Title: Effects of carbon dioxide on the physiology and biochemistry of photosynthesis in soybean /
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Title: Effects of carbon dioxide on the physiology and biochemistry of photosynthesis in soybean /
Physical Description: xii, 181 leaves : ill. ; 28 cm.
Language: English
Creator: Campbell, William J., 1951-
Publication Date: 1986
Copyright Date: 1986
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Subject: Plants, Effect of carbon dioxide on   ( lcsh )
Soybean -- Physiology   ( lcsh )
Photosynthesis   ( lcsh )
Agronomy thesis Ph. D
Dissertations, Academic -- Agronomy -- UF
Genre: bibliography   ( marcgt )
non-fiction   ( marcgt )
 Notes
Thesis: Thesis (Ph. D.)--University of Florida, 1986.
Bibliography: Bibliography: leaves 161-180.
Statement of Responsibility: by William J. Campbell Jr.
General Note: Typescript.
General Note: Vita.
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Bibliographic ID: UF00099327
Volume ID: VID00001
Source Institution: University of Florida
Holding Location: University of Florida
Rights Management: All rights reserved by the source institution and holding location.
Resource Identifier: alephbibnum - 000897362
notis - AEK6031
oclc - 015504470

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EFFECTS OF CARBON DIOXIDE ON THE PHYSIOLOGY AND
BIOCHEMISTRY OF PHOTOSYNTHESIS IN SOYBEAN












BY

WILLIANI J. CAMPBELL, JR.


A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA INJ
PARTIAL FULFILLN1ENT OF THE REQUIREMENTS
FOR THE DEGREE OF DOCTOR OF PHILOSOPHY



UNIVERSITY OF FLORIDA


1986





























It is not too much to say that a comparatively
sudden increase of carbon dioxide in the air to an
extent of but two or three times the present
amount, would result in the speedy destruction of
nearly all our flowering plants.

H. T. Brown and F. Escombe (1902)















ACKNOKLEDGMENTTS


I wish to express my appreciation to Dr. L. H. Allen, Jr., for

serving as chairman of my supervisory committee and for his generous

support during my graduate study. I sincerely thank Dr. George Bowes

for allowing me to spend two and one-half rewarding years in his

laboratory as well as for serving on my supervisory committee. The

time spent in his laboratory was most beneficial to my education. I

would also like to thank Drs, K. J. Boote, J. W. Jones, and T. R.

Sinclair, for their time and efforts as supervisory committee members.

I wish to thank Dr. Pierce Jones for assistance during several of

the experiments and for years of interesting conversations. The

assistance of Dr. Klaus Heimburg in deciphering the leaf gas exchange

system was indispensable and is gladly acknowledged. In addition, I

would like to thank Drs. Gabriel Hfolbrook and J. C. V. Vu, for their

instruction and discussion concerning laboratory techniques. The

assistance of Mr. Paul Lane in calibrating the IR gas analyzers and

the helpful suggestions of Dr. Julia Reiskcind and Mr. William Spencer

are gratefully acknowledged. This research was supported in part by

USDOE-USDA Interagency Agreement No. DE-AI01-81ER60001, and funding

for the graduate assistantship was provided in part by USDA\-ARS and in

part by the USDOE-USDAZ Interagency Agreement.

Finally, I would like to thank Susie for her constant

encouragement and patience.


Ill















TABLE OF CONTENTS

PAGE

ACKNOWLEDGMENTS ............. ............. ................. iii

LIST OF TABLES......................................... vi

LIST OF FIGURES........................................ vii

KEY TO ABBREVIATIONS.................................. ix

ABSTRACT............... .................... .................. xi

CHAPTER I INTRODUCTION: A REVIEW OF PHOTOSYNTHETIC
CARBON ASSIMILATION IN C3 FLANTS.... ............ 1

Photosynthetic Carbon Reduction Cycle........... 2
Photorespiratory Carbon Oxidation Cycle......... 10
RuBP Carboxylase/0xygenase........ .............. 13
Experimental Approach........................... 18

CHAPTER II THE EFFECTS OF SHORT-1TERM EXPOSURES TO CO, ON
LEAF PHOTOSYNTHETIC RATE, RuBP CARBOXYLASE
ACTIVITY ANiD RuBP LEV EL ................... ...... 22

