) treatments suggesting that the threshold for pCO2 uptake had been

reached at 180 Pa pCO2. Longer-term studies on the interaction of CO2 and pressure









on plant growth in hypobaria will provide further insight into the mechanisms of plant

adaptation to hypobaria that can be used to develop decision support tools for growing

plants on future space exploration.








Fluorescent lamps

ZI\Z I!I\Z I!I\


Balance


Figure 4-1. Schematic of the low pressure growth chambers used for experiments.
Each chamber has 0.09 m3 total internal volume. The data logger (CR10)
recorded electrical signals from temperature, CO02, 02, balances (n=3),
pressure, relative humidity, and light sensors for each chamber.



































Figure 4-2. Radish (26 day-old) grown for six days in super elevated C02 (180 Pa) at
101 (first row), 66 (second row), or 33 (third row) kPa total pressure. Side
view is shown on left side of figure with corresponding top view in the right
panel. All plants were acclimated in the chamber for one day prior to
experiments.










S40 Pa CO2
S100 Pa CO2
O180 Pa CO2


1.6


q 1.2
E
0
0.8


0.4


40 Pa CO2
S100 Pa CO2
S180 Pa CO2


101


Total Pressure (kPa)


Figure 4-3. Total leaf area of radish grown at various total pressures and C02 levels.
(A) Ratio of leaf area from the last day of the experiment, at day 7 (D7) to the
first day (D1) for plants grown at 40, 100 or 180 Pa CO02 and at 33, 66 or 101
kPa total pressure. (B) Leaf surface area at D7 of treatment for plants grown
for six days at 40,100 or 180 pCO2 and at 33, 66 or 101 kPa total pressure.
Bars represent the mean STDEV from three experiments (n = 3) with each
experiment having three plants.


il


-I-


0
13
Q
0
CU
=or




,-
03
-0


1J


- T


0

300

250

200

150

100


I I


Mmm


.A.".


1 0


m












40 y= -5.6x + 41 66 kPa
R- R= 0.99 101 kPa
30 Ty = 39.9e-O.24x
RI = 0.95
20

10
y = 49.6e-o67x
R_ = 0.99
D 0
SOO B 100 Pa CO2
100
O 1 y=-7.2x + 100


60
y =-12.8x + 105
40 R 0.99 33 kPa

S-= -17.4x + 106 66 kPa
R2 = 0.99

0 111

200 C 180 Pa CO2

160
01kPa
y = -5.0x + 184
120 R20.98
y= -10.6x + 181 66 kPa
80 R = 0.99 /
y= -15.64x + 179
40 -R2= 0.99

0
0 1 2 3 4 5

Time (h)

Figure 4-4. C02 drawdown curves on the final day of the experiment of 26 day-old
radish grown for six days at 33, 66, or 101 kPa. The CO2 levels (partial
pressure) were either low (40 Pa; A); high (100 Pa; B) or super elevated (180
Pa, C). Bars represent the mean STDEV from three experiments (n =3) with
each experiment having three plants.








60
50 A 40 Pa CO2 :,r
40
0 3366 kPa ,,'
40 66kPa ..L
._ 30 101 kPa
-, 20 I
C
E 10 -


EW 60o
SB 100 Pa CO2
50
0 33 kPa
40
0 66 kPa
C 30
30 4 101 kPa
S 20

10

03 60
E 5o C 180 Pa CO2
U 40 0 33 kPa
U 66 kPa
30 101 kPa ,.,
20 -


0
0 2 4 6 8

Figure 4-5. Water loss due to transpiration of 26 day-old radish grown for 7 days for low
(40 Pa; A), high (100 Pa; B) or super-elevated C02 (180 Pa; C) at 33, 66, or
101 kPa total pressure. Values represent the mean of three experiments (n =
3) with each experiment having three plants. Standard deviation bars have
been included only for every 1 hour for clarity. Arrows indicate the start of
pressure treatments.









Table 4-1. Set points for the environmental conditions
Parameter Set point
Air Temperatures 22/20 1C day/night
Relative Humidity >85%
PAR 250 pmol m-2 s-1
Total Pressure 33 2, 66 2 or 101 kPa
pO2 21 1 kPa
CO2 40, 100, and 180 Pa











Table 4-2. The effect of pressure and C02 on plant growth (dry and fresh weight), water content, CA and dark respiration
(DR) and transpiration rates of 26-day-old radish plants grown for six days at the given pressure treatment. All
values except CA and DR are based on mean of three experiments with three plants per experiment (n = 3
STDEV). CA and DR are based on three replicated chambers.


Treatment
Press pCO2
(kPa) (Pa)
33 40
100
180
66 40
100
180
101 40
100
180
P
C02
PX C02


Parameters


Fresh Wt.
(g plant-1)
3.78 0.11
3.26 1.08
3.26 0.01
3.36 0.17
3.31 0.16
4.65 1.16
3.00 .0.85
2.82 0.41
3.66 0.30
N.S.
N.S.
N.S.


Dry Wt.
(g plant-1)
0.49 0.07
0.57 0.07
0.62 0.21
0.45 0.10
0.61 0.25
0.65 0.16
0.42 0.05
0.47 0.12
0.50 0.04
**


N.S.


Water CA


(%)
88 2
81 4
80 4
88 3
85 8
87 1
85 5
86 6
86 1
N.S.
N.S.
N.S.


(pmole m-2s-1)
7.24 1.44
6.84 1.10
3.58 1.69
4.28 0.99
12.42 2.86
6.48 3.20
4.32 0.65
4.43 1.59
3.44 1.04
**
**
**


(mol m2s-1)
1.71 0.43
1.31 0.17


1.66
1.61
1.91
1.19
1.15
0.84
0.60
**.
N.S.
N.S.


0.76
0.12
0.61
0.33
0.20
0.23
0.15


Transpiration
(mLm-2day-1)
584 39
339 51
344 59
488 112
337 106
298 50
209 27
134 12
89 23
**
N.S.
N.S.


Significance was determined by two-way ANOVA
*p < 0.05, ** p < 0.01






100


CHAPTER 5

THE EFFECTS OF SHORT-TERM AND LONG-TERM ACCLIMATION OF RADISH TO
HYPOBARIA

Introduction

Water is an extremely important resource for any agricultural system. Therefore, it

is not surprising that plants adjust their growth in response to the differing amounts of

water that is available to optimize their growth and survival. In water-limiting conditions,

plants adjust their growth to promote water uptake and minimize water loss. This

adjustment in growth depends on the duration and the extent of the water stress. For

example, short-term responses to limited water availability include the closure of

stomata (Ehleringer and Cooper, 1992), increased gene regulation of stress pathways

(Knight and Knight, 2001), osmotic adjustment in the roots (Rodriguez et al., 1995),

changes in signal transport (Morgan, 1984) and inhibition of growth (Schulze, 1986). For

longer-term responses to water stress, plants may increase root growth relative to shoot

growth (Rodriguez et al., 1995), reduce the transpiration area (Jackson et al., 2000),

and have osmotic adjustments to their root systems (Morgan, 1984).

For long-term space missions, the conservation and recycling of water by plants

that are used as part of an Advanced Life Support system will be important for the

success of any mission to distant planets or moons. In these missions, plants will likely

be grown at reduced pressures relative to Earth sea level pressure which can result in

enhanced transpiration/evapotranspiration and photosynthesis (Iwabuchi et al., 1996;

Goto et al., 1996; Daunicht and Brinkjans, 1992, 1996; Corey et al., 1996, 2002;

Wilkerson, 2005). This enhanced transpiration can result in a variety of stress

responses if water is not readily available to the plant. For example, radish grown at


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ambient total pressure immediately wilted when transferred to hypobaria at 25 kPa total

pressure (Wilkerson 2005). These plants recovered within several hours after being

transferred to the low pressure environment although at reduced transpiration rates

prior to the transplant suggesting that plants partially closed their stomata. In other

cases, radish did not wilt after being transferred to low pressure (33kPa; Wehkamp

2009; Gohil et al., 2010). These plants likely had more water available to them since

they were grown in hydroponic systems compared to the wilted plants that were grown

in soil pots. Arabidopsis plants that were exposed to hypobaria also showed no sign of

wilting when transferred to low pressure but they had over 200+ genes that were

differentially regulated compared to plants that were maintained in ambient pressures

(Paul et al.,, 2004; Paul and Ferl, 2006). These genes included several that are involved

in water-stress-related biochemical pathways such as abscicic acid. In many cases,

plants acclimated to hypobaria by adjusting their transpiration over time to levels that

were similar to plants grown in ambient pressure (Iwabuchi and Kurata, 2003;

Wehkamp 2009). The early adjustment or acclimation to hypobaria appears to involve a

decrease in stomatal aperture reducing transpiration and the longer-term adaptation

mechanisms including decreases in stomatal size (Iwabuchi and Kurata, 2003). Young

leaves exposed to hypobaria or emerging in hypobaria are likely to go through some

other physiological changes that older leaves do not go through but this response is not

well studied. Taken together, it appears that plants respond to hypobaria and enhanced

transpiration similar to a water stress but the extent of this is unclear and may depend

on the type of substrate used to grow the plant. To understand the adaptation

mechanisms used by plants to hypobaria, the CO2 assimilation, transpiration, dry


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weight, plant growth, leaf area and stomatal density were compared for plants that were

grown both in long and short term hypobaria.



Materials and Methods

Plant Material
Seeds of radish (Raphanus sativus L. cv. Cherry Bomb) were germinated and

grown in 350 mL flasks filled with about 300 mL of nutrient solution of 1/4 strength MS

(Murashige and Skoog, 1962), pH adjusted to 6.8 with HCI (1 mM) or NaOH (1 M).

Rockwool stoppers (35 cm diameter x 40 cm height; Grodan Delta, Aurora, CO) and

absorption paper rolls 2 cm X 10 cm (Anchor paper, St. Paul, MN) served as a wicking

system. Plants were grown for total of 28 days. The volume of flask was large enough

for the 4 week experimental studies that the level of nutrient solution at the end of the

experiment was well above the majority of roots. The roots were well-developed and

reached far into the flask at this growth stage. Each stopper was soaked in a nutrient

solution of 1/4 strength MS for 24 hours prior to the experiment setup and the pH was

maintained around 6.8 through the use of 1 mM of HCI or 1 M NaOH. Each seed was

rolled inside absorption paper, placed inside the center of the rockwool, and then placed

so that the wick was ~ 7 cm within the nutrient solution. In order to avoid as much

evaporation of the solution, the top of the rockwool was covered in plastic wrap. Each

flask was then wrapped in foil to reduce light exposure to the nutrient solution. For the

five experimental treatments (Treatment A-E; Table 5-1), seeds were germinated and

grown at 101 kPa or 33 kPa and then transferred to 33 kPa (0.1 kPa pp CO2, 21 kPa pp

02) on days 7, 14, 21 or 26 (Table 5-1). During the hypobaric studies the environmental

conditions are described in Table 5-2.


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Growth Chambers and Environmental Control

The experiments were performed in three chambers (Figure 5-1) of total volume of

0.09 m3 each with a fan for air circulation (Delta Model, P/N BFB0512M, Silicon Valley

Compucycle, San Jose, CA). The chambers had sensors for temperature (DS10 Dallas

Semiconductors, Dallas, TX), pressure (P/N ASCX15AN, Sensym ICT, Milpitas, CA),

CO2 (OEM ultrasonic, 6004, Goleta, CA), 02 (MAX-250, Maxtec Co., Salt Lake, UT),

relative humidity (HH-4602-L-CPREF, Honeywell, Morristown, NJ), light (LI-190, Li-Cor,

Lincoln, NB) and three balances for weight determination per chamber (LPS-0.6 kg,

Celtron Tech. Inc. Colvina, CA). Calibration of 02 and CO2 sensors was performed

according to Richards et al., (2006). CO2 and 02 levels were adjusted daily at 4 hours

into the photoperiod. CO2 assimilation studies were started at the same time in the

photoperiod for all tests and were repeated daily. After the acclimation period, the gas

and pressure composition were brought to set points daily (Table 5-2). Environmental

conditions for the one day acclimation in the growth chamber were 22 2 C, 12 hour

photoperiod, at 250 [[mol m-2 s-1 using cool-white, florescent lamps and 85% relative

humidity. The pO02 was maintained at -21 kPa with other environmental conditions

described (Table 5-2).

Each chamber contained one to two individual plants. Pressure was maintained

with a vacuum pump (1/2 HP, JB Industries, Chicago, IL) and chambers had leakage

rates of ~0.03% (chamber volume/h). Data collection and controls were performed using

a CR10 data logger (Campbell Scientific, Logan, UT) that was connected to a PC.

Experiments were performed in triplicate.


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Gas Exchange Rates

The C02 assimilation rates (CA) were calculated by performing draw down curves

for 5 hours period once CO2 levels were adjusted at a set point (0.12 kPa) at noon. The

slope of the regression equation represented the CA rate per hour which was converted

into pmol m-2 s-1. The range of CO02 over a 24h period was (100 22). The transpiration

rates were calculated by performing draw down curves of load cell values for 5 hours

period once CO2 levels were adjusted at set point (0.12 kPa) at noon. The slope of the

regression equation represented the transpiration rate per hour which was converted

into pmol m-2 s-1.

Plant Biomass and Leaf Area

After the experimental treatments in the chambers, plants were removed from the

chamber blotted dry and analyzed for fresh weight, leaf area, and dry weight. Dry weight

analysis was performed by placing plants on pre-weighed pans at 60C for 48h and

reweighing the contents. Leaf area was measured on the last day of experiment with

scanned digital images using Image Pro Plus software (ver. 6.0 Media Cybernetics,

Bethesda, MD).

Scanning Electron Microscopy

Radish leaves were fixed in 2.5% glutaraldehyde in 0.1 M sodium cacodylate, pH

7.24 and stored overnight at 4 C. The fixed leaves were processed with the aid of a

Pelco BioWave laboratory microwave (Ted Pella, Redding, CA, USA). Samples were

washed in 0.1M sodium cacodylate pH 7.24, post fixed with 2% buffered osmium

tetroxide, water washed and dehydrated in a graded ethanol series 25, 50, 75, 95, and

100% and critical point dried (Bal-Tec CPD030, Leica Microsystems, Bannockburn, IL,

USA). Samples were mounted on carbon adhesive tabs on aluminum specimen mount,


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Au/Pd sputter coated (Deskll, Denton Vacuum, Moorestown, NJ, USA) examined and

high resolution digital micrographs acquired with field-emission scanning electron

microscope (S-4000, Hitachi High Technologies America, Inc. Schaumburg, IL, USA).

Leaf Stomatal Density and Stomatal Index

The surface electron microscopy images were used to calculate leaf stomatal

density, i.e., the total number of stomata per unit leaf area (Radoglou and Jarvis, 1990).

Fully grown, young leaves from the plants used for measuring gas exchange rates were

selected. Leaf discs of 8 mm diameter from the area about 1 cm from the mid rib were

punched out from an attached leaf using a plunger and immediately transferred to the

buffer solution. Three samples, two samples of hypobaria treatment and one sample for

control (101 kPa), were analyzed at the electron microscopy facility at the University of

Florida. The number of stomata (s) and epidermis cells (e) were counted from the

scanned images. The leaf stomatal index (LSI) was calculated using the formula [s / (e

+ s)] X 100. The stomatal size was calculated by averaging the total length (pm) of

primary axis of all the guard cells of the scanned images.

Results

Seed Germination and Plant Growth in Hypobaria

The germination of radish was significantly affected by the partial pressure of

oxygen (pO02) in hypobaria (33 kPa total pressure) with no germination at 1.5 kPa pO2

and only 45% of the seeds germinating at 4 kPa pO2. This suggests that the minimum

pO2 required to induce seed germination with the flask system at 33kPa total pressure

is between 1.5-4 kPa (Figure 5-1). Interestingly, as pO2 increased to 6 and 10 kPa,

germination increased to 62 and 82%, respectively. In fact, at 10 kPa pO2, the


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germination was not statistically different from pO02 of 21 kPa (at either 33 kPa or 101

kPa total pressures; Figure 5-1).

To study the effects of the age of the plants at the time of transfer to hypobaria on

plant growth and acclimation, five treatments were performed. These treatments

consisted of transferring seedlings after 0, 7, 14, 21, or 26 days of normal atmospheric

pressures into hypobaria at 33kPa total pressure (Table 5-1; Treatments A-E).

Seedlings appeared healthy except in the case for the seedlings that were transferred to

hypobaria (33 kPa) after one week where the plants all had stunted growth or died

(Treatment B; Figure 5-2). This suggests that the developmental stage of growth during

which plants are transferred to low pressure is important for health and survival of a

crop in hypobaria. Due to poor growth and in several cases the death of plants from the

one-week-old stage treatment (Treatment B, Table 5-1), no further analyses were

performed on these plants.

As far as the growth of the plants during the 28 day experiments, plants from the

four treatments (A, C, D, and E) all had similar fresh and dry weights and root to shoot

ratios (Table 5-3). The longer the plants were exposed to hypobaria, the smaller the leaf

area of the plants and the thicker the leaves at the end of the experiment (Figure 5-3C;

Table 5-4). The greatest leaf area was observed from plants grown with 2 days

exposure to hypobaria (at 82 cm-2) followed by leaves from plants grown at normal

pressure (at 78 cm-2) and the lowest leaf area was observed for plants grown

completely in hypobaria (at 58 cm-2; Figure 5-3C; Table 5-4).

C02 Assimilation Rates (CA)

In general, the longer the time that plant remained in hypobaria, the greater the

carbon dioxide assimilation rates (CA) on the last day of the experiment (Fig. 5-3A).


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However, the increase was significant only at p< 0.1 and not at p<0.05 value. The

highest CA was observed for Treatment A (28 days in hypobaria) at 23 [mol m-2 s-1 and

lowest for Treatment D (2 days in hypobaria) at 14 [[mol m-2 s-1 (Figure 5-3A). This

suggests that plants have a rapid adjustment in CA during the initial stages of plant

transfer to hypobaria indicated by the slight drop in CA for the two days treatment in

hypobaria (Treatment D). Interestingly, when comparing CA over the course of the

experiment, there was very little difference in CA between plants that were grown

entirely in hypobaria and those grown in normal atmospheric pressure (Figure 5-4A).

This may be a result of the limited amount of light used in these studies (250 [mol m-2 S-

1) and not by CO2 availability since CO2 diffuses at a faster rate in hypobaria than

normal pressures and CA would be expected to be higher in the hypobaria treatments.

Further studies that compare CA in higher light conditions in hypobaria and normal

pressure are required to determine if that is the case.

Transpiration Rates (T)

Plants grown entirely in hypobaria (Treatment A) had significantly higher (p< 0.05)

transpiration rates on the last day of the experiment compared to those plants exposed

to just two days of hypobaria (Treatment D; Figure 5-3B). The highest transpiration

rates were observed for these plants grown entirely in hypobaria at 2.60.5 mmol m-2 s-1

followed by the next longest treatment in hypobaria (Treatment C) at 2.30.6 mmol m-2

s-1. The lowest transpiration rates were found for plants exposed to two days in

hypobaria (Treatment D) at 1.7 0.4 mmol m-2 S-1 (Figure 5-3B). Plants that were grown

entirely in hypobaria had higher transpiration rates than those grown entirely in normal

pressure (Treatment E) for the entire experiment except for day 21 suggesting that


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plants did not acclimate to the enhanced water loss in the hypobaria treatments at least

in the first two weeks of growth (Figure 5-4B).

Water Use Efficiency (WUE)

Water use efficiency (WUE) of plants grown in hypobaria was calculated as a ratio

of transpiration rate over CA, i.e., amount of water transpired per amount of CO2

assimilated. The WUE were calculated on days 7, 14, 21 and 28 (last day) of the

experiment (Figure 5-4C). The WUE of a plants grown in hypobaria was similar to WUE

of plants grown at normal pressure at 21 and 28 days of growth but lower for days 7 and

14 (Figure 5-4C). Since CA between these two treatments were similar (Figure 5-4C),

this suggests that the increase in transpiration (T) on these days (Figure 5-4B) and not

CA (Figure 5-4A) was the cause for this lower WUE since in both cases, the WUE

increased over time until a maximum rate was reached after 21 days with approximately

8.6x1 0-3 [mol of C02 assimilated per [[mol H20 transpired per leaf area (Figure 5-4C).

The WUE of 24 days old spinach was ~ 4x1 0-3 [mol of CO2 assimilated per [[mol H20

grown in hypobaria at 25 kPa and was similar to that of plants grown at normal pressure

(Iwabuchi and Kurata, 2003).

Stomata Development

The stomatal density (i.e. number of stomata per cm2 of leaf) on the abaxial side of

leaf was similar between plants grown entirely in hypobaria (Treatment A) compared to

plants grown entirely at normal pressure (Treatment E; Table 5-4). The average

stomatal frequency of 1.42 X 105 per cm-2 was found for plants grown in hypobaria

compared to 1.28 X 105 per cm-2 for plants grown at normal pressure. Other parameters


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such as epidermal cell density, stomatal index, and length of stomata pore were not

significantly different between the two treatments (Table 5-4).

Discussion

Seed Germination

In one of the earliest hypobaria studies, rye seeds did not germinate at 3 kPa

total pressure with Mars level pO2 (~0.3 kPa) and pCO2 (-0.7 kPa; Siegel et al., 1963).

Here, radish seeds did not germinate in hypobaria at 1.5 kPa pO02 (33 kPa total

pressure) and only had 40% germination at 4 kPa pO02 (Figure 5-1). Mustard seeds did

not germinate below 5 kPa pO02 (Musgrave et. al., 1988). In other recent studies with the

same cultivar of radish, only 2-5 % radish seeds germinated at 2 kPa pO02 in hypobaria

(25 and 50 kPa total pressures; Wehkamp, 2009). However, 30% of the seeds

germinated at 5 kPa pO02 in hypobaria (25 kPa total pressures). The lack of oxygen

availability in low oxygen environments inhibits respiration in the seeds which prevents

germination (Bewley and Black, 1994). The composition of the seed coat and availability

of nutrient reserves within seeds could also affect seed germination and thus the level

of oxygen required for germination will likely be species dependent. AI-Ani et al. (1985)

reported that no germination of radish occurred at 2 kPa of pO02 in ambient pressure but

did germinate at 7 kPa of pO02. Here, at 10 kPa pO02 (33 kPa pTotal) the germination was

about 82 % compared to 75 % reported for radish at 10 kPa pO2 with lower total

pressures of 25 kPa total pressure (Wehkamp 2009). These results suggest that the

lower limit for radish germination in hypobaria or in ambient pressure is at approximately

2 kPa pO2 although higher levels >10kPa pO2 are required for germination at levels near

normal pressure and oxygen conditions.


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Seedling Growth in Hypobaria

Radish plants that were exposed to hypobaria (33kPa) one week after germination

(Treatment B) exhibited severe stress or died within a few days in hypobaria (Figure 5-

2). However, the plants from the rest of the treatments even those that were germinated

in hypobaria appeared healthy and there was no indication of water stress or leaf

damage. The poor growth and death of plants after one week of transplant is likely due

to the fragile stage of the seedlings at the time of transfer where only the cotyledons

were the main area for photosynthesis and transpiration. Indeed, the cotyledons from

plants from the one-week-old treatment curled and wilted within the first few days of low

pressure exposure (Figure 5-2). In contrast, three-day-old radish seedling that were

grown under ambient atmospheric pressure then exposed to hypobaria (25 kPa) grew

without any detrimental effects (Wehkamp, 2009). However, those seedlings were

grown at higher light conditions than the seedlings from this study and may have

already developed primary leaves or may have had a larger root/shoot ratio to keep up

with the enhanced transpiration that occurs in hypobaria (Gohil et al., 2010). There may

be a critical root to leaf balance that is required for successful acclimation of a plant to

hypobaria and this may be met at either the early transfer period of less than one week

or after the primary leaves are established at approximately two weeks.

The radish plants that were germinated and grown in hypobaria for the entire

duration of the experiment (Treatment A) had similar growth (fresh weight, dry weight

and total dry weight) when compared to the control or plants grown for short-term

hypobaria (Treatment D, Table 5-3). Previous studies also found no significant

differences in the biomass accumulation between radish grown in hypobaria and those

grown in ambient atmospheric pressure (Levine et al., 2008; Wehkamp 2009). Other


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species such as lettuce (Sparnarkel and Drew, 2002; He et al., 2003, 2007) and spinach

(Iwabuchi; Goto et al., 1996) had similar growth between those grown in hypobaria and

those in normal pressure.

Interestingly, the leaf area of plants was significantly affected by reduced pressure

with a decrease in leaf area the longer the time the plants remained in hypobaria

(Figure 5-3C). The leaves from plants grown entirely in hypobaria (Treatment A) were

approximately 30 % smaller compared to leaves from plants grown for a short-term in

hypobaria (i.e., 2 days, Treatment D) and those grown completely at ambient pressure

(Treatment E). The leaves from plants grown in hypobaria for longer-term were also

thicker (Table 5-4). Reduced leaf growth can be caused by ethylene accumulation in

closed chambers (He et. al., 2003, 2007). Even though ethylene was not measured in

the present study, the absence of leaf senescence, leaf epinasty, and leaf pigmentation

due to damaged chlorophyll apparatus, all indicators of ethylene build up, suggests that

ethylene was not the cause of the reduced leaf area observed in these studies. In

addition, other factors that are responsible for mitigating the negative effects of ethylene

in hypobaria include the enhanced eviction of ethylene from mesophyll tissues in low

pressure environments (Burg and Burg, 1966), the higher CO2 concentration (0.1 kPa)

in low pressure acts as a competitive inhibitor of the ethylene and the larger chamber

volume (0.1 m-3) used in these studies compared to other reports. Also, studies on gene

expression of Arabidopsis grown in hypobaria compared to plants grown at ambient

pressure found no regulation of ACC synthase or ACC oxidase genes which are

precursor to ethylene synthesis (Paul et al., 2004; Richards et al., 2006). Therefore, it is

likely that the reduced leaf area we observed for plants grown longer terms in hypobaria


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was not caused by ethylene and could be a result of enhanced diffusion of C02 to the

leaf surface or other effects of hypobaria environment such as enhanced transpiration.

Plants may acclimate to hypbobaria by reducing leaf area tominimize transpiration.

Gas Exchange, C02 Assimilation, and Transpiration in Hypobaria

At the end of the short-term exposure to hypobaria (i.e., 2 days in hypobaria,

Treatment D), the CO2 assimilation rates (CA) of plants were not significantly different

compared to plants grown in ambient pressure (Figure5-3A; Table 5-3). Similar results

were found in a previous study with mature radish (26 day old), where after 2 days the

CA rates for plants grown at 33 and 66 kPa total pressure were similar to those of plants

grown in ambient pressure for the same CO2 levels (Gohil et al., 2010). However, for

those studies, after seven days in hypobaria, the mature radish plants had increased CA

rates compared to plants grown entirely at ambient pressure. Here, there was no

significant difference in the CA during the first three weeks of growth in hypobaria

compared to the CA of plants grown entirely in ambient pressure aside from an increase

for the last week in the experiment( p<0.1 ;Figure 5-3A; Figure 5-4A). The CA of plants

grown in hypobaria increased by 25% at the end of the fourth week when compared to

the CA from plants that had only been exposed to two days of hypobaria (Figure 5-3A).

Similar results were reported in lettuce grown entirely in hypobaria where CA rates

increased only during the last week of the experiment (day 25 to 30) when compared to

plants grown at ambient pressure (Sparnarkel and Drew, 2002). Several studies have

also reported increased CA along with increased biomass for plants that are grown in

hypobaria however, these studies were of various durations and gas treatments in

hypobaria. (Andre and Massimino 1992, Corey et al. 1996, Sparnakel and Drew 2002,

Goto et al. 2002). Here, the CA reached a saturation level at approximately 21 days after


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growth (Figure 5-4A) which is similar to results reported for previous studies with radish

grown in hypobaria (Wehkamp 2009). Radish plants reached compensation points at a

faster rate as total pressure reduced (100, 70, 50, 25, 10 kPa; Wehkamp 2009).

However, after 16 h in hypobaria, Arabidopsis plants exhibited nearly identical CO2

draw down curves at various low pressure treatments implying that there is an

acclimation response (Richards et al., 2006). Taken together, it appears that CA

increases in hypobaria for plants that are transferred to hypobaria from ambient

pressures as a short-term response, but this enhancement may not last after a few days

or several weeks of growth in hypobaria.

In contrast to CA, transpiration rates were significantly higher for plants grown in

hypobaria (p< 0.05) on the last day of the experiment suggesting that transpiration from

the leaves was more sensitive to hypobaria treatments than the CA. Also, during the first

two weeks, the transpiration rates of plants that were grown in hypobaria were higher

compared to plants grown at ambient atmospheric pressure (Figure 5-4B) but reached a

maximal rate by the third week. Wehkamp (2009) also reported that the transpiration

rates of radish reached a maximal rate at the end of the third week. In spinach, the

transpiration rates were slightly higher during the short-term exposure (1 day) to

hypobaria but after 10 days in hypobaria both the CA and transpiration rates were

similar to those of plants grown in ambient pressure (Iwabuchi and Kurata, 2003). This

suggests that plants undergo adaptation during the long-term exposure to hypobaria

which results in reduced transpiration rates.

Water use efficiency (WUE), based on the rate of CO2 assimilated over the rate of

water lost, increased at a much faster rate for plants grown in hypobaria compared to


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plants grown in ambient pressure (Figure 5-4C). However, the overall WUE remained

higher for plants grown at ambient pressure throughout the duration of the experiment,

except for day 21, compared to plants grown in hypobaria (Figure 5-4C). The WUE of

spinach grown for 10 days in hypobaria (25 kPa) was similar to that from plants grown

at ambient pressure (Iwabuchi and Kurata, 2003). For many plant species, increases in

WUE are observed when water deficit is mild irrespective of the cause of stomata

closure (Raven 2002). Changes in WUE of plants could be another acclimation

response to water deficit sensing in hypobaria. This may be a concern for plants grown

in substrates where water availability is restricted (i.e. soil or agar). In this study, plants

were grown in nutrient liquid thus water was not likely limited.

Short-term Acclimation to Hypobaria

Reduced pressure in a closed system increases diffusivity of gases which may

result in increased gas exchange rates for plants grown in hypobaria (Rygalov et al.,

2004). Although increased CA is beneficial for plant growth up to limit, the increased

transpiration rates may result in water stress if the rate of water uptake is slower than

the rate of water lost. For example, for radish that were grown at ambient pressure in

soil and then transferred to hypobaria (25kPa total Pressure) plants wilted (Wilkerson

2005). However, these plants were able to recover after several hours in hypobaria.

After short-term exposure to hypobaria, Arabidopsis plants showed no wilting or

dehydration responses, however, gene expression analyses revealed that almost 200

genes were differentially expressed in response to hypobaria and hypoxia of which

about 100 genes were unique to hypobaria (Paul and Ferl, 2006). Of these hypobaria-

related genes, about 20 (e.g. LATE EMBRYOGENESIS ABUNDANCE (LEA), COLD

RESPONSIVE (COR78), DEHYDRATION RESPONSIVE (DR29), ABSCISIC ACID


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(ABA)) were related to dehydration or water stress. These studies suggest that plant

perceive water stress when exposed to hypobaria. Therefore, it is not surprising that the

response mechanisms that plants use to acclimate to short-term hypobaria stress may

be similar to responses that deal with water deficit, high temperatures or very low

relative humidity (Iwabuchi and Kurata, 2003). In a review by Chaves et al. (2003) these

early mechanisms of adaptation to these stresses are described and summarized here

in Figure 5-6. For example, short-term hypobaria such as reduced CA and stomatal

response (Figure 5-6) were observed in the present study whereas other responses

including gene-level responses and multi stress sensing have been reported in previous

studies for plants grown in hypobaria (Paul and Ferl, 2006).

There are different pathways for stress perception and stress responsive genes.

The plant hormone ABscisic Acid (ABA) has been identified as an important chemical

signal in regulation of stomata in response to water deficit (Schulze 1986). The ABA

signal is localized in roots as well as shoots. Some of the genes are activated are

involved in pathways that protect against such stress may be activated for plants

transferred from normal pressure to lower pressure. Research on the water deficit

sensing and signaling mechanisms suggests that signaling pathways are

interconnected and cross talk occurs between the different types of abiotic stresses

(Knight and Knight, 2001). For example, hypobaria may result in increased vapor

pressure deficit (VPD), a drop in leaf water potential, a reduced leaf temperature

(Iwabuchi and Kurata, 2003; Wilkerson, 2005) and an increase in leaf transpiration

(Richards et al., 2006; Gohil, 2010). All of these stresses may occur simultaneously in

hypobaria. In particular, the drop in leaf temperature in hypobaria may be responsible


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for inducing the cold resistance genes that were found for Arabidopsis (e.g. COR78;

Paul et al., 2004). A temperature drop of up to 4 C was reported for leaves of radish

when chamber pressure was lowered to 10 kPa (Wilkerson, 2005) and this may induce

a cold stress response.

To understand the effects of short-term exposure to hypobaria on plant

transpiration, Sparnarkel and Drew (2002) exposed mature lettuce plants to hypobaria

(70 kPa) and ambient pressure on alternate days from day 30 to day 38. They found

that transpiration rates were higher at ambient pressure and lower at reduced pressure

and the pattern was repeating during next 6 days. The reversible trend suggests that

short-term effects are not due to morphological changes taking place during the growth

but due to stomatal response. Since changes in stomatal density are not possible for

short term acclimation to hypobaria, the closure of stomata in response to enhanced

transpiration is likely in this study.

Long-term Acclimation to Hypobaria

When mature radish plants were exposed to hypobaria (33 kPa) and similar C02

levels in these studies for seven days, the transpiration rates of these plants were

almost twice that of plants grown at ambient pressure (Gohil et al., 2010). Based on

those studies, the gas exchange rates of radish grown completely in hypobaria

treatment were expected to be higher than plants grown in ambient pressures. Although

slightly higher transpiration rates were seen for these plants during the first two weeks

of growth and on the last day of the experiment, they were only increased by ~20 %

(Figure 3-2, Table 5-3). This suggests that plants adjusted their growth so that their

transpiration rates were minimized as they grew in hypobaria. Indeed, plants that were

grown entirely in hypobaria had reduced leaf area compared to plants grown in ambient


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pressure (Figure 5-3C). This reduced leaf area along with increased specific leaf area

(SLA), and thus increased leaf thickness, may be a result of a perceived water deficit in

hypobaria. Others have also reported increased leaf thickness for plants grown in

hypobaria although the leaf area was not reported (Wehkamp, 2009). A reduced leaf

surface may result in less water lost through surface evaporation so that plants may

decrease leaf area in response to the perceived water stress in hypobaria. However, the

decrease in leaf area in hypobaria may also be in response to other environmental

conditions such as increased diffusion of CO2 into the leaf surface in hypobaria.

The stomata are a common gate for CO2 assimilation and water vapor exit through

the leaf air space. Therefore, it would not be surprising that stomatal number and

stomatal control would be influenced by reduced pressure since both CO2 and H20

diffuse at a faster rate in low pressure. Although leaf area was reduced at 33 kPa, The

stomatal density was similar for plants grown in low pressure and the controls in this

study. In contrast, Wehkamp (2009) reported highly significant (p<0.001) correlation

between reduced pressure and stomatal density for radish. The number of stomata

increased as total pressure reduced (10, 33, 66, and 98 kPa) and as pO02 reduced was

reduced with up to 44 % increase in stomatal density at 10kPa total pressure and 2 kPa

pO02 compared to plants grown at ambient pressure. The difference in stomata numbers

reported previously and in this study might be due to the differences in the methods

(leaf imprints vs. SEM analysis), the location and age of the leaf for counts, the different

lighting and gas conditions, and the duration of hypobaria treatments. Why plants would

increase their stomatal density in response to increased transpiration of hypobaria is not

clear but for rice (Meng et al., 1999) and perennial grass species (Xu and Zhou, 2006),


117






118


a moderate increase in stomatal density with moderate drought was reported. Xu and

Zhua (2006) reported that stomatal density and guard cell size have plasticity in

response to large variation in water status. Thus, increase in stomatal density could be

a part of adaptation strategy in response to hypobaria stress similar to a water stress. In

Arabidposis, differential gene expression profile under hypobaria and hypoxia also

included genes associated with stomatal development and regulation (e.g., STOMATAL

DENSITY AND DISTRIBUTION ; SDD1) supporting this possibility (Berger and

Altmann, 2000; Paul and Ferl, 2006).

Alternatively, changes in stomata development may be attributed to other

environmental factors in hypobaria such as enhanced diffusion of CO2 to the leaf. An

increase in CO2 has shown decrease in stomata density (Knapp et al., 1994) so the

reports by Wehkamp (2009) for increase in stomata density in hypobaria do not support

this hypothesis. In addition, Case et al., (1998) compared the effects of CO2

concentration (370 and 680 ppm) on twelve wild radish plants and found that the

elevated CO2 did not significantly affect stomata index or the guard cell length.

Therefore, the changes in stomata observed in previous studies (Wekhamp 2009) are

not likely due to the enhanced CO2 in hypobaria.

Shoot or leaf water content has been suggested as a direct indicator of

physiological functioning (Sinclair and Ludlow, 1985). In the present study, the percent

moisture content of plants grown entirely at hypobaria 86% was similar to moisture

content of plants grown at ambient pressure at 88% so that the water status of the

plants does not appear to be affected by pressure. Studies to compare this response

with different substrates to grow plants in hypobaria i.e., soil or agar, are required.


118






119


Radish plants grown in hypobaria for long-term had reduced leaf area but

enhanced transpiration compared to plants grown short-term in hypobaria or grown

entirely at ambient pressures. The acclimation to hypobaria may include adaption

mechanisms that overlap with those for water deficit responses such as reducing the

area for transpiration or inducing biochemical pathways that are involved in water stress

responses. Further studies on the gene expression and growth of plants in hypobaria

are required to identify if acclimation to hypobaria is truly a drought response

Summary

The gas phase in hypobaria can result in reduced germination of radish if oxygen

levels are not above 10kPa. Plants acclimate to hypobaria through short-term and long-

term mechanisms. For example, short-term responses (few hours to days) to hypobaria

include a decrease in stomata aperture (Iwabuchi and Kurata, 2003) and the reduction

of gas exchange rates (Sparnarkel and Drew, 2002; Figure 5-3A). Here, the biomass of

plants grown long-term in hypobaria was similar to plants grown in ambient pressures.

However, the CA (p<0.1) and the transpiration rates (p<0.05) were enhanced. Although

transpiration was enhanced for plants grown entirely in hypobaria compared to plants

grown entirely in ambient pressure, this enhancement was at much reduced levels than

that for adult plants that were grown for seven days in hypobaria. For example, long-

term acclimated plants to hypobaria had a 20% increase in transpiration (Figure 5-3B)

compared to plants that were acclimated for seven days that had 200% increase in

transpiration relative to plants grown in ambient pressure (Gohil, 2010).

Long-term exposure to hypobaria may result in the activation of water deficit

defense mechanism in plants such as reducing their leaf area. Here, acclimation to


119






120


hypobaria resulted in increased WUE as transpiration was reduced after three weeks of

growth in hypobaria. Such response has also been reported in several plant species

under mild water deficit (Chaves et al., 2003).


120









I 101 kPa


1.5 4 6 10 21 21
p02 (kPa)


Figure 5-1. Germination of radish seedlings at 33 kPa total pressure and various pO2.
Bars represents the mean STDEV (n=2)


121


33 kPa






122


Figure 5-2. Radish plants after four weeks of growth Treatment A (28 days at 33 kPa),
Treatment B (21 days at 33 kPa), Treatment C (14 days at 33 kPa), and
Treatment D (2 days at 33 kPa). All plants in treatment B died within one
week of low pressure exposure.


122






123


30

^T 25 A a
ab*
20 ab*
E b*
7 15




0
3
B a
2.5
ab
S 2 b ab
E1.5


0.5

0
100

80
O C b

so a a

40

20

0
A C D E
Treatment

Figure 5-3. Carbon dioxide assimilation (CA;A), Transpiration rates (B) and leaf area (C)
on the last day of the experiment. The bar represent the mean STDEV from
three experiments (n=3) each having two or three plants. Bars with different
letters indicate statistically different means as calculated by multiple t-test for
comparing two samples for means (p<0.05). The indicates significant
difference at p<0.1.




123






124



25

4 20 A
C',
E 15 B

E 10 o
=-I- -ELONGTERM (A)
< -9--CONTROL (E)
0





1.5
0


0.5

1-


E 8 C




04





(n=3) each having at least two plants.
2124



0 7 14 21 28
Time (Days)

Figure 5-4. CA (A) and Transpiration rates (B) and WUE (C) on the day 7, 14, 21 and
28 of the experiment. The values are mean STDEV from three experiments
(n=3) each having at least two plants.






124






125


- 200 pm


200 pm


Figure 5-5. Scanning electron microscopy (SEM) image from a youngest leaf of a plant
grown entirely in hypobaria (A; 33 kPa) or in normal pressure (B; 101 kPa).


125


*


33 kPa


104-,kRa.k












Long term


Short term


Shoot
Shoot growth inhibition
*Reduced transpiration area
Gene response
Metabolic acclimation
Osmotic adjustment


Root


Turger maintenance
Sustained root growth
Increased root:shoot ratio
Increased absorption area


Shoot
Stomata closure
*Reduced CA
Multi stress sensing
Gene response
Inhibition of growth


Root


Signal transport
Xylem hydraulic
changes
Assimilate transport
Cell drought signaling
Osmotic adjustment
Gene response


Figure 5-6. The long-term and short-term responses of the shoot or root to water deficit,
low humidity and high temperature (adapted from Chaves et al., 2003). The *
marked responses were observed in present study with the bold indicated in
other reports (Paul et al., 2004; Paul and Ferl, 2006).


126


126






127


Table 5-1. Number of days plants exposure to normal pressure and hypobaria.
Treatment Days of treatment
(101 kPa) (33 kPa)
A 0 days 28 days
B* 7 days 21 days
C 14 days 14 days
D 26 days 2 days
E 28 days 0 days
Plants were severely damaged thus not analyzed for further studies.


127






128


Table 5-2. Environmental conditions used for the experiments.


Parameter
Total Pressure
Light
Air Temperature
Relative Humidity
CO2
02


Set Point
101 or 33 1 kPa
250 pmol m-2 s-1
22/20 o1C day/night
>85%
0.1 to 0.02 kPa
21 kPa


128






129


Table 5-3. Average fresh weight (FW), dry weight (DW) of shoots, roots, hypocotyls in hypobaria treatments (A-D) and
control treatment (E). The values are mean STDEV from three experiments (n=3) each having at least two
plants. ANOVA p<0.05
Treatment Parameters
Days Fresh weight (g plant-1) Dry weight (g plant-1) Root/Shoot
101/33 Shoot Root Hypo Total Shoot Root Hypocotyl Total ratio
A 0/28 3.61.3 0.80.11 2.41.2 6.8 0.7 0.520.11 0.530.2 0.250.06 1.30.2 1.020.12
*B 14/14 N/A N/A N/A N/A N/A N/A N/A N/A N/A
C 14/14 3.90.6 0.90.23 2.0 0.6 6.8 0.5 0.500.10 0.520.1 0.230.03 1.30.1 1.040.08
D 26/2 3.1 0.3 0.70.19 2.1 0.3 5.9 0.5 0.370.15 0.340.1 0.290.02 1.00.1 0.920.11
E 28/0 3.2+1.1 1.2+0.30 2.2+1.1 6.6 08 0.390.12 0.33+0.1 0.280.04 1.00.1 0.850.06


N.S.


N.S.


N.S. N.S.


N.S.


* Plants were severely damaged thus not analyzed for further statistics.


N.S.


N.S.


N.S.


N.S.


129













Table 5-4. Average specific leaf area, stomata density, stomata index and average
stomata pore length from leaf of hypobaria (33 kPa) and normal pressure
(101 kPa)
33 kPa 101 kPa
Leaf area (cm2) 58 2.8a 82 10.2b
Specific leaf area (cm2 g-1) 115 12a 208 10b
Stomata density ( 105 cm-2 ) 1.42 0.11a 1.28 0.16a
Stomata per SLA (105 cm-2 g-1) 19.2 1.8a 34.8 2.7b
Epidermal cell density (cm-2) 31 2a 29 2a
Stomata index 55 2a 52 2a
Average stomata pore length (pM) 9.63 2.2a 9.98 2.9a
Values with different letters indicate statistically different means as calculated by t-test
for comparing two samples for means (p<0.05).


130









CHAPTER 6

TRANSPIRATION MODEL PERFORMANCE AT REDUCED PRESSURE


Introduction

Low pressure environments have increased gas diffusivity which can result in

increased gas exchange rates of plants and thus increased transpiration compared to

plants grown in normal pressure. Previously Wilkerson (2005) applied the Penman-

Monteith model to predict transpiration rates of radish in hypobaria. Though the model

performed well, it was not very effective at very low pressure (10 kPa), was fairly

complex, and was developed for evapotranspiration for field grown plants and not

transpiration of individual plants. A model developed by Sinclair (1998) described in

Chapter 3 is simpler and requires fewer assumptions compared to the Penman-

Monteith model (Wilkerson, 2005). Therefore, the simplified model was used here to

predict transpiration of radish in low pressure environments. The model incorporated the

environmental conditions (gas phase composition, pressure, the temperature, and air

velocity) and also the physiology of the leaf stomataa density, stomata dimensions, leaf

length etc.). A sensitivity analysis of the model for each parameter was performed to

identify the parameters that influenced transpiration to the greatest extent. Many of

these parameters could be controlled to minimize water loss and maximize plant

growth. The prediction of transpiration based on the model was compared with the

observed from plants that were grown in either 33 or 101 kPa (Chapter 5) were

compared to the model.









Materials and Methods

A list of the environmental parameters that were used in the model is provided in

Table 6-1. The parameters that were adjusted to compare against reference values are

listed in Table 6-2. The sensitivity of transpiration predictions to pressure (which also

affected gas diffusivity), vapor pressure deficit (VPD) and stomatal width and number at

22 oC 1 m s-1 air velocity, and 100 Pa pCO2 was determined by varying one parameter

at a time while the remaining parameters were held constant (Table 6-2). Transpiration

rate was calculated by equation 3-15.

Sensitivity Analysis

Table 6-3 lists the parameters that were evaluated and their reference (101 kPa,

0.6 kPa VPD, 20C, 100 Pa pCO2, and 200 [[mol m-2 s-1 light intensity, and 21 kPa p02

by comparing the % change. The percent change in transpiration was calculated by

equation 6-1 for each parameter perturbation (Wilkerson, 2005).

T_ T (6-1)
% change = T 0 (6-1)
T

Where T = Transpiration rate with one parameter varied, g m-2 S-1

T = Transpiration calculated at reference parameter value, g m-2 s-1

The evaluation of the model was done by comparing the calculated values with

the measured values for transpiration as presented in Chapter 5 which are averages of

three replicated experiments. The VPD was varied by -50 and +50% from the reference

value (0.6 kPa). The pressure values were varied from -67% of the reference value

(101 kPa). The stomatal width was varied by -0.0001, -0.0002 -0.0003 and stomatal

number by -50, +50% from the reference value (n =13500).


132









Results and Discussion

Transpiration rate was predicted as a function of stomata dimensions, stomata

number and the diffusivity of water, as described by Sinclair's (1998) and Chapter 3.

Although transpiration rates were calculated at over a range of pressures, they were

compared with measured transpiration rate from data from plants grown as described in

Chapter 5. These plants were grown for 28 days at 33 and 101 kPa, 20-22 oC, >85%

relative humidity and 100 Pa of pCO2. Measured transpiration rate were on average

2.16 g m-2 min-1 and 2.76 g m-2 min1 for plants grown at 101 kPa and 33 kPa

respectively, suggesting that transpiration rate increased by 25 % as total pressure

reduced. The model predicted an increase in transpiration rates as total pressure

reduced from 101 to 33 kPa (Figure 6-1). The model very closely predicted transpiration

at 101kP at 2.2 g m-2 min1. However, the model predicted that the transpiration should

be 80 % higher at approximately at 4 g m-2 min-' at 33 kPa. The predicted transpiration

rates at various stomatal widths (0.0001, 0.0002 and 0.0004 cm) are given in Figure 6-

2. The predicted transpiration rate at 33 kPa total pressure and 0.0004 cm stomatal

width was 4 g m-2 min-' which after adjusting the stomatal width to 0.0003 cm was 3.9 g

m-2 min1 and at 0.0001 cm width was 3.8 g m-2 min- suggesting that stomatal width

adjustment by these values had little effect on the transpiration rates. At normal

pressure changing the stomatal width from 0.0004 to 0.0003 and 0.0001 changed

transpiration rate only by -0.5 and -0.9%.

To study the effects of vapor pressure deficit (VPD) on transpiration rates, two

different values of VPD (0.6 kPa low VPD and 0.9 kPa high VPD) were adjusted in

equation 2-15. The VPD had significant effect on the transpiration rate. At lower VPD

and at normal pressure, transpiration rate was similar to that of predicted using the


133









model. However, at higher VPD transpiration rate increased by 45 % and 164 % at 101

kPa and 33 kPa respectively compared to measured transpiration rate at 33 and 101

kPa (Figure 6-3). The variability in transpiration could be accounted due to the number

of stomata at various pressures. According to Figure 6-4 increases in stomata number

from approximately 1350 to 10000 resulted in predicted increased transpiration however

further increase in number of stomata did not significantly increased transpiration as the

curve approached a maximal transpiration. Thus increased transpiration was restricted

beyond stomata number 10000 as a result of limitations in stomatal conductance. When

number of stomata was reduced by 50 % in Model, the transpiration rate was reduced

by approximately 14% at 101 kPa and 33 kPa (Table 6-3). However, when number of

stomata were increased by 50 % (20,500) in model, the transpiration rates was same to

reference value at 101 kPa and 82% higher at 33 kPa, suggesting that there is a limit to

increase in transpiration due to higher number of stomata, and increasing the stomatal

number beyond 13,500 did not further increased transpiration rate.

Conclusion

The transpiration model (Sinclair, 1998) incorporating stomatal conductance,

stomatal aperture, diffusivity and VPD performed well at normal pressure. Increased

VPD resulted in increased transpiration. At reduced pressure the parameters such as

stomatal width and in some cases stomatal number are most likely to change the

transpiration rate due change gas diffusivity at reduced pressure.


134




















.E 3.5
E

E
0

O.2
o o




2
0


1 .5 1i i -i i i i
30 40 50 60 70 80 90 100 110
Pressure [kPa]


Figure 6-1. Predicted transpiration rates from 33 to 101 kPa with observed data, 'o',
from plants grown at 33kPa and 101 kPa as described in Chapter 5 of this
dissertation.


135














^ 0.0001 cm
E -A-- 0.0003 cm
E


02


,-



0
0 33 66 99
Pressure (kPa)

Figure 6-2. Predicted transpiration rate at various total pressures (33, 66, 101 kPa) and
various stomatal widths (0.0001, 0.0003 and 0.0004 cm).


136












--low VPD (0. 6 kPa)
-3- high VPD (0.9 kPa)


Pressure (kPa)


Figure 6-3. Predicted transpiration rate at three different pressures (33, 66 and 101
kPa) and two different vapor pressure deficit (VPD) levels (0.6 kPa and 0.9
kPa).


a---


---~--0











4.5
33 kPa
E- 4-

E 3.5-
0)



C 2.5-

1-
2.
0 1 2 3
Number of stomata x 104

Figure 6-4. Predicted transpiration rates as stomata number varies from 1350 to 27000
at 33kPa.

























138











Table 6-1. Parameters and reference values used in the model.
Reference value
Parameter Variable (units) (at 101 kPa)
Stomata Length a (cm) 0.0010
Stomata Width b (cm) 0.0002
Stomata Depth d (cm) 0.0001
Diffusivity of Water D (cm2 s-1) 0.8331
Number of. stomata n (no. cm-2) 13500
Stomatal Conductance hs (cm s-1) 17.7
Boundary Layer Conductance hb (cm s-) 1.6
Constant 0.6450
Thermal Diffusivity of Water K (cm2 S-1) 10
Length of leaf L (cm) 0.4250
Kinematic Viscosity Kv (cm2 S-1) 100
Air Speed u (cm s-1) 2353
Reynolds Number Re 0.0002
Viscosity of Air p ( g cm-1 s-1) 0.0004
Density of Air p (g cm-3) 0.5101
Schmidt Number Sc 0.507


139










Table 6-2. Parameter description and reference values used for sensitivity analysis
Parameter Description Reference value


Atmospheric pressure
Diffusivity of gases
vapor pressure deficit
Stomatal width
Stomatal number


101 kPa
According to Figure 3-1
0.6 kPa (Low)
0.0004 cm
13500


140


D
VPD
b
n











Table 6-3. Sensitivity analysis of the transpiration model at various conditions. Given
are the transpiration rates and percent change from the reference conditions
(101 kPa, 0.6 kPa VPD, and stomatal width of 4 x 10-4 cm and stomatal
number of 13500) when one parameter is varied. The reference transpiration
is 2.2 g m-2 min-1.
Tran (g m-2 min1) % change
Pressure
101 kPa 2.2 0 (reference value)
33 kPa 4 82
VPD air-leaf
0.6 kPa
101kPa 2.1 -4
33kPa 3.9 77
0.6 kPa
101kPa 2.2 0 (reference value)
33kPa 4 82
0.9 kPa
101 kPa 3.2 45
33 kPa 5.8 163
Stomatal width
0.0001 cm
101 kPa 2 -9
33 kPa 3.8 72
0.0003 cm
101 kPa 2.1 -5
33 kPa 3.9 78
0.0004 cm
101 kPa 2.2 0 (reference value)
33 kPa 4 82
Stomatal number
6750 1.9 -14
3.7 68
13500
101 kPa 2.2 0 (reference value)
33 kPa 4 82
20250
101 kPa 2.2 0
33 kPa 4 82









CHAPTER 7

SUMMARY AND FUTURE WORK

Plants will be an integral part of an Advanced Life Support (ALS) system for space

exploration for many reasons but particularly as a source of fresh food. The plant growth

facility in an ALS will most likely be maintained at reduced pressure due to the lower

costs associated with low pressure facilities compared to those that would be run at

normal pressures. The overall growth of plants will depend on the environment within

the chambers. Important environmental factors that will be monitored and controlled

include temperature, relative humidity, light, total pressure, and overall gas composition

(CO2, 02, ethylene among other gas volatiles). Here, the effects of reduced pressure on

radish gas exchange, growth and adaptation to hypobaria were studied. These studies

were performed using unique environmental chambers that could monitor all of the

important environmental conditions that would be needed for growing plants on long-

term missions to the Moon or Mars in hypobaria. However, these chambers had

limitations since the relative humidity, gas phase composition, light and temperature

were not controlled by the chambers themselves but by either a larger environmental

chamber in which they were housed (temperature and light) or through manual control

mechanisms such as the use of saturating salt solutions to maintain humidity levels

within the chamber or through manual injection of gas into the chamber on daily

intervals to maintain various gas phase concentrations set points. Also, the nutrient

solution was not monitored except for the first and final days of the experiments.

Improvements to the system would include developing an automated system for

controlling humidity and gas phase composition as well as a hydroponics system that

could monitor and control nutrient levels, pH and be adjusted throughout the growth of


142









plants. Despite these limitations of the chambers, radish plants grew well in low

pressure and their growth and gas exchange rates could be monitored.

Radish plants grown short term (1 week) in hypobaria had increased biomass

(DW), CO2 assimilation, dark respiration (DR), and transpiration compared to plants

grown in ambient pressures. Transpiration was reduced and growth enhanced by

enrichment of the gas phase with CO2 for all pressure treatments (33, 66, and 101kPa).

Plant transpiration rates remained constant over the seven days in hypobaria

suggesting that plants grown short term in hypobaria did not acclimate to hypobaria by

reducing stomata aperture during this period. Very high pCO2 (180 Pa) when combined

with hypobaria (33 or 66 kPa) induced leaf damage. The leaf damage was not found at

lower pCO2 (100 Pa) treatments suggesting that the threshold for pCO2 uptake had

been reached at 180 Pa pCO2. However, the cause of this leaf damage is unknown.

Further studies to identify the cause of this damage in low pressure are required. These

studies should include the analysis that compare chlorophyll content, rubisco activity,

and starch accumulation in the leaves of damaged plants.

In addition, the stage at which plants are transplanted from high to low pressure

can affect the growth and survival of the plant in hypobaria since plants at early stages

may not have the proper root to shoot balance to deal with the increased transpiration

that often occurs in hypobaria. Overall, it appears that radish plants do acclimate to

hypobaria long term by reducing leaf area and increasing leaf thickness. This may be

an adaption to water stress caused by enhanced transpiration, an adaption to the

increased diffusion to CO2 in low pressure or other adaptations to the interactions of the

gas phase and other conditions such as lower leaf temperature in hypobaria. Further


143









studies on these interacting factors are required to understand the mechanisms of plant

adaption to hypobaria.

Transpiration and photosynthesis are both sensitive to the total pressure of the

atmosphere. Due to the increases in diffusivity of gases in hypobaria, and hence gas

flux, plant growth and water loss can be influenced by the pressure of the growth

chamber. If water flux, for example, is a critical aspect of a water purification system or

for water conservation, the transpiration rate of the plants can be increased or

decreased by simply adjusting the plant chamber pressure or pCO2. Models predict

that both CO2 assimilation and transpiration will increase in hypobaria due to the

increased diffusivity of gases. However, these models still require improvements since

many assumptions are required. For example, the stomatal dimensions and densities

are difficult to measure for plants grown in hypobaria and further studies are required

that compare stomata and leaf development from plants grown long term in hypobaria

with those grown at normal atmospheric conditions.

Although utilizing plants as part of a BLSS seems promising, it is far from mature.

More studies are required to identify the effects of the challenging environments on

plant growth and development that will be encountered on space missions including

increased radiation and reduced water supplies. Gene expression analyses suggest

that plants are undergoing stress in hypobaria. This stress response could divert some

of the metabolic energy from plant growth to other pathways that may result in reduced

food supply. Further integration of molecular information in response to hypobaria may

provide information to breed or genetically engineer plants that can mitigate these

stresses in hypobaria. The knowledge gained from research on plant adaption to low


144









pressures for long-term space missions can be applied for terrestrial agriculture

particularly in areas of the world where water and other resources are limited.


145











;{CR10};


01: 1 Execution Interval
Reference Temp Reading
1: Internal Temperature (P17)
1: 1 Loc [ temp_int ]
2: Z=X*F (P37)
1:1 X Loc [temp_int ]
2: 1.0 F
3: 35 Z Loc [ temp_int2 ]
3: Z=X+F (P34)
1:35 X Loc [ temp_int2 ]
2:0.0 F
3: 36 Z Loc [ temp_int3 ]
02 Reading


(seconds)


4: Volt (SE) (P1)
1: 1 Reps
2: 15 2500 mV Fast Range
3: 1 SE Channel
4: 21 Loc [ 02sensor ]
5: 1.0 Mult
6: 0.0 Offset
02 Calibration

5: Z=X*F (P37)
1:21 X Loc [ 02sensor ]
2:419.43 F
3: 47 Z Loc [ 02sensor2 ]
6: Z=X*F (P37)
1:47 X Loc [ 02sensor2 ]
2: 1.07 F
3:54 Z Loc [ mult ]
7: Z=X+F (P34)
1:54 X Loc [mult ]
2: 487.07 F
3: 48 Z Loc [ 02sensor3 ]
8: Z=X*F (P37)
1:43 X Loc [Prs3 ]
2: 1.2975 F
3: 49 Z Loc [ factor ]
9: Z=X*F (P37)
1:43 X Loc [Prs3 ]


146


APPENDIX
CR10 PROGRAM


Table 1 Program









2:2.4183 F
3: 50 Z Loc [ factor ]
10: Z=X+F (P34)
1:50 X Loc [ factor ]
2: -7.1664 F
3:51 Z Loc [ factor ]
11: Z=X+Y (P33)
1:48 X Loc [ 02sensor3 ]
2:49 Y Loc factor1 ]
3: 52 Z Loc [ factor ]
12: Z=X+F (P34)
1:52 X Loc [ factor ]
2: -164.56 F
3: 55 Z Loc [ factor ]
13: Z=X/Y (P38)
1:55 X Loc [ factor ]
2:51 Y Loc [ factor ]
3: 53 Z Loc [ 02output ]
RH Reading 1

14: Volt (SE) (P1)
1: 1 Reps
2: 15 2500 mV Fast Range
3: 3 SE Channel
4:22 Loc [ RH ]
5: 1.0 Mult
6: 0.0 Offset
RH Calibration

15: Z=X*F (P37)
1:22 X Loc [ RH ]
2: 1 F
3:65 Z Loc [ RH3 ]
16: Z=X+F (P34)
1:65 X Loc [RH3 ]
2: -.793 F
3:63 Z Loc [ RH2 ]
17: Z=X*F (P37)
1:63 X Loc [RH2 ]
2: .03 F
3: 64 Z Loc [ RHoutput ]
Pressure Reading

18: Volt (SE) (P1)
1: 1 Reps
2: 15 2500 mV Fast Range
3: 4 SE Channel









4: 24 Loc [ Prs
5: 1.0 Mult
6: 0.0 Offset
Pressure Calibration

19: Z=X+F (P34)
1:24 X Loc [Prs
2: -122.59 F
3: 42 Z Loc [ Prs2

20: Z=X*F (P37)
1:42 X Loc [Prs2
2: .0456 F
3: 43 Z Loc [ Prs3
Pump Relay Control


21: If (X<=>F) (P89)
1:43 X Loc [ Prs3 ]
2:3 >=
3:-5 F
4:46 Set Port 6 High
22: If (X<=>F) (P89)
1:43 X Loc [ Prs3 ]
2:4 <
3:-7 F
4:56 Set Port 6 Low
;solenoid control (normally closed)
23: If (X<=>F) (P89)
1:43 X Loc [ Prs3 ]
2:3 >=
3:-5 F
4:48 Set Port 8 High
24: If (X<=>F) (P89)
1:43 X Loc [ Prs3 ]
2:4 <
3:-7 F
4: 58 Set Port 8 Low
25: Do (P86)
1: 10 Set Output Flag High
; IRt/c
26: Thermocouple Temp (DIFF) (P14)
1: 1 Reps
2: 14 250 mV Fast Range
3:6 DIFF Channel
4: 3 Type K (Chromel-Alumel)
5: 1 Ref Temp (Deg. C) Loc [ temp_int ]
6: 66 Loc [ IRtc ]


148









7:1.0 Mult
8: 0.0 Offset
CO2 Reading

27: Volt (Diff) (P2)
1: 1 Reps
2: 15 2500 mV Fast Range
3: 04 DIFF Channel
4:25 Loc [ CO2 ]
5: 1.0 Mult
6: 0.0 Offset
Light Reading

28: Volt (SE) (P1)
1: 1 Reps
2: 15 2500 mV Fast Range
3:05 SE Channel
4: 26 Loc [ light ]
5: 1.0 Mult
6: 0 Offset
Light Calibration

29: Z=X*F (P37)
1:26 X Loc [ light ]
2: 0.0353 F
3:29 Z Loc [ light ]
30: Z=EXP(X) (P41)
1:29 X Loc [ light ]
2:30 Z Loc [ light ]
31: Z=X*F (P37)
1:30 X Loc [ light ]
2: 0.0027 F
3:31 Z Loc [ light ]
Thermocouple Reading

32: Thermocouple Temp (DIFF) (P14)
1: 1 Reps
2: 14 250 mV Fast Range
3: 05 DIFF Channel
4: 1 Type T (Copper-Constantan)
5: 1 Ref Temp (Deg. C) Loc [ temp_int ]
6: 37 Loc [ thermocou ]
7: 1.0 Mult
8: 0.0 Offset
33: Z=X*Y (P36)
1:68 X Loc [ 02ppa ]
2:43 Y Loc [ Prs3 ]


149









3:69 Z Loc [ 02pp ]
34: Z=X*F (P37)
1:53 X Loc [ 02output ]
2: 0.01 F
3: 68 Z Loc [ 02ppa ]
*Program

02: 600 Execution Interval (seconds)
1: Do (P86)
1: 10 Set Output Flag High
2: Real Time (P77)
1: 110 Day,Hour/Minute (midnight = 0000)
3: Sample (P70)
1: 1 Reps
2: 43 Loc [ Prs3 ]
4: Sample (P70)
1: 1 Reps
2: 37 Loc [ thermocou ]
5: Sample (P70)
1: 1 Reps
2: 53 Loc [ 02output ]
6: Sample (P70)
1: 1 Reps
2: 31 Loc [ light ]
7: Sample (P70)
1: 1 Reps
2:25 Loc [ C02 ]
8: Sample (P70)
1: 1 Reps
2: 64 Loc [ RHoutput ]
9: Sample (P70)
1: 1 Reps
2: 69 Loc [ 02pp ]
*Subroutines
End Program
-Input Locations-
1 temp_int 1 3 1
2 100
3 000
4 000
5 000
6 000
7 000
8 000
9 000
10 000
11 000


150









12 000
13 000
14 000
15 100
16 000
17 000
18 000
19 000
20 temp 1 0 0
21 02sensor 1 1 1
22 RH 1 1 1
23 CO2sensor 1 0 0
24 Prs 1 1 1
25 C02 1 1 1
26 light 1 1 1
27 exponent 1 0 0
28 lightcorr 1 0 0
29 light 1 1 1
30 light 1 1 1
31 light 1 1 1
32 temp2 1 0 0
33 temp3 1 0 0
34 temp4 1 0 0
35 temp_int2 1 1 1
36 temp_int3 1 0 1
37 thermocou 1 1 1
38 expone 1 0 0
39 therm2 1 0 0
40 _light__ 1 0 0
41 lig 1 00
42 Prs2 1 1 1
43 Prs3 1 8 1
44 light 1 0 0
45 light 1 0 0
46 light 1 0 0
47 02sensor2 1 1 1
48 02sensor3 1 1 1
49factor1 1 1 1
50factor2 1 1 1
51 factor 1 1 1
52 factor 1 1 1
53 02output 1 2 1
54 mult 1 1 1
55 factor 1 1 1
56 C02 1 0 0
57 RH 1 00









58 C02 1 0 0
59 o2ave 1 0 0
60 o2ave2 1 0 0
61 mult2 1 0 0
62 o2ave3 1 0 0
63 RH2 1 1 1
64 RHoutput 1 1 1
65 RH3 1 1 1
66 IRtc 1 0 1
67 Prs3 1 0 0
68 02ppa 1 1 1
69 02pp 1 1 1
-Program Security-
0000
0000
-Mode 4-
-Final Storage Area 2-


152









LIST OF REFERENCES


AI-Ani, A., Bruzau, F., Raymond, P., Saint-Ges, V., Leblacnc, J. M. Germination,
respiration and adenylate energy charge of seeds at various oxygen partial
pressures. Plant Physiol. 79, 855-890, 1985.

Andre, M., Massimino, D. Growth of plants at reduced pressures: experiment in wheat-
technological advances and constraints. Adv. Space Res. 12, 97-106, 1992.

Arnold, J. H. Studies in diffusion, Industrial and Engineering Chemistry 22, 1091-1095,
1930.

Assmann, S.M. The cellular basis of guard cell sensing of rising CO2. Plant Cell Environ.
22, 629-37, 1999.

Baker, D. The History of Spaceflight. Crown Publishers, Inc., New York, 1981.

Berger, D., Altman, T. A subtilisin-like serine protease involved in the regulation of
stomatal density and distribution in Arabidopsis thaliana. Genes Dev. 14, 1119-
1131, 2000.

Bewley, J.D., and Black, M. Seeds: Physiology of Development and Germination. New
York: Plenum Press, 1994.

Bierhuizen, J.F., Slatyer, R.O., Photosynthesis of cotton leaves under a range of
environmental conditions in relation to internal and external diffusive resistance.
Aust. J. Biol. Sci. 17, 348-359, 1964a.

Bird, R.B., Stewart, W.E., Lightfoot, E.N. Transport Phenomena. John Wiley, New York.
1960.

Brinckmann, E. ESA hardware for plant research on the International Space Station Adv
Space Res 36, 1162-1166, 2005.

Bucklin, R.A., Fowler, P.A., Rygalov, V.Y., Wheeler, R., Mu, Y., Hublitz, I., Wilkerson,
E.G. Greenhouse design for the mars environment: development of a prototype,
deployable dome. ActaHort. 659, 127-34, 2004.

Burg, S.P., Burg, E.A. Fruit storage at sub-atmospheric pressures. Sci. 153,314-15,
1966.

Burg, S.P. Post harvest physiology and hypobaric storage of fruits. CABI publishing,
Cambridge, MA, 2004.


153









Case, A. L., Curtis, P.S., Snow, A.A. Heritable variation in stomatal responses to
elevated CO2 in wild radish, Raphanus raphanistrum (Brassicaceae) Am. J. Bot.
85, 253-255, 1998.

Chapman, S., Cowling, T. G. The Mathematical Theory of Non uniform Gases. 3rd ed,
Cambridge. 1970.

Chaves, M.M., Maroco, J.P., Pereira, J.S. Understanding plant response to drought-
from genes to whole plant. Func. Plant Bio. 30, 239-264, 2003.

Corey, K.A., Bates, M.E., Adams, S.L. Carbon dioxide exchange of lettuce plants under
hypobaric conditions. Adv. Space Res. 18, 265-72, 1996.

Corey, K.A., Barta, D.J., Henninger, D.L. Photosynthesis and respiration of wheat stand
at reduced atmospheric pressure and reduced oxygen. Adv. Space Res. 20, 1869-
1877, 1997.

Corey, K.A., Barta, D.J., Edeen, M.A., Henninger, D.L. Atmospheric leakage and
method for measurement of gas exchange rates of a crop stand at reduced
pressure. Adv. Space Res. 20, 1861-1887, 1997a.

Corey, K.A., Barta, D.J., Wheeler, R.M. Toward Martian agriculture: responses of plants
to hypobaria. Life Support Biosph. Sci. 8, 103-114, 2002.

Cowles, J. R., Scheld, H.W., Lemay R., Peterson C. Growth and lignification in
seedlings exposed to eight days of microgravity, Ann. Bot., 54, 33-49, 1984.

Cramer, D. R., Reid, D. H., Klein, H. P. The first dedicated life sciences mission-
Spacelab 4. Adv. Space Res, 3, 143-151, 1984.

Daunicht, H.J., Brinkjans, H.J. Gas exchange and growth of plants under reduced
pressure. Adv. Space Res. 12, 107-114, 1992.

Daunicht H.J., Brinkjans, H.J. Plant responses to reduced air pressure: advanced
techniques and results. Adv. Space Res. 18, 273-281, 1996.

Drake, B. G, Gonzalez-Meler, M. A, Long, S. P. More efficient plants: a consequence of
rising atmospheric C02? Annu. Rev. Plant Physiol. Plant Mol. Biol. 48, 609-639,
1997.

Drysdale A.E. Life support trade studies involving plants. SAE Technical Paper Series
2001-01-2362, 2001.

Ehleringer, J. R., Cooper, T. A. On the role of orientation in reducing photo inhibitory
damage in photosynthetic twig desert shrubs. Plant Cell and Environ. 15, 301-306,
1992.


154









Fingers B., Strayer R.F., Garland J.L., Mackowiak C.L., Atkinson, D.F., Planning for the
Rapid Aerobic Bioreactor Integration Test (Rabit) at the Kennedy Space Center's
advanced life support breadboard project, International Conference on
Environmental Systems, Monterey, CA, USA, 1996.

Finlayson, S.A., Gohil, H.L., Kato-Noguchi, H., Lee I-J., Morgan, P.W. Circadian
ethylene synthesis in Sorghum bicolor: Expression and control of the system at the
whole plant level. J. Plant Growth Reg., 23, 29-36, 2004.

Fuller, E.N., Schettler, R.P., Giddings, J.D. New method for prediction of binary gas-
phase diffusion coefficients. Ind. Eng. Chem. 58, 18-27, 1966.

Gale, J. Availability of carbon dioxide for photosynthesis at high altitudes: Theoretical
considerations. Ecology 53, 494-497, 1972.

Gebhart, B.J. Transient natural convection from vertical elements. J. Heat Trans. 83,
61-70, 1961.

Gohil, H.L., Bucklin, R. A., Correll, M.J. The effects of CO2 and hypobaria on growth and
transpiration of radish (Raphanus sativus). Adv. Space Res. 45, 823-831, 2010.

Goto, E., Iwabuchi, K., Takakura, K. Effect of reduced total air pressure on spinach
growth. J. Agric. Meteorol. 51, 139-145, 1995.

Goto, E., Ohta, H., Iwabuchi, K., Takakura, T. Measurement of net photosynthetic and
transpiration rates of spinach and maize plants under hypobaric condition. J. Agric.
Meteorol. 52, 117-23, 1996.

Goto, E., Arai, Y., Omasa, K. Growth and development of higher plants under hypobaric
conditions. International Conference of Environmental Systems Soc Autom Eng,
San Antonio, TX. Meeting Paper No 2002-01-2439, 2002.

Grimwood, J. M. Project Gemini technology and operations A chronology, NASA,
NASA SP-4002, Wash., DC, 1969.

Halstead, T. W., Dutcher F.R., Status and prospects in experiments on plant growth in
space, Ann.Bot. 54, 3-18, 1984.

He, C., Davies, F.T., Lacey, R.E., Drew, M.C., Brown, D.L. Effect of hypobaric
conditions on ethylene evolution and growth of lettuce and wheat. J. Plant Physiol.
160, 1341-1350, 2003.

He, C., Davies, F.T., Lacey, R.E. Separating the effects of hypobaria and hypoxia on
lettuce: growth and gas exchange. Physiol. Plant. 131, 226-240, 2007.

He, C., Davies, F.T., Lacey, R.E. Ethylene reduces gas exchange and growth of lettuce
plants under hypobaric and normal atmospheric conditions. Physiol. Plant. 135,
258-271, 2009.