Introduction.................. .................. 22
Materials and M~ethods........................... 28
Results....... .................................. 37
Discussion.................................. 57

CHAPTER III RESPONSE OF PHOTOSYNTHETIC BIOCHEMISTRY ANTD
PHYSIOLOGY TO LONG-TERM EXPOSURE TO
SUBATMOSPHERIC AND SUPERATMOSPHERIC CO2
CONCENTRATIONS.................... .....:.......... 66

Introduction................................ 66
Materials and Methods........................... 71
Results. ................. ................ ....... 77
Discussion.................................. 106

CHAPTER IV EFFECTS OF TEMPERATURE ON PHOTOSYNTH~ESIS AND
RuBP CARBOXYLASE AT TWO GROWTH CO2
CONCENTRATIONS.................... .............. 116

Introduction........... ....... ......... ....... 116
Materials and Methods. .................... ...... 119
Results..................................... 123
Discussion.................................. 133











PAGE


CHAPTER V GENERAL SUMMARY AND CONCLUSIONS................. 139

APPENDIX A LEAF AND CANOPY PHIOTOSYNTHETIC RATE RESPONSES
TO LIGHT AT TWO CO2 CONCENTRATIONS.............. 143

APPENDIX B EFFECT OF LEAF SAMPLE SIZE ON IN VITRO RuBP
CARBOXYLASE ACTIVITY.............................. 150

APPENDIX C LINEAR REGRESSION PARAMETERS ................... ... 158

LITERATURE CITED........................................ 161

BIOGRAPHICAL SKETCH...................................... 181














LIST OF TABLES


TABLE PAGE

2.1 Effects of growth CO2coenatnonlf
characteristics................... 38

2.2 Effects of growth CO2 concentration an pod weight
and total green leaf area per plant................. 40

2.3 Effects of growth CO2coenrtoonRBas
activity in leaves collected following i-hour
exposures to six different CO2 concentrations....... 52
2.4 Effects of growth CO, concentration on RuBP levels
in leaves collected following 1-hour exposures to
six different CO2 concentrations........ ........... 58

3.1 Effect of growth CO2 concentration on SLW, LAI,
chlorophyll, and total leaf soluble protein......... 78

3.2 Effect of growth CO2 concentration on chlorophyll
and total leaf soluble protein expressed on a dry
weight basis...................................... 80

3.3 Effect of growth CO2 concentration on apparent
K (CO ), Vma an"d dissolved free CO2 at the
mesophyll cell wall................. 105

4.1 Effect of growth air temperature on maximum
canopy net photosynthetic rates.................... 125

C.1 Linear regression parameters (for short-term
CO2 concentrations) for data in Chapter II.......... 158

C.2 Linear regression parameters (for growth CO2
concentration) for data in Chapter III....... 159

C.3 Linear regression parameters (for growth air
temperature) for data in Chapter IV................. 160













LIST OF FIGURES

FIGURE PAGE

1.1. A non-stoichiometric diagram of the PCR cycle in C3
chloroplasts (after Bassham, 1979).......... 5

1.2. A non-stoichiometric diagram of the integration of
the PCR and PCO cycles in C3 chloroplasts (after
Lorimer, 1981).. . . . . . . . . . 12

2.1. Intercellul~ar CO2 concentration versus ambient CO2
concentration for leaves grown at two CO2
concentrations................................. 42

2.2. Leaf photosynthetic rate versus intercellular C92
concentratio fo eae grown at 330 pl CO21
(A) and 660 pl CO2 1 (B).............. 44

2.3. Mean leaf photosynthetic rate versus mean
intercellular CO2 concentration for leaves grown
at 330 pl CO2 1 and 660 Ul CO2 1 ......... 47

2.4. Leaf RuBPCase activity versus CO2coenrtnfr
samples collected following 1 hour exposures to six
different CO2 concentrations. ........................ 50

2.5. Activation status of RuBPCase versus CO -
concentration f~o leaves grown at 330 iil CO2 11
or 660 ul CO2 1 ... .... .. .. .. .. ... 54