155









Hirshfelder, J.O., Curtis, F., Bird, R.B. Molecular Theory of Gases and Liquids. John
Wiley, New York, 1948

Hublitz, I., Heat and mass transfer of a low pressure Mars greenhouse: simulation and
experimental analysis, Ph.D. Dissertation, The University of Florida, 2006.

Iwabuchi, K., Goto, E., Takakura, T. Effect of 02 partial pressure under low Air pressure
on net photosynthetic rate of spinach. Acta Hort. 399, 101-106, 1994.

Iwabuchi, K., Goto, E., Takakura, T. Germination and growth of spinach under
hypobaric conditions. Environ. Control. Biol. 34, 169-178, 1996.

Iwabuchi, K., Kurata, K. Short-term and long-term effects of low total pressure on gas
exchange rates of spinach. Adv. Space Res. 31, 241-44, 2003.

Jackson, R. B., Sperrry, J.S., Dawson, T. E., Root water uptake and transport: Using
physiological processes in global predictions. Trends in plant science. 5, 482-488,
2000.

Jarvis, P.G. The interpretation of the variations in leaf water potential and stomatal
conductance found in canopies in the field. Phil. Trans. R. Soc. Lond. B. 273, 593-
610, 1996.

Johnson, A. T. Biological Process Engineering. John Wiley, New York. 1999.

Jones, H. Breakeven mission durations for physicochemical recycling to replace direct
supply life support, SAE Paper No. 2007-01-3221, International conference on
environmental systems, Chicago, Illinois, 2007.

Katchalsky, A., Curran, P.F. Nonequilibrium Thermodynamics in Biophysics. Harvard
Uni. Press, Cambridge, MA. 1967.

Klein, H.P. US biological studies in space, ActaHorticulture. 8, 927-938, 1981.

Knapp, A. K., Cocke, M., Hamerlynck, E. P., Owensby, C. E., Effect of Elevated CO2 on
Stomatal Density and Distribution in a C4 Grass and a C3 Forb under Field
Conditions. Ann. Bot. 74: 595-599, 1994.

Knight, H., Knight, M. Abiotic signaling pathways: Specificity and cross-talk. Trends
Plant Sci. 6, 262-267, 2001.

Kordyum, E.L., Sytnik, K.M., Chernyaeva, 1.1. Peculiarities of genital organ formation in
Arabidopsis thaliana under spaceflight conditions. Adv. Space Res. 3, 247-250,
1983.

Korner, C., Paulsen, J. A world-wide study of high altitude treeline temperatures.
Journal of Biogeography 31: 713-732, 2004.


156









Kuang, A., Musgrave M.E., Matthews, S.W. Cytochemical localization of reserves
during seed development in Arabidopsis thaliana under spaceflight conditions,
Ann. Bot. 78, 343-351, 1996.

Levine, L.H., Bisbee, P.A., Richards, J.T., Birmele, M.N., Prior, R.L., Perchonok, M.,
Dixon, M., Yorio, N.C., Stutte, G.W., Wheeler, R.M. Quality characteristics of the
radish grown under reduced atmospheric pressure. Adv. Space Res. 41, 754-762,
2008.

Levine, L.H., Richards, J.T., Wheeler, R.M. Super-elevated CO2 interferes with stomatal
response to ABA and night closure in soybean (Glycine max). J. Plant. Physiol.
166, 903-913, 2009.

Levinskikh, M.A., Sychev, V.N., Derendyaeva, T.A.,Signalova, O.B., Salisbury, F.B.,
Campbell, W.F., Bingham, G.E., Bubenheim, D.L., Jahns, G. Analysis of the
spaceflight effects on growth and development of super dwarf wheat grown on the
space station Mir. J. Plant Physiol. 156, 522-529, 2000.

Link, B.M., Durst, S.J., Zhou, W., Stankovic, B. Seed-to-seed growth of Arabidopsis
thaliana on the International Space Station. Adv. Space Res. 31(10), 2237-2243,
2003.

Long, S.P., Ainsworth, E.A., Rogers, A., Ort, D.R. Rising atmospheric carbon dioxide:
plants FACE the future. Ann. Rev. Plant Bio. 55, 591-628, 2004.

Malashenkov, D., World Space Congress in Houston, Texas, 2002.

Mansell, R.L, Rose, G. W, Richardson, B., Miller, R.L. Effects of prolonged reduced
pressure on the growth and nitrogen content of turnip (Brassica rapa L.). SAM-TR-
68-100. School of Aerospace Medicine Technical Report, pp 1-13, 1968.

Martin, C.E., McCormick, A.K, Air handling and atmosphere conditioning systems for
manned spacecraft. ICES Paper No. 921350. SAE International. 1992.

Mashinskiy, A., G. Nechitailo. Birth of space agriculture. Tek Molodezhi 4, 2-7, 1993.

Mashinsky, A., Ivanova, I., Derendyaeva, T., Nechitailo, G. and Salisbury, F. From
'seed-to-seed' experiment with wheat plants under space-flight conditions. Adv.
Space Res. 14, 13-19, 1994.

Massimino, D., Andre, M. Growth of wheat under one-tenth of the atmospheric
pressure. Adv. Space Res. 24, 293-296, 1999.

Meng, L., Li, L., Chen, W., Xu, Z., Liu, L. Effect of water stress on stomatal density,
length, width and net photosynthetic rate in rice leaves. J. Shenyang Agric. Uni.
30, 477-480, 1999.









Merkys, A.I., Laurinavicius, R.S, Complete cycle of individual development of
Arabidopsis thaliana (L.) Heynh. plants on board the Salyut-7 orbital station.
Dokladi Akademii Nauk SSSR 271, 509-512, 1983.

Morgan, J. M., Osmoregulation and water stress in higher plants. Trends Plant Physiol.
Plant mol. Biol., 35, 299-319, 1984.

Mott, K. A., Parkhurst, D.F. Stomatal responses to humidity in air and helox. Plant, Cell,
and Env. 14, 509-515, 1991.

Mu, Y. A distributed control system for low pressure plant growth chambers. Ph.D
Dissertation. The University of Florida, 2005.

Murashige, T., Skoog, F. A revised medium for rapid growth and bioassays with tobacco
tissue cultures. Physiol. Plant. 15, 473-497, 1962.

Musgrave, M. E., Gerth, W, A., Scheld, H. W, Strain, B, R. Growth and mitochondrial
respiration of mungbeans (Phaseolus aureus Roxb.) germinated at low pressure,
Plant. Physiol., 86, 19-22, 1988.

Musgrave ME., Seeds in space. Seed Sci. Res. 12:1-16, 2002.

NASA/TM-210785. Guidelines and Capabilities for Designing Human Missions. 2003.

Nechitailo, G.S., Mashinsky, A.L. Space biology: Studies at orbital station. Moscow, Mir
Publishers. 1993.

Nobel, P.S. Physicochemical and Environmental Plant Physiology. Academic Press,
San Diego, CA.1999.

Oberg J., Uncovering Soviet Disasters: "Dead Cosmonauts", pp 156-176, Random
House, New York, 1988, retrieved 14 October 2009.

Parfenov, G.P., Abramova, V.N. Blossoming and maturation of Arabidopsis seed:
Experiment on Biosatellite Kosmos 1129. Dokladi Akademii Nauk. SSR. 256, 254.
1981.

Parlange, J., Wagoner, P.E. Stomatal dimension and resistance to diffusion. Plant
Physiol. 46, 337-342, 1970.

Paul, A.L., Schuerger, A.C., Popp, M.P., Richards, J.T., Manak, M.S., Ferl, R.J.
Hypobaric biology: Arabidopsis gene expression at low atmospheric pressure.
Plant Physiol. 134, 215-223, 2004.

Paul, A.L., Ferl, R.J. Biology in low atmospheric pressure: implication for exploration
mission design and advance life support system. Gravi. Space Biol. Bull. 19, 3-17,
2006.


158









Porterfield, D.M., Neichitailo, G.S., Mashinski, A.L., Musgrave, M.E. Complete plant
growth systems in space. Adv. Space Res. 2002.

Purswell, J. L. Engineering design of a hypobaric plant growth chamber. Master of
Science Thesis: Biological and Agricultural Engineering. Texas A&M University,
College Station, TX; 2002.

Radoglou, K.M., Jarvis, P.G. Effects of CO2 enrichment on four poplar clones. II. leaf
surface properties. Ann. Bot. 65, 627-632, 1990.

Rajapakse, N. C., He, C., Cisneros-Zevallos, L., Davies F.T. Hypobaria and hypoxia
affects growth and phytochemical contents of lettuce. Sci. Hort. 122: 171-178,
2009.

Rand, R.H. Gaseous diffusion in the leaf interior. Trans. Am. Soc. of Agri. Eng. 20, 701-
704, 1970.

Raven JA. Selection pressure on stomatal evolution. New Phyto. 153: 171-386, 2002

Richards, J.T., Corey, K.A., Paul, A.L., Ferl, R.J., Wheeler, R.M., Schuerger, A.C.
Exposure of Arabidopsis thaliana to hypobaric environments: implications for low-
pressure bioregenerative life support systems for human exploration missions and
terraforming on Mars. Astrobio. 6, 851-866, 2006.

Rodrigues, M. L., Pachico, C. M. Chaves, A. M. Plant Soil relations, root distributions
and biomass partitioning in Lupinus Albus. M. under drought conditions. J Expt.
Bot. 46: 947- 956, 1995.

Rule, D.E., Staby, G.L. Growth of tomato seedlings at sub-atmospheric pressures.
HortSci. 16, 331-332, 1981.

Rygalov, V.Y., Fowler, P.A., Metz, J.M., Wheeler, R.M., Bucklin, R.A. Water cycles in
closed ecological systems: effects of atmospheric pressure. Life Supp. Biosph.
Sci. 8, 125-135, 2004.

Salisbury, F.B., Bingham, G.E., Campbell, W.F., Carman, J.G., Hole, P., Gillespie, L.S.,
Sychev, V.N., Berkovitch, Yu., Podolsky, I.G. and Levinskikh, M. Growing super-
dwarf wheat on the Russian space station Mir. ASGSB Bulletin 9, 63. 1995.

Schulze, E.D. Carbon dioxide and water vapor exchange in response to drought in the
atmosphere and the soil. Ann. Rev. Plant physiol. 37, 247-274, 1986.

Schwartzkopf, S. H., Mancinelli R. L. Germination and growth of wheat in simulated
martian atmospheres. Acta Astronautica 25(4), 245-247, 1991.


159









Schwartzkopf, S. H., Grote, J. R., Stroup, T.L. Design of a low atmospheric pressure
plant growth chamber. SAE Technical Paper No. 951709. Warrendale, P.A.:
Society of Automotive Engineers, 1995.

Siegel, S. M., Rosen, L. A., Giumarro, C. Plants at sub-atmospheric oxygen-levels.
Nature 198: 1288-1290. 1963.

Simpson, M. S.; Young, J. S. A plant growth structure for Martian derived atmosphere.
SAE Technical Paper No. 981901. Warrendale, PA.: Society of Automotive
Engineers; 1998.

Sinclair, T.A. Theoretical considerations in the description of evaporation and
transpiration. In: Stewart, D. A., Nielson D.R. (Eds.), Irrigation of Agricultural
Crops. pp.343-361.1980.

Sinclair, T.R., Ludlow, M.M. Who taught plants the thermodynamics of water? The
unfulfilled potential of plant water potential. Aust. J. Plant Physiol. 12, 213-217,
1985.
Slattery, J.C., Bird, R.B. Calculation of the diffusion coefficients of dilute gases and of
the self-diffusion coefficient of dense gases. Ind. Eng. Chem. 4, 137-143, 1958.

Smolders, P., Soviets In Space, Kluwer Agemene Vitgaven, Holland, 1973.

Spanarkel, R., Drew, M.C. Germination and growth of lettuce (Lactuca sativa) at low
atmospheric pressure. Physiol. Plant. 116, 468-477, 2002.

Stanghellinni, C., Bunce, J.A. Response of photosynthesis and conductance to light,
C02, temperature and humidity in tomato plants grown at ambient and elevated
CO2. Photosynthetica. 29, 487-497, 1993.

Svetlana S, Slaveyko N, Tania I, Plamen K, Iliana I Monitoring of plant growth
environment in the SVET-3 space greenhouse: Measurement of Plant shoot
environment Sozopol, In: Proceeding of the 14 In: International Scientific
Conference ELECTRONICS ET. 21-23 Sept, Sozopol, 3, 13-18. 2005.

Sychev V.N., Levinskikh M, A., Sergey A. G., Bingham G. E., Podolsky I.G. Spaceflight
effects on consecutive generations of peas grown on board the Russian segment
of International Space Station. Acta Astronautica, 60, 426-432, 2007.

Thimann, K.V. Biosatellite II experiments: Preliminary results. Proc. Nat. Acad. Sci. 60
(2), 347-361, 1968.

Tisserat, B., Herman, C., Silman, R., Bothast, R.J. Using ultra high CO2 levels enhances
plantlet growth in vitro. HortTech. 7, 282-289, 1997.


160









Usuda, H., Shimogawara, K. The effects of increased atmospheric carbon dioxide on
growth, carbohydrates, and photosynthesis in radish, Raphanus sativus. Plant Cell
Physiol. 39, 1-7, 1998.

Wehkamp, C. Plant age affects the long-term growth response to reduced total pressure
and oxygen partial pressure. Ph.D. Dissertation. The University of Guelph, 2009.

Wheeler, R.M., Mackowiak, C.L., Yorio, N.C., Sager, J.C. Effects of CO2 on stomatal
conductance: do stomata open at very high CO2 concentrations? Ann. Bot. 83,
243-251, 1999.

Wheeler, R.M., Stutte, G.W., Subbarao, G.V., Yorio, N.C. Plant growth and human life
support for space travel, in: Pessarakli M (ed) Handbook of Plant and Crop
Physiolog, 2nd Edn. Marcel Dekker Inc., New York, USA, pp. 925-941, 2001.

Wheeler, R.M. Horticulture for Mars. ActaHort. 642, 201-215, 2004.

Wilkerson, E.G. Plant evapotranspiration in a greenhouse on Mars. Ph.D. Dissertation.
University of Florida, 2005.

Xu, Z.Z., Zhou, G,S. Nitrogen metabolism and photosynthesis in Leymus chinensis in
response to long-term soil drought. J Plant Growth Regul 25, 252-266, 2006.










BIOGRAPHICAL SKETCH

Hemant Gohil was born (1976) in Santarampur, a small town in a western state of

India, Gujarat. He did his schooling in Ahmedabad city and completed his Bachelors

degree in Agriculture in 1999 from Gujarat Agricultural University, Anand. He came to

the United States pursue his Masters in Molecular and Environmental Plant Science

from Texas A&M University in May 2002. He worked at the Indian Agricultural Research

Institute, New Delhi from 2003 to 2005. He came to UF for his Ph.D. in Applied Science

in Agricultural and Biological Engineering. He finished his Ph.D. in August 2010.


162





PAGE 1

1 GAS EXCHANGE AND ACCLIMATION OF RADISH IN REDUCED PRESSURE ENVIRONMENTS By HEMANT LAXMANSINH GOHIL A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2010

PAGE 2

2 Hemant L Gohil

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3 Dedicated in memory of Kalpana Chawla

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4 ACKNOWLEDGMENTS I am thankful to the Agricultural and Biological Engineering Department at the University of Flor ida for access to resources and facilities as well as providing me my research assistantship. I am also thankful to NASA for funding my research studies. I express my deepest gratitude to Dr. Melanie Correll who served as chair of my committee. I would li ke to thank her for her guidance, support and patience during my course work and research and I am thankful to her for helping me to improve in scientific and technical writing. It was a great learning experience working with Dr. Correll. I am thankful to my committee members; Dr. Ray Bucklin for his friendly guidance and enlightening discussions (academic and non academic); Dr. Thomas Sinclair, for his keen interest in improving my fundamental understanding of gas exchange in plants; Dr. Robert Ferl for broadening the perspective of my research and Dr. John Sager for his valuable guidance whenever it was required. Without the help of John Truett, a distinct coll eague and a friend, the low pressure growth chambers for my research would not have been so rel iable I am also grateful to him for teaching me mechanical and electrical aspects of instrumentation. I am also thankful to Alex Stim p son for his help in programming at the beginning of my research. It was great fun working with Samantha, Stella and Derek I am grateful to Samantha for her help with experimentation and to Stella for seed germination studies. I am thankful to our technical staff, especially Veronica Campbell for meticulous proofreading of my manuscripts and ordering research materials. I am grateful to Billy Duckworth, Steve Feagle, Deniel Preston and Helena Niblack for technical assistance

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5 whenever it was needed. I am also thankful to our departmental staff, Mary Hall, Robin Snyder and Dawn Mendoza for their assistance with official and p aperwork. Throughout my research there were many friends and people who directly or indirectly helped me, I am thankful to all of them. I am grateful to my parents who stood by me whenever I made an important decision. My humble gratitude goes to shri Chi nu Bapji and Sadhaguru Jaggi Vasudev for their blessings and spiritual energy. My deepest gratitude goes to my loving wife, Hiral whose perennial care and courage motivated me through the ups and downs in research and in day to day life. I also benefited a lot by discussing research problems with her. I feel extremely luc ky to have her endless support, without which, finishing Ph.D. would have been very stressful.

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6 TABLE OF CONTENTS ACKNOWLEDGMENTS ...................................................................................................... 4 page LIST OF TABLES ................................................................................................................ 9 LIST OF FIGURES ............................................................................................................ 10 LIST OF PARAMETERS ................................................................................................... 12 ABSTRACT ........................................................................................................................ 13 CHAPTER 1 ATMOSPHERIC CONDITIO NS USED FOR BIOLOGIC AL SYSTEMS DURING SPACE MISSIONS: IMPL ICATIONS FOR HYPOBAR IC PLANT BIOLOGY .......... 17 Advanced Life Support Systems (ALS) ..................................................................... 17 Gas Phase for Biological Life Support ....................................................................... 18 The International Space Station (ISS) ....................................................................... 26 Advanced Plant Studies on the ISS ........................................................................... 27 Hypobaric Plant Biology ............................................................................................. 28 The Effects of Hypobaria on Seed Germination and Plant Growth .......................... 28 Summary ..................................................................................................................... 32 St ructure of the D issertation ....................................................................................... 35 2 DESIGN AND CONSTRUCT ION OF LOW PRESSURE GROWTH CHAMBERS (LPGCS) ...................................................................................................................... 40 System Description ..................................................................................................... 43 Gas Leakage Tests ..................................................................................................... 44 Data Acquisition and Control System ........................................................................ 45 Sensors and Their Calibrations .................................................................................. 46 Pressure Sensor .................................................................................................. 46 Relative Humidity (RH) Sensor ............................................................................ 47 Oxygen Sensor (O2) ............................................................................................. 47 Carbon Dioxide (CO2) Sensor ............................................................................. 48 Load Cell .............................................................................................................. 49 Light Sensor Calibration ...................................................................................... 50 Temperature Sensors .......................................................................................... 50 Summary and Future Improvements ......................................................................... 51 3 MODELING GAS DIFFUSI VITY, PHOTOSYNTHESIS AND TRANSPIRATION UNDER HYPOBARIA ................................................................................................. 63 Plants in Hypobaria ..................................................................................................... 64

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7 Binary Diffusion of Gases ........................................................................................... 65 The Effects of Background Gases on Overall Diffusivity ........................................... 67 Diffusivity of CO2 and H2O in Hypobaria .................................................................... 68 Gas Diffusion in Leaves .............................................................................................. 70 Leaf Transpiration ................................................................................................ 71 Le af Photosynthesis ............................................................................................. 75 Conclusion .................................................................................................................. 75 4 THE INTERACTING EFFE CTS OF CO2 AND HYPOBARIA ON GRO WTH AND TRANSPIRATION OF RADISH ( RAPHANU S SATIVUS ) ........................................ 78 Introduction ................................................................................................................. 79 Materials and Methods ............................................................................................... 82 Plant Mat erial ....................................................................................................... 82 Growth Chambers and Environmental Control ................................................... 83 Plant Harvest ........................................................................................................ 84 Statistical Analysis ............................................................................................... 84 Results ........................................................................................................................ 84 Plant Growth ......................................................................................................... 84 CO2 As similation .................................................................................................. 85 Transpiration Rates .............................................................................................. 86 Discussion ................................................................................................................... 87 Summary ..................................................................................................................... 91 5 THE EFFECTS OF SHORT -TERM AND LONG -TERM ACCLIMATION OF RADISH TO HYPOBARIA ........................................................................................ 100 Introduction ............................................................................................................... 100 Materials and Methods ............................................................................................. 102 Growth Chambers and Environmental Control ................................................. 103 Gas exchange R ates ......................................................................................... 104 Plant B iomass and L eaf Area ............................................................................ 104 Scanning Electron Microscopy .......................................................................... 104 Leaf S tomatal D ensity and Stomatal Index ....................................................... 105 Results ...................................................................................................................... 105 Seed Germination and Plant Growth in Hypobaria ........................................... 105 CO2 Assimilation Rates (CA) .............................................................................. 106 Transpiration Rates (T) ...................................................................................... 107 Water U se Efficiency (WU E) ............................................................................. 108 Stomata Development ....................................................................................... 108 Discussion ................................................................................................................. 109 Seed G ermination .............................................................................................. 109 Seedling G rowth in H ypobaria ........................................................................... 110 Gas E xchange, CO2 assimilation, and Transpiration in H ypobaria .................. 11 2 Short -term Acclimation to H ypobaria ................................................................. 114 Longterm Acclimation to H ypobaria ................................................................. 116 Summary ................................................................................................................... 119

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8 6 TRANSPIRATION MODEL PERFORMANCE AT REDUC ED PRESSURE .......... 131 Introduction ............................................................................................................... 131 Materials and Methods ............................................................................................. 132 Sensitivity Analysis ................................................................................................... 132 Results and Discussion ............................................................................................ 133 Conclusion ................................................................................................................ 134 7 SUMMARY AND FUTURE W ORK .......................................................................... 142 APPENDIX: CR10 PROGRAM ..................................................................................... 146 O2 Reading ................................................................................................................ 146 O2 C alibration ............................................................................................................ 146 RH Reading 1 ........................................................................................................... 147 RH C alibration ........................................................................................................... 147 Pressure Reading ..................................................................................................... 147 Pressure C alibration .......................................................................................... 148 Pump Relay Control ........................................................................................... 148 CO2 Reading ............................................................................................................. 149 Light Reading ............................................................................................................ 149 Light C alibration ........................................................................................................ 149 Thermocouple Reading ............................................................................................ 149 Program .................................................................................................................... 150 LIST OF REFERENCES ................................................................................................. 153 BIOGRAPHICAL SKETCH .............................................................................................. 162

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9 LIST OF TABLES Table page 1 -1 Total pressure and gas composition used to support life in various space missions. ................................................................................................................. 38 1 -2 Hypobaria studies at various total pressures and gas composition ..................... 39 4 -1 Set points for the environmental conditions .......................................................... 98 4 -2 The effect of pressure and (carbon dioxide) CO2 on plant growth (dry and fresh weight), water content, CA and dark respiration (DR) and transpiration rates of 26 -day old radish plants grown for six days at the given pressure treatment.. ............................................................................................................... 99 5 -1 Number of days plants exposure to normal pressure an d hypobaria. ............... 127 5 -2 Environmental conditions used for the experiments. .......................................... 128 5 -3 Average fresh weight (FW), dry weight (DW) of sh oots, roots, hypocotyls in hypobaria treatments (A -D) and control treatment (E). ....................................... 129 5 -4 Average specific leaf area, stomata density, stomata index and average stomata pore length from leaf of hypobaria (33 kPa) and normal pressure (101 kPa) .............................................................................................................. 130 6 -1 Parameters and reference values used in the model. ....................................... 139 6 -2 Parameter description and reference values used for sensitivity analysis ........ 140 6 -3 Sensitivity analysis of the transpiration model at various conditions.. .............. 141

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10 LIST OF FIGURES Figure page 1 -1 Theoretical depiction of a future Martian farm. Courtesy: nasa.gov ..................... 36 1 -2 Simplified equatio ns showing human respiration (top) and plant photosynthesis (bottom).. ....................................................................................... 37 2 -1 Low Pressure Growth Chamber (LPGC) ............................................................... 53 2 -2 Low Pr essure Growth Chambers developed for this objective with gas tanks, datalogger (CR10) and PC interface. .................................................................... 54 2 -3 Calibration of the pressure sensor comparing pressure gauge reading against mill ivolts readings. ..................................................................................... 55 2 -4 Calibration of the relative humidity sensor against known saturated salt solutions. ................................................................................................................. 56 2 -5 Calibration curves of an oxygen sensor at different pressures (5A) and curves of slope and intercept (5B). ........................................................................ 57 2 -6 Calibration curves for a CO2 sensor at different pressures (A) and curve of slope and in tercept (B). .......................................................................................... 58 2 -7 CO2 sensor reading against the equivalent syringe volume ................................. 59 2 -8 Calibration for a load cell used to measur e the weight of the flask containing plants. ..................................................................................................................... 60 2 -9 Calibration curve for light sensor readings against handheld light meter ............ 61 2 -10 Pressure (~33 kPa), O2 (~20 kPa) and temperature (~23 C) recordings for five hour for chamber A (top panel), chamber B (middle panel) and chamber C (bottom panel). .................................................................................................... 62 3 -1 The individual mass diffusivity of CO2 and H20 calculated from the binary mass diffusivity using the empirical formula given by Fuller et al. (1966).. .......... 77 4 -1 Schematic of the low pressure growth chambers used for experiments. Each chamber has 0.09 m3 total internal volume.. ........................................................ 93 4 -2 Radish (26 day old) grown for six days in super elevated CO2 (180 Pa) at 101 (first row), 66 (second row), or 3 3 (third row) kPa total pressure.. ................ 94 4 -3 Total leaf area of radish grown at various total pressures and CO2 levels.. ........ 95

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11 4 -4 C O2 drawdown curves on the final day of the experiment of 26 day old radish grown for six days at 33, 66, or 101 kPa.. ............................................................. 96 4 -5 Water loss due to transpiration of 26 day old radish grown for 7 d ays for low (40 Pa; A), high (100 Pa; B) or super elevated CO2 (180 Pa; C) at 33, 66, or 101 kPa total pressure. .......................................................................................... 97 5 -1 Germination of radish seedlings at 33 kPa total pressure and various pO2. Bars represents the mean STDEV (n=2) ......................................................... 121 5 -2 Radish plants after four weeks of growth Treatment A (28 days at 33 kPa), Treatment B (21 days at 33 kPa), Treatment C (14 days at 33 kPa), a nd Treatment D (2 days at 33 kPa). .......................................................................... 122 5 -3 Carbon dioxide assimilation (CA;A), Transpiration rates (B) and leaf area (C) on the last day of the experiment. ....................................................................... 123 5 -4 CA (A) and Transpiration rates (B) and W ater U se Efficiency (WUE) (C) on the day 7, 14, 21 and 28 of the experiment. ........................................................ 124 5 -5 Scanning electron microscopy (SEM) image from a youngest leaf of a plant grown entirely in hypobaria (A; 33 kPa) or in normal pressure (B; 101 kPa). .... 125 5 -6 The long-term and short -term responses of the shoot or root to water deficit, low humidity and high temperature (adapted from Chaves et al., 2003). .......... 126 6 -1 Predicted transpiration rates from 33 to 101 kPa. ............................................... 135 6 -2 Predicted transpiration rate at various total pressures (33, 66, 101 kPa) and various stomatal widths (0.0001, 0.0003 and 0.0004 cm). ................................. 136 6 -3 Predicted transpirat ion rate at three different pressures (33, 66 and 101 kPa) and two different vapor pressure deficit (VPD) levels (0.6 kPa and 0.9 kPa). ... 137 6 -4 Predicted transpiration rates at stomata number s. ............................................. 138

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12 LIST OF PARAMETERS P VPD RH pCO2 pO2 L t P0 Pi Ca Ci CA DR T FW DW WUE k m AB a b d D n hs hb K L Kv u Re D Sc pressure (kPa) vapor pressure deficit (kPa) relative humidity (%) partial pressure of CO2 (Pa) partial pressure of O2 (Pa) leak rate (% Vol/day) time interval (h) initial pressure (kPa) end pressure (kPa) CO2 concentration outside leaf air space (Pa) CO2 concentration inside leaf air space (Pa) CO2 assimilation rate (mol m2 s1) Dark respiration rate (mol m2 s1) transpiration (mmol m2 s1) fresh weight (g) dry weight (g) water use efficiency (mol CO2 mol H2O1) the Boltzmann constant, ergs/K molecular weight ( g mol1) collision diameter, cm stomata l ength (cm) stomata width (cm) stomata depth (cm) diffusivity (cm2 s1) num b er of stomata stomatal conductance (cm s1) boundary layer conductance (cm s1) thermal diffusivity (cm2 s1) length (cm) kine tic v isco sity (cm2 s1) air speed (cm s1) R eynolds number viscosity ( g cm1 s1) density (g cm3) diffusivity (cm2 s1) Schmidt number

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13 Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy GAS EXCHANGE AND ACCLIMATION OF RADISH IN REDUCED PRESSURE ENVIRONMENTS By Hemant Laxmansinh Gohil August 2010 Chair: Melanie Correll Major: Agricultural and Biological Engineering Plants grown on longterm space missions will li kely be grown in low pressure environments (i.e., hypobaria). In general, the fundamental growth and photosynthesis of plants grown in hypobaria are similar to plants grown at normal atmospheric conditions with some exceptions. For example, transpiration rates can be elevated in low pressure resulting in plant wilting if water is not readily available. Plants may also exhibit poor growth and germination if the partial pressure s of critical gases are not maintained to certain levels (e.g., pO2 for seed ger mination and pCO2 for photosynthesis) Since the gas phase of the environment is so critical for successful growth of plants in both normal and low pressure environments the effects of the gas phase composition, particularly CO2, on the gas exchange and growth of radish (Raphanus sativus var. Cherry Bomb II) in hypobaria w ere studied Low pressure growth chambers were built that could monitor the environmental parameters for these studies The fresh weight (FW), leaf area, dry weight (DW), CO2 assimilatio n rates (CA), dark respiration rates (DR), and transpiration rates from 26 day old radish plants that were grown for an additional seven days at different total pressures (33, 66 or 101 kPa)

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14 and pCO2 (40 Pa, 100 Pa and 180 Pa) were measured. In general, th e dry weight of plants was enhanced with CO2 enrichment and with decreased total pressure. In limited pCO2 (40 Pa), the transpiration for plants grown at 33 kPa was over twice that of controls (101 kPa total pressure with 40 Pa pCO2). Increasing the pCO2 f rom 40 Pa to either 100 or 180 Pa reduced the transpiration rates for plants grown in hypobaria and at normal atmospheric pressures. Plants grown at lower total pressures (33 and 66 kPa total pressure) and super elevated pCO2 (180 Pa) had evidence of leaf damage. Taken together, radish growth can be enhanced and transpiration reduced in hypobaria by enriching the gas phase with CO2 although at high levels of CO2 leaf damage can occur. Since the diffusivities of gas increases as the atmospheric pressure dro ps, it is expected that transpiration and CO2 assimilation in plants would increase as plants grow in hypobaria. A mathematical relationship based on the principle s of thermodynamics was developed for calculating the transpiration and photosynthesis for plants. Stomatal conductance is sensitive to total pressure. At 33 kPa total pressure, stomatal conductance increases with the boundary increasing by a factor of 1.7, thus the boundary layer thickness conductance increases by 7 0%. Since the leaf conductanc e is a function of both stomatal conductance and the boundary layer conductance, the overall conductance will increase resulting in significantly higher levels of transpiration as the pressure drops The conductance of gases is also regulated by stomatal aperture in an inverse relationship. Stomatal aperture is directly influenced by concentration of CO2 inside the leaf space. The higher CO2 concentration inside the leaf air space during low pressure treatments may result in stomata closing partially or ful ly which may reduce the excessive transpiration caused by increased diffusivity. Therefore, a reduced

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15 pressure environment with high CO2 may be an ideal scenario for minimizing transpiration and maximizing the plant biomass yield in BLSS. The application o f this model to data for plants acclimated long term to hypobaria (33 kPa) and reference values (101kPa) suggest that for transpiration the model predicts transpiration for plants that are grown at normal pressure fairly well, but for those grown at 33kPa the model over predicts transpiration observed data by almost T o understand the mechanisms of plant adaptation to hypobaria plants were transferred to low pressure at different stages of growth ( 0, 2, 14, and 26 days after germination ) The growth, CA, transpiration and stomatal density were compared between these plants Plants exposed short term to hypobaria (< 2 days in hypobaria) responded differently than those exposed to long -term hypobaria (>2 day s) For example, plants that were grown entirely i n hypobaria had smaller and thicker leaves compared to plants that were exposed for 2 days or less to hypobaria. These long -term treated plants also had higher CA (p<0.1) and transpiration rates (p< 0.05) even though their overall growth (FW and DW) was n ot significantly affected by hypobaria The stomatal density of plants grown long term in hypobaria was not significantly different than plants grown short term to hypobaria. Therefore, it appears that plants may respond to enhanced gas exchange in hypobaria by reducing their leaf area. Further studies on the mechanisms of plant adaptation are required to identify other biological or physiological mechanisms of plant acclimation to hypobaria. Some of the engineering constraints to grow plants in Martian plant growth facilities can be offset by growing plants at reduced atmospheric pressure. Characterization of hypobaria response at reduced pressure can provide data which

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16 can help in explaining some of the response which plants may encounter on Martian faci lity. These studies are important for understanding mechanism by which plant control water relations.

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17 CHAPTER 1 ATMOSPH ERIC CONDITIONS USED FOR BIOLOGICAL SYSTE MS DURING SPACE MISSIONS: IMPLICATIO NS FOR HYPOBARIC PLANT BIOLOGY Advance d Life Support System s (ALS) Longterm, space exploration with humans will require an A dvanced L ife S upport system (ALS) that provides air, water, and food to explorers in a sustainable manner This system will consists of the latest technologies for atmos phere revitalization, water supply and food and fiber production as well as the recycling of these valuable resources These technologies may be based on physico -chemical (P -C) or biological approaches. The P -C approach uses mechanical or chemical mechanisms to provide an ALS whereas the biological approach, the Biological L ife Support S ystem (BLSS), uses biological systems (e.g., plants, algae or microbes) to supply the requirements for ALS. The efficiency of each approach has been compared and estimates of the time until the system has reached sustainability have been modeled. Th is time to reach sustainability is an important aspect for cost analysis and as an indicator to how long a space colony can be supported without re -supply from Earth. Unfortunate ly, the results of these studies are conflicting as to which method is more efficient for long-term space missions. For example, Alan Drysdale (Boeing Corporation; Drysdale, 2001 ) suggests that the BLSS may take three years for sustainability making it a v iable option for ALS. In contrast, Harry Jones (NASAAmes: Jones, 2007 ) suggests that the BLSS could take up to ten years to reach a sustainable state and therefore he supports a P -C approach. Barry Fingers ( Dynamac Corp., Fingers et al., 1996) suggests t he ideal scenario is the hybrid of both the approaches, where P -C approach is used for the initial one to two years until the BLSS can become sustainable. The P -C approach has

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18 already been used on the International Space Station (ISS) with much success but the costs associated with transport of materials for P -C systems are considerably lower for the station that is ~ 375 kilometers above earth than for distant planets. Arguments for the P -C approach suggest that the advancements in water and air recovery an d biomass processing make the P C more cost effective and reliable than BLSS Whereas the s upporters of the BLSS suggest that eventually a biological system for material recycling will be required for longduration missions and for their benefits to explo rers living with plants and having a regular supply of fresh food ( Wheeler, 2004 ). As one of the major components of developing a BLSS the environment including the gas phase, temperature and pressure used to support the biological system must be careful ly monitored and controlled. This chapter discusses the environments (i.e., gas phase composition and pressures) that have been used in the past to support life on space missions and describes the effects of these types of environments on plants as part of a BLSS. Gas Phase for Biological Life Support Here, a brief history of the environments that have been used for life support is described with emphasis on those systems that have been used for humans and plants. The first biological system to go up in spa ce was a dog named Laika sent by the U nited States of S oviet R ussia ( USSR ) on Sputnik 2 on Oct ober 4, 1957. The pressure and atmospheric composition of the vessel was controlled with an oxygen generator CO2 and vapor scrubbing system with pressures and ga s concentrations (CO2, N2 and O2) maintained to levels of Earth sea level (Table 1 -1 ). Unfortunately, the inadequate temperature control system resulted in the heatstroke and the death of Laika within a few hours ( Malashenkov, 2002 ). After Laika, the USSR sent several dogs and cats in to

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19 space of which some returned safely. Shortly thereafter ( 1958 -62) during the Mercury missions, the US sent monkeys into space with a total atmospheric pressures of 34 kPa at 100% oxygen. The advantage of using the low pressure in this case was that it required less total gas to be transported from Earth to support life and thus increased mission duration. I n addition, the low pressure required a much lighter hull for the craft since a smaller pressure difference between th e inside of the craft and the vacuum of space was maintained. H igh levels of O2, greater than 35 kPa ( Baker 1981) can be toxic to humans and animals, thus 34 kPa of total pressure of O2 was chosen as the highest pressure. Th is atmospheric composition and pressure was used for the Mercury, Gemini, and Apollo space missions ( Martin and McCormick, 1992 ). The success of th e se first missions with animals led to more challenging human missions by the USSR and the USA. On April 12, 1961, the first human, Yuri G agarin, was sent to space by the USSR in Vostok 1. This vessel had sea level atmospheric conditions (100 kPa total pressure, 0.04 to 0.2 Pa CO2, and 21kPa O2). The gas composition and high pressure of the Vostok missions required strong materials for the cabin construction and the necessity for prolonged de nitrogenation before any extra vehicular operation since suits on these missions were much lower than cabin pressure. As a benefit of this gas composition and pressure, the lower oxygen concentration of the atmosphere prevented fire hazards and allowed for a more e arthlike environment for biological studies ( Baker, 1981). The oxygen was provided by potassium superoxide CO2 and excess water was absorbed by lithium hydroxide. These atmospheric conditions were used throughout the USSR program up until M ir (Baker, 1981). Shortly after the USSR manned space mission on May 5, 1961 the US sent Alan Shepherd to

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20 space on Mercury Freedom 3 in similar atmospheric conditions as w ere used for the monkeys in the past Mercury missions ( 34 kPa at 100% oxygen). A total of six manned missions were performed during the Mercury period using this enriched oxygen, low pressure environment. T he first two-person mission and the first mission with the cosmonauts without a spa cesuit were in Voskhod 1 (Oct 1964 ). The Voskhod spacecraft were modified Vostok vessels that had a parachute system removed to allow for a second cosmonaut. The atmospheric conditions were similar to those of the Vostok missions (100 kPa, 0.04 to 0.2 Pa C O2, and 21kPa O2) Voskhod 2 ( Mar 1965) was the first mission that included a human spacewalk by Alexei Leonov. This required for extra vehicular activities the cosmonaut had to reduce the pressure in his suit to either 40.6 kPa or 27.4 kPa total pressures before exiting the vehicle which put the cosmonaut in danger of getting the bends (Baker, 1981). There was some difficulty entering the airlock on the vessel due to the rigidity of his pressurized suit relative to the airlock. The suit was designed to provide 45 minutes of oxygen for breathing and cooling and allowed for venting of gas and vapor to space. After the success with upper orbit (Earth) flights, missions to the Moon became the focus of space exploration. However, before the astronauts could lan d on the moon several manned Gemini missions (196566) were used to survey the Moon and test the capabilities of the rockets and for landing. As a part of Biostake series of experiments, corn and mustard seeds were carried by a stronaut Ed White in his spac e suit (at 25.5kPa, 100% oxygen ; Grimwood et al., 1969) during the first US extravehicular activity (EVA) on Gemini 4 (1967). Though seeds were germinated during subsequent

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21 growth studies on E arth at ambient pressure, abnormality in the plants was observed likely due to heavy ion radiation ( Paul and Ferl, 2006). After Gemini, the Apollo missions were developed to bring astronauts to the moon. Unfortunately on January 27, 1967, the Apollo 1 mission ended in disaster with a fire that killed the three astronauts on board while on E arth (Edward White, Virgil Grissom, and Roger Chaffee). The fire was caused by the high flammability of a pure oxygen environment inside the capsule (at 100 kPa; Apollo 204 Review Board Report, 1967). This high pressure was requir ed during launch since the craft were not built to withstand high pressure differences between the environments inside and outside of the vessel. A similar incident occurred earlier in March 1961 in the USSR which claimed the life of Soviet cosmonaut train ee, Valentin Bondarenko when a fire started in the pure oxygen atmosphere in the chamber he had been in although this incident was concealed from the public for years (Oberg, 2009). As a result of the Apollo fire, NASA set new criteria for gas phase comp osition for space missions. These included that the gas phase would consist of 60% oxygen and 40% nitrogen at roughly sea-level pressure at launch, then the pressure was lowered by releasing gas to maintain about a 40kPa difference between the inside and outside of the vessel during ascent until the atmosphere reached approximately 34kPa and 100% oxygen during the first 24 hours in space. The astronauts were acclimated in space suits from the time of launch at 100% oxygen (34 kPa) approximately three hours before launch to prevent the bends during the depressurization and ascent Once the craft reached the set poi nt atmospheric conditions the astronauts could remove their suits and move about the cabin. These Apollo missions ran from 196772. Unfortun ately, the hazards of oxygen flammability

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22 were realized again during the Apollo -13 mission, when, on the return flight to Earth the oxygen gas tank was damaged and ended up starting a fire. To address this, the Apollo 14 mission had several modifications t o the gas tank systems. Interestingly, t he Apollo 14 mission had one of the first long-term treatments of seed in space when astronaut Stuart Roosa, the flight commander, carried 500 seeds in his personal belongings and brought them back to Earth. The seeds were of several tree species including sycamore, pines, and fir. After the 9day mission these seeds were given to several educational institutes including the University of Florida, the trees germinated from these seeds appear to have no signs that th e mission to the moon had negatively affected their growth ( Klein, 1981 ). During the Biosatellite program (19661969) NASA sent the Bion series of satellites with specimens of fruit fly, frog eggs, bacteria and wheat seedlings to study the effects of weig htlessness on living organism. Total pressure was maintained at approximately 10 0 kPa and oxygen partial pressure was 21 kPa. In the Biosatellite II pepper plant experiment, a camera recorded the positions of plant leaves with respect to time to study th e effect of microgravity on plant movement (Thimann, 1968). In order to conduct longterm research on living systems in space as well as to establish a space laboratory for small animals, plants, microorganisms, and humans, the USSR launched Salyut (19718 2 ) and the USA launched Skylab (197379). Salyut was at approximately sea level pressure and atmosphere composition. Salyut carried the Oasis, the first of its kind plant growth system with a cinematic recording system (Neichitaleo and Mashinski, 1993). It was used to cultivate Brassica capitata, Linum usitatissium and Allium porrum The biological experiments also included variety of

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23 species including spiders, worms, fishes, and frogs Many of the experiments were performed to study the effects of radiati on and space environment on biological systems. In contrast to Salyut, the Skylab platform was operated at 34 kPa total pressure but the atmospheric composition was 70% oxygen and 30% nitrogen. Nitrogen helped reduce the risk of pure oxygen while still mai ntaining the health of the Astronauts from hypoxic conditions. One of the plant experiments (Skylab e xperiment ED -61/62) studied phototropism of tomato in low gravity. This was the first time the seeds were germinated in agar in space microgravity. These s tudies also compared various light intensities that are required to produce photosynthesis for plants grown in space (Cramer et al., 1984) Interestingly, a combined Skylab-Salyut station was proposed by the US and the USSR. But, as noted above, each stati on had different gas mixes and pressures. Since each station would need to make modifications it was proposed that a 55kPa total pressure environment, slightly enriched in oxygen would be used to compromise and minimize the modifications required for one station. However, this combined station was never realized. To replace Salyut, the USSR built Mir (1980 -98). The M ir the Russian word for world or peace, station had an atmosphere of 101 kPa total pressure and earth ambient concentration of gases (21 kPa O2, 80 kPa N2 and 0.040.2 kPa CO2). Seed germination experiments on board Mir were done with variety of small, closed or ventilated/partially ventilated chambers, from simple a beaker containing moist soil (Kosmos 1129 ; P arfeenov and Arbanova, 1981) t o a sophisticated greenhouse, called S vet, the Russian word for cosmos (Nechitai lo and Mashinsky, 1993). During the early

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24 experiments on seed germination, plants exhibited poor growth and stress (Kordyum et al., 1985). This was possibly due to the lack of natural convection of air movement in microgravity leading to formation of stagnant air layers around seedlings within the closed chamber (Musgrave et al., 1988 ). Another attempt to germinate seeds and complete the life cycle in space resulted in a failed experiment ( Mashinsky et al, 1994). However, a complete plant life cycle was performed with wheat grown in the Svet greenhouse which had a better control system for the gas phase (Svetlana et al., 2005). However, after t hese plants were returned to Earth (STS-81, 1997) it appeared that the wheat flowers had sterile seeds. Initially, microgravity was assumed to be the cause of the seed sterility but when researchers grew a dwarf wheat variety at the same levels of atmospheric composition that the super dw arf was exposed to in space, they found higher concentration of ethylene on the station to be the cause ( Salisbury 1995 ). Plants tolerate up to 45 ppb concentration of ethylene, with anything higher resulting in reduced plant growth. Salisbury et al. ( 199 5 ) found that the ethylene in the growth chamber was as high as 12 ppm, ~1000 times higher than the tolerable limit for plants Since ethylene produced by plants was indicated as the source of ethylene and since convection is limited in microgravity, thes e researchers developed a dwarf variety of wheat (Apogee) that is insensitive to higher concentrations of ethylene. The Apogee wheat produced non-sterile flowers and viable seeds on board Mir and the ISS suggesting that ethylene and not microgravity was th e cause for flower sterility found in previous missions ( Levinskikh et al., 2000). Due to the problems with high ethylene and CO2 concentrations in the cabin of Mir which were harmful to plant growth, it was decided that an independent,

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25 controllable growt h chamber was required to conduct specialized experiments which would protect plants from exposure to cabin level CO2 and ethylene. Therefore the Svet greenhouse (1990-2000) was built by Russia and was the first automated plant growth facility The greenh ouse was at 101 kPa total pressure and atmospheric composition similar to Earth sea level except CO2 levels were up to 0.2 kPa. However, it had open type air ventilation system that had air in contact with the Mir cabin atmosphere (Svetlana et al., 2005). Improvements to the plant growth chambers were made to SVET in the late 1990s which included the ability to monitor the plant growth in real time by measurement of CO2 and H2O exchange rates, temperature, and relative humidity. This allowed the measurement s of photosynthesis and transpiration rates based on the current plant CO2, vapor, temperature and relative humidity levels. However, the open air system was no t ideal for careful control of gas phase surrounding plants. After the Biosatellite program was cancelled in 1968, it was not until 1982 during the third space flight program (STS -3) that the first Plant Growth Unit (PGU) was launched by the USA to perform s eedling growth experiments ( Cow les et al., 1984 ). PGU served for 15 years for use on plant growth studies before Astroculture was introduced. The Astroculture G rowth C hamber (ASC -GC) was the first of its kind It was a completely automat ed growth chamber developed in the early 19 90s to provide support system for plant growth in closed environment. It was developed by the Wisconsin Center for Space Robotics and Automation at the University of Wisconsin, Madison. The CO2 was maintained in the range of 300 2000 ppm and the ethylene concentration could be reduced to less than 50 ppb using a catalytic ethylene scrubber. The main objective of ASC -GC was to perform short and long-term plant experiments

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26 in microgravity to study seedto -seed cycle in space with automatic control for up to 30 days. The ASC -GC hardware was efficiently used for studies on Ar abidopsis (STS68). In one study, the reproductive ability of pre germinated Arabidopsis seedlings in various gas compositions and ventilation regimes were studied. At a CO2 concentrations of 300 2000 ppm Arabidopsis had sterile pollen and embryos, and at very high CO2 concentration (8000 ppm) plants exhibited early abortion of ovules and mature pollen, and the release of pollen from anthers was restricted preventing fertilization (Kuang et al., 1996) The Mir station was in operation for fifteen years unti l March 23, 2001, when it was deliberately de orbited, breaking apart during atmospheric re entry over the South Pacific Ocean. The International Space Station (ISS) The International Space Station ( ISS ) is a joint project of several space agencies across the world led by NASA. The ISS construction began in 1998 and has had continuous human presence since Nov ember 2000. The ISS platform runs at 101 kPa total pressure with E arth sea level concentration of the gases. Advanced Astroculture made its debut flight during the second ISS increment to study the effects of microgravity on seed to seed development of Arabidopsis thaliana. The m ain objective of this study was to grow a second generation of plants using the first generation of seeds and harvesting the living plant tissues for gene expression analysis (Fourth and Fifth increment ; Link et a l., 2003). The c ompletion of a complete life cycle by Brassica rapa L and wheat plants of Apogee variety in the Svet greenhouse on board Mir indicated that plants can be grown in consecutive generations in space ( Levienskikh et al., 2000; Musgrave et al., 2002 ). During March 2003 to April 2005, a Russian group led by Sychev studied five

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27 consecutive generations of genetically engineered dwarf green peas in a greenhouse called LADA in the Russian module of ISS. The LADA has mainly e arthlike atmospheric pressu re and gas concentrations of CO2, N2 and O2; though CO2 concentration can reach up to 0.1 kPa. They reported that pea plants grown over a complete ontogeny cycle on board ISS were similar to ground controls in terms of plant development and genetic charact eristics ( Sychev et al., 2007). Advance d Plant Studies on the ISS The ISS now has more sophisticated plant growth chambers such as the European Modular Cultivation System (EMCS). The EMCS was launched in Feb ruary 20 07; a s one of the European Space Agency (ESA) contribution s to the ISS. It is the part of European Columbus L ab section of the ISS. It has two centrifuge rotors, which can provide different gravitational accelerations from 0.001 to 2 g (Brinckmann, 2005). The advantage of EMCS is that the h ardwar e can be experiment specific. The incubation chamber control s relative humidity down to 30 %, oxygen at 0 -21 % and CO2 from 200 2000 ppm The EMCS also has an ethylene scrubber which prevents ethylene accumulation. Th e EMCS was originally designed for pla nt experiments but due to the recent budget cuts to the space program it has also been used for other organisms such as fruit flies (Brinckmann, 2005) The US and the USSR have chosen different levels of gas phase composition and total pressures to maint ain environments for life support In the future, it is likely that life support systems will be maintained at lower pressures for the cost savings in transporting valuable gases and reduction in the cost of materials to maintain structural integrity of ch ambers used to house living systems. The effects of low pressure on plant growth are described in the next section.

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28 Hypobaric Plant Biology Plants will be an integral part of BLSS for human space exploration since they can provide a source of food, can revitalize air and water for human use, and offer psychological benefits to space travelers during long-term missions away from Earth (Fig ure 52 ). During many of these missions, plants will likely be grown at reduced atmospheric pressure in environmentally controlled chambers (Paul and Ferl, 2006). The low pressure environment will minimize the amount of atmospheric gases that need to be transported from Earth and the low pressure will require less structural mass of the chambers to withstand the low pressur e/vacuum environments found in space or on many planets or m oons. The study of plants in low pressure is termed hypobaric plant biology and, on Earth, it includes the study of plants at high altitudes. The existence of flora and fauna at very high altitude s, up to 5000 m (total atmospheric pressure of 55 kPa) with warmer temperatures suggests that plants have existing mechanisms for growth at low pressure at least down to approximately of an atmosphere ( Korner, 2004). Because plants will likely be grown i n low pressure environments on the Moon or Mars, a brief history of hypobaric plant biology as it relates to space biology is described. The Effects of Hypobaria on Seed Germination and Plant Growth Successful crop growth in hypobaria depends on a variety of interacting factors such as the relative gas composition (particularly O2 and CO2), absolute total pressure, vapor pressure deficit (VPD) of the atmosphere, and the temperature, among other factors. Many studies have been performed to identify the effec ts of low pressure on germination, plant growth and development and many of these studies have recently been reviewed (Paul and Ferl, 2006) but are described here briefly and summarized in

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29 Table 1 2 In one of the first experiments on germination in hypobaria, r ye seeds that were grown in approximately 3 kPa total pressure with Mars level carbon dioxide partial pressure (0.45.kPa) without oxygen had no seed germination (Siegel, 1963 ). However, at 3kPa of pure oxygen the seeds germinated. Other studies also report seed germination at partial pressure of oxygen of 5 -8 kPa at various total pressures (Musgrave et al., 1988; Schwar t zkopf and Mancinelli; 1991). These studies indicate that for many species the lower limit for germination in a pure oxygen environment is at approximately 5kPa. Although seed germination appears to be restricted to a small window of partial pressure of oxygen t he results on the effects of these low pressures on plant growth are conflicting. For example, tomato plants had reduced growt h in 40 kPa compared to control (93 kPa) in one study ( Daunicht and Brinkjans, 1992), but showed enhanced growth at 33kPa compared to control in another ( Rule and Staby, 1981 ). Mungbean growth was also enhanced in hypobaria at 22 kPa total pressure ( Musgra ve et al., 1988). These differences in responses were attributed to the differences in composition of the atmosphere with respect to CO2 and O2. Reduced growth may have been due to the lower partial pressure of oxygen ( pO2at ~8.4 kPa) which can induce hypoxia stress in plants and result in poor growth (Musgrave et al., 1988) In general, for short -term studies in hypobaria, plants have increased growth if enough oxygen and water are supplied. This increased growth is partially attributed to enhanced photo synthetic rates of the plants as a result of reduced pO2 or alternatively, due to the increases in diffusion of CO2 (Iwabuchi et al., 1996 ; Corey et al., 1996, 2002; Goto et al., 1996; Richards et al., 2006). Lower pO2 inhibits photorespiration since CO2 h as less competition with O2

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30 for the enzyme Ribulose Bisphosphate Carboxylase/Oxygenase (RUBISCO) the first step in carbon fixation ( Drake et al., 1997). The increased photosynthesis at low pressure and low pO2 was decreased by injecting O2 into the environment suggesting that indeed the ratio of CO2 to O2 was the cause of the increased growth in some studies (Corey et al 2002 ). Even under normal pressures (101 kPa) Arabidopsis had increased photosynthetic rates when plants were grown in hypoxic conditions (2.1 kPa ppO2; Richards et al., 2006). Further studies with Arabidopsis with greater range of pressures showed less of a difference in carbon dioxide assimilation (CA) after 16 hours in hypobaria relative to controls at 100 kPa compared to plants that w ere exposed to hypobaria for only 1 hour, this suggest s a possible adaptive response after 16h in hypobaria (Richards et al., 2006 ). In rice, the negative effects of hypoxia on growth at 25kPa total pressure were alleviated by maintaining the pO2 at 10 kPa (Goto et al., 2002). Therefore, the benefits of low pO2 for increased photosynthesis in hypobaria need to offset the negative effects of hypoxia on plant growth. Others suggest that the e nhanced diffusion of CO2 in hypobaria to the leaf allows for improv ed CO2 fixation and this may account for the increased growth for plants grown in hypobaria (Goto et al., 1995; Daunicht and Brinkjans, 1996; Massimino and Andr, 1999). In contrast, some long -term experiments reported that plants had similar rates of photosynthesis in hypobaria as those grown in normal atmospheric conditions and had no increase in growth (Iwabuchi and Kurata, 1996; Spanarkel and Drew, 2002). This may be a result of reduced stomata apertures in acclimated leaves during the long -term hypob aria treatments thus reducing CO2 diffusion into the leaf (Iwabuchi and Kurata, 1996 ).

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31 Further studies on the dynamics of CO2 diffusion to the leaf and its effects on plant growth are required to understand this response. Other gases in the hypobaria envi ronment also diffuse at a greater rate relative to high pressures environments and these include ethylene and other plant volatiles Ethylene is known to cause a variety of responses in plants including enhanced leaf senescence and reduced overall growth (Finlayson et al., 2004). To study the effects of ethylene in hypobaria He et al. (2007and 2009 ) exposed lettuce and wheat to 25 and 101 kPa total pressure at various oxygen concentrations and monitored ethylene. Their results suggested that hypobaria doe s not affect the plant growth and that ethylene concentration was the same in both the hypobaric conditions and in the normal pressure controls. However, t his low level of ethylene may be a result of the reduced oxygen partial pressure since this can reduc e ethylene synthesis in plants (Burg 2004). The effects of hypobaria on gene regulation w ere studied in Arabidopsis plants grown at 10 and 101 kPa total pressure and at 2 and 21 kPa pO2 (Paul et al., 2004). Th is study identified genes that were regulated i n low pressure but were not caused by low levels of oxygen i.e., not due to hypoxia. These genes that were specifically regulated by low pressure included genes involved in desiccation -related pathways such as dehydrins, ABA related proteins, and cold-re sponsive (COR )-related proteins (Paul et al., 2004). This suggests that plants do undergo stress in hypobaria, particularly water stress. Future studies on the gene expression of plant grown in hypobaria are required to identify the downstream pathways of this response and the effects of long term acclimation on gene regulation.

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32 Recent studies on plant growth for radish that were grown entirely in hypobaric conditions ( Levine et al., 2008; Wehkamp, 2009 ) suggest that plants reduce their excessive water l oss over time in hypobaria by reducing their transpiration and CO2 assimilation rates to levels similar to plants grown at ambient pressure. In spinach, the transpiration rates were slightly higher during the short -term exposure (1 day) to hypobaria but af ter 10 days in hypobaria, both the CO2 assimilation and transpiration rates were similar to those of plants grown in ambient pressure ( Iwabuchi and Kurata, 2003 ) However, it is still not clear how plants acclimate to longterm exposure. Groundbased studi es have also been performed to test the fruit and nutritional quality of radish grown at low pressures ( 33, 66 kPa ) and these studies found that the biochemical composition of the radish was not significantly different from plants grown at ambient levels f or the compounds tested (Levine et al., 2008). Summary Maintaining life in space is a challenge. Throughout the history of the space program a careful balance of keeping the gas composition at pressures that allow life to thrive at economical levels while minimizing the dangers of these atmospheres to life has been a primary goal of engineers As we advance to the next step of bringing life to distant planets, further modifications in the atmospheric conditions to support life are likely. Due to the cost s avings for growing plants in reduced pressures, low pressure growth chambers are likely to be used Although the exact level of total atmospheric pressure has not been set, it is suggested that plants will be grown at approximately 55kPa in a M oon or Mars greenhouse ( personal communication, Ray Wheeler, NASA). Studies on seed germination, CO2 enrichment, ethylene evolution, vegetative growth and even gene expression profiles in low pressure on E arth and in space suggest that

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33 plants can be grown at low press ures and be a viable food source for explorers These studies also give ample evidence that the fundamental biological processes of photosynthesis and growth are essentially the same in low pressure and Earth sea level pressures The use of plants in a biological life support system (BLSS ) seems to be a promising approach, although it is far from mature. Since the inception of the concept salad machine extensive research has been performed to study the plant response to various environmental factors whic h have contributed to the development of the hardware that is currently used in space for plant growth. H owever there are several technological and operational issues associated with ALS which will need to be addressed before humans colonize space. For example, how does a low pressure environment affect plants over several generations ? T he genetic studies suggest that hypobaria may result in a metabolic drain on plants and therefore they may not be physiologically healthy and over generations these may result in poor growth and reproductive health C omprehensive stud ies on the interacting effects of all environmental parameters in hypobaria on plant growth and development has not been performed. As we plan for the future to grow plants on the Moon or Mars the proper selection of pressure and environmental parameters is required to ensure a sustainable plant growth system The range of gas compositions that are likely to be selected will depend on not only the species but also the stage of plant development since O2 requirements are different in the vegetative and reproductive periods (Wheeler 2004). In addition, gas composition may be dictated by the available resources on each planet. For example, CO2 may be used to pressurize plant growth chambers on Mars due to its

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34 availability. The task of optimizing gas phase composition and pressures for plant growth for space exploration will require many more studies. During the last decade, our understanding of the effects of hypobaria on plant growth from the m olecular level to whole plant level has improved (Paul et al., 2004; Wehkamp 2009) Studies have shown that seed germination, seedling growth and plant development can occur in hypobaria when pO2, pCO2, and pH2O are at levels that support growth. Gas excha nge studies on plants grown in low pressure suggest that plants may have increased CO2 assimilation and transpiration but this may depend on the duration of exposure to hypobaria ( Rule and Staby, 1981; Andre and Massimino, 1992; Daunicht and Brinkjans, 1996; Corey et al., 1997; Iwabuchi and Kurata, 2003). On Mars, CO2 is readily available and can be used to pressurize the chambers for plant growth but the effects of very high CO2 concentration on plant growth in hypobaria are not known. Also only a few e xperiments have stud ied the effects of long -term hypobaria on plant growth, thus the acclimation mechanisms that plants utilize to adapt to hypobaria are unclear. Therefore, for this dissertation, the gas exchange rates of radish in hypobaria in various levels of CO2 are studied and the effects of long-term hypobaria on plant acclimation are explored. Radish was chosen as the model plant for these studies since it is selected by NASA as one of the salad crops ( Wheeler, 2001), it has been used in several hyp obaria studies (Wilkerson, 2005; Levine et al., 2008; Wehkamp 2009) and the anatomy of radish lends itself to perform transpiration studies. The radish roots and leaves are also edible and provide excellent nutri ti onal value to future space explorers ( Le vine et al., 2008 ). To study the gas exchange and acclimation of radish in hypobaria, the following four objectives were addressed:

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35 1 Design and build four, mid -size, low pressure growth chambers (LPGC) that can m onitor temperature, pressure, humidity, CO2, O2, light and plant weight. 2 Predict the effects of pressure on the diffusivity of H2O and CO2 and the implication of these on plant growth. 3 Study the interacting effects of hypobaria and p CO2 on plant growth, photosynthesis and transpiration. 4 Compare g rowth, transpiration photosynthesis and stomata between long and short -term acclimated plants in hypobaria Structure of the D issertation This dissertation is organized into topical research areas that address the above objectives Chapter 2 describes th e construction and testing of low pressure growth chamber s (LPGCs) that can monitor environmental parameters important for plant growth. Chapter 2 also describes the procedure s for sensor calibration and data management using data acquisition The predicti on of the effects of reduced pressure on the diffusivity of H2O and CO2 and the implication of these on plant growth is described in C hapter 3 Chapter 4 describes the interacting effects of total pressure (33, 66 and 101 kPa) and partial pressure of CO2 ( 0.04, 0.1 and 0.18 kPa) on growth, photosynthesis and transpiration of radish plants. Chapter 5, describes studies on the effects of long -term ( 1 4 weeks in hypobaria ) and short -term ( 2 day s in hypobaria ) acclimation to hypobaria on growth, photosynthesis and transpiration of r adish plants. In C hapter 6, the transpiration model described in C hapter 3 i s applied to predict transpiration rate at various reduced pressures, vapor pressure deficit s (VPD) and stomatal widths Finally, in Chapter 7, the general conclusions and recommendations for future studies are stated. The program for sensor and parameter control is included in the appendices.

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36 Figure 1-1 Theoretical depiction of a future Martian farm. Courtesy: nasa.gov

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37 (Adapted from Wheeler et al., 200 4 ) Figure 12. Simplified equations showing human respiration ( t op) and plant photosynthesis ( b ottom). The products of photosynthesis are oxygen (O2), which can be used by explorers and carbohydrate (CH2O), wh ich can be used for food. Through transpiration, plants can be used to purify wastewater, i.e., the transpired water can be condensed as clean water.

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38 Table 1 1. Total pressure and gas composition used to support life in various space missions. Year Atm ospheric composition Comment 1957, Sputnik Sea level gas concentration and total pressure (101 kPa) Dog ( Laika ) was the first to enter into space: died of heat stroke (Maleshankov, 2002) 1961 63, Vostok Sea level gas concentration and total pressure (10 1 kPa) First human Yuri Gagarin entered into space (Baker, 1981) 1958 63, Mercury Total pressure 34 kPa, Pure O 2 Monkeys and Chimpanzees (Martin and McCormick, 1992) 196465, Voslhod Sea level gas concentration and total pressure (101 kPa) Space walk by c osmonauts (Baker, 1981) 1965 66, Gemini Total pressure 34 kPa, Pure O2 Corn (Baker, 1981) 1968, Biosatellite Sea level gas concentration and total pressure (101 kPa) First complete plant growth system by USA, Gravitropism studies on pepper plants ( Bak er, 1981 ) 1967 72, Apollo Total pressure 34 kPa, Pure O2 First Lunar Landing: s eed material exposed to lunar atmosphere (Klein, 1981) 197379, Skylab Total pressure 34 kPa, 70 % O2, 30 % N2 First US Space Station with Astronauts (Klein, 1981) 197183 Salyut 1 Sea level gas concentration and total pressure (101 kPa) Orchids for psychological benefit and p eas, Onion cultivated on board (Smolders, 1973) 1980 98, Mir I Sea level gas concentration and total pressure (101 kPa) Plant complete life cycle on s everal plant species (Salisbury 199 5 ) 1998 ISS Sea level gas concentration and total pressure (101 kPa) Advanced plant level studies ( eg. Gene expression) (Brin c kmann, 2005 )

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39 Table 1 -2. Hypobaria studies at various total pressures and gas composi tion. Reference Crop CO 2 (Pa) O 2 (kPa) Pressure kPa Comment Seigel et al., 196 3 r ye 0.24 % 0.1 % 3, 10, 50, 100 Seeds fail to germinate at 3 kPa total pressure Mansell e t al., 1968 t urnip 40 21 50, 93 Transpiration rate increased at 50 kPa Rule and Staby, 1981 tomato 14,28,40 3,7,10 17, 34, 51 Hypobaria increased C A and biomass Musgrave et al., 1988 mungbean 40 2,5,8,21 21 23 No adverse effects of 22 kPa on growth Goto et al., 1995 spinach 50, 100 2,8,15,21 50, 75, 101 No difference in growth at 50, 75 & 100 kPa Corey et al., 1996 lettuce 40, 80 10, 21 50, 101 At 50 kPa Pn a nd biomass increased by 25 % Daunicht, Brinkjans, 1996 tomato 40 40 40, 70, 100 At 40 kPa tomato plant had reduced growth Iwabuchi et al., 1996 spinach 40 21 25, 101 Growth was same in hypobaria and 101 kPa Corey et al., 1997 a wheat 120 14,21 70 Pn ra tes slightly increased (15%) at 70 kPa Massimino and Andre 1999 wheat 65, 100 8, 18 10, 20 dry mass increased by 75 % at 10 kPa Goto et al., 2002 rice 50, 100 21 50 ,70, 101 Growth normal at 50 kPa, reduced at 34 kPa Sparnakel and Drew, 2002 lettuc e 70 21 70, 100 Slightly enhanced growth at 70 kPa He et al., 2003 lettuce, Wheat 100 6, 12, 21 30 Ethylene synthesis down by 65% at 30 kPa Iwabuchi and Kurata, 2003 spinach 40 20 25 At 25 kPa stomata pore size, aperture reduced Paul et al., 2004 Arab idopsis 100 2,21 10, 100 over expression of drought related genes Wilkerson, 2005 radish 100 21 33, 100 Transpiration enhances at reduced pressure Paul and Ferl, 2006 Arabidopsis 100 2, 21 25, 100 Expression of A DH gene under hypoxia Richards et al., 2006 Arabidopsis 40, 100 5,10,15,21 25,50,75,100 Pn rates and growth increased at hypobaria He et al., 2007 lettuce 100 6, 12, 21 25 Oxygen below 12 kPa reduced plant growth Levine at al., 2008 radish 100 21 33, 66, 101 Little effects of hypobaria on nutritional quality He et al., 2009 lettuce 100 12, 21 25, 100 Ethylene reduces growth under hypobaria Wehkamp, 2009 radish 100 2,6,12,21 10,33,66,101 Long term hypobaria reduced transpiration loss Raj a pakse et al., 2009 lettuce 100 6, 10,21 25, 100 Hy poxia and hypobaria increases phyto chemical

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40 CHAPTER 2 DESIGN AND CONSTRUCT ION OF LOW PRESSURE GROWTH CHAMBERS (LPGCS) In order to grow plants at low pressures, a chamber cap able of maintaining low pressure for long periods and a system to control and monitor environmental parameters such as temperature, light, relative humidity (RH) and gas composition is required. The objective of this chapter is to describe the design and c onstruction of four, medium -scale; low pressure plant growth chambers (LPGCs) that were used for the experiments that are described in Chapters 4 and 5 of this dissertation. Maintaining a plant growth facility on Mars or the Moon at reduced atmospheric pr essure (hypobaria) compared to Earth sea level pressure will reduce the cost of launching and transporting the components to build the facility since a smaller pressure difference between the inside and the outside of the facility would require materials w ith less structural mass. This low pressure facility would also need less gas to pressurize t he chamber thus reducing the amount of valuable gases that would be supplied from Earth. These lighter structural materials will promote ease of construction but t hey must also withstand the harsh conditions of a distant planet or moon. Such a LPGC should be small enough to be efficient in modulating the thermal heat transfer and promote mixing of the gases but minimize the re -supply of gases such as CO2 for maintai ning plant growth. In contrast, the chamber should be large enough to allow for plant growth without hindrance to leaf and plant expansion. According to one estimate, the required area to grow a crop to fulfill 100 % of the food requirements for one person is 50 m2 ( Wheeler et al., 2001). Since building of such a large system is costly, the objective of this project was to build smaller -scale research chambers (0.09 m3) that could

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41 accommodate three plants while being able to fit all three chambers into a l arger environmentally controlled incubator at UF (Environmental Growth Chambers, Chagrin Falls, Ohio) Sensors in the LPGC were designed to monitor O2, CO2, relative humidity, temperature, pressure, light and accommodate three load cells for monitoring tra nspiration and plant growth. Other LPGCs have been built in the past to study plant growth but unfortunately, for most of these, there is little documentation or details as to the design and construction of the systems ( Purswell, 2002). Most of the LP GCs had limitations in terms of the range of experiments that could perform either due to the small size or due to other drawbacks in their construction. For example, Corey et al. (199 6 ) used a system which had very high leak rates thus the plant growth ch amber could not be operated below 70 kPa. Daunicht and Brinkja n s (199 6 ) used a mass flow controller to regulate the air flow and gas composition and mechanical precision vacuum controller to maintain low pressure; however specifics about the chamber were n ot given. In their system, excess CO2 and ethylene was removed by constantly ventilating by bringing in the outside air at normal pressure. Schwarzkopf et al. (1991) designed a LPGC which could be operated down to 1 kPa with leak rates of 1% chamber volume/h but the type of sensors used to measure various parameters was not reported. Simpson and Young (1998) devised a growth chamber to simulate the Martian atmosphere which could operate down to ~0.133 kPa but the data collection system was not described. Re cent reviews on the design and construction of other LPGCs are described elsewhere (Mu 2005, Wilkerson 2005, Hublitz, 2006).