2.6. Leaf RuBP levels versus CO2 concentration in samples
collected following 1-hour exposures to six different
CO2 concentrations............................... 56
3.1. The soluble protein/chlorophyll ratio versus growth
CO2 concentration................................ 82
3.2. Canopy net photosynthesis (on a land area basis)
versus solar irradiance for canopies grown at 6
different CO2 concentrations. ........................ 84

3.3. Maximum canopy net photosynthetic rate versus
growth CO2 concentration............................ 87

3.4. A. RuBPCase activity versus growth CO, concentration.
B. RuBPCase activation versus growthC2
concentration. ..........................f............ 90










FIGURE


PAGE


3.5. Levels of RuBP versus growth CO2 concentration......., 92

3.6. RuBPCase activity versus HCO, concentration in
leaf tissue grown at 160 pl CO ......... 95

3.7. RuBPCase activity versus HCO, concentration in
leaf tissue grown at 280 pl C20 1 .......... 97

3.8. RuBPCase activity versus HCO concentration in
leaf tissue grown at 330 plCO 1 .......... 99

3.9. RuBPCase activity versus HCOz can entration in
leaf tissue grown at 660 plCO 1 .......... 101

3.10. RuBPCase activity versus HCO, coqnentration in
leaf tissue grown at 990 pl CO2 1 .......... 103

6.1. Initial RuBPCase activity versus grow h air
temperature for 330 and 660 pl CO2 1 grown
plants............ ................ ................... 128

4.2. Total RuBPCase activity versus growthlair
temperature for 330 and 660 ill CO2 1 grown
plants......................... .................~~~~~ 130

4.3. RuBPCase activation (%) versus growth air
temperatu e for plants grown at 330 or 660
pl CO2 1 .. .. .. . .. .. .. . . .. 132

4.4. Levels of RuBP versus growth air tempera ure
for plants grown at 330 or 660 pl CO2 1 ....... 135

A.1. Leaf net photosynthesis versus quantum flux density
for plant~ grown and measured at 330 and 660
ill CO2 1 .. .. .. . .. . .. . . 146

A.2. Canopy net photosynthesis versus quantum flux density
for canop es grown and measured at 330 and 660
ul CO2 1 .......................................... 148

B.1. Initial and total RuBPCase activity versus leaf
sample size used in assay. ........................... 153

B.2. Percent activation of RuBPCase versus leaf sample
size used in assay......... .........................~~ 155











KEY TO ABBREVIATIONS


C Stromal concentration of CO2

CA Carbonic anhydrase

Ca CO2 concentration ambient to leaf

Ci CO2 concentration in air in leaf intercellular spaces (ul 1 1)

Ci' Percent of CO2 in air in leaf intercellular spaces (v/v)

Cin CO2 concentration of air entering leaf chamber

Cout CO2 concentration of air leaving leaf chamber

ACO2 Activator CO2 in Rubisco activation
DAP' Days after planting

DHAP Dihydroxyacetone Phosphate

diPGA 1,3-diphosphoglycerate

DTT Dithiothreitol

E Enzyme

E4P Erythrose 4-phosphate

EDTA Ethylenediaminetetraacetic acid

FBP Fructose 1,6-bisphosphate

F6P Fructose 6-phosphate

GAP Clyceraldehyde 3-phosphate

Kc Michaelis constant for CO2

KctEnzyme tunmover number (s-1)
K Michaelis constant

Ko Michaelis constant for 02
LAI Leaf area index

M Metal cation for enzyme activation










0 Stromal concentration of 02

P Atmospheric pressure

PCO Photorespiratory carbon oxidation

PCR Photosynthetic carbon reduction

PGA 3-phosphoglycerate

P-GLY 2-phosphoglycolate

Pi Inorganic phosphate

pK' First ionization constant

Pn Net photosynthetic rate

PVP-40 Polyvinylpyrrolidone

RSP Ribose 5-phosphate

Rleaf Total leaf resistance to water vapor diffusion
RuBP Ribulose 1,5-bisphosphate

Ru5P Ribulose 5-phosphate

S7P Sedobeptulose 7-phosphate

SBP Sedobeptulose 1,7-bisphosphate

SLW Specific leaf weight

Tris Tris hydroxymethyll) aminomethane

Tris-HC1 Hydrochloride of Tris

V Standard molar gas volume

Vc Vma of carboxylation reaction

Vo Vmax of oxygenation reaction

c Velocity of carboxylation reaction

Vmax Theoretical maximum velocity of enzyme catalyzed reaction

Vo Velocity of oxygenation reaction
X5P Xylulose 5-phosphate

a Solubility coefficient in water














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


EFFECTS OF CARBON DIOXIDE ON THE PHYSIOLOGY AND
BIOCHEMISTRY OF PHOTOSYNTHESIS IN SOYBEAN

By

WILLIAM J. CAMPBELL, JR.