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42 The low pressure chambers that were built for this project are based on the d esigns of previous LPGCs by Wilkerson (2005) and Hubli t z ( 2006) with minor modifications. The largest of these systems used for hypobaria studies was constructed to fit inside a large stainless steel vacuum chamber (1.2 m X 1.2 m X 1.0 m) that simulated the Martian atmosphere of 0.6 kPa (Hublitz, 2006). The LPGC was constructed with a polycarbonate hemispherical dome (1 m diameter) that served as the top of a Mars greenhouse and an aluminum flat plate as a base. This chamber was housed within the larger vacuum chamber. The primary purpose of this LPGC was to study the heat and mass transfer in two of the main Martian conditions, i.e., reduced pressure and extremely low temperature. An industrial freezer (~ 2m X 2m X 2 m) brought the temperature of the vacuum chambers to as low as 22 C. Hublitz (2006) and used mass flow controllers and solenoid valves to regulate the gases and pressure and grew lettuce plants for approximately seven days in these chambers. A visual interface ( LabView software, National Instruments, TX ) was used to collect the data and control gas and pressure (Hublitz, 2006). However, localized temperature gradients within the chamber and difficulties in collecting condensate resulted in large ice blocks that accumulated in the lower portion of the chamber. To date, this is the only LPGC that simulated the actual pressure conditions that would be used in a LPGC on Mars, i.e., the chamber at higher pressures than the surrounding environment but still much lower than Earth sea level. Wilkerson (2005) used a simplified bell jar system which had an aluminum base to house wiring, sensors, a cooling coil and a humidifier with a glass bell -jar top to house the plants. This chamber was 22 cm in diameter and 38 cm high. These

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43 chambers were mainly used to study plant growth and transpiration in low pres sure. Although they had a relatively small area for plant growth and were awkward to instrument, the major advantage of these systems were the flexibility in their design with many ports and the o -r ing seal for the bell jar was leak proof. The top was also easy to replaced before and after experiments. These chambers were used to grow radish at various pressures for one day. The design of LPGCs described here are larger versions (i.e., 6 times the volume) of the chambers of Wilkerson (2005). System Descrip tion The chambers designed for these studies (Figure 2 1) consist of two main components, a transparent Plexiglas tube for the cylindrical wall of the cover (1.2 cm thick, at 20 cm in diameter and 60 cm tall) that was topped with a circular (3 cm thick, 20 cm diameter) acrylic disk (Professional Plastics, Fullerton, CA). The top disc had a groove cut out that had an O -ring ( 2.57 mm diameter) which provided a gas tight fit with the tube The top of an aluminum cube (25 cm X 25 cm X 25 cm) was welded to a cir cular aluminum disk (46 cm diameter ) that had a square (25 cm X 25 cm) cut out to open the upper chamber to the lower cube A circular groove (1.9 cm length) in the aluminum disk housed a rubber gasket (Gainesville rubber & Co. Inc., FL ) and provided a gas -tight seal when the Plexiglas tube was placed on top and placed under low pressure. The transparent cover makes it easy to monitor plant growth and allows for light transmission. These chambers can be run at pressures as low as 2 kPa. The chamber is large enough to contain three (7.4 cm X 7.4 cm) pots and all the various sensors to monitor the environment. The aluminum base houses the cooling fan, wires and circuitry for the many sensors. Four LPGCs (internal volume of 0.09 m3) were

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44 assembled; one for tes ting and calibrating of the sensors and three for experimental treatments (Figure 21). Six feed-thru (type K) ports in the sides of LPGC allowed wires to pass from the inside to the outside of the chamber ( PFT2NPT -4CU Omega Inc, Stamford, CT). I n the st udies reported here, only three of the ports were utilized. The chambers have sensors for temperature (DS -75, DS10 Dallas Semiconductors, Dallas, TX), pressure ( MPXH6115AC6U ASCX15AN, Sensym ICT, Milpitas, CA), CO2 (T -6004, OEM ultrasonic, 6004, Goleta, C A), O2 (MAX -250, Matec Co., Salt Lake, UT), relative humidity (HH -4602 -L -CPREF, Honeywell, Morristown, NJ), light (LI -190, Li -Cor, Lincoln, NB) and three load cell (LPS 0.6 kg, Celtron Tech. Inc. Colvina, CA). A vacuum pump (373 watts) was used to remove g ases from the chambers to maintain the low pressure environment. Gas Leakage Tests One of the objectives for the LPGC was to minimize the amount of gas leakage. Leaks in the chambers result in the loss of gasses and difficulty in maintaining set points for experimental procedures. The leak rate will also determine how often the vacuum pump must be operated to keep pressures at the set levels. Leaks were prevented by applying vacuum grease to the gaskets that was in the aluminum plate of the base, and through the use of TeflonTM tape around all screw threads. The use of plastic tubing instead of copper tubing that was previously used in other chambers (Wilkerson, 2005) also improved this chamber to make an almost airtight system. These LPGCs are able to hold a pressure as low as 1.3 kPa pressure with the leak rate of 0.03 kPa/h. This low level of leakage required minimal use of the vacuum pump during the experimental procedures ( often only once a week ). Solenoid valves ( F -822 -G -4 24VDC Gulf Controls

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45 Company LCC, Gainesville, FL) were fitted in the vacuum line for air tight closing of the system. The following formula was used to calculate the leak rate, L = ( Pi Po)* 100 1440 Pi t (2 1) Where, L = Leak rate (% Vol/day) Pi = Initial pressure (kPa) Po = Final Pressure (kPa) t = Time interval (hr) 1 Day = 1440 minutes Data Acquisition and Control System Wilkerson et al. (2005) used Opto 22 interface hardware ( SNAP ultimate brain, Opto22, Temecula, CA) to monitor plant growth and collect data. Al though the hardware is very effective for monitoring and controlling sensors, it is bulky and is not easy to modify. The present research used a CR10 data logging system ( Campbell Scientific Inc, Logan, USA). The CR10 is a centralized control system which requires fewer components to interface with sensors, it is easy to use and scalable. The data logger is a small computer combined with a sensitive voltmeter that records the voltages (raw analog signals) from the sensors (Figure 22) and can be connected w ith a PC via a cable interface. The PC used software PC208 (Campbell Scientific, Logan, Utah) to convert the voltage data into sensor readings. For example, a 0 to 5 volt signal range can represent either 0 -100 % relative humidity or 0 100 % oxygen. It provides the input voltage of environmental parameters at small time interval (programmable) and data can be saved and logged either on the data logger or on the PC. When an

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46 excitation voltage is applied to a sensor, the sensor transmits a voltage signal t o the CR10 based on the environmental condition. The PC208W software (Campbell Scientific Inc, Logan, Utah) converted voltage reading into a final reading that is appropriate for the parameter being measure (e.g., relative humidity in %). The software ass isted with creating the program, m onitoring real -time measurements and r etrieving stored data. The standard CR10X has 12 single ended inputs which can also be used as 6 differential inputs. It can measure DC voltage up to 2.5 volts. For each chamber, ther e were two differential type of sensors (a load cell and light sensor ) and four, single ended channels for CO2, O2, RH and pressure sensors. The additional channels were not required for the environmental parameters measured in the experiments presented in this dissertation. A singleended input measures the difference of a single conductor relative to ground; whereas a differential input measures the voltage difference between two conductors. The CR10 requires a nominal 12 volt DC power supply, which was p rovided by a 12 volt battery. The battery was continuously charged using a 12 volt battery charger. Sensors and Their Calibrations Pressure Sensor The pressure sensors are small integrated circuit sensors. The pressure sensors (ASCX15AN, Sensym ICT) were c alibrated against a precision pressure gauge (Digiquartz T60 series, Paroscientific Inc. Redmond, WA) following a method used by Wilkerson (2005). They were calibrated for a range from 0 kPa to 101 kPa. All the sensors were calibrated by linear regression analysis. The response of the sensor was linear with respect to voltage and an example of this calibration for one of the four

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47 sensors is shown in Figure 23. The program for controlling pressure 2 kPa via solenoid valve is contained in Appendix A. Relati ve Humidity Sensor Relative humidity values are not dependent on total pressure, thus calibration at normal pressure was conducted. R elative humidity sensors (HH 4602 -L -CPREF, Honeywell, Morristown, NJ) were calibrated in a closed container using saturated salts (Greenspan, 1977). Five containers each had a 500 mL beaker containing saturated salt solutions to maintain specific level of relative humidity at steady state and were incubated for 24 48 h. They were saturated solutions of MgCl2 (32.8% RH), NaBr (57.6 % RH), NaCl (75.3 % RH), KBr (81.8% RH) and K2SO4 (97.3% RH). One sensor at a time was calibrated. The sensor was kept inside the container for a period of time until the reading stabilized. Sensors were connected to the CR10 for recording the signals and then the voltage reading of a sensor was recorded. The known relative humidity vs. voltage reading was plotted for each chamber. A slope equation gives an offset values and a multiplier values which were then input into the PC208 program. During the t rials all four RH sensors varied <1% of the range compared to each other. Figure 2-4 is an example of one of the RH sensor calibration plots. Oxygen S ensor Oxygen concentration was measured by galvanic cell type oxygen sensors (MAX 250, Maxtec, Salt lack c ity, UT). The Maxtec MAX -250 senses between 0 and 100% oxygen. Due to the pressure differences that were used in this experiment, the factory calibration data was not valid for the low pressure studies used here. The sensor was calibrated following a met hod used by Wilkerson (2005) and Mu (2005). At the start of the calibration, the chambers were purged twice using nitrogen gas to remove any

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48 residual oxygen. The sensors were calibrated using different mixtures of oxygen and nitrogen. For example, for a 10% oxygen concentration a chamber would be filled to 90 kPa with nitrogen, and an addition 10kPa of oxygen for a total pressure of 100 kPa. This was repeated for other total pressure and oxygen levels and is shown in Figure 25A. The slopes and intercepts w ere then plotted against pressure and regression curves were performed using Excel (Microsoft Seattle, WA 2003). The data from calibration of one of the three oxygen sensor is this is shown in Figure 2-5B. These lines show the slope and intercept as a f unction of pressure over time, and can be applied to the sensor readings to determine the oxygen percent at any given signal and pressure using equation 2-2. Where slope is defined by equation 2-3 and intercept is defined by equation 2-4. O2 [%] = Slope (P)*mvolts + Intercept (P) (2 -2) Slope (P) = 367.15*P1.1475 (2 -3) Intercept (P) = 0.0012*P2 0.1581*P +3.4498 (2 -4) Carbon D ioxide Sensor The CO2 sensors used for these studies were the OEM -6004 module (T -6004, OEM ultrasonic, 6004, Golet a, CA). These cost effective modules had a range of 02000 ppm. Because these sensors are pressure sensitive, they had to be calibrated at different pressures following a method used by Wilkerson (2005). At the start of calibration, chambers were purged t wice using nitrogen gas to get rid of any residual CO2. The sensor was calibrated using different mixtures of nitrogen (ranging 20 to 100 kPa) and carbon dioxide ranging 0.04 to 0. 15 kPa CO2. Since the carbon dioxide portion was so small, it did not affect the total pressure. The CO2 gas was added using known

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49 amount of syringe volume (based on the total volume of chamber). Data at these percentages were collected at different total pressures, ranging from 20 kPa to 100 kPa, and plotted against the pressure in units of millivolts. These data were compared on the basis of pressure and a linear regression of each pressure at different percentages was found. These results are shown in Figure 2 6A. The slopes and intercepts were then plotted against pressure and regression curves were performed using Excel (Microsoft Seattle, WA ). The data from an example calibration of one of the three CO2 sensor are shown in Figure 26B. These lines show the slope and intercept as a function of pressure over time, and can be applied to the sensor readings to determine the carbon dioxide percentage at any given signal and pressure using equation 2-5. Where the slope is defined by equation (2 6 ) and intercept is defined by equation ( 2 -7 ). Later CO2 sensor readings were compared against the known syringe volume of CO2 gas in to chamber (Figure 27) CO2 [%] = Slope (P)*mvolts + Intercept (P) (2 -5) Slope (P) = y = 225.51*P1.2942 (2 -6) Intercept (P) = y = 0.0845*P2 + 16.533*P 928.34 (2 -7) Load Cell The voltage ou tput from the load cells used in these experiments to record the weight of the plants and the flasks did not depend on the total pressure of the environment, thus, the load cells were calibrated at normal pressure following a method used by Wilkerson (2005 ). A total of nine load cells (LPS -0.6 kg, Celtron Tech. Inc. Colvina, CA)), three per each chamber, were used for the experimental chambers. Each sensor was factory calibrated in terms of FSO (Full Scale Output), which is mV/V output. Since the excitation voltage of CR10 was 5 volts, FSO was multiplied by 5. For

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50 example, a load cell with FSO sensitivity of 1.0 mV/V has full scale = 5.1 mv The multiplier was calculated by dividing 600g (full scale weight) with FSO. For this example, it w as 6 00/5.1 = 115.88 grams The offset value is a reading by the sensor when nothing is on the load cell. Load cell readings were tested for accuracy with standard weights (Figure 2-8). Light Sensor Calibration The light sensors (LI 190SA, Li -Cor Inc, Lincoln, NE) were insensitive to variation in temperature, pressure and gas composition. Thus, the factory calibration was used for the light sensor following a method used by Wilkerson (2005). Each sensor had certified values of an offset and multiplier. Since the sensor output is in the millivolt range, the signal was amplified in order to be read correctly. A LI -COR millivolt adapter was used to amplify the mvolts into voltage data. The light readings were compared against the reading by a hand held light meter (LI -250A, Li -Co r Inc, Lincoln, NE) from the same company that had been calibrated at the factory. The plot of the hand held vs. connected sensor is given in Figure 29. Temperature Sensors The temperature sensor ( DS10 Dallas Semiconductors Dallas, TX) is factory calibra ted and does not require further calibration even at very low pressures. The temperature sensors were shielded with aluminum foil as they are sensitive to ambient radiation. Leaf temperature was measured by fine gaugetype -K thermocouples (OS36SM K-140F, Omega; Stamford, CT). The performance of temperature sensor at constant temperature in all three experimental chambers is given in Figure 210.

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51 Summary and Future Improvements Four LPGC s were designed and met the specifications required. The chambers de signed for these studies are suited for studying physiological aspects of plant growth and development in low pressures, including transpiration and the effects of the gas phase on plant growth and development. The LPGSs were able to maintain pressure, O2 concentration, air temperature and relative humidity to given set points while measuring the plant weight and CO2 uptake rates. Vacuum feed through ports successfully prevented air leak in to the chambers. There were a few limitations to this system. For example, nutrient and water supply was not controlled. A c irculating hydroponics system would be a better alternative to the flask system that was used here. In addition, water that was transpired, collected on the walls of the Plexiglas top and at the bo ttom of the metal base for initial studies and this was improved with salt saturated solutions that absorbed excess water Future improvements would include a mechanism for collecting condensate using a cooled coil or other mechanisms. Humidity control was not available for values below 85%. Future improvements would include a humidity control system to study the interacting effects of humidity and low pressure on plant growth. For these studies, the the l ight levels were at 250 mol m2 s1 using cool whi te, florescent lamps to avoid hazards in environmental chambers associated with other lamps However, on the surface of planets the light levels may be very high at or above 400600 mol m2 s1 so improvements to the lighting system may be required. Alth ough the CR 10 data logging system is easy to use, it requires battery power supply which needs to be charged at regular intervals The limited number of ports on the CR10 prevented the addition of more sensors A c entralized data logging system will be mo re effective as it

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52 requires less wiring and is less complex and could readily be adapted to connect to user friendly software for programming such as LabView (National Instruments, Austin, TX) and scale up of the instrumentation would be relatively easy An efficient way to maintain and control concentration of nitrogen, oxygen and carbon dioxide is required and a mass flow controller which automatically controls the gas levels is needed Ethylene can be scrubbed by circulating a chamber air through stainl ess stee l tube containing potassium permanganate (He et al., 2009) Although, in the present study temperature control within a chamber was not required, it will be necessary to have temperature control for self -contained chambers that would be used on the Moon or Mars

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53 Figure 21. L ow Pressure G rowth C hamber (LPGC)

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54 Figure 22. Low Pressure Growth Chambers developed for this objective with gas tanks, datalogger (CR10) and PC interface. CR 10 Vacuum Interface N 2 O 2 CO 2 Light s

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55 Figure 23. Calibration of the pressure sensor comparing pressure gauge reading against millivolts readings. y = 21.924x + 122.43 R2 = 1 0 500 1000 1500 2000 2500 0 20 40 60 80 100 Digital reading (kPa) CR10 reading (mvolts)

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56 Figure 24. Calibration of the relative humidity sensor against known saturated salt solutions. y = 0.0255x + 0.9798 R2 = 0.9974 0 0.5 1 1.5 2 2.5 3 3.5 4 0 20 40 60 80 100 120

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57 Figure 25. Calibrat ion curves of an oxygen sensor at different pressures (5A ) and curve s of slope and intercept (5B). y = 385.52x-1.1503R2 = 0.9973 y = 0.0023x2 0.3554x + 13.051 R2 = 0.9826 -2 0 2 4 6 8 10 12 14 0 20 40 60 80 100 Slope Intercept Total pressure (kPa) Slope, Intercept B y = 0.584x 2.8929 y = 0.3556x 2.0423 y = 0.2471x 1.6221 y = 0.1281x 1.046 0 5 10 15 20 25 30 0 10 20 30 40 50 60 40 kPa 60 kPa 80 kPa 101 kpa Oxygen % CR10 reading (mvolts) A

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58 Figure 26. Calibration curves for a CO2 sensor a t different pressures (A) and curve of slope and intercept (B). CO 2 (ppm) Total pressure (kPa) 0 400 800 1200 1600 2000 0 500 1000 1500 2000 2500 3000 100kPa 80kPa 60kPa 40kPa 20kPa y = 225.51x-1.2942R2 = 0.9982 y = -0.0845x2 + 16.533x 928.34 R2 = 0.9977 -650 -550 -450 -350 -250 -150 -50 50 0 20 40 60 80 100 Slope Intercept Slope, intercept Sensor reading (mvolts) A B

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59 Figure 27. CO2 sensor reading against the equivalent syringe volume 0 1000 2000 3000 4000 5000 6000 0 0.05 0.1 0.15 0.2 33 kPa 66 kPa 101 kPa CO 2 (kPa) CO 2 (ppm)

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60 Figure 28. Calibration for a load cell us ed to measure the weight of the flask containing plants. y = 0.0083x + 0.2995 R2 = 0.9901 0 1 2 3 4 5 6 0 100 200 300 400 500 600 700 Voltage Read Load Cell (volts) Weight (g)

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61 Figure 29. Calibration curve for l ight sensor readings against handheld light meter y = 28.271Ln(x) + 164.76 R2 = 0.9965 0 50 100 150 200 250 300 0 10 20 30 40 50 60 70 80 Light sensor calibration Log. (Light sensor calibration) Handheld unit reading ( mol s 1 m 2 ) Light Sensor Readings

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62 Figure 210. Pressure (~33 kPa), O2 (~20 kPa) and temperature (~23 C) recordings for five hour for chamber A (top panel), chamber B (middle panel) and chamber C (bottom panel). 0 10 20 30 40 50 0 5 10 15 20 25 Pressure Oxygen Temperature 0 10 20 30 40 50 0 5 10 15 20 25 0 10 20 30 40 50 0 1 2 3 4 5 0 5 10 15 20 25 Time (h) Total Pressure (kPa) Oxygen (kPa) Temp erature (C) A B C

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63 CHAPTER 3 MODELING GAS DIFFUSIVITY PHOTOSYNTHESIS AND TRANSPIRATION UNDER HYPOBARIA As an in tegral part of Bioregenerative Life Support System (BLSS) for longterm space missions, plants will likely be grown at reduced pressure. At reduced pressures, the diffusivity of gases increases. This will affect the rates at which CO2 is assimilated and water is transpired through stomata. To understand the effects of reduced pressure on plant growth, the diffusivities of CO2 and H2O at various total pressures (101, 66, 33, 22, 11 kPa) and CO2 concentrati ons (0.04, 0.1 and 0.18 kPa) were calculated. The dif fusivity is inversely proportional to total pressure and shows dramatic increase at pressures below 33 kPa (1/3 atm ). A mathematical relationship based on the principle s of thermodynamics was developed for calculating the transpiration and photosynthesis for plants. Stomatal conductance is sensitive to total pressure. At 33 kPa total pressure, stomatal conductance increases with the boundary increasing by a factor of 1.7, thus the boundary layer thickness conductance increases by 7 0%. Since the leaf conductance is a function of both stomatal conductance and the boundary layer the overall conductance will increase resulting in significantly higher levels of transpiration as the pressure drops The conductance of gases is also regulated by stomatal aperture in an inverse relationship. Stomatal aperture is directly influenced by concentration of CO2 inside the leaf space. The higher CO2 concentration inside the leaf air space during low pressure treatments may result in stomata closing partially or fully which may reduce the excessive transpiration caused by increased diffusivity. Therefore, a reduced pressure environment with high CO2 may be an ideal scenario for minimizing transpiration and maximizing the plant biomass yield in BLSS.

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64 Plants in Hypobaria Longterm missions to Moon and Mars will require a well -studied and predictable Advanced Life Support System (ALS) for space explorers. Among other components such as like waste treatment and energy production, bio-regeneration (BLSS) through the use of plants has been identified as an integral part of ALS. Plants will be utilized for CO2 removal from the air that is respired from explorers, for oxygen regeneration and water purification systems, and as a source of food for human use (NASA, 2003). There is no a tmospheric pressure on the Moon with very little of an atmosphere on Mars (less than 1 kPa total pressure). Chambers used to grow plants will likely be maintained at reduced total pressure (i.e., hypobaria). Operating the chamber at reduced pressure will m itigate the negative effects caused by large pressure differences between the inside of the chamber and the vacuum/low pressure environments of the Moon or Mars (Bucklin et al., 2004) The reduced pressure will also reduce consumable and structural components of ALS, thus launch cost. Another advantage of lower pressure would be reduced leakage of valuable gases requiring less total gas to pressurize the chamber. Richards et al. (2006) and Paul and Ferl (2006) reviewed the advantages of low pressure environ ments for plant growth on space missions. One of th e fundamental differences in hypobaria com pared to normal atmosphere is the diffusivity of gases (Rygalov et al., 2004), which may influence rates of water vapor loss and carbon dioxide (CO2) assimilation by plants. The effects of diffusivity on the plant transpiration at normal atmosphere have been studied (Mott and Parkhurst, 1991) but there are very few studies (Gale, 1972; Goto et al., 1996) on the effects of diffusivity of CO2 and water on plant growth at hypobaria. This article reviews the physical relationships that

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65 influence the diffusivity of H2O and CO2 in hypobaria, and the consequent implications for plant growth. Binary Diffusion of Gases The general relationships defining gaseous diffusion in physical, chemical and biological systems have been developed, although not applied for understanding gas exchange by plants in hypobaria. Gas flux density, according to Ficks law is directly proportional to the driving force, i.e. partial pressure gradient, and the conductance of the gas in the gas mixture. Gas conductance is dependent on the mass diffusivity of the binary gas system ( DAB) as a function of temperature, pressure and the gas composition. In a binary gas system, DAB is proportional to tempera ture and inversely proportional to pressure. It is the sensitivity of DAB to pressure that is of special interest in growing plants in hypobaria chambers. The earliest attempts to derive the mathematical relationship to calculate binary gas diffusivity at reduced pressure used a StephanMaxwell hard-sphere model (Hirshfelder et al., 1948; Bird et al., 1954). They presented the mathematical expression of binary gas diffusion based on the kinetic theory describing constant motion of atoms, ions and molecules at temperature above absolute zero. The diffusion coefficient of the gases at reduced pressure can be estimated from the intermolecular forces of the fluxes (Slattery and Bird, 1958). To improve upon the calculations based on theoretical properties and ge t more accurate gas phase coefficient Bird et al. (1960) simplified the derivation of Slattery and Bird (1958) by using the Chapman-Enskog model (Equation 4 1; Chapman and Cowling, 1952), which defines the binary diffusion coefficient of gas A in gas B ( DAB, cm2 s1) in terms of potential energy of interaction between a pair of molecules in the gas. They presented the theoretical diffusion over the range of pressures from 10 to 100 kPa and

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66 compared it with experimental data. Though, the predicted values w ere within 5% of the calculated values, it required considerable calculations. 2 / 1 21 1 8 32 3 B A AB ABm m kT n D (3 -1 ) where n = total concentration of both species, mol cm3 T = temperature, K k = the Boltzmann constant, ergs/K mA, mB = molecular mass, g m ol1 AB = collision diameter, separation between molecular centers of unlike pairs upon collision, cm In deriving Equation 31, the following assumptions were made, The gases are non reactive. The gases as a whole are assumed to be at rest but the molecular motion is taken into account. All molecules have velocities, representing the region of last collision. Temperature profile of gas corresponds to the Fouriers law of heat conductance. Thermal conductivity for polyatomic gas es is calculated at low density. Equation 3 1 has two main limitations because it is based on the hard-sphere model (Fuller et al., 1966). There is a a narrow temperature r Arnolds (1930) original suggestions to overcome these limitations were implemented by Fuller et al. (1966) by, Replacing the temperature dependence with Sutherland temperature corrections to improve the tem perature dependence. -Bas atom ic volume parameter which is an easily measured property of a diffusing substance.

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67 An empirical analysis done by Fuller et al. (1966) described DAB by the following general equation. 3 2 1 2 / 11 1 B A B A b ABV V p m m T c D (3 -2 ) w here c = an empirical constant p = pressure Pa b = empirical temperature power dependence Vi = special diffusion parameters to be summed over atoms of diffusing species at normal boiling point, m3 kmol1 here, Vair =20 .1, VH2O = 12.7, VCO2 = 26.7 123 = empirical exponents to the diffusion volumes. Fuller et al. (1966) used results from about 300 experimental values to obtain the empirical coefficients for various gas mixtures by using a non -linear least square analysis. Optimization to obtain the smallest standard deviation resulted in the following empirical relationship. 2 3 / 1 3 / 1 2 / 1 75 1 31 1 10B A B A ABV V p m m T D (3 -3) The Effects of Background Gases on Overall Diffusivity Generally air is composed of multi gas components and can be considered one sing le gas if the concentration of individual gases does not change. However, in a system where total pressure and the partial pressures of the individual gases are varying, considering air as a single gas may not give correct value s of overall diffusivity of CO2 and H2O. In complex biological systems such as alveolar regions of lungs, closed plant/animal growth chamber s fermentation vessels and plant leaf stom ata, the air cannot be represented by binary gas constituents (Johnson, 1999). The partial

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68 pressure o f the background gases directly affects the overall diffusivity of a gas. For example, diffusivity of oxygen in a mixture of nitrogen will not be the same as in a mixture of helium, even though the total pressure and pO2 remains the same (Mott and Parkhurs t, 1991). Higher values of mass diffusivity mean larger amounts of mass transfer. Fuller et al. (1966) proposed a method to calculate the individual constituent mass diffusivity (or overall diffusivity of particular gas) in a multi-component gas at different mole fractions. The calculation of mass diffusivity of a constituent gas (for e.g. CO2 in air composed of N2, CO2, O2 and H2O) requires first the calculation of binary diffusivity of that gas (CO2) i n all gas components (Equation 3-3). 1 1 A N j AB jD D (3 -4) w here, DA = mass diffusivity of a constituent A in multi gas (m2 s1). j = the mole fraction of gaseous component DAB = Binary mass diffusivity of constituent A in a gas B (m2 s1). Eq. 5 can be applied to our case to define DCO 2 in a chamber composed of CO2, O2, N2 and H2O. 12 2 2 2 2 2 2 2 2 2 2 2 2 N CO N O CO O O H CO O H COD D D D DCO CO CO (3 -5 ) Diffusivity of CO2 and H2O in Hypobaria The earlier studies on various aspects of plant growth under reduced pressure have suggested that plant growth is possible down to 10 kPa (Musgrave et al., 1988). Also, high partial pressure s of CO2 up to 200 Pa can be beneficial to plant growth. Therefore, for clarity, the diffusivity of H2O and CO2 in binary gas system s was calculated at various total pressures and pCO2 using equati on (E quation 3 4). The

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69 pH2O and temperature were held constant for all calculations at 2.64 kPa and 22 C, respectively. As shown in Figure 3 1, the diffusivity of both H2O and CO2 is highly sensitive to pressure. Diffusivity of the gases increases as pressure decreases, reflecting the diminished interaction among gas molecules. The increase in diffusivity is not linear but shows a dramatic increase at low pressures of below 20 kPa. Increasing the CO2 partial pressure in air does not significantly ch ange the diffusivity from physiological levels. For example, at 33 kPa total pressure, when CO2 is increased from 40 to 180 Pa, the mole fraction increases from 0.001 to 0.005 (mol kmol1), which is much less when compared to the sum of mole fraction of remaining gases (pO2 at 21 kPa = 0.606 mol kmol1 and pN2 at 12 kPa = 0.313 mol kmol1). The ratio of the diffusivity of H2O to CO2 is interesting because of the impact of the exchange of gas by plants for these two gases. The diffusivity of H2O under 101 kPa total pressure is generally about 1.6 times higher than CO2 due to two main reasons (i) t he difference in the molecular weight of water (18 g mol1) and CO2 (44 g mol1) and (ii) the atomic volume of water (12.7 g mol1) and CO2 (26.7 g mol1). Since these two factors are not sensitive to pressure, the ratio of diffusivity of the two gases is virtually ins ensitive to pressure (inset Figure 3 1). At standard temperature and pressure, the ratio of gaseous phase resistance of water and CO2 in air is 1.7 ( Bieurhuizen and Slayer, 1964a). The calculation of the ratio based on assumptions by Gale et al. (1972) also agrees on value of the diffusive resistance around of 1.7 though their assumptions for the diffusive resistance ratio equation did not include the low pressure.

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70 Gas Diffusion in Leaves The growth and health of plants is dependent on their ability to transpire H2O and assimilate CO2. Both of these gas exchange processes by leaves is dependent on the sensitivity of gas diffusivity. This can be shown by examining the basic equations defining leaf gas flux density. The fundamental description of mass flux density for a leaf is the Onsager expression for coupled nonequilibrium flow as a result of water vapor and temperature gradients (Katchalsky and Cur ran, 1967). T L L JT v v v (3 -6 ) w here, J = Vapor flux density (mol cm2 s1) Lv= Onsager coefficient of vapor LVT = Onsager vapor temp coupling coefficient v = vapor chemical potential difference across the diffusion path. (J mol1) = Temperature difference across the diffusion path (K) Since in most plant systems there is a small temperature gradient and a comparatively small coupling coefficient, Equation 3 6 reduces to the familiar simplified expression of flux density of individual gas species under steady state conditions. In Equation 3 7 the Onsager coefficient is expressly defined by vm J (3 -7 ) w here m = mobility co efficient, cm 2 mol J1 s1 v = vapor density, mol cm 3 = chemical potential of vapor, J mol1 x = distance, cm For the short distances of vapor flux between leaves and the bulk atmosphere, the chemical potential is defined by the vapor pressure gradient (J cm3). When the atm osphere adjacent to the liquid surface is saturated with vapor ( Pv*) v vP P x m J (3 -8 )

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71 The mobility coefficient can be explicitly defined as a function of gas diffusivity. a aP D m (3 -9 ) w here, Pa = Pre ssure of air, J cm3 a = density of air, mol cm3 On the molar basis, the evaporation rate can be written simply as a v a aP P P x D E (3 10) The D/ 1) for vapor flux from a surface at saturated vapor pressure. Leaf T ranspiration Transpiration is more complicated than simple diffusion because the surface for evaporation is buried inside a leaf at the cell walls. Transpiration requires the diffusion along the pathway from the cell walls through stomatal pores and then through the leaf boundary layer. The conductance limitation to vapor in this pathway from inside leaves is predominantly stomatal (Rand, 1977) at about 90 % or more of the limitation (Noble, 1990). Therefore, the conductance for transpiration must account for gas conduc tance through stomata pores. The stomatal conductance ( hs, cm s1) is directly dependent on gas diffusivity as well as pore aperture and pore depth (P arlange and Wagoner, 1970). Equation 411 describes this relationship. b a b d n D b a hs4 ln 2 (3 11) w here a, b = length of major and minor axis of pore aperture, respectively, cm d = pore depth, cm D = diffusivity of water vapor (cm2 s1) n = stomata density (number cm2)

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72 The value b (pore width) is dependent on conditions in the leaf and commonly varies from 0 to 4*104 cm (Sinclair, 1980). Environmental factors such as light, CO2 concentration and vapor pressure deficit can have large influences on pore width. Eq uation 3 11 also clearly demonstrates that stomata conductance is dependen t on gas diffusivity. As shown previously, the value of D is a function of a total pressure, and hence total pressure will have direct influence on stomatal conductance. Under low pressure situations Figure 3 1 shows that D is increased, which according to Eq uation 3 -11 means stomata conductance is increased. For example, for plant s grown at one third of the standard earth atmospheric pressure stomatal conductance will increase by the ratio of mass diffusivity at 0 .3 a tm to that of at 1 atm (Figure 31) thus increas ing in stomatal conductance by approximately a factor of 3. Thus, transpiration rate and CO2 assimilation rates would be substantially increased under hypobaria conditions. The conductance of the boundary layer surrounding the leaf is also dependent on gas diffusivity, although in a complex manner because of the convective movement of air. The theoretical considerations used for the flat plate evaporative loss through boundary layer can be applied for the plant leaf (Sinclair, 1980). The effec ts of boundary layer on a flat plate of an infinite length (Gebhart, 1961) can be estimated by the following equation. 3 / 1 2 / 1Re 664 0 Sc L K hbl (3 12) w here, hb l = vapor boundary layer conductance, cm s1 K = fluid thermal diffusivity (k/ 0.215 cm2 s1 at 20 C Where, k = thermal conductivity (W.m1.K1), Cp = specific heat (J kg1 K1) = the fluid density (kg m3) Re = Reynolds number Sc = Sc h mi dt number

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73 The fluid thermal diffusivity is a function of 1/ whereas the thermal conductivity and specific heat will remain almost constant for constant temperature and low pressure (p < 2 atm; Salazar, 2003) For example, at 33 kPa the K will be 3 times the value of K at 101 kPa. Re = Reynolds number ( uL/Kv) w here L = length of flat surface, cm u = air speed, cm s1 Kv = kinematic viscosity at 0.15 cm2 s1 at 20 C. The Re is inversely of related to kinematic viscosity ( Thus the Reynolds number is significantly affected by pressure. T he dynamic viscosity does not significantly change with pressure. For example at 33 kPa, the Re will decrease by a factor of 3 compared to normal atmospheric pressure since the density of gases is 1/3 of the normal pressure. Sc = Schmidt number ( ) where = fluid viscosity (N s m2) D = mass diffusivity, m2 s1 The Sc is a function of fluid density and diffusivity. At 33 kP a, the diffusivity of gases increase s by three times co mpared to normal atmospheric pressure but this value is offset by the decrease in the density of air The value of Sc at 101 kPa as well as at 33 kPa (20 C) equals approximately 0.63 for water vapor Overall, at 33 kPa the value of hbl will be increased b y a factor of 1.7 compared to normal atmospheric pressure (101kPa). Combining the constants at 20 C, the boundary layer conductance (hbl) for both sides of leaf in a lamina r flow can be predicted from Equation 313. Thus, the diffusion coefficient also i nfluences the boundary layer conductance through the Schmidt

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74 number. However, the fact that the Schmidt number is taken to the cube root and the boundary layer conductance offers little limitation to gas flux density, the following simplified equation is generally appropriate (Sinclair 1980). L u hbl63 0 (3 13) where L is the length (cm) of the leaf u is the wind velocity (cm2 s1). At 33 kPa, the co efficient value will be 1.096, which is 1.74 times the co efficient for 101 kPa. Th e combined conductance ( s tomatal and boundary layer) can be designated as h (cm s1). bl s bl sh h h h h (3 -1 4 ) This equation suggests that the combined conductance will increase as pressure decreases. For example for h at 33 kPa, the values would be 3 and about 1.7 times the normal atmospheric pressure values, for hs and hbl, respectively This is assuming similar stomatal open geometries and air velocities between 33kPa and 101kPa. Inserting the stomatal and boundary layer conductance into an equ a tion i n the general form of Equation 3-15, the transpiration ( T units) can be defined by the following equation (Sinclair, 1980). (3 15) where, PL = saturated vapor pressure at lea f temperature, Pa Pv = vapor pressure outside the leaf air space, Pa It should be noted that as transpiration rates increase in lower pressure the surface leaf temperature will drop due to latent heat of vaporization. This drop in ) ( Pv P h TL

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75 temperature will result in a lower vapor pressure deficit in low pressure compared to higher pressures. This would decrease transpiration rate below the initial estimate. Leaf P hotosynthesis Photosynthesis is a function of the partial pressure carbon dioxide gradient but the di rection of diffusion is opposite of that of transpiration. Similar to T photosynthesis is a function of the CO2 partial pressure (Pa) difference between the atmosphere and the inside of the leaf and t otal gas conductance (Equation 316 ; Sinclair 1980). ) ( 442 2 i CO a COP P h A (3 16) A is the photosynthetic rates and 44 is the molecular weight of CO2. The PCO2a and PCO2i are the leaf outer and internal leaf partial pressure s (Pa) of CO2, respectively. The h is the total conductance for CO2 whi ch was described for transpiration and would increase as pressure drops Stomatal conductance is a function of the stomata aperture, which in turn is dependent on the diffusion coefficient of CO2 (Montieth and Unsworth, 1990). Because the mass diffusivity is a function of a pressure and the molecular weight, the effects of reduced pressures should be reflected in its effects on plant growth (i.e., the CO2 assimilation and the transpiration rates). At reduced pressure the diffusion limitation to CO2 and water transfer through stomata should be reduced proportionately (Mott and Parkhurst, 1991). Thus, resulting in enhanced photosynthesis and reduced transpiration. Conclusion There is a strong correlation between the total pressure and the diffusivity of CO2 a nd H2O (Figure 3 -1). At reduced pressure, the diffusivity of individual gases increases. The increased diffusion should result in increased number of CO2 molecules transferred

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76 through stomata thus increasing the plant biomass. Higher CO2 levels in conjunct ion with reduced pressure can enhance the plant growth. The CO2 assimilation via photosynthesis (A ) is also subject to the stomatal regulation which implies that there is a threshold limit of CO2 beyond which stomatal aperture will tend to close preventing further A and it is expected that this value be lower at lower pressures compared to higher pressures. Similarly, due to increased diffusion more water molecules should move out of the stomatal pore as less resistance for water in liquid phase to reach th e equilibrated gas phase water. Thus, transpiration rates will also increase. H igh CO2 partial pressure combined with reduced pressure can decrease transpiration through stomatal clos ure. Further studies in low pressures with various gas compositions is re quired to identify the threshold level of each gas to enhance the plant growth and reduce transpiration rates without adverse effects. This analysis of the sensitivity of the diffusivity of CO2 and H2O to pressure shows that engineers designing ALS system s have two variables to consider. Transpiration and photosynthesis are both sensitive to the partial pressure of each gas in the bulk atmosphere around the plants and the total pressure of the atmosphere. Due to the high sensitivity of the diffusion coef ficients at hypobaria pressure, and hence gas flux, plant growth and water loss can be influenced by the pressure of the growth chamber. If water flux, for example, is a critical aspect of a water purification system, transpiration rate of the plants can be increased or decreased by simply adjusting the plant chamber pressure. Similarly, plant growth will be responsive to the pressure changes as enhanced CO2 assimilation is predicted.

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77 Figure 31. The individual mass diff usivity of CO2 and H20 calculated from the binary mass diffusivity using the empirical formula given by Fuller et al. (1966). Inset: The ratio of diffusivity of H2O over CO2. 0.0 0.5 1.0 1.5 2.0 2.5 3.0 0 20 40 60 80 100 D D H2O CO2 0 0.5 1 1.5 2 0 30 60 90 Total pressure (kPa) Diffusivity (cm 2 s 1 ) Total pressure (kPa) DH 2 O/ DCO 2

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78 CHAPTER 4 THE INTERACTING EFFECTS OF CO2 AND HYPOBARIA ON GROWTH AND TRANSPIRATION OF RADISH ( RAPHANUS SATIVUS ) Plants grown on longterm space missions will likely be grown in low pressure environments (i.e., hypobaria). However, transpiration rates can be elevated in low pressure resulting in plant wilting or stress. It is poss ible to reduce transpiration by increasing the partial pressure of CO2 (pCO2), but the effects of altering CO2 to super elevated levels on plant growth and transpiration in hypobaria are not known. Here, the interacting effects of pCO2 and total atmospheri c pressure on the growth and transpiration of radish ( Raphanus sativus var. Cherry Bomb II) were studied. This material also appears in Gohil et al., (2010). The fresh weight (FW), leaf area, dry weight (DW), CO2 assimilation rates (CA), dark respiration r ates (DR), and transpiration rates from 26 day old radish plants that were grown for an additional seven days at different total pressures (33, 66 or 101 kPa) and pCO2 (40 Pa, 100 Pa and 180 Pa) were measured. In general, the dry weight of plants was enhanced with CO2 enrichment and with decreased total pressure. In limited pCO2 (40 Pa), the transpiration for plants grown at 33 kPa was over twice that of controls (101 kPa total pressure with 40 Pa pCO2). Increasing the pCO2 from 40 Pa to either 100 or 180 P a reduced the transpirations rates for plants grown in hypobaria and at normal atmospheric pressures. However, plants grown at lower total pressures (33 and 66 kPa total pressure) and super elevated pCO2 (180 Pa) had evidence of leaf damage. Radish growth can be enhanced and transpiration reduced in hypobaria by enriching the gas phase with CO2 although at high levels of CO2 leaf damage can occur.

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79 Introduction Plants will be an integral part of an Advanced Life Support system for longterm, human space exploration. Plants will be utilized for CO2 removal from the air that is respired from explorers, they will act as oxygen regeneration and water purification systems, and they will be a source of food and fiber for human use (NASA, 2002). On planets with lit tle to no atmospheres, the chambers that will be used to grow plants will likely be maintained at reduced total pressure (i.e., hypobaria). The low pressure inside the chamber will mitigate the negative effects caused by large pressure differences between the inside of the chamber and the vacuum/low pressure environments of the planet or moon (Bucklin et al., 2004). Maintaining the chambers at low pressure will also reduce costs associated with transporting heavy materials used to build chambers that could withstand large pressure differences and the lower pressure would reduce leakage of valuable gases and require less total gas to pressurize the chamber. Since transport costs associated with supplies are expected to be one of the major limitations in longterm space missions, any mechanisms to reduce these costs should be explored. For review on the advantages of low pressure environments for plant growth on space missions see Richards et al., ( 2006) and Paul and Ferl (2006). On Earth, many species of plants grow at high altitudes with low total pressure ( 55 kPa). This suggests that plants can adapt to hypobaria. Since pressures for growing plants on the Moon or Mars will be even lower (i.e., 33 kPa, Paul and Ferl 2006) than that found on Earth, studies on the effects of these lower pressures on plant growth have been performed. The results of these studies have shown that seed germination (Musgrave et al., 1988), plant growth and development (Iwabuchi and

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80 Kurata 1996; Goto et al., 2002; Spanarkel and Drew, 2002; Richards et al., 2006; He et al., 2007), and fruit ripening (Burg and Burg, 1966) are possible in hypobaric conditions for a broad range of species with species surviving in pressures as low as 10 kPa provided there is enough pO2, pCO2, and pH2O ( Massimino and Andre, 1999; Goto et al., 2002). Although it is well known that plants can grow and develop in hypobaria, the effects of low pressure on the photosynthesis, biomass accumulation, and transpiration rates of plants are unclear. For example, som e studies report increased biomass, photosynthesis or transpiration in hypobaria (Rule and Staby, 1981; Andre and Massimino, 1992; Daunicht and Brinkjans, 1996; Corey et al., 1997; Iwabuchi and Kurata, 2003) while others report reduced growth or no change in either plant biomass, photosynthesis or transpiration in hypobaric conditions (Goto et al., 1995; 1996; Iwabuchi et al., 1996; Spanarkel and Drew, 2002; Iwabuchi and Kurata, 2003; Richards et al., 2006; He et al., 2007). The differences in the experimental procedures between these studies make it hard to interpret the effects of hypobaria on plant growth and development. For example, the plant species, pCO2, pO2, pH2O, and the duration of the experiments all varied between experiments and likely account for the differences in plant responses to hypobaria. In order to develop decision support tools for growing plants on the Moon or Mars, further studies on the effects of low pressure environment on plant growth responses are required. As total atmospheric pressure decreases, the diffusivity of water vapor and gases increase (Gale, 1971). The enhanced transport of water from the leaf surface and CO2 toward the plant from the surrounding gas phase may account for the enhanced evapotranspiration/transpiration and photosynthesis that was found in some studies for

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81 plants grown in hypobaria (Iwabuchi et al., 1996; Goto et al., 1996; Daunicht and Brinkjans, 1992, 1996; Corey et al., 1996, 2002; Wilkerson, 2005). Enhanced transpiration in hypobaria can have signifi cant negative effects on plants if the water is not readily replaced. For radish, the increased evapotranspiration in hypobaria resulted in severe wilting of radish within 30 minutes of transfer from high (101k Pa) to lower pressure (25k Pa, Wilkerson, 2005). This water stress in hypobaria appears to cause the differential expression of drought -related genes (Paul et al., 2004). These changes in gene expression may trigger downstream responses in plants to induce stomata closure to reduce water loss. For ex ample, stomata apertures from spinach grown in hypobaria long -term (10 days) were smaller with similar transpiration rates as plants grown at normal atmospheric pressure (Iwabuchi and Kurata, 2003). Increasing the pCO2 can also reduce stomata aperture and thus reduce transpiration rates even in hypobaria. For example, a twenty percent decrease in transpiration rates for tomato grown in hypobaria was reported when CO2 was increased from 40 to 100 Pa (Daunicht and Brinkjans, 1996). The CO2 levels for Advanced Life Support systems may be at super elevated levels ( 0.6 kPa or higher, Wheeler et al., 1999) and CO2 may be used as a pressurizing gas for growing plants on Mars which would result in very high pCO2 (Wheeler, 2001). Unfortunately, the interacting effec ts of high levels of pCO2 and pressure on plant growth, transpiration and photosynthesis are unclear. Therefore, the objectives of the present study were to study the interacting effects of CO2 and pressure on growth ( dr y weight, DW, and fresh weight, FW), CO2 assimilation (CA), and transpiration rates of radish. These studies provide insight into the detrimental effects of

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82 high levels of CO2 on plant development at low pressures and identify methods for reducing transpiration and improving growth in hypobaria with CO2 enrichment. Materials and Methods Plant Material Seeds of radish (Raphanus sativus L. cv. Cherry Bomb) were germinated and grown in pots containing soil ( MetroMix 300, Sun Grow Horticulture, Bellevue, WA) with nutrient solution of 1/6 stren gth MS (Murashige and Skoog, 1962) medium applied three-times a week. Plants were grown for 20 days at 22C with a 12h photoperiod with lighting at 150 mol m2 s1 from cool white -florescent lambs in environmentally controlled chambers at 85% relative hum idity. After growth in pots, plants were rinsed in ddH2O and transferred to 250 mL flasks containing ~300 mL of 1/6 strength MS medium, pH adjusted to 6.8 with HCl (1 mM) or NaOH (1 M). The volume of flask was large enough for the short -term studies that t he level of nutrient solution at the end of the experiment was well above the majority of roots. The roots were well -developed and reached far into the flask at this growth stage. Plants were placed in the holes of the stoppers and surrounded by putty (Plu mbers Putty, P/N 43048, Ace Hardware, IL) to minimize water loss during experiments. Plants were grown in flasks under similar conditions of pots for 4 to 5 days before being transferred to experimental growth chambers for acclimation. The fresh weight (FW ) of plants was measured on the acclimation date at 4.0 0.9 g. The nutrient level was then filled to ~300 mL prior to acclimation. Plants were then acclimated in low -pressure chambers at normal atmospheric pressure (101 kPa with 40, 100 or 180 Pa pCO2 an d 21 kPa pO2 and 2.2 kPa pH2O) for one day prior to experimental treatments. Plants were grown for an

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83 additional seven days under pressure and gas treatments then removed for plant harvest. Growth Chambers and Environmental Control The experiments were pe rfo rmed in three chambers (Figure 4-1) of total volume of 0.09 m3 with a fan (Delta Model, P/N BFB0512M, Silicon Valley Compucycle, San Jose, CA). The chambers had sensors for temperature (DS10 Dallas Semiconductors, Dallas, TX), pressure (P/N ASCX15AN, Sensym ICT, Milpitas, CA), CO2 (OEM ultrasonic, 6004, Goleta, CA), O2 (MAX 250, Maxtec Co., Salt Lake, UT), relative humidity (HH 4602L -CPREF, Honeywell, Morristown, NJ), light (LI -190, Li -Cor, Lincoln, NB) and three balances for weight determination per ch amber (LPS 0.6 kg, Celtron Tech. Inc. Colvina, CA). Calibration of O2 and CO2 sensors was performed according to Richards et al., (2006). CO2 and O2 levels were adjusted daily at 4 hours into the photoperiod. CO2 assimilation studies were started at the same time in the photoperiod for all tests and were repeated daily. After the acclimation period, the gas and pressure composition were brou ght to set points daily (Table 4-1). Environmental conditions for the one day acclimation in the growth chamber were 2 2 2 C, 12 hour photoperiod, at 250 mol m2 s1 using cool white, florescent lamps and 85% relative humidity. The pO2 was maintained at 21 kPa with other environment al conditions described (Table 4-1). Each chamber contained three individual plants. P ressure was maintained with a vacuum pump (1/2 HP, JB Industries, Chicago, IL) and chambers had leakage rates of ~0.03% (chamber volume/h). Data collection and controls were performed using a CR10 datalogger (Campbell Scientific, Logan, UT) that was connec ted to a PC. Experiments were performed in triplicate.

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84 Plant Harvest After the experimental treatments in the chambers, plants were removed from the chamber blotted dry and analyzed for fresh weight, leaf area, and dry weight. Dry weight analysis was perf ormed by placing plants on pre weighed pans at 60C for 48h and reweighing the contents. Leaf area was measured on day 7 with digital images using Image Pro Plus software (ver. 6.0 Media Cybernetics, Bethesda, MD). Statistical Analysis For CO2 and pressure treatments, the analysis was treated as a two-factor analysis of variance (ANOVA) using SAS (SAS institute NC, USA) and the mean of three individual plants of independent experiments (here, n=3). Results Plant Growth Plants grown with 40 or 100 Pa pCO2 appeared healthy at all total pressure treatments (33, 66, or 101 kPa). However, at 180 Pa pCO2, leaves from plants grown at 33 and 66 kPa pressures were yellow, had red speckles and leaf tip burn, and appeared to have reduced chlorophyll content. Thi s leaf damage was not observed for plants grown at 180 Pa pCO2 and 101 kPa pressure (Figure 4 -2). Despite this apparent leaf damage, plant dry weight was the greatest in the 180 Pa pCO2 treatments for all pressu res (33, 66, or 101 kPa, Table 4-2). The pr essure and pCO2 both significantly affected the dry weight (p < 0.01) but the interaction between these two parameters was not significant (p > 0.05). The dry weight of plants was maximal at 66 kPa total pressure and 180 Pa pCO2 with 0.65 g per plant and lowest at 101 kPa total pressure and 40 Pa pCO2 at 0.42 g per plant (Table 4-2). For all pressure treatments, increases in pCO2 resulted in increases in dry weight (Table 4-2). Radish fresh weight and % water content

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85 were not significantly affected by tot al pressure, pCO2, or the interaction bet ween the two treatments (Table 42 ). Leaf area decreased as pCO2 levels increased for plants grown at 33 kPa total pressures (Figure 43). This reduction in leaf area with increasing pCO2 levels was also observed fo r plants grown at 66kPa and 101 kPa pressures but t o a much lesser extent (Figure 43). The reduction in leaf area may have been in part due to the senescence of the hypocotyl leaves since this was accelerated in higher pCO2. Plants grown in low pCO2 (40 P a) and low pressure (33 kPa) had the largest leaf area ( 240 cm2 per plant) compared to other pCO2 a nd pressure treatments (Figure 4-3). CO2 Assimilation The CO2 assimilation rates (CA) for plants remained approximately constant from day 1 to day 7 for 40 and 100 Pa pCO2 treatments for all pressures however, at 180 Pa pCO2, the CA decreased with each passing day (data not shown). This reduction in CA was greater at 33 and 66 kPa than for plants grown at 101 kPa. The pressure and the pCO2 treatments as well as their interaction significantly affected CA with the greatest CA at 100 Pa pCO2 and 66 kPa at 12.42 mol m2 s1 and the lowest at 180 Pa pCO2 and 101 kPa at 3.44 mol m2 s1(Table 4-2). The CO2 uptake rates at low pCO2 (40 Pa) and 33 kPa total pres sure was greater than the uptake rates from plants grown at similar pCO2 levels with 66 kPa or 101 kPa total pressure (Figure 4-4A). This corresponded to a greater leaf area in the low pCO2, low pressure treatments compared to plants from other pressure an d CO2 combinations (Figure 4-3). At 100 Pa pCO2, plants grown at 66 kPa had the greatest CO2 uptake (Figure 4-4B) which corresponded to the maximal CA (based on leaf area) and the greatest dry weight of all experimental treatments (Table 42). For plants grown at 101 kPa, the CA (based on leaf area) was

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86 similar for all CO2 treatments (Table 4 2) despite having an increased slope in CO2 uptake as pCO2 levels increased (Figure 4-4). At super elevated levels of CO2 (180 Pa), the CO2 uptake rate was the lowest for plants grown at 33 kPa total pressure compared to plants from other treatment combinations (Figure 4 -4C). Dark respiration rates (DR) were significantly (p < 0.01) affected by total pressure but not by the CO2 or inter actions between the two (Table 4-2). In general, as total pressure was reduced from 101 kPa to 33 kPa, the DR increased for plants grown at all pCO2 levels. The exception to this was at 100 Pa pCO2 and 66 kPa total pressures where the DR was maximal at 1.91 mol m2 s1. This corresponde d with plants with the greatest dry weight (Table 4-2). In contrast, the lowest DR was found with plants grown at 180 Pa pCO2 and 101 kPa total pressure at 0.6 mol m2 s1 that corresponded to the lowest CA (Table 4 -2). Transpiration Rates Plants grown at reduced pressures (33 or 66 kPa) had greater transpiration rates than plants grown at 101 kPa, regardless of the CO2 treatment (Table 42). In fact, plants grown at 33 kPa total and 40 Pa pCO2 had over twice the transpiration rate ( 580 mL m2 day1) as plants grown at 101 kPa total and 40 Pa pCO2 ( 210 mL m2 day1, Table 4 2). Transpiration rates were reduced by increasing pCO2 from 40 to 100 Pa for all pressure treatments but a further increase in pCO2 to 180 Pa only reduced transpiration rates for pl ants grown at 101 kPa (Table 42, Figure 4-5). The cumulative water loss per plant was greatest in low pCO2 (40 Pa) and low pressure (33 kPa) with 50 mL of water lost per plant over the seven days in the chambers (1 day acclimation, 7 day treatment) compared to plants grown at 101 kPa total pressure with 180 Pa pCO2

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87 which lost the least amount of water for any treatments over the experiment at 12 mL per plant (Figure 4 5). Transpiration was minimal at night as indicated by the plateaus on the curves of cumulative water loss for all pressure and pCO2 treatments (Figure 4 5). Discussion Radish plants appeared healthy with no signs of water stress or leaf damage for most of the pressure and pCO2 combinations that were used in these experiments. The exception to this was for plants grown at lower pressures (66 or 33 kPa) and super elevated pCO2 (180 Pa, Figure 4 2). These plants had yellow leaves with red speckles, and leaf tip damage. Previous studies with radish in hypobaria did not report any leaf damage a t similar pressures (33, 66, and 96 kPa) even after 21 days in treatments with 120 Pa pCO2 (Levine et al., 2008). The differences in leaf appearance between this and the previous study may be that the 180 Pa pCO2 used here was at a high enough concentration to induce leaf damage at low pressure. Low pressures enhance diffusion rates of CO2 (Gale, 1971) therefore, the detrimental effects of elevated CO2 may occur at lower concentrations in hypobaria. Since plants appeared healthy at 101 kPa and 180 Pa pCO2, it suggests that this level of pCO2 is not detrimental to growth at normal atmospheric pressures. Previous studies at super elevated levels of pCO2 (500 and 1000 Pa) reported leaf bleaching in potatoes, wheat and soybeans at normal atmospheric pressure (Ti sserat et al., 1997; Wheeler et al., 1999). In addition, high pCO2 can cause accelerated leaf senescence (Usuda and Shimogawara, 1998). It is also possible that ethylene buildup in the system could have caused the leaf damage in our studies since ethylene was not scrubbed from the gas phase. Ethylene, which can

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88 accumulate in closed systems with plants, can reduce chlorophyll levels in leaves and promote leaf senescence (He et al., 2009). The much larger chambers that were used in previous studies with radis h in hypobaria (Levine et al., 2008) may have prevented high concentrations of ethylene from accumulating near the plant thus preventing leaf damage. Further studies with the removal of ethylene from the chambers may clarify the roles of ethylene in inducing leaf damage at super elevated pCO2 in hypobaria. Despite the leaf damage in hypobaria with super elevated pCO2, plants from these treatments had enhanced dry weights compared to plants grown at lower pCO2 levels (40 or 100 Pa pCO2, Table 4-2). Super el evated pCO2 can greatly enhance starch accumulation in leaves (Levine et al., 2009) which may account for the increased dry weights that were observed. In general, CO2 enrichment (100 or 180 Pa) increased the dry weight of plants for all pressure treatment s compared to plants grown at limiting pCO2 (40 Pa, Table 4 2). Many experiments have demonstrated that CO2 enrichment at normal pressures causes significant increases in photosynthetic uptake of CO2 and increases in biomass for a variety of plant species including radish (Usuda and Shimogawara, 1998; Long et al., 2004). Radish had an increase in biomass (dry weight) in hypobaria compared to plants grown in normal atmospheric pressures (Table 4 2). While some studies report similar increases in biomass from plants grown in hypobaria (Andre and Massimino 1992; Corey et al., 1996; Spanarkel and Drew, 2002; Goto et al., 2002), others report no change or a decrease in biomass (Daunicht and Brinkjans, 1992; Iwabuchi et al., 1994; Goto et al., 1995; He et al., 2007, 2009). Not surprisingly, the photosynthetic rates reported for plants grown hypobaria also conflicts, with some studies reporting

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89 enhanced photosynthetic rates and others reporting no change (Iwabuchi et al., 1994; Goto et al., 2002; Corey et al., 1997; Spanarkel and Drew, 2002; Iwabuchi and Kurata, 2003; Richards et al., 2006). For Arabidopsis, photosynthetic rates were increased with decreasing pressure for limiting pCO2 (40 Pa) but were unchanged in response to hypobaria at non limiting pCO2 (70 -100 Pa, Richards et al., 2006). In this case, the pO2 was dropped as pressure was lowered thus resulting in lower pO2/pCO2 ratio. A lower pO2/pCO2 ratio can result in enhanced photosynthesis. The pO2 was maintained in our experiments at ambient levels (21 kPa) yet plants had enhanced photosynthetic rates in hypobaria even in non-limiting CO2 (Table 4 2 Figure 44). A previous study with radish also reported both an increase in shoot biomass and an increase in photosynthesis for plants grown at low pressure (33 kPa) with normal atmospheric levels of pO2 (21 kPa; Levine et al., 2008). Therefore, it appears that for radish the biomass and photosynthetic rates are enhanced in hypobaria when the pO2 is maintained at normal atmospheric levels. The dark respiration rates (DR) were enhanced for radish in hypobaria (Table 42) which correlates with results found for Arabidopsis and lettuce (Spanarkel and Drew 2002; Richards et al., 2006). In contrast, others report a lower DR for lettuce that was grown longer -term in hypobaria (He et al., 2007). Since plants may adapt to low pressure it may be possible that plants adapted and reduced their DR in response to hypobaria. Transpiration rates of radish were higher in hypobaria regardless of CO2 levels compared to plants grown in normal atmospheric pressures (Table 42, Figure 45). Enhanced evapotranspiration or transpiration rates have been reported in hypobaria for

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90 a few species (Daunicht and Brinkjans, 1996; Iwabuchi and Kurata, 2003; Wilkerson, 2005; Richards et al., 2006; Levine et al., 2008). The increased diffusivity of gases in low pressure can result in rapid water removal from leaves and promote wilting of plants that are transferred from high pressure to low pressure environments (Rygalov et al., 2004; Wilkerson, 2005). Drought related genes are differentially regulated in response to hypobaria suggesting that plants do respond to the enhanced water loss in hypobaria. Approximately 200 drought -stress-related genes were differentially regulated in Arabidopsis in re sponse to hypobaria and included genes involved in desiccation related pathways such as dehydrins and ABA related proteins (Paul et al., 2004) Similar to the results of photosynthetic rates in hypobaria, the evapotranspiration rates in Arabidopsis studies were increased in hypobaria for low pCO2 treatments but were similar to plants grown at normal atmospheric pressures when pCO2 levels were nonlimiting (Richards et al., 2006). For radish, enhanced transpiration rates in hypobaria (33 or 66 kPa) occurred even in nonlimiting CO2 (Table 4 2, Figure 4-5). These differences may be accounted for the different mechanisms of acclimation to hypobaria for different species with Arabidopsis responding quicker to the hypobaria or the differences in the measured parameter evapotranspiration versus transpiration. However, for spinach grown in non-limiting CO2 (100 Pa) and short -term in hypobaria the transpiration rates were enhanced compared to plants grown at normal atmospheric pressure but after ten days acclimation in hypobaria plants had similar rates of transpiration as plants grown at normal atmospheric pressure (Iwabuchi and Kurata, 2003). The stomata apertures from the spinach that were acclimated long -term to hypobaria were smaller than those that were grown at higher pressure. It may be that the radish had not yet acclimated to

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91 hypobaria even after sev en days (Table 42, Figure 4-5). Transpiration rates were reduced by increasing pCO2 from 40 to 1 00 Pa for all pressures (Table 42, Figure 4-5). However, an incr ease in pCO2 from 100 to 180 Pa only reduced transpiration for plants grown in normal atmospheric pressure (101 kPa) and not for plants grown in h ypobaria (33 or 66 kPa; Figure 4-5). Many studies have shown that at normal atmospheric pressure, stomata clos ure occurs in plants as pCO2 is increased from 40 to 100 Pa but above 100 Pa there is little further decrease in the stomata aperture (Jarvis 1976; Stanghellini and Bunce, 1993; Assmann, 1999; Wheeler et al., 1999). This may explain why a further increase in pCO2 from 100 to 180 Pa did not reduce transpiration for plants grown in hypobaria. Taken together, it appears that plants acclimate to hypobaria and the enhanced transpiration by closing stomata when CO2 is not limited. However, in limited CO2, stomata remain open in hypobaria provided that water is non-limiting. Further studies on the development and response of stomata in hypobaria may lead to identifying the mechanisms of adaptation for plants to the low pressure environment. Summary Radish plants gr own in hypobaria had increased biomass (DW), CO2 assimilation, dark respiration (DR), and transpiration compared to plants grown in ambient pressures. Transpiration was reduced and growth enhanced by enrichment with CO2 for all pressure treatments. Plant t ranspiration rates remained constant over seven days of hypobaria treatments suggesting that plants did not acclimate to hypobaria by reducing stomata aperture during this period. Very high pCO2 (180 Pa) when combined with hypobaria (33 or 66 kPa) induced leaf damage. The leaf damage was not found at lower pCO2 (100 Pa) treatments suggesting that the threshold for pCO2 uptake had been reached at 180 Pa pCO2. Longer -term studies on the interaction of CO2 and pressure

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92 on plant growth in hypobaria will provide further insight into the mechanisms of plant adaptation to hypobaria that can be used to develop decision support tools for growing plants on future space exploration.

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93 Balance Solenoid Valve P CO2 O2N2 Data logger Vacuum Pump Fluorescent lamps Figure 4 1. Schematic of the low pressure growth chambers used for experiments. Each chamber has 0.09 m3 total internal volume. The data logger (CR10) recorded electrical signals from temperature, CO2, O2, balances (n=3), pressure, relative humidity, and light sensors for each chamber.

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94 Figure 42. Radish (26 day old) grown for six days in s uper elevated CO2 (180 Pa) at 101 (first row), 66 (second row), or 33 (third row) kPa total pressure. Side view is shown on left side of figure with corresponding top view in the right panel. All plants were acclimated in the chamber for one day prior to experiments

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95 0 0.4 0.8 1.2 1.6Leaf Area Ratio [ D7/D1] (cm2/cm2) 40 Pa CO2 100 Pa CO2 180 Pa CO2 0 50 100 150 200 250 300 33 66 101Leaf Area (cm2)Total Pressure ( kPa) 40 Pa CO2 100 Pa CO2 180 Pa CO2A B Figure 43. Total leaf area of radish grown at various total pressures and CO2 levels. (A) Ratio of leaf area from the last day of the experiment, at day 7 (D7) to the first day (D1) for plants grown at 40, 100 or 180 Pa CO2 and at 33, 66 or 101 kPa total pressure. (B) Leaf surface area at D7 of treatment for plants grown for six days at 40,100 or 180 pCO2 and at 33, 66 or 101 kPa total pressure. Bars represent the mean STDEV from three experiments (n = 3) with each experiment having three plants.

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96 y = 49.6e 0.67x R = 0.99 y = 39.9e 0.24x R = 0.95 y = 5.6x + 41 R = 0.99 0 10 20 30 40 50 33 kPa 66 kPa 101 kPa y = 12.8x + 105 R = 0.99 y = 17.4x + 106 R = 0.99 y = 7.2x + 100 R = 0.99 0 20 40 60 80 100 y = 5.0x + 184 R = 0.98 y = 15.64x + 179 R = 0.99 y = 10.6x + 181 R = 0.99 0 40 80 120 160 200 0 1 2 3 4 5 A B C 40 Pa CO2100 Pa CO2180 Pa CO2 33 kPa 66 kPa 101 kPa 101 kPa 33 kPa 66 kPa pCO2(Pa)Time (h) Figure 4 4. CO2 drawdown curves on the final day of the experiment of 26 day old radish grown for six days at 33, 66, or 101 kPa. The CO2 levels (partial pressure) were either low (40 Pa; A); high (100 Pa; B) or super elevated (180 Pa, C). Bars represent the mean STDEV from three experiments (n =3) with each experiment having three plants.

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97 Figure 45 Water loss due to transpiration of 26 day old radish grown for 7 days for low (40 Pa; A), high (100 Pa; B) or super el evated CO2 (180 Pa; C) at 33, 66, or 101 kPa total pressure. Values represent the mean of three experiments (n = 3) with each experiment having three plants. Standard deviation bars have been included only for every hour for clarity. Arrows indicate the start of pressure treatments

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98 Table 4 1. Set points f or the environmental conditions Parameter Set point Air Temperatures 22/20 1C day/night Relative Humidity >85% PAR 2 s 1 Total Pressure 33 2, 66 2 or 101kPa pO 2 21 1 kPa CO 2 40, 100 and 180 Pa

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99 Table 4 2 The effect of pressure and CO2 on plant growth (dry and fresh weight ), water content, CA and dark respiration (DR) and transpiration rates of 26 -day old r adish plant s grown for six days at the given pressure treatment. All values except CA and DR are based on mean of three experiments with three plants per experiment (n = 3 STDEV). CA and DR are based on three replicated cham bers. Treatment Parameters Press pCO 2 Fre sh Wt. Dry W t. Water C A DR Transpiration (kPa) (Pa) (g plant 1 ) (g plant 1 ) (% ) (mole m 2 s 1 ) (mol m 2 s 1 ) (mL m 2 day 1 ) 33 40 3.78 0.11 0.49 0.07 88 2 7.24 1.44 1.71 0.43 584 39 100 3.26 1.08 0.57 0.07 81 4 6.84 1.10 1.31 0.17 339 51 180 3.26 0.01 0.62 0.21 80 4 3.58 1.69 1.66 0.76 344 59 66 40 3.36 0.17 0.45 0.10 88 3 4.28 0.99 1.61 0.12 488 112 100 3.31 0.16 0. 61 0.25 85 8 12.42 2.86 1.91 0.61 337 106 180 4.65 1.16 0.65 0.16 87 1 6.48 3.20 1.19 0.33 298 50 101 40 3.00 .0.85 0.42 0.05 85 5 4.32 0.65 1.15 0.20 209 27 100 2.82 0.41 0.47 0.12 86 6 4.43 1.59 0.84 0.23 1 34 12 180 3.66 0.30 0.50 0.04 86 1 3.44 1.04 0.60 0.15 89 23 P N.S. ** N.S. ** ** ** CO 2 N.S. ** N.S. ** N.S. N.S. P X CO 2 N.S. N.S. N.S. ** N.S. N.S. Significance was determined by two way ANOVA *p < 0.05, ** p < 0. 01

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100 100 CHAPTER 5 TH E EFFECTS OF SHORT -TERM AND LONG -TERM ACCLIMATION OF RADISH TO HYPOBARIA Introduction Water is an extremely important resource for any agricultural system. Therefore, it is not surprising that plants adjust their growth in response to the differing amount s of water that is available to optimize their growth and survival. In water -limiting conditions, plants adjust their growth to promote water uptake and minimize water loss. This adjustment in growth depends on the duration and the extent of the water stre ss. For example, short -term responses to limited water availability include the closure of stomata (Ehleringer and Cooper, 1992), increased gene regulation of stress pathways (Knight and Knight, 2001), osmotic adjustment in the roots (Rodriguez et al., 199 5), changes in signal transport ( Morgan, 1984) and inhibition of growth (Schulze, 1986). For longer -term responses to water stress, plants may increase root growth relative to shoot growth (Rodriguez et al., 1995), reduce the transpiration area (Jackson et al., 2000), and have osmotic adjustments to their root system s (Morgan, 1984). For longterm space missions, the conservation and recycling of water by plants that are used as part of an Advanced L ife S upport system will be important for the success of a ny mission to distant planets or moons. I n these missions, plants will likely be grown at reduced pressures relative to Earth sea level pressure which can result in enhanced transpiration/evapotranspiration and photosynthesis (Iwabuchi et al., 1996; Goto e t al., 1996; Daunicht and Brinkjans 1992, 1996; Corey et al., 1996, 2002; Wilkerson, 2005). This enhanced transpiration can result in a variety of stress responses if water is not readily available to the plant For example, radish grown at

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101 101 ambient total pressure immediately wilted when transferred to hypobaria at 25 kPa total pressure (Wilkerson 2005). These plants recovered within several hours after being transferred to the low pressure environment although at reduced transpiration rates prior to the tr ansplant suggesting that plants partially closed their stomata. In other cases, radish did not wilt after being transferred to low pressure (33kPa; Wehkamp 2009; Gohil et al., 2010). These plants likely had more water available to them since they were grow n in hydroponic systems compared to the wilted plants that were grown in soil pots. Arabidopsis plants that were exposed to hypobaria also showed no sign of wilting when transferred to low pressure but they had over 200+ genes that were differentially regulated compared to plants that were maintained in ambient pressures (Paul et al., 2004; Paul and Ferl, 2006). These genes included several that are involved in water -stress-related biochemical pathways such as abscicic acid In many cases plants acclimated to hypobaria by adjusting their transpiration over time to levels that were similar to plants grown in ambient pressure ( Iwabuchi and Kurata, 2003; Wehkamp 2009). The early adjustment or acclimation to hypobaria appear s to involve a decrease in stomatal aperture reducing transpiration and the longer -term adaptation mechanisms including decreases in stomatal size (Iwabuchi and Kurata, 2003). Young leaves exposed to hypobaria or emerging in hypobaria are likely to go through some other physiological changes that older leaves do not go through but this response is not well studied. Taken together, it appears that plants respond to hypobaria and enhanced transpiration similar to a water stress but the extent of this is unclear and may depend on the type of substrate used to grow the plant. To understand the adaptation mechanism s used by plants to hypobaria, the CO2 assimilation transpiration, dry

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102 102 weight, plant growth, leaf area and stomatal density were compared for plants that were grown both in long and sho rt term hypobaria. Materials and Methods Plant Material Seeds of radish (Raphanus sativus L. cv. Cherry Bomb) were germinated and grown in 350 mL flasks filled with about 300 mL of nutrient solution of 1/ 4 strength MS (Murashige and Skoog, 1962) pH ad justed to 6.8 with HCl (1 mM) or NaOH (1 M). Rockwool stoppers (35 cm diameter x 40 cm height; Grodan D elta, Aurora, CO) and absorption paper rolls 2 cm X 10 cm (Anchor paper, St Paul, MN) served as a wicking system. Plants were grown for total of 28 days The volume of flask was large enough for the 4 week experimental studies that the level of nutrient solution at the end of the experiment was well above the majority of roots. The roots were well developed and reached far into the flask at this growth st age. Each stopper was soaked in a nutrient solution of 1/4 strength MS for 24 hours prior to the experiment setup and the pH was maintained around 6.8 through the use of 1 mM of HC l or 1 M NaOH. Each seed was rolled inside absorption paper, placed inside the center of the rockwool, and then placed so that the wick was ~ 7 cm within the nutrient solution. In order to avoid as much evaporation of the solution, the top of the rockwoo1 was covered in plastic wrap. Each flask was then wrapped in foil to reduc e light exposure to the nutrient solution. For the five experimental treatments (Treatment A E; Table 51) seeds were germinated and grown at 101 kPa or 33 kPa and then transferred to 33 kPa (0.1 kPa pp CO2, 21 kPa pp O2) on days 7, 14, 21 or 26 (Table 5 1 ). During the hypobaric studies the environmental conditions are described in Table 5 2

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103 103 Growth Chambers and Environmental Control The experiments were performed in three chambers ( Figure 5-1) of total volume of 0.09 m3 each with a fan for air circulat ion (Delta Model, P/N BFB0512M, Silicon Valley Compucycle, San Jose, CA). The chambers had sensors for temperature (DS10 Dallas Semiconductors, Dallas, TX), pressure (P/N ASCX15AN, Sensym ICT, Milpitas, CA), CO2 (OEM ultrasonic, 6004, Goleta, CA), O2 (MAX 250, Maxtec Co., Salt Lake, UT), relative humidity (HH 4602 -L -CPREF, Honeywell, Morristown, NJ), light (LI -190, Li -Cor, Lincoln, NB) and three balances for weight determination per chamber (LPS 0.6 kg, Celtron Tech. Inc. Colvina, CA). Calibration of O2 and CO2 sensors was performed according to Richards et al., (2006). CO2 and O2 levels were adjusted daily at 4 hours into the photoperiod. CO2 assimilation studies were started at the same time in the photoperiod for all tests and were repeated daily. After t he acclimation period, the gas and pressure composition were brought to set points daily ( Table 5-2). Environmental conditions for the one day acclimation in the growth chamber were 22 2 C, 12 hour photoperiod, at 250 mol m2 s1 using cool white, flor escent lamps and 85% relative humidity. The pO2 was maintained at 21 kPa with other environmental conditions described ( Table 5 2). Each chamber contained one to two individual plants Pressure was maintained with a vacuum pump (1/2 HP, JB Industries, Chicago, IL) and chambers had leakage rates of ~0.03% (chamber volume/h). Data collection and controls were performed using a CR10 data logger (Campbell Scientific, Logan, UT) that was connected to a PC. Experiments were performed in triplicate.