December 1986

Chairman: L. H. Allen, Jr.
Major Department: Agronomy

In three consecutive years (1983, 1984, and 1985) soybeans

(Glycine max L. Merr. cy Bragg) were grown from seed to maturity in

six outdoor environmentally controlled plant growth chambers under

natural solar irradiance. The CO2 concentrations inside the chambers

were controlled to various levels during these studies. Both field

and laboratory measurements were made to investigate the effects of

CO2 concentration on photosynthesis. Emphasis was placed on the

response to CO2 of ribulose 1,5-bisphosphate (RuBP) and RuBP

carboxylase (RuBPCase), the substrate and enzyme of the carbon

fixation reaction in soybean.

Following growth at 330 (atmospheric concentration) or 660

ul CO2 1-1, leaflet photosynthetic rates were always greater for the

elevated CO2 grown plants when measured over a wide range of CO2

concentrations. This enhanced capacity for photosynthesis was

possibly a result of changes in internal leaf anatomy, or to greater










assimilate demand, or both, in the high CO2 grown plants. The RuBP

concentration decreased with increasing CO2, but still appeared to be

greater than the active site concentration of RuBPCase. The RuBPCase

activity, expressed on an area basis, was not affected by growth CO2

concentration. It appears that RuBPCase and RuBP are thus not

involved significantly in the enhanced photosynthetic capacity.

Evaporative cooling kept leaf temperatures from reaching the

higher air temperatures during studies on temperature effects on

soybean grown at atmospheric and twice atmospheric concentrations of

CO2. Although air temperatures were increased by approximately 5 and
100C, leaf temperatures were usually not increased more than

approximately 2.5 and 4.50C, respectively. These leaf temperature

increases were not great enough to affect canopy photosynthesis or

RuPBCase activity (on a chlorophyll basis) in either CO2 treatment.

Canopy photosynthesis was, however, greater at the higher CO2
concentration. The concentration of RuBP was reduced at higher

temperatures.

Increasing growth CO2 concentrations (from 160 to 990 pl CO2 1-1)

resulted in decreasing RuBPCase activities and RuBP levels, when both

were expressed on a chlorophyll basis. At the higher CO2

concentrations, the concentration of RuBP appeared to approach the

concentration of RuBPCase active sites. Both the apparent Km(CO2) and

Vmax of RuBPCase showed small, but statistically significant,

decreases with increasing CO2.


xii














CHAPTER I
INTRODUCTION: A REVIEW OF PHOTOSYNTH~ETIC
CARBON ASSIMILATION IN C3 PLANTS

Photosynthesis is the process in which green plants and certain

bacteria assimilate inorganic carbon into organic compounds. Light is

the source of energy for this process and is absorbed in the plant by

various pigments. The photochemical reactions involved in absorbing

and transferring light energy are referred to as the "light reactions"

while reactions responsible for the fixation of inorganic carbon and

its subsequent metabolism are often referred to as the "dark

reactions." Since several of the enzymes of photosynthetic carbon

assimilation are light-activated, the "dark reactions" are not

completely independent of light.

Under conditions of high quantum flux density, several processes

can be identified as being potentially involved in regulation of

photosynthetic carbon assimilation. One of the more marked of these

processes is the CO2 fixation reaction. Characteristics of this

reaction have been used to assign plants to various photosynthetic

categories. Terrestrial plants have been divided into four

photosynthetic categories based on the path of carbon during

photosynthesis, physiological characteristics, and leaf anatomy. In

Cg plants the initial product of the carbon fixation reaction is a

three-carbon phosphorylated compound, whereas in Cq plants it is a

four-carbon organic acid. Crassulacean acid metabolism (CAM) is a











photosynthetic pathway in which the initial carbon fixation product is

a four-carbon compound, however, most of the carbon fixation occurs at

night. Characteristics of these three pathways are reviewed by Black

(1973). The fourth category, C3-Cq intermediates, exhibit

physiological and anatomical characteristics intermediate between C3

and Cq species. Holaday and Chollet (1984) have recently reviewed the

photosynthetic characteristics of plants in this category.