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104 104 Gas E xchange R ates The CO2 assimilation rates (CA) were calculated by performing draw down curves for 5 hours period once CO2 levels were adjusted at a set point (0.12 kPa) at noon. The slope of the regression equation represented the CA rate per hour which was conver ted into mol m2 s1. The range of CO 2 over a 24h period was (100 22 ). The transpiration rates were calculated by performing draw down curves of load cell values for 5 hours period once CO2 levels were adjusted at set point (0.12 kPa) at noon. The slope of the regression equation represented the transpiration rate per hour which was converted into mol m2 s1. Plant B iomass and L eaf A rea After the experimental treatments in the chambers, plants were removed from the chamber blotted dry and analyzed f or fresh weight, leaf area, and dry weight. Dry weight analysis was performed by placing plants on pre weighed pans at 60C for 48h and reweighing the contents. Leaf area was measured on the last day of experiment with scanned digital images using Image Pr o Plus software (ver. 6.0 Media Cybernetics, Bethesda, MD). Scanning Electron M icroscopy Radish leaves were fixed in 2.5% glutaraldehyde in 0.1M sodium cacodylate, pH 7.24 and stored overnight at 4 C. The f ixed leaves were processed with the aid of a Pelc o BioWave laboratory microwave (Ted Pella, Redding, CA, USA). Samples were washed in 0.1M sodium cacodylate pH 7.24, post fixed with 2% buffered osmium tetroxide, water washed and dehydrated in a graded ethanol series 25, 50, 75, 95, and 100% and critical point dried (Bal -Tec CPD030, Leica Microsystems, Bannockburn, IL, USA). Samples were mounted on carbon adhesive tabs on aluminum specimen mount,

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105 105 Au/Pd sputter coated (DeskII, Denton Vacuum, Moorestown, NJ, USA) examined and high resolution digital micro graphs acquired with fieldemission scanning electron microscope (S 4000, Hitachi High Technologies America, Inc. Schaumburg, IL, USA). Leaf Stomatal D ensity and Stomatal Index The surface electron microscopy images were used to calculate leaf stomatal density, i.e., the total number of stomata per unit leaf area ( Radoglou and Jarvis, 1990) Fully grown young leaves from the plants used for measuring gas exchange rates were selected. Leaf discs of 8 mm diameter from the area about 1 cm from the mid rib were punched out from an attached leaf using a plunger and immediately transferred to the buffer solution. Three samples, two samples of hypobaria treatment and one sample for control (101 kPa) were analyzed at the electron microscopy facility at the Univ ersity of Florida. The number of stomata (s) and epidermis cells (e) were counted from the scanned images. The leaf stomatal index (LSI) was calculated using the formula [s / (e + s)] X 100. The stomatal size was calculated by averaging the total length ( m ) of primary axis of all the guard cells of the scanned images. Results Seed G ermination and Plant Growth in Hypobaria The germination of radish was significantly affected by the partial pressure of oxygen (pO2) in hypobaria (33 kPa total pressure) with no germination at 1.5 kPa pO2 and only 45% of the seeds germinating at 4 kPa pO2. This suggests that the minimum pO2 required to induce seed germination with the flask system at 33kPa total pressure is between 1.54 kPa (Figure 51). Interestingly, as pO2 increased to 6 and 10 kPa, germination increased to 62 and 82%, respectively. In fact, at 10 kPa pO2, the

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106 106 germination was not statistically different from pO2 of 21 kPa (at either 33 kPa or 101 kPa total pressures; Figure 51). To study the effects of th e age of the plants at the time of transfer to hypobaria on plant growth and acclimation, five treatments were performed. These treatments consisted of transferring seedlings after 0, 7, 14, 21, or 26 days of normal atmospheric pressures into hypobaria at 33kPa total pressure (Table 5 -1; Treatments A -E). Seedlings appeared healthy except in the case for the seedlings that were transferred to hypobaria (33 kPa) after one week where the plants all had stunted growth or died (Treatment B; Figure 52). This sug gests that the developmental stage of growth during which plants are transferred to low pressure is important for health and survival of a crop in hypobaria. Due to poor growth and in several cases the death of plants from the one week old stage treatment (Treatment B, Table 5-1) no further analyses were performed on these plants. As far as the growth of the plants during the 28 day experiments, plants from the four treatments (A, C, D, and E) all had similar fresh and dry weights and root to shoot ratios (Table 5 3). The longer the plants were exposed to hypobaria, the smaller the leaf area of the plants and the thicker the leaves at the end of the experiment (Figure 5-3C; Table 5 4). The greatest leaf area was observed from plants grown with 2 days expos ure to hypobaria (at 82 cm2) followed by leaves from plants grown at normal pressure (at 78 cm2) and the lowest leaf area was observed for plants grown completely in hypobaria (at 58 cm2; Figure 5 -3C; Table 5 4). CO2 A ssimilation Rates (CA) In general the longer the time that plant remained in hypobaria, the greater the carbon dioxide assimilation rates (CA) on the last day of the experiment (Fig. 5-3A).

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107 107 However, the increase was significant only at p< 0.1 and not at p<0.05 value. The highest CA was o bserved for Treatment A (28 days in hypobaria) at 23 mol m2 s1 and lowest for Treatment D (2 days in hypobaria) at 14 mol m2 s1 (Figure 53A). This suggests that plants have a rapid adjustment in CA during the initial stages of plant transfer to hypo baria indicated by the slight drop in CA for the two days treatment in hypobaria (Treatment D). Interestingly, when comparing CA over the course of the experiment, there was very little difference in CA between plants that were grown entirely in hypobaria and those grown in normal atmospheric pressure (Figure 54A). This may be a result of the limited amount of light used in these studies (250 mol m2 s1) and not by CO2 availability since CO2 diffuses at a faster rate in hypobaria than normal pressures and CA would be expected to be higher in the hypobaria treatments. Further studies that compare CA in higher light conditions in hypobaria and normal pressure are required to determine if that is the case. Transpiration Rates (T) Plants grown entirely in hyp obaria (Treatment A) had significantly higher (p< 0.05) transpiration rates on the last day of the experiment compared to those plants exposed to just two days of hypobaria (Treatment D; Figure 5-3B). The highest transpiration rates were observed for these plants grown entirely in hypobaria at 2.6 0.5 mmol m2 s1 followed by the next longest treatment in hypobaria (Treatment C) at 2.3 0.6 mmol m2 s1. The lowest transpiration rates were found for plants exposed to two days in hypobaria (Treatment D) at 1. 7 0.4 mmol m2 s1 (Figure 5-3B). Plants that were grown entirely in hypobaria had higher transpiration rates than those grown entirely in normal pressure (Treatment E) for the entire experiment except for day 21 suggesting that

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108 108 plants did not acclimate to the enhanced water loss in the hypobaria treatments at least in the first two weeks of growth (Figure 54B). Water U se E fficiency (WUE) Water use efficiency (WUE) of plants grown in hypobaria was calculated as a ratio of transpiration rate over CA, i.e amount of water transpired per amount of CO2 assimilated. The WUE were calculated on days 7, 14, 21 and 28 (last day) of the experiment (Figure 5 -4C). The WUE of a plant s grown in hypobaria was similar to WUE of plants grown at normal pressure at 21 and 28 days of growth but lower for days 7 and 14 (Figure 5-4C). Since CA between these two treatments were similar (Figure 54C), t his suggests that the increase in transpiration (T) on these days (Figure 54B) and not CA (Figure 5-4A) was the cause for this lower WUE since i n both cases, the WUE increased over time until a maximum rate was reached after 21 days with approximately 8.6x103 mol of CO2 assimilated per mol H2O transpired per leaf area (Figure 5-4C). The WUE of 24 days old spinach was ~ 4x103 mol of CO2 assimilated per mol H2O grown in hypobaria at 25 kPa and was similar to that of plants grown at normal pressure (Iwabuchi and Kurata, 2003). Stomata Development The stomatal density (i.e. number of stomata per cm2 of leaf) on the abaxial sid e of leaf was similar between plants grown entirely in hypobaria (Treatment A) compared to plants grown entirely at normal pressure (Treatment E; Table 54). The average stomatal frequency of 1.42 X 105 per cm2 was found for plants grown in hypobaria comp ared to 1.28 X 105 per cm2 for plants grown at normal pressure. Other parameters

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10 9 109 such as epidermal cell density, stomatal index, and length of stomata pore were not significantly different between the two treatments (Table 54). Discussion Seed G erminatio n In one of the earliest hypobaria studies, rye seeds did not germinate at 3 kPa total pressure with Mars level pO2 (~0.3 kPa) and pCO2 (~0.7 kPa; Siegel et al., 1963). Here, radish seeds did not germinate in hypobaria at 1.5 kPa pO2 (33 kPa total pressu re) and only had 40% germination at 4 kPa pO2 (Figure 5 -1). M ustard seeds did not germinate below 5 kPa pO2 (Musgrave et. al., 1988). In other recent studies with the same cultivar of radish, only 2-5 % radish seeds germinated at 2 kPa pO2 in hypobaria (2 5 and 50 kPa total pressures; Wehkamp, 2009). However, 30% of the seeds germinated at 5 kPa pO2 in hypobaria (25 kPa total pressures). The lack of oxygen availability in low oxygen environments inhibits respiration in the seeds which prevents germination (Bewley and Black, 1994). The composition of the seed coat and availability of nutrient reserves within seeds could also affect seed germination and thus the level of oxygen required for germination will likely be species dependent. Al -Ani et al. (1985) rep orted that no germination of radish occurred at 2 kPa of pO2 in ambient pressure but did germinate at 7 kPa of pO2. Here, at 10 kPa pO2 (33 kPa pTotal) the germination was about 82 % compared to 75 % reported for radish at 10 kPa pO2 with lower total press ures of 25 kPa total pressure (Wehkam p 2009). These results suggest that the lower limit for radish germination in hypobaria or in ambient pressure is at approximately 2 kPa pO2 although higher levels >10kPa pO2 are required for germination at levels near normal pressure and oxygen conditions.

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110 110 Seedling G rowth in H ypobaria Radish plants that were exposed to hypobaria (33kPa) one week after germination (Treatment B) exhibited severe stress or died within a few days in hypobaria (Figure 52). However, the pl ants from the rest of the treatments even those that were germinated in hypobaria appeared healthy and there was no indication of water stress or leaf damage. The poor growth and death of plants after one week of transplant is likely due to the fragile sta ge of the seedlings at the time of transfer where only the cotyledons were the main area for photosynthesis and transpiration. Indeed, t he cotyledons from plants from the one week old treatment curled and wilted within the first few days of low pressure ex posure (Figure 5-2). In contrast, threeday old radish seedling that were grown under ambient atmospheric pressure then exposed to hypobaria (25 kPa) grew without any detrimental effects (Wehkamp, 2009). However, those seedlings were grown at higher light conditions than the seedlings from this study and may have already developed primary leaves or may have had a larger root/shoot ratio to keep up with the enhanced transpiration that occurs in hypobaria (Gohil et al., 2010). There may be a critical root to leaf balance that is required for successful acclimation of a plant to hypobaria and this may be met at either the early transfer period of less than one week or after the primary leaves are established at approximately two weeks. The radish plants that w ere germinated and grown in hypobaria for the entire duration of the experiment (Treatment A) had similar growth (fresh weight, dry weight and total dry weight) when compared to the control or plants grown for short -term hypobaria (Treatment D, Table 5-3). Previous studies also found no significant differences in the biomass accumulation between radish grown in hypobaria and those grown in ambient atmospheric pressure (Levine et al., 2008; Wehkamp 2009). Other

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111 111 species such as lettuce (Sparnarkel and Drew, 2 002; He et al., 200 3 2007) and spinach (Iwabuchi; Goto et al., 1996) had similar growth between those grown in hypobaria and those in normal pressure. Interestingly, the leaf area of plants was significantly affected by reduced pressure with a decrease in leaf area the longer the time the plants remained in hypobaria (Figure 53C). The leaves from plants grown entirely in hypobaria (Treatment A) were approximately 30 % smaller compared to leaves from plants grown for a short term in hypobaria (i.e., 2 days Treatment D) and those grown completely at ambient pressure (Treatment E). The leaves from plants grown in hypobaria for longer term were also thicker ( Table 5-4 ). Reduced leaf growth can be caused by ethylene accumulation in closed chambers (He et. al., 2003, 2007). Even though ethylene was not measured in the present study, the absence of leaf senescence, leaf epinasty, and leaf pigmentation due to damaged chlorophyll apparatus, all indicators of ethylene build up, suggests that ethylene was not the cause of the reduced leaf area observed in these studies. In addition, other factors that are responsible for mitigating the negative effects of ethylene in hypobaria include the enhanced eviction of ethylene from mesophyll tissues in low pressure environments ( Burg and Burg, 1966 ), the higher CO2 concentration (0.1 kPa) in low pressure acts as a competitive inhibitor of the ethylene and the larger chamber volume (0.1 m3) used in these studies compared to other reports. Also, studies on gene expression of Ar abidopsis grown in hypobaria compared to plants grown at ambient pressure found no regulation of ACC synthase or ACC oxidase genes which are precursor to ethylene synthesis (Paul et al., 2004; Richards et al., 2006). Therefore, it is likely that the reduced leaf area we observed for plants grown longer terms in hypobaria

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112 112 was not caused by ethylene and could be a result of enhanced diffusion of CO2 to the leaf surface or other effects of hypobaria environment such as enhanced transpiration Plants may acclim ate to hypbobaria by reducing leaf area tom inimize transpiration. Gas E xchange CO2 A ssimilation and Transpiration in H ypobaria At the end of the short -term exposure to hypobaria (i.e., 2 days in hypobaria, Treatment D), the CO2 assimilation rates (CA) o f plants were not significantly different compared to plants grown in ambient pressure (Figure5-3A; Table 53). Similar results were found in a previous study with mature radish ( 26 day old), where after 2 days the CA rates for plants grown at 33 and 66 kP a total pressure were similar to those of plants grown in ambient pressure for the same CO2 levels (Gohil et al., 2010). However, for those studies, after seven days in hypobaria, the mature radish plants had increased CA rates compared to plants grown ent irely at ambient pressure. Here, there was no significant difference in the CA during the first three weeks of growth in hypobaria compared to the CA of plants grown entirely in ambient pressure aside from an increase for the last week in the experiment ( p <0.1 ; Figure 53A; Figure 5-4A). The CA of plants grown in hypobaria increased by 25% at the end of the fourth week when compared to the CA from plants that had only been exposed to two days of hypobaria ( Figure 5-3A ). Similar results were reported in lett uce grown entirely in hypobaria where CA rates increased only during the last week of the experiment ( d ay 25 to 30) when compared to plants grown at ambient pressure (Sparnarkel and Drew, 2002). Several studies have also reported increased CA along with increased biomass for plants that are grown in hypobaria however, these studies were of various durations and gas treatments in hypobaria. (Andre and Massimino 1992, Corey et al. 1996, Sparnakel and Drew 2002, Goto et al. 2002). Here, the CA reached a saturation level at approximately 21 days after

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113 113 growth (Figure 5-4A) which is similar to results reported for previous studies with radish grown in hypobaria (Wehkamp 2009). Radish plants reached compensation points at a faster rate as total pressure reduced (1 00, 70, 50, 25, 10 kPa; Wehkamp 2009 ). However, after 16 h in hypobaria, Arabidopsis plants exhibited nearly identical CO2 draw down curves at various low pressure treatments implying that there is an acclimation response (Richards et al., 2006). Taken tog ether, it appears that CA increases in hypobaria for plants that are transferred to hypobaria from ambient pressures as a short -term response, but this enhancement may not last after a few days or several weeks of growth in hypobaria. In contrast to CA, tr anspiration rates were significantly higher for plants grown in hypobaria (p< 0.05) on the last day of the experiment suggesting that transpiration from the leaves was more sensitive to hypobaria treatments than the CA. Also, during the first two weeks th e transpiration rates of plants that were grown in hypobaria were higher compared to plants grown at ambient atmospheric pressure (Figure 5 -4B) but reached a maximal rate by the third week. Wehkamp (2009) also reported that the transpiration rates of radis h reached a maximal rate at the end of the third week. In spinach, the transpiration rates were slightly higher during the short -term exposure (1 day) to hypobaria but after 10 days in hypobaria both the CA and transpiration rates were similar to those of plants grown in ambient pressure (Iwabuchi and Kurata, 2003). This suggests that plants undergo adaptation during the long-term exposure to hypobaria which results in reduced transpiration rates. Water use efficiency (WUE), based on the rate of CO2 assimi lated over the rate of water lost, increased at a much faster rate for plants grown in hypobaria compared to

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114 114 plants grown in ambient pressure (Figure 5-4C). However, the overall WUE remained higher for plants grown at ambient pressure throughout the durati on of the experiment, except for day 21, compared to plants grown in hypobaria (Figure 54C). The WUE of spinach grown for 10 days in hypobaria (25 kPa) was similar to that from plants grown at ambient pressure (Iwabuchi and Kurata, 2003). For many plant species, increases in WUE are observed when water deficit is mild irrespective of the cause of stomata closure ( Raven 2002). Changes in WUE of plants could be another acclimation response to water deficit sensing in hypobaria This may be a concern for plants grown in substrates where water availability is restricted (i.e. soil or agar). In thi s study, plants were grown in nutrient liquid thus water was not likely limited. Short -term A cclimation to H ypobaria Reduced pressure in a closed system increases dif fus ivity of gases which may result in increased gas exchange rates for plants grown in hypobaria (Rygalov et al., 2004). Although increased CA is beneficial for plant growth up to limit, the increased transpiration rates may result in water stress if the r ate of water uptake is slower than the rate of water lost. For example, for radish that were grown at ambient pressure in soil and then transferred to hypobaria (25kPa total Pressure) plants wilted (Wilkerson 2005). However, these plants were able to recover after several hours in hypobaria. After short -term exposure to hypobaria, Arabidopsis plants showed no wilting or dehydration responses, however, gene expression analyses revealed that almost 200 genes were differentially expressed in response to hypob aria and hypoxia of which about 100 genes were unique to hypobaria (Paul and Ferl, 200 6 ). Of these hypobaria related genes, about 20 (e.g. LATE E MBRYOGENESIS ABUNDANCE (LEA), COLD RESPONSIVE (COR78), DEHYDRATION RESPONSIVE (DR29), ABSCISIC ACID

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115 115 (ABA)) were related to dehydration or water stress. These studies suggest that plant perceive water stress when exposed to hypobaria. Therefore, it is not surprising that the response mechanisms that plants use to acclimate to short -term hypobaria stress may be simi lar to responses that deal with water deficit, high temperatures or very low relative humidity (Iwabuchi and Kurata, 2003). In a review by Chaves et al. (2003) these early mechanisms of adaptation to these stresses are described and summarized here in Figu re 5 -6. For example, short -term hypobaria such as reduced CA and stomatal response (Figure 5-6) were observed in the present study whereas other responses including gene-level responses and multi stress sensing have been reported in previous studies for pl ants grown in hypobaria (Paul and Ferl, 2006). There are different pathways for stress perception and stress responsive genes. The plant hormone ABscisic Acid (ABA) has been identified as an important chemical signal in regulation of stomata in response t o water deficit ( Schulze 1986 ). The ABA signal is localized in roots as well as shoots. Some of the genes are activated are involved in pathways that protect against such stress may be activated for plants transferred from normal pressure to lower pressure. Research on the water deficit sensing and signaling mechanisms suggests that signaling pathways are interconnected and cross talk occurs between the different types of abiotic stress es (Knight and Knight, 2001). Fo r example, hypobaria may result in increased vapor pressure deficit (VPD), a drop in leaf water potential, a reduced leaf temperature (Iwabuchi and Kurata, 2003; Wilkerson, 2005) and an increase in leaf transpiration (Richards et al., 2006; Gohil, 2010). All of the s e stresses may occur simultane ously in hypobaria. In particular, the drop in leaf temperature in hypobaria may be responsible

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116 116 for inducing the cold resistance genes that were found for Arabidopsis (e.g. COR78; Paul et al., 2004). A temperature drop of up to 4 C was reported for leaves of radish when chamber pressure was lowered to 10 kPa (Wilkerson, 2005) and this may induce a cold stress response. To understand the effects of short term exposure to hypobaria on plant transpiration, Sparnarkel and Drew (2002) exposed mature lettuce plants to hypobaria (70 kPa) and ambient pressure on alternate day s from day 30 to day 38. They found that transpiration rates were higher at ambient pressure and lower at reduced pressure and the pattern was repeating during next 6 days. The reversible trend suggests that short -term effects are not due to morphological changes taking place during the growth but due to stomatal response. Since changes in stomatal density are not possible for short term acclimation to hypobaria, the closure of stomata in resp onse to enhanced transpiration is likely in this study. Long -term A cclimation to H ypobaria When mature radish plants were exposed to hypobaria (33 kPa) and similar CO2 levels in these studies for seven days, the transpiration rates of these plants were a lmost twice that of plants grown at ambient pressure (Gohil et al., 2010). Based on those studies, the gas exchange rates of radish grown completely in hypobaria treatment were expected to be higher than plants grown in ambient pressures. Although slightly higher transpiration rates were seen for these plants during the first two weeks of growth and on the last day of the experiment, they were only increased by ~20 % (Figure 3 2 Table 5-3). This suggests that plants adjusted their growth so that their tran spiration rates were minimized as they grew in hypobaria. Indeed, plants that were grown entirely in hypobaria had reduced leaf area compared to plants grown in ambient

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117 117 pressure ( Figure 53C). This reduced leaf area along with increased specific leaf area (SLA), and thus increased leaf thickness, may be a result of a perceived water deficit in hypobaria. Others have also reported increased leaf thickness for plants grown in hypobaria although the leaf area was not reported (Wehkamp, 2009). A reduced leaf su rface may result in less water lost through surface evaporation so that plants may decrease leaf area in response to the perceived water stress in hypobaria. However, the decrease in leaf area in hypobaria may also be in response to other environmental conditions such as increased diffusion of CO2 into the leaf surface in hypobaria. The stomata are a common gate for CO2 assimilation and water vapor exit through the leaf air space. Therefore, it would not be surprising that stomatal number and stomatal cont rol would be influenced by reduced pressure since both CO2 and H2O diffuse at a faster rate in low pressure. Although leaf area was reduced at 33 kPa, T he stomatal density was similar for plants grown in low pressure and the controls in this study In contrast, Wehkamp (2009) reported highly significant (p<0.001) correlation between reduced pressure and stomatal density for radish The number of stomata increased as total pressure reduced (10, 33, 66, and 98 kPa) and as pO2 reduced was reduced with up to 4 4 % increase in stomatal density at 10kPa total pressure and 2 kPa pO2 compared to plants grown at ambient pressure. The difference in stomata numbers reported previously and in this study might be due to the differences in the methods (leaf imprints vs. S EM analysis), the location and age of the leaf for counts, the different lighting and gas conditions, and the duration of hypobaria treatments. Why plants would increase their stomatal density in response to increased transpiration of hypobaria is not clea r but for rice ( Meng et al., 1999) and perennial grass species (Xu and Zhou, 2006 ),

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118 118 a moderate increase in stomatal density with moderate drought was reported. Xu and Zhua (2006 ) reported that stomatal density and guard cell size have plasticity in respons e to large variation in water status. Thus, increase in stomatal density could be a part of adaptation strategy in response to hypobaria stress similar to a water stress. In Arabidposis differential gene expression profile under hypobaria and hypoxia also included genes associated with stomatal development and regulation ( e.g., STOMATAL DENSITY AND DISTRIBUTION ; SDD1 ) supporting this possibility ( Berger and Altmann, 2000; Paul and Ferl, 2006). Alternatively, changes in stomata development may be attribu ted to other environmental factors in hypobaria such as enhanced diffusion of CO2 to the leaf An increase in CO2 has shown decrease in stomata density (Knapp et al., 199 4 ) so the reports by Wehkamp (2009) for increase in stomata density in hypobaria do no t support this hypothesis In addition, Case et al., (1998) compared the effects of CO2 concentration (370 and 680 ppm) on twelve wild radish plants and found that the elevated CO2 did not significantly affect stomata index or the guard cell length. Theref ore, the changes in stomata observed in previous studies (Wekhamp 2009) are not likely due to the enhanced CO2 in hypobaria. Shoot or leaf water content has been suggested as a direct indicator of physiological functioning ( Sinclair and Ludlow, 1985). In the present study, the percent moisture content of plants grown entirely at hypobaria 86% was similar to moisture content of plants grown at ambient pressure at 88% so that the water status of the plants does not appear to be affected by pressure. Studies to compare this response with different substrates to grow plants in hypobaria i.e., soil or agar, are required

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119 119 Radish plants grown in hypobaria for longterm had reduced leaf area but enhanced transpiration compared to plants grown short term in hypobar ia or grown entirely at ambient pressures The acclimation to hypobaria may include adaption mechanisms that overlap with those for water deficit responses such as reducing the area for transpiration or inducing biochemical pathways that are involved in w ater stress responses. Further studies on the gene expression and growth of plants in hypobaria are required to identify if acclimation to hypobaria is truly a drought response Summary The gas phase in hypobaria can result in reduc ed germination of radish if oxygen levels are not above 10kPa. Plants acclimate to hypobaria through short -term and longterm mechanisms. For example, short -term responses (few hours to days) to hypobaria include a decrease in stomata aperture (Iwabuchi and Kurata, 2003) and the reduction of gas exchange rates (Sparnarkel and Drew, 2002; Figure 5-3A). Here, the biomass of plants grown longterm in hypobaria was similar to plants grown in ambient pressures. However, the CA (p<0.1) and the transpiration rates (p<0.05) were enhanced Although transpiration was enhanced for plants grown entirely in hypobaria compared to plants grown entirely in ambient pressure, this enhancement was at much reduced levels than that for adult plants that were grown for seven days in hypobaria. For example, long term acclimated plants to hypobaria had a 20% increase in transpiration (Figure 5-3B ) compared to plants that were acclimated for seven days that had 200% increase in transpiration relative to plants grown in ambient pressure (Gohil, 2010). L ongterm exposure to hypobaria may result in the activation of water deficit defense mechanism in plants such as reduc ing their leaf area. Here, acclimation to

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120 120 hypobaria resulted in increased WUE as transpiration was reduced after three week s of growth in hypobaria. Such response has also been reported in several plant species under mild water deficit (Chaves et al., 2003). 0/2 14/ A

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121 121 Figure 5 1 Germination of radish seedlings at 33 kPa total pressure and various pO2. Bars represents the mean STDEV (n=2) 0 20 40 60 80 100 1.5 4 6 10 21 21 101 kPa pO 2 (kPa) Germination (%) 33 kPa

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122 122 Figure 52 Radish p lant s after four weeks of growth Treatment A (28 days at 33 kPa), T reatment B (21 days at 33 kPa), T reatm ent C (14 days at 33 kPa), and T reatment D (2 days at 33 kPa). All plants in treatment B died within one week of low pressure exposure. A D C B 3.8 cm 3.8 cm 3.8 cm 3.8 cm D C Transpiration (ml m 2 s 1 )

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123 123 Figure 5 3 Carbon dioxide assimilati on ( CA; A), Transpiration rates (B ) and leaf area (C) on the last day of the experiment The bar represent the mean STDEV from three experiments (n=3) each having two or three plants. Bars with different letters indicate statistically different means as c alculated by multiple t -test for comparing two samples for means (p<0.05). The indicates significant difference at p<0.1. 0 5 10 15 20 25 30 0 0.5 1 1.5 2 2.5 3 T ( m mol m 2 s 1 ) B a b a b* A 0 20 40 60 80 100 Leaf area (m 2 plant 1 ) C a a b b ab* ab* ab ab Treatment A C D E CA ( mol m 2 s 1 )

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124 124 Figure 54 CA (A) and Transpiration rates ( B) and WUE ( C ) on the day 7, 14, 21 and 28 of the exper iment The values are mean STDEV from three experiments (n=3) each having at least two plants 0 2 4 6 8 10 0 7 14 21 28 0 0.5 1 1.5 2 2.5 3 Time (Days) WUE (10 3 mol/ mol) T (mmol m 2 s 1 ) C A (mol m 2 s 1 ) 0 5 10 15 20 25 LONGTERM (A) CONTROL (E) A B C

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125 125 Figure 55. Scanning electron microscopy (SEM) image from a youngest leaf of a plant grown entirely in hyp obaria (A; 33 kPa) or in normal pressure (B; 101 kPa). 200 m 200 m 33 kPa 101 kPa

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126 126 Figure 56. The longte rm and short term response s of the shoot or root to water deficit, low humidity and high temperature ( a dapted from Chaves et al., 2003). T he marked responses were observed in present study with the bold indicated in other reports ( Paul et al., 2004; Paul and Ferl, 2006). Long term Short term Shoot Shoot growth inhibition *Reduced transpiration a rea Gene response Metabolic acclimation Osmotic adjustment Shoot Stomata closure *Reduced CA Multi stress sensing Gene response Inhibition of growth Root Turger maintenance Sustained root growth Increased root:shoo t ratio Increased absorption area Root Signal transport Xylem hydraulic changes Assimilate transport Cell drought signaling Osmotic adjustment Gene response

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127 127 Table 5 1. Number of days plants exposure to normal pressure and hypobaria. Treatment Days of treatment (1 01 kPa) (33 kPa) A 0 days 28 days B 7 days 21 days C 14 days 14 days D 26 days 2 days E 28 days 0 days Plants were severely damaged thus not analyzed for further studies.

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128 128 Table 5 2. Environmental conditions used for the experiments. Parameter Set Point Total Pressure 101 or 33 1 kPa Light 2 s 1 Air Temperature 22/20 1C day/night Relative Humidity >85% CO 2 0.1 to 0.02 kPa O 2 21 kPa

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129 129 Table 5 3. Average fresh weight ( FW ), dry weight ( DW ) of shoots, roots, hypocotyls in hypobaria treatments (A -D) and control treatment (E). The values are mean STDEV from three experiments (n=3) each having at least two plants. ANOVA p<0.05 Treatment Parameters Days Fresh weight (g plant 1 ) Dry weight (g plant 1 ) Root/Shoot 101/ 33 Shoot Root Hypo Total Shoot Root Hypocotyl Total ratio A 0 / 28 3.6 1.3 0.8 0.11 2.4 1.2 6.8 0.7 0.52 0.11 0.53 0.2 0.25 0.06 1.3 0.2 1.02 0.12 *B 14 /14 N/A N/A N/A N/A N/ A N/A N/A N/A N/A C 14 /14 3.9 0.6 0.9 0.23 2.0 0.6 6.8 0.5 0.50 0.10 0.52 0.1 0.23 0.03 1.3 0.1 1.04 0.08 D 26 / 2 3.1 0.3 0.7 0.19 2.1 0.3 5.9 0.5 0.37 0.15 0.34 0.1 0.29 0.02 1.0 0.1 0.92 0.11 E 28 / 0 3.2 + 1. 1 1.2 + 0.30 2.2 + 1.1 6.6 08 0.39 0.12 0.33 + 0.1 0.28 0.04 1.0 0.1 0.85 0.06 N.S. N.S. N.S. N.S. N.S. N.S. N.S. N.S. N.S. Plants were severely damaged thus not analyzed for further statistics

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130 Table 5 4. Average s pe cific leaf area, stomata density, stomata index and average stomata pore length from leaf of hypobaria (33 kPa) and normal pressure (101 kPa) 33 kPa 101 kPa Leaf area (cm 2 ) 58 2.8 a 82 10.2 b Specific leaf area (cm 2 g 1 ) 115 12 a 208 10 b Stomata d ensity ( 10 5 cm 2 ) 1 .42 0.1 1 a 1 2 8 0. 16 a Stomata per SLA (10 5 cm 2 g 1 ) 19.2 1. 8 a 34.8 2.7 b Epidermal cell density (cm 2 ) 31 2 a 29 2 a Stomata index 55 2 a 52 2 a 9.63 2.2 a 9.98 2.9 a Values with different letters indicate statistically different means as calculated by t -test for comparing two samples for means (p<0.05).

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131 CHAPTER 6 TRANSPIRATION MODEL PERFORMANCE AT REDUC ED PRE SSURE Introduction Low pressure environment s have increased gas diffusivity which can result in increased gas exchange rates of plants and thus increased transpiration compared to plants grown in normal pressure. Previously Wilkerson (2005) applied the P enman Monteith model to predict transpiration rates of radish in hypobaria. Though the model performed well, it was not very effective at very low pressure (10 kPa) was fairly complex and was developed for evapotranspiration for field grown plants and no t transpiration of individual plants A model developed by Sinclair (1998) described in Chapter 3 is simpler and requires fewer assumptions compared to the PenmanMonteith model (Wilkerson, 2005). Therefore, the simplified model was used here to predict t ranspiration of radish in low pressure environments. The model incorporated the environmental conditions ( gas phase composition, pressure, the t emperature, and air velocity ) and also the physiology of the leaf (stomata density, stomata dimensions, leaf len gth etc. ). A sensitivity analysis of the model for each parameter was performed to identify the parameters that influenced transpiration to the greatest extent Many of these parameters could be controlled to minimize water loss and maximize plant growth. T he prediction of transpiration based on the model was compared with the observed from plants that were grown in either 33 or 101 kPa (Chapter 5) were compared to the model.

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132 Materials and Methods A l ist of the environmental par ameters that were used in th e model is provided in Table 6 1. The parameters that were adjusted to compare against reference values are listed in Table 6 -2 The sensitivity of transpiration predictions to pressure (which also affected gas diffusivity), vapor pressure deficit (VPD) and stomatal width and number at 22 C 1 m s1 air velocity, and 100 Pa pCO2 was determined by varying one parameter at a time while the remaining parameters were held constant (Table 6 -2). Transpiration rate was calculated by equation 3-15. Sensitivity A nalysis Table 6 3 lists the parameters that were evaluated and their reference (101 kPa, 0.6 kPa VPD, 20C, 100 Pa pCO2, and 200 mol m2 s1 light intensity, and 21kPa pO2 by comparing the % change The percent change in transpiration was calculated by eq uation 6-1 for each parameter perturbation (Wilkerson, 2005). 0 0 % T T T change (6 1 ) Where T = Transpiration rate with one parameter varied, g m2 s1 T = Transpiration calculated at reference parameter value, g m2 s1 The evaluation of the model was done by comparing the calculated values with the measured values for transpiration as presented in Chapter 5 which are averages of three replicat ed experiments. The VPD was varied by -50 and +50% from the refer ence value (0.6 kPa). The pressure values were varied from -67% of the reference value (101 kPa). The stomatal width was varied by 0.0001, 0.0002 0.0003 and stomatal number by -50, +50% from the reference value ( n = 13500).

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133 Results and Discussion Trans piration rate was predicted as a function of stomata dimensions stomata number and the diffusivity of water as described by Sinclairs ( 1998 ) and Chapter 3. Although transpiration rates were calculated at over a range of pressures they were compared wit h measured transpiration rate from data from plants grown as described in Chapter 5. These plants were grown for 28 days at 33 and 101 kPa, 2022 C, >85% relative humidity and 100 Pa of pCO2. Meas ured transpiration rate were on average 2.16 g m2 min1 an d 2.76 g m2 min1 for plants grown at 101 kPa and 33 kPa respectively, suggesting that transpiration rate increased by 25 % as total pressure reduced. The model predicted an increase in transpiration rates as total pressure reduced from 101 to 33 kPa (Fi gure 6 1). The model very closely predicted transpiration at 101kP at 2.2 g m2 min1. However the model predicted th at the transpiration should be 80 % higher at approximately at 4 g m2 min1 at 33 kPa The predicted transpiration rates at various st omatal widths (0.0001, 0.0002 and 0.0004 cm) are given in F igure 62. The predicted transpiration rate at 33 kPa total pressure and 0.0004 cm stomatal width was 4 g m2 min1 which after adjusting the stomatal width to 0.0003 cm was 3.9 g m2 min1 and at 0.0001 cm width was 3.8 g m2 min1 suggesting that stomatal width adjustment by these values had little effect on the t ranspiration rates At normal pressure changing the stomatal width from 0.0004 to 0.0003 and 0.0001 changed transpiration rate only by 0.5 and -0.9% To study the effects of vapor pressure deficit (VPD) on transpiration rate s, two different values of VPD (0.6 kPa low VPD and 0.9 kPa high VPD) were adjusted in equation 2-15. The VPD had significant effect on the transpiration rate. At lower VPD and at normal pressure, transpiration rate was similar to that of predicted using the

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134 model. However, at higher VPD transpiration rate increased by 45 % and 1 64 % at 101 kPa and 33 kPa respectively compared to measured transpiration rate at 33 and 101 kPa (Figure 63). The variability in transpiration could be accounted due to the number of stomata at various pressures. According to F igure 64 increase s in stomata number from approximately 1350 to 10000 resulted in predicted increased transpirati on however further increase in number of stomata did not significantly increased transpiration as the curve approached a maximal transpiration. Thus increased transpiration was restricted beyond stomata number 10000 as a result of limitations in stomatal c onductance. When number of stomata was reduced by 50 % in Model, the transpiration rate was reduced by approximately 14% at 101 kPa and 33 kPa (Table 63). However, when number of stomata were increased by 50 % (20,500) in model, the transpiration rates was same to reference value at 101 kPa and 82% higher at 33 kPa, suggesting that there is a limit to increase in transpiration due to higher number of stomata, and increasing the stomatal number beyond 13,500 did not further increased transpiration rate. Conclusion The transpiration model (Sinclair, 1998) incorporating stomatal conductance, stomatal aperture, diffusivity and VPD performed well at normal pressure. Increased VPD resulted in increased transpiration. At reduced pressure the parameters such as sto matal width and in some cases stomatal number are most likely to change the transpiration rate due change gas diffusivity at reduced pressure.

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135 30 40 50 60 70 80 90 100 110 1.5 2 2.5 3 3.5 4 4.5 Pressure [kPa]Transpiration g m2 min1 Figure 61. Predicted transpiration rates from 33 to 101 kPa with observed data, o, from plants grown at 33kPa and 101 kPa as described in Chapter 5 of this dissertation

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136 Figure 62. Predicted transpiration rate at various total pressures (33, 66, 101 kPa) and various stomatal widths (0.0001, 0.0003 and 0.0004 cm). 0 1 2 3 4 0 33 66 99 0.0001 cm 0.0003 cm 0.0004 cm Pressure (kP a) Transpiration (g m 2 min 1 )

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137 Figure 63. Predicted transpiration rate at three different pressures (33, 66 and 101 kPa) and two different vapor pressure deficit (VPD) levels (0.6 kPa and 0.9 kPa). 0 1 2 3 4 5 6 0 33 66 99 low VPD (0. 6 kPa) high VPD (0.9 kPa) Pressure (kPa) Transpiration (g m 2 min 1 )

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138 0 1 2 3 x 104 2 2.5 3 3.5 4 4.5 Number of stomataTranspiration g m2 min133 kPa Figure 64. Predicted transpiration rates as stomata number varies from 1350 to 27000 at 33kPa.

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139 Table 6 1. Parameters and reference values used in the model. Parameter Variable (units) Reference value (at 101 kPa) Stomata Length Stomata Width Stomata Depth Diffusivity of Water Number of. stomata Stomatal Conductance Boundary Layer Conductance Constant Thermal Diffusivity of Water Length of leaf Kinematic Viscosity Air Speed Reynolds Number Viscosity of Air Density of Air Schmidt Number a (cm) b (cm) d (cm) D (cm2 s1) n (no. cm2) hs (cm s1) hb (cm s1) K (cm2 s1) L (cm) Kv (cm2 s1) u (cm s1) Re ( g cm1 s1) 3) Sc 0.0010 0.0002 0.0001 0.8331 13500 17.7 1.6 0.6450 10 0.4250 100 2353 0.0002 0.0004 0.5101 0.507

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140 Table 6 2. Parameter description and reference values used for sensitivity analysis Parameter Description Reference value P D VPD b n Atmospheric pressure Diffusivity of gases vapor pressure deficit Stomatal width Stomatal number 101 kPa According to Figure 3-1 0.6 kPa (Low) 0.0004 cm 13500

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141 Table 6 3. Sensitivity analysis of the transpiration model at various conditions. Given are the transpiration rates and percent change from the reference conditions (101 kPa, 0.6 kPa VPD, and stomatal width of 4 x 104 cm and stomatal number of 13500) when one parameter is varied. The reference transpiration is 2.2 g m2 min1. Tran (g m 2 min 1 ) % change 101 kPa Pressure 33 kPa 0.6 kPa VPD air leaf 101kPa 33kPa 0.6 kPa 101kPa 33kPa 0.9 kPa 101 kPa 33 kPa 0.0001 cm Stoma tal width 101 kPa 33 kPa 0.0003 cm 101 kPa 33 kPa 0.0004 cm 101 kPa 33 kPa 6750 Stomatal number 13500 101 kPa 33 kPa 20250 101 kPa 33 kPa 2.2 4 2.1 3.9 2.2 4 3.2 5.8 2 3.8 2.1 3.9 2.2 4 1.9 3.7 2.2 4 2.2 4 0 (reference value) 82 -4 77 0 (reference value) 82 45 163 -9 72 -5 78 0 (reference value) 82 -14 68 0 (reference value) 82 0 8 2

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142 CHAPTER 7 SUMMARY AND FUTURE W ORK Plants will be an integral part of an Advanced Life Support (ALS) system for space exploration for many reasons but particularly as a source of fresh food. The plant growth facility in an ALS will most likely be maintained at reduced pressure due to the lower costs associated with low pressure facilities compared to those that would be run at normal pressure s. The overall growth of plants will depend on the environment within the chambers. Important environmental factors that will be monitored and controlled include temperature, relative humidity, light, total pressure, and overall gas composition (CO2, O2, e thylene among other gas volatiles). Here, the effects of reduced pressure on radish gas exchange, growth and adaptation to hypobaria were studied. These studies were performed using unique environmental chambers that could monitor all of the important envi ronmental conditions that would be needed for growing plants on longterm missions to the Moon or Mars in hypobaria. However, these chambers had limitations since the relative humidity, gas phase composition, light and temperature were not controlled by th e chambers themselves but by either a larger environmental chamber in which they were housed (temperature and light) or through manual control mechanisms such as the use of saturating salt solutions to maintain humidity levels within the chamber or through manual injection of gas into the chamber on daily intervals to maintain various gas phase concentrations set points. Also, the nutrient solution was not monitored except for the first and final days of the experiments. Improvements to the system would include developing an automated system for controlling humidity and gas phase composition as well as a hydroponics system that could monitor and control nutrient levels, pH and be adjusted throughout the growth of

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143 plants. Despite these limitations of the ch ambers, radish plants grew well in low pressure and their growth and gas exchange rates could be monitored. Radish plants grown short term (1 week) in hypobaria had increased biomass (DW), CO2 assimilation, dark respiration (DR), and transpiration compa red to plants grown in ambient pressures. Transpiration was reduced and growth enhanced by enrichment of the gas phase with CO2 for all pressure treatments (33, 66, and 101kPa). Plant transpiration rates remained constant over the seven days in hypobaria s uggesting that plants grown short term in hypobaria did not acclimate to hypobaria by reducing stomata aperture during this period. Very high pCO2 (180 Pa) when combined with hypobaria (33 or 66 kPa) induced leaf damage. The leaf damage was not found at lo wer pCO2 (100 Pa) treatments suggesting that the threshold for pCO2 uptake had been reached at 180 Pa pCO2. However, the cause of this leaf damage is unknown. Further studies to identify the cause of this damage in low pressure are required. These studies should include the analysis that compare chlorophyll content, rubisco activity, and starch accumulation in the leaves of damaged plants. In addition, the stage at which plants are transplanted from high to low pressure can affect the growth and survival of the plant in hypobaria since plants at early stages may not have the proper root to shoot balance to deal with the increased transpiration that often occurs in hypobaria. Overall, it appears that radish plants do acclimate to hypobaria long term by redu cing leaf area and increasing leaf thickness. This may be an adaption to water stress caused by enhanced transpiration, an adaption to the increased diffusion to CO2 in low pressure or other adaptations to the interactions of the gas phase and other condi tions such as lower leaf temperature in hypobaria. Further

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144 studies on these interacting factors are required to understand the mechanisms of plant adaption to hypobaria. Transpiration and photosynthesis are both sensitive to the total pressure of the atmo sphere. Due to the increases in diffusivity of gases in hypobaria, and hence gas flux, plant growth and water loss can be influenced by the pressure of the growth chamber. If water flux, for example, is a critical aspect of a water purification system or for water conservation, the transpiration rate of the plants can be increased or decreased by simply adjusting the plant chamber pressure or pCO2. Models predict that both CO2 assimilation and transpiration will increase in hypobaria due to the increased diffusivity of gases. However, these models still require improvements since many assumptions are required. For example, the stomatal dimensions and densities are difficult to measure for plants grown in hypobaria and further studies are required that c ompare stomata and leaf development from plants grown long term in hypobaria with those grown at normal atmospheric conditions. Although utilizing plants as part of a BLSS seems promising, it is far from mature. More studies are required to identify the effects of the challenging environments on plant growth and development that will be encountered on space missions including increased radiation and reduced water supplies. Gene expression analyses suggest that plants are undergoing stress in hypobaria. Th is stress response could divert some of the metabolic energy from plant growth to other pathways that may result in reduced food supply. Further integration of molecular information in response to hypobaria may provide information to breed or genetically engineer plants that can mitigate these stresses in hypobaria. The knowledge gained from research on plant adaption to low

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145 pressures for long -term space missions can be applied for terrestrial agriculture particularly in areas of the world where water and other resources are limited.

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146 APPENDIX C R10 PROGRAM ;{CR10} ; Table 1 Program 01: 1 Execution Interval (seconds) ; Reference Temp Reading 1: Internal Temperature (P17) 1: 1 Loc [ temp_int ] 2: Z=X*F (P37) 1: 1 X Loc [ temp_int ] 2: 1.0 F 3: 35 Z Loc [ temp_int2 ] 3: Z=X+F (P34) 1: 35 X Loc [ temp_int2 ] 2: 0.0 F 3: 36 Z Loc [ temp_int3 ] O2 Reading 4: Volt (SE) (P1) 1: 1 Reps 2: 15 2500 mV Fast Range 3: 1 SE Channel 4: 21 Loc [ O2sensor ] 5: 1.0 Mult 6: 0.0 Offset O2 C alibration 5: Z=X*F (P37) 1: 21 X Loc [ O2sensor ] 2: 419.43 F 3: 47 Z Loc [ O2sensor2 ] 6: Z=X*F (P37) 1: 47 X Loc [ O2sensor2 ] 2: 1.07 F 3: 54 Z Loc [ mult ] 7: Z=X+F (P34) 1: 54 X Loc [ mult ] 2: 487.07 F 3: 48 Z Loc [ O2sensor3 ] 8: Z=X*F (P37) 1: 43 X Loc [ Prs3 ] 2: 1.2975 F 3: 49 Z Loc [ factor1 ] 9: Z=X*F (P37) 1: 43 X Loc [ Prs3 ]

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147 2: 2.4183 F 3: 50 Z Loc [ factor2 ] 10: Z=X+F (P34) 1: 50 X Loc [ factor2 ] 2: 7.1664 F 3: 51 Z Loc [ factor3 ] 11: Z=X+Y (P33) 1: 48 X Loc [ O2sensor3 ] 2: 49 Y Loc [ factor1 ] 3: 52 Z Loc [ factor4 ] 12: Z=X+F (P34) 1: 52 X Loc [ factor4 ] 2: 164.56 F 3: 55 Z Loc [ factor5 ] 13: Z=X/Y (P38) 1: 55 X Loc [ factor5 ] 2: 51 Y Loc [ factor3 ] 3: 53 Z Loc [ O2output ] RH Reading 1 14: Volt (SE) (P1) 1: 1 Reps 2: 15 2500 mV Fast Range 3: 3 SE Channel 4: 22 Loc [ RH ] 5: 1.0 Mult 6: 0.0 Offset RH C alibration 15: Z=X*F (P37) 1: 22 X Loc [ RH ] 2: 1 F 3: 65 Z Loc [ RH3 ] 16: Z=X+F (P34) 1: 65 X Loc [ RH3 ] 2: .793 F 3: 63 Z Loc [ RH2 ] 17: Z=X*F (P37) 1: 63 X Loc [ RH2 ] 2: .03 F 3: 64 Z Loc [ RHoutput ] Pressure Reading 18: Volt (SE) (P1) 1: 1 Reps 2 : 15 2500 mV Fast Range 3: 4 SE Channel

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148 4: 24 Loc [ Prs ] 5: 1.0 Mult 6: 0.0 Offset Pressure C alibration 19: Z=X+F (P34) 1: 24 X Loc [ Prs ] 2: 122.59 F 3: 42 Z Loc [ Prs2 ] 20: Z=X*F (P 37) 1: 42 X Loc [ Prs2 ] 2: .0456 F 3: 43 Z Loc [ Prs3 ] Pump Relay C ontrol 21: If (X<=>F) (P89) 1: 43 X Loc [ Prs3 ] 2: 3 >= 3: 5 F 4: 46 Set Port 6 High 22: If (X<=>F) (P89) 1: 43 X Loc [ Prs3 ] 2: 4 < 3: 7 F 4: 56 Set Port 6 Low ; solenoid control (normally closed) 23: If (X<=>F) (P89) 1: 43 X Loc [ Prs3 ] 2: 3 >= 3: 5 F 4: 48 Set Port 8 High 24: If (X<=>F) (P89) 1: 43 X Loc [ Prs3 ] 2: 4 < 3: 7 F 4: 58 Set Port 8 Low 25: Do (P86) 1 : 10 Set Output Flag High ; IRt/c 26: Thermocouple Temp (DIFF) (P14) 1: 1 Reps 2: 14 250 mV Fast Range 3: 6 DIFF Channel 4: 3 Type K (Chromel -Alumel) 5: 1 Ref Temp (Deg. C) Loc [ temp_int ] 6: 66 Loc [ IRtc ]

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149 7: 1.0 Mult 8: 0.0 Offset CO2 Reading 27: Volt (Diff) (P2) 1: 1 Reps 2: 15 2500 mV Fast Range 3: 04 DIFF Chan nel 4: 25 Loc [ CO2 ] 5: 1.0 Mult 6: 0.0 Offset Light Reading 28: Volt (SE) (P1) 1: 1 Reps 2: 15 2500 mV Fast Range 3: 05 SE Channel 4: 26 Loc [ light ] 5: 1.0 Mult 6: 0 Offset Light C alibration 29: Z=X*F (P37) 1: 26 X Loc [ light ] 2: 0.0353 F 3: 29 Z Loc [ light2 ] 30: Z=EXP(X) (P41) 1: 29 X Loc [ light2 ] 2: 30 Z Loc [ light3 ] 31: Z=X*F (P37) 1: 30 X Loc [ light3 ] 2: 0.0027 F 3: 31 Z Loc [ light4 ] Thermocouple Reading 32: Thermocouple Temp (DIFF) (P14) 1: 1 Reps 2: 14 250 mV Fast Range 3: 05 DIFF Channel 4: 1 Type T (Copper -Constantan) 5: 1 Ref Temp (Deg. C) Loc [ temp_int ] 6: 37 Loc [ thermocou ] 7: 1.0 Mult 8: 0.0 Offset 33: Z=X*Y (P36) 1: 68 X Loc [ O2ppa ] 2: 43 Y Loc [ Prs3 ]

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150 3: 69 Z Loc [ O2pp ] 34: Z=X*F (P37) 1: 53 X Loc [ O2output ] 2: 0.01 F 3: 68 Z Loc [ O2ppa ] Program 02: 600 Execution Interval (seconds) 1: Do (P86) 1 : 10 Set Output Flag High 2: Real Time (P77) 1: 110 Day,Hour/Minute (midnight = 0000) 3: Sample (P70) 1: 1 Reps 2: 43 Loc [ Prs3 ] 4: Sample (P70) 1: 1 Reps 2: 37 Loc [ thermocou ] 5: Sample (P70) 1: 1 Reps 2: 53 Loc [ O2output ] 6: Sample (P70) 1: 1 Reps 2: 31 Loc [ light4 ] 7: Sample (P70) 1: 1 Reps 2: 25 Loc [ CO2 ] 8: Sample (P70) 1: 1 Reps 2: 64 Loc [ RHoutput ] 9: Sample (P70) 1: 1 Reps 2: 69 Loc [ O2pp ] Subroutines End Program -Input Locations 1 temp_int 1 3 1 2 _________ 1 0 0 3 _________ 0 0 0 4 _________ 0 0 0 5 _________ 0 0 0 6 _________ 0 0 0 7 _________ 0 0 0 8 _________ 0 0 0 9 _________ 0 0 0 10 _________ 0 0 0 11 _________ 0 0 0

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151 12 _________ 0 0 0 13 _________ 0 0 0 14 _________ 0 0 0 15 _________ 1 0 0 16 _________ 0 0 0 17 _________ 0 0 0 18 _________ 0 0 0 19 _________ 0 0 0 20 temp 1 0 0 21 O2sensor 1 1 1 22 RH 1 1 1 23 CO2sensor 1 0 0 24 Prs 1 1 1 25 CO2 1 1 1 26 light 1 1 1 27 exponent 1 0 0 28 lightcorr 1 0 0 29 light2 1 1 1 30 light3 1 1 1 31 light4 1 1 1 32 temp2 1 0 0 33 temp3 1 0 0 34 temp4 1 0 0 35 temp_int2 1 1 1 36 temp_int3 1 0 1 37 thermocou 1 1 1 38 expone 1 0 0 39 therm2 1 0 0 40 __light__ 1 0 0 41 lig 1 0 0 42 Prs2 1 1 1 43 Prs3 1 8 1 44 light5 1 0 0 45 light 6 1 0 0 46 light7 1 0 0 47 O2sensor2 1 1 1 48 O2sensor3 1 1 1 49 factor1 1 1 1 50 factor2 1 1 1 51 factor3 1 1 1 52 factor4 1 1 1 53 O2output 1 2 1 54 mult 1 1 1 55 factor5 1 1 1 56 CO2_____ 1 0 0 57 RH_______ 1 0 0

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152 58 CO2______ 1 0 0 59 o2ave 1 0 0 60 o2ave2 1 0 0 61 mult2 1 0 0 62 o2ave3 1 0 0 63 RH2 1 1 1 64 RHoutput 1 1 1 65 RH3 1 1 1 66 IRtc 1 0 1 67 _____Prs3 1 0 0 68 O2ppa 1 1 1 69 O2pp 1 1 1 -Program Security 0000 0000 -Mode 4-Final Storage Area 2

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153 LIST OF REFERENCES Al -Ani, A., Bruzau, F., Raymond, P., Saint -Ges, V., Leblacnc, J. M. Germination, respiration and adenylate energy charge of s eeds at various oxygen partial p ressures. Plant Physiol. 79, 855890, 1985 Andre, M Massimi no D Growth of plants at reduced pressures : e xperiment in wheat t echnological advances and constraints. Adv Space Res 12 97 -106 1992 Arnold, J. H. Studies in diffusion, Industrial and Engineering Chemistry 22, 1091 1095, 1930. Assmann, S.M. The cel lular basis of guard cell sensing of rising CO2. Plant Cell Environ. 22, 62937 1999. Baker, D The History of Spaceflight. Crown Publishers, Inc., New York 1981 Berger, D., Altman, T A subtilisin -like serine protease involved in the regulation of stomatal density and distribution in Arabidopsis thaliana Genes Dev. 14, 1119 1131, 2000. Bewley, J.D., and Black, M. Seeds: Physiology of Development and Germination. New York : Plenum Press, 1994. Bierhuizen, J.F., Slatyer, R.O., Photosynthesis o f cotton leaves under a range of environmental conditions in relation to internal and external diffusive resistance. Aust. J. Biol. Sci. 17, 348 -359, 1964a. Bird, R.B., Stewart, W.E., Lightfoot, E.N. Transport Phenomena. John Wiley, New York. 1960. Brinckm ann, E. ESA hardware for plant research on the International Space Station Adv Space Res 36, 1162 1166, 2005. Bucklin R A. Fowler P. A. Rygalov V Y Wheeler R Mu Y. Hublitz I Wilkerson E. G Greenhouse de sign for the mars environment: d evelopm ent of a prototype, deployable dome. ActaHort 659, 12734 2004. Burg S. P. Burg, E. A. Fruit storage at subatm ospheric pressures. Sci. 153, 314 15 1966. Burg, S.P. Post harvest physiology and hypobaric storage of fruits. CABI publishing, Cambridge, MA, 2004.

PAGE 154

154 Case, A. L., Curtis, P.S., Snow A.A. Heritable variation in stomatal responses to elevated CO2 in wild radish, Raph anus raphanistrum (Brassicaceae) Am. J. Bot 85, 253255, 1998. Chapman, S., Cowling, T. G. The Mathematical Theory of Non uniform Gases. 3rd ed, Cambridge. 1970. Chaves M M Maroco, J P., Per eira, J S. Understanding plant response to drought from genes to w hole plant. Func. Plant Bio. 30, 239264, 2003 Corey, K.A., Bates, M.E., Adams, S.L. Carbon dioxide exchange of lettuce plan ts under hypobaric conditions. Adv Space Res 18, 26572, 1996 Corey K. A. Barta, D J Henninger D L Photosynthesis and respiration of wheat stand at reduced atmospheric pressure and reduced oxygen. Adv Space Res 20, 1869 1877, 1997 Corey K. A. B arta, D J Edeen, M A. Henninger D L Atmospheric leakage and method for measurement of gas exchange rates of a crop stand at reduced p ressure. Adv Space Res 20, 1861 -1887, 1997 a Corey K. A. Barta, D J Wheeler R M Toward Martian agriculture: res ponses of plants to hypobaria. Life Support Biosph. Sci. 8 103-114, 2002. Cowles, J. R., Scheld, H.W., Lemay R., Peterson C. Growth and lignification in seedlings exposed to eight days of microgravity, Ann. Bot ., 54, 3349, 1984. Cramer, D. R. Reid, D H. Klein, H. P. The first dedicated life sciences mission Spacelab 4. Adv. Space Res, 3, 143-151, 1984. Daunicht, H.J., B rinkjans, H.J. Gas exchange and growth of plants under reduced pressure. Adv. Space Res 12, 107114, 1992. Daunicht H.J., Brinkjans, H.J. Plant responses to reduced air pressure: advanced techniques and results. Adv Space Res 18 273-2 81 1996 Drake, B G, Gonzlez -Meler, M. A, Long, S. P More efficient plants: a consequence of rising atmospheric CO2? Annu. Rev Plant Physiol Plant Mol Biol 48, 609-639 1997. Drysdale A.E. Life support trade studies involving plants. SAE Technical Paper Series 2001012362, 2001. Ehleringer, J. R., Cooper, T. A. On the role of orientation in reducing photo inhibitory damage in photosynthetic twig desert shrubs. Plant Cell and Environ. 15, 301 -306, 1992.

PAGE 155

155 Fingers B., Strayer R.F., Garland J.L., Mackowiak C.L., Atkinson, D.F., Planning for the Rapid Aerobic Bioreactor Integration Test (Rabit) at the Kennedy Space Centers advanced life support breadboard project, International Conference on Environmental Systems, Monterey, CA, USA, 1996. Finlayson, S A. Gohil H L Ka to -Noguchi H Lee I -J., Morgan, P .W. Circadian ethylene s ynthesis in S orghum bicolor : Expression and control of the system at the whole plant level. J Plant Growth Reg., 23, 29 -36, 2004. Fuller, E.N., Schettler, R.P., Giddings, J.D. New method for pred iction of binary gas phase diffusion coefficients. Ind. Eng. Chem. 58, 18-27, 1966. Gale, J. Availability of carbon dioxide for photosynthesis at high altitudes: Theoretical considerations. Ecology 53, 494 -497, 1972. Gebhart, B.J. Transient natural convection from vertical elements. J. Heat Trans. 83, 6170, 1961. Gohil, H.L., Bucklin, R. A., Correll, M.J The effects of CO2 and hypobaria on growth and transpiration of radish ( Raphanus sativus ). Adv. Space Res. 45, 823 831, 2010 Goto, E. Iwabuchi K. Takakura, K. Effect of reduced total air pressure on spinach growth. J Agri c. Meteorol 51, 139 -145, 1995. Goto, E. Ohta H Iwabuchi K. Takakura, T Measurement of net photosynthetic and transpiration rates of spinach and maize plants under hypobaric condition. J. Agric. Meteorol 52 11 7 23, 1996. Goto, E., Arai, Y., Omasa, K. Growth and development of higher plants under hypobaric conditions. International Conference of Environmental Systems Soc Autom Eng, San Antonio, TX. Meeting Paper No 200201 -2 439, 2002. Grimwood, J. M. Project Gemini technology and operations A chronology, NASA, NASA SP -4002, Wash., DC, 1969. Halstead, T. W., Dutcher F.R., Status and prospects in experiments on plant growth in space, Ann.Bot. 54, 3 -18, 1984. He, C., Davies, F.T., Lacey, R.E., Drew, M.C., Brown, D.L Effect of hypobaric conditions on et hylene evolution and growth of l et tuce and w he at. J. Plant Physiol. 160, 13411350, 2003. He C Davies F T Lacey R E. Separating the effects of hypobaria and hypoxia on let tuce: growth and gas exchange. Physiol Plant 131, 2262 40 2007. He C Davies F T Lacey R E. Ethylene reduces gas exchange and growth of lettuce plants under hypobaric and normal atmospheric conditions Physiol. Plant 135, 258-2 71 2009.

PAGE 156

156 Hirshfeld er, J.O., Curtis, F., Bird, R.B. Molecular Theory of Gases and Liquids. John Wiley, New York 1948 Hublitz, I., Heat and mass transfer of a low pressure Mars greenhouse: simulation and experimental analysis, Ph.D. Dissertation, The University of Florida, 2006. Iwabuchi, K., Goto, E., Takakura, T. Effect of O2 p artial p ressure under low Air pressure on n et photosynthetic rate of spinach. Acta Hort. 399, 101 106, 1994. Iwa buchi, K., Goto, E., Takakura, T. Germination and growth of spinach under hypobaric conditions. Environ Control Biol 34 169178, 1996 Iwabuchi, K., Kurata, K. Short term and long-term effects of low total pressure on gas exchange rates of spinach. Adv Space Res 31, 24144, 2003. Jackson, R. B., Sperrry, J.S., Dawson, T. E., Root water uptake and transport: Using physiological processes in global predictions. Trends in plant science. 5, 482488, 2000. Jarvis, P.G. The interpretation of the variations in leaf water potential and stomatal conductance found in canopies in the field. Phil T rans R Soc Lond B. 273, 593610, 1996. Johnson, A. T. Biological Process Engineering. John Wiley, New York. 1999. Jones, H. Breakeven mission durations for physicochemical recycling to replace direct supply life support, SAE Paper No. 2007-01 -3221, In ternational conference on environmental systems, Chicago, Illinois, 2007. Katchalsky, A., Curran, P.F. Nonequilibrium Thermodynamics in Biophysics. Harvard Uni. Press, Cambridge, MA. 1967. Klein, H.P. US biological studies in space, ActaHorticulture. 8, 92 7 938, 1981. Knapp, A. K., Cocke, M., Hamerlynck, E. P., Owensby, C. E., Effect of Elevated CO2 on Stomatal Density and Distribution in a C4 Grass and a C3 Forb under Field Conditions. Ann. Bot. 74: 595-599, 1994 Knight, H., Knight M. Abiotic signaling pathways: Specificity and cross -talk. Trends Plant Sci. 6, 262-267, 2001 Kordyum, E.L., Sytnik, K.M., Chernyaeva, I.I. Peculiarities of genital organ formation in Arabidopsis thaliana under spaceflight conditions. Adv. Space Res. 3, 247 250, 1983. Krner C., Paulsen, J. A worldwide study of high altitude treeline temperatures. Journal of Biogeography 31: 713-732 2004.

PAGE 157

157 Kuang, A., Musgrave M.E., Matthews, S.W. Cytochemical localization of reserves during seed development in Arabidopsis thaliana under spaceflight conditions, Ann. Bot. 78, 343 -351, 1996. Levine L H Bisbee, P. A. Richards J T Birmele, M N Prior, R L Perchonok M Dixon M Yorio N C ., Stutte, G.W., Wheeler, R.M. Quality characteristics of the radish grown under reduced atmospheric pressure. Adv Space Res 41, 7547 62 2008. Levine, L.H., Richards, J.T., Wheeler, R.M. Super elevated CO2 interferes with stomatal response to ABA and night closure in soybean ( Glycine max ). J. Plant. Physiol. 166, 903-913, 2009. Levinskikh, M.A., Sy chev, V.N., Derendyaeva, T.A.,Signalova, O.B., Salisbury, F.B., Campbell, W.F., Bingham, G.E., Bubenheim, D.L., Jahns, G. Analysis of the spaceflight effects on growth and development of super dwarf wheat grown on the space station Mir. J. Plant Physiol. 1 56, 522 529, 2000. Link B. M Durst S. J Zhou, W Stankovic B. Seed-to -seed growth of Arabidopsis thaliana on the International Space Station. Adv. Space Res. 31(10), 22372243, 2003. Long, S. P Ainsworth E. A. Rogers A. Ort, D.R. Rising atmospher ic carbon dioxide: plants FACE the future. Ann. Rev. Plant Bio. 55, 591628, 2004. Malashenkov, D., World Space Congress in Houston, Texas 2002. Mansell R L, Rose G W, Richa rdson, B. Miller R L Effects of prolonged reduced pressure on the growth and nitrogen content of t urnip ( Brassica rapa L.). SAM -TR 68 100. School of Aerospace Medicine Technical Report, pp 1 -13 1968. Martin, C.E., McCormick, A.K, Air handling and atmos phere conditioning systems for manned spacecraft. ICES Paper No. 921350. SAE International. 1992. Mashinskiy, A., G. Nechita i lo. Birth of space agriculture. Tek Molodezhi 4, 2-7, 1993. Mashinsky, A., Ivanova, I., Derendyaeva, T., Nechitailo, G. and Salisbury, F. From seed-to -seed experiment with wheat plants under space -flight conditions. Adv. Space Res. 14, 13 19, 1994. M assimino, D., Andre, M. Growth of w heat under one-tenth of the atmospheric p ressure. Adv. Space Res 24 293296, 1999 Meng L Li L Chen, W Xu Z Liu L. Effect of water stress on stomatal density, length, width and net photosynthetic rate in rice leaves. J. Shenyang Agric Uni. 30, 477 480, 1999.

PAGE 158

158 Merkys, A.I., Laurinavicius, R.S, Complete cycle of individual development of Arabidopsis thaliana (L.) Heynh. plants on board the Salyut -7 orbital station. Dokladi Akademii Nauk SSSR 271, 509 512, 1983. Morgan, J. M., Osmoregulation and water stress in higher plants. Trends Plant P hysiol. Plant mol. Biol. 35, 299319, 1984 Mott, K. A., Parkhurst, D.F. Stomatal responses to humidity in air and helox. Plant, Cell, and Env. 14, 509515, 1991. Mu, Y. A distributed control system for low pressure plant growth chambers. Ph.D Dissertation. The University of Florida, 2005 Murashige, T., Sk oog, F. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physio l. Plant 15, 473497, 1962 Musgrave, M. E., Gerth, W, A., Scheld, H. W, Strain, B, R. Growth and mitochondrial respiration of mungbeans (Phaseolus aureus Roxb.) g erminated at low pressure, Plant. Physiol., 86, 1922, 1988. Musgrave ME., Seeds in s pace. Seed Sci. Res. 12:116, 2002. NASA/TM 210785. Guidelines and Capabilities for Designing Human Missions. 2003. Nechitailo, G.S., Mashinsky, A.L. Space biology: Studies at orbital station Moscow, Mir Publishers. 1993. N obel, P.S. Physicochemical and E nvironmental Plant Physiology. Academic Press, San Diego, CA.1999. Oberg J., Uncovering Soviet Disasters : "Dead Cosmon auts", pp 156176, Random House, New York, 1988, retrieved 14 October 2009. Parfenov, G.P., Abramova, V.N. Blossoming and maturation of Arabidopsis seed: Experiment on Biosatellite Kosmos 1129. Dokladi Akademii Nauk SSR 256, 254. 1981. Parlange, J., Wag oner, P.E. Stomatal dimension and resistance to diffusion. Plant Physiol. 46, 337 342, 1970. Paul A .L., Schuerger A. C Popp M P Richards, J.T., Manak, M.S., Ferl, R.J. Hypobaric biology: Arabidopsis gene expression at low atmospheric pressure. Plant Physiol 134, 215223, 2004. Paul, A.L., Ferl, R.J. Biology in low atmospheric pressure: i mplication for exploration mission design and advance life support system. Gravi Space B iol Bull. 19, 3 17 2006.

PAGE 159

159 Porterfield, D.M., Neichitailo, G.S., Mashinski, A .L., Musgrave, M.E. Complete plant growth systems in space. Adv. Space Res. 2002. Purswell, J. L. Engineering design of a hypobaric plant growth chamber. Mas ter of Science Thesis: Biological and Agricultural Engineering. Texas A&M University, College Stati on, TX; 2002. Radoglou K M Jarvis P. G. Effects of CO2 enrichm ent on four poplar clones. II. l eaf surface properties. Ann. Bot. 65, 627 632, 1990. Rajapakse, N. C., He, C., Cisnero s -Zevallos, L., Davies F.T Hypobaria and hypoxia affects growth and phytochemical contents of lettuce. Sci. Hort. 122: 171-178, 2009. Rand, R.H. Gaseous diffusion in the leaf interior. Trans. Am. Soc. of Agri. Eng. 20, 701 704, 1970. Raven JA. Selection pressure on stomatal evolution. New Phyto. 153: 171386, 2002 Richards, J.T., Corey, K.A., Paul, A.L Fe rl, R.J., Wheeler, R.M., Schuerger, A.C. Exposure of Arabidopsis thaliana to hypobaric environments: implications for low pressure bioregenerative li fe support systems for human exploration missions and terraforming on M ars. Astrobio 6, 851866, 2006. Rodr igues, M. L., Pachico, C. M. Chaves, A. M. Plant Soil relations, root distributions and biomass partitioning in Lupinus Albus. M. under drought cond itions. J Expt. Bot. 46: 947956, 1995 Rule, D E., Staby, G.L. Growth of tomato seedlings at subatmospheric pressures. HortSci. 16 3313 32 1981. Rygalov V. Y. Fowler P. A. Metz J M Wheeler R M Bucklin R A. Water cycles in closed ecological sy stems: e ffects of atmospheric pressure. Life Supp. B iosph Sci 8, 125 -135, 200 4 Salisbury, F.B., Bingham, G.E., Campbell, W.F., Carman, J.G., Hole, P., Gillespie, L.S., Sychev, V.N., Berkovitch, Yu., Podolsky, I.G. and Levinskikh, M. Growing super dwarf wheat on the Russian space station Mir. ASGSB Bulletin 9, 63. 1995 Schulze, E D. Carbon dioxide and water vapor exchange in response to drought in the atmosphere and the soil. Ann. Rev. Plant physiol. 37, 247 -274, 1986. Schwar t zkopf S H., Mancinelli R. L. Germination and g rowth of wheat in simulated martian atmospheres. Acta Astronautica 25(4), 245247, 1991.

PAGE 160

160 Schwar t zkopf S. H., Grote, J. R., Stroup, T.L. Design of a low atmospheric pressure plant growth chamber. SAE Technical Paper No. 951709. Warren dale, P.A.: Society of Automotive Engineers, 1995. Siegel, S. M., Rosen L. A., Giumarro, C. Plants at subatmospheric oxygen -l evels. Nature 198: 1288-1290. 1963. Simpson, M. S.; Young, J. S. A plant growth structure for Martian derived atmosphere. SAE Technical Paper No. 981901. Warrendale, PA.: Society of Automotive Engineers; 1998. Sinclair, T.A. Theoretical considerations in the description of evaporation and transpiration. In: Stewart, D. A., Nielson D.R. (Eds.), Irrigation of Agricultural Crops. pp.343361.1980. Sinclair, T.R., Ludlow M.M. Who taught plants the thermodynamics of water? The unfulfilled potential of plant water potent ial. Aust. J. Plant Physiol. 12, 213 217, 1985. Slattery, J.C., Bird, R.B Calculation of the diffusion coefficient s of dilute gases and of the self -diffusion coefficient of dense gases. Ind. Eng. Chem 4, 137 143, 1958. Smolders, P., Soviets In Space, Kluwer Agemene Vitgaven, Holland, 1973. Spanarkel, R., Drew, M.C. Germination and growth of lettuce ( Lactuca sativa) at low atmospheric pressure. Physiol. Plant. 116, 4684 77 2002. Stanghellinni C Bunce J A Response of photosynthesis and conductance to light, CO2, temperature and humidity in tomato plants grown at ambient and elevated CO2. Photosynthetica. 29 48 7 -4 97 1993 Svetlana S, Slaveyko N, Tania I, Plamen K, Iliana I Monitoring of plant growth environment in the SVET -3 space greenhouse: Measurement of Plant shoot environment Sozopol, In: Proceeding of the 14 In: International Scientific Conference ELECTR ONICS ET. 21 23 Sept, Sozopol, 3, 13-18. 2005. Sychev V.N., Levinskikh M, A., Sergey A. G., Bingham G. E., Podolsky I.G. Spaceflight effects on consecutive generations of peas grown on board the Russian segment of International Space Station. Acta Astronautica 60, 426-432 2007. Thimann, K.V. Biosatelli te II experiments: Preliminary r esults. Proc. Nat. Acad. Sci. 60 (2), 347-361, 1968. Tisserat B Herman, C., Silman, R., Bothast, R.J. Using ultra high CO2 levels enhances plantlet growth in vitro. HortTech. 7 2822 89 1997

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161 Usuda, H., Shimogawara, K. The effects of increased atmospheric carbon dioxide on growth, carbohydrates, and photosynthesis in radish, Raphanus sativus Plant Cell Physiol. 39 1 7, 1998. Wehkamp, C. Plant age affects the longterm growth response to reduced total pressure and oxygen partial pressure. Ph.D. Dissertation. The University of Guelph, 2009. Wheeler R M Mackowiak C L Yorio, N C ., Sager, J.C. Effects of CO2 on stomatal conductance: do stomata open at very high CO2 c oncentrations? Ann Bot 83, 243-2 51 1999. Wheeler R.M., Stutte, G.W., Subbarao, G.V., Yorio, N.C. Plant growth and human life support for space travel, i n: Pessarakli M (ed) Handbook of Plant and C rop Physiolog, 2nd Edn. Marcel Dekker Inc., New York, USA, pp. 9259 41, 2001. Wheeler R.M. Horticulture for Mars. ActaHort 642 201 -215, 2004. Wilkerson, E.G. Plant evapotranspiration in a greenhouse on Mars. Ph.D. Dissertation. University of Florida 2005. Xu Z .Z., Zhou, G S. Nitrogen metabolism and photosynthesis in Leymus chinensis in response to long -term soil drought. J Plant Growth Regul 25, 252 266, 2006.

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162 BIOGRAPHICAL SKETCH Hemant Gohil was born (1976) in Santarampur, a small town in a western state of India, Gujarat. He did his schooling in Ahmedabad city and completed his Bachelors degree in Agriculture in 1999 from Gujarat Agricultural University, Anand. He came to the U nited States pursue his Masters in Mole cular and Environmental Plant Science from Texas A&M Univ ersity in May 2002. He worked at the Indian Agricultural Research Institute, New Delhi from 2003 to 2005. He came to UF for his Ph.D. in Applied Science in Agricultural and Biological Engineering. He finished his Ph.D. in August 2010