One of the main objectives of the research described in the

following chapters was to investigate the CO2 fixation reaction in

soybean, a C3 type plant, by examining the enzyme and substrates
involved. Prior to discussing specific objectives and the general

experimental approach, CO2 fixation in C3 type plants is reviewed.

This review covers CO2 fixation and the subsequent regeneration of the

CO2 acceptor, the competitive photorespiratory cycle, and the enzyme
responsible for catalyzing the initial reactions in both pathways.

Proposed sites of regulation other than the carboxylation reaction are

also discussed.


Photosynthetic Carbon Reduction Cycle


Description of the Cycle


The photosynthetic carbon reduction (PCR) cycle (also known as

the reductive pentose phosphate or Calvin cycle) is the biochemical

pathway in which CO2 is converted to a number of sugar phosphates

including the regeneration of the CO2 acceptor ribulose 1,5-

bisphosphate (RuBP) (Bassham et al., 1954). This biochemical pathway

is apparently present in all photosynthetic green plants (Bassham,











1979). The 13 enzyme-catalyzed reactions of this cycle occur in the

chloroplast. These reactions are catalyzed by 11 different enzymes,

as it is currently believed that the two aldolase reactions are

catalyzed by the same enzyme as are the two transketolase reactions

(Robinson and Walker, 1981; Latzko and Kelly, 1979). A non-

stoichiometric schematic diagram of the PCR cycle is presented in

Figure 1.1. Carbon enters the cycle when CO2 is combined with RuBP to

produce two three-carbon compounds. This carboxylation reaction is

catalyzed by the enzyme RuBP carboxylase (RuBPCase). Carbon passes

through the cycle to regenerate the CO2 acceptor. At two key points

in the cycle carbon compounds may be removed and either utilized in

starch synthesis or exported from the chloroplast to be metabolized in

the cytosol. Both of these pathways represent net carbon gain for the

photosynthetic cell. The ATP and NADPH consumed in the PCR cycle are

generated during photosynthetic electron transport, and production of

both requires light energy (Arnon et al., 1954). In addition to

combining with CO2, RuBP can combine with 02 in an oxygenation

reaction catalyzed by RuBP oxygenase (Bowes et al., 1971). The

carboxylation and oxygenation reactions are catalyzed by the same

enzyme RuBP carboxylase/oxygenase (Rubisco), which functions both as a

carboxylase and an oxygenase. Further discussion of this enzyme and

its regulation is presented later.


























Fig. 1.1. A non-stoichiometric diagram of the PCR cycle in
C3chloroplasts (after Bassham, 1979).
Abreviations: RuBP, ribulose 1,5-bisphosphate;
PGA, 3-phosphoglycerate; diPGA, 1,3-
diphosphoglycerate; GAP, glyceraldehyde 3-
phosphate; DHAP, dihydroxyacetone phosphate; FBP,
fructose 1,6-bisphosphate; F6P, fructose 6-
phosphate; E4P, erythrose 4-phosphate; SBP,
sedobeptulose 1,7-bisphosphate; S7P,
sedobeptulose 7-phosphate; X5P, xylulose 5-
phosphate; R5P, ribose 5-phosphate; Ru5P,
ribulose 5-phosphate. Sites of potential
metabolic regulation are: (1) RuBP carboxylase;
(2) GAP dehydrogenase; (3) fructose 1,6-
bisphosphatase; (4) sedobeptulose 1,7-
bisphosphatase; (5) phosphoribulokinase; (6)
pathway for starch synthesis in the chloroplast;
(7) phosphate translocator facilitating exchange
of certain metabolites between chloroplast and
cytosol.











Regulation of the PCR Cycle


Five of the PCR cycle enzymes have been identified as being

light-activated. These are RuBPCase, glyceraldehyde 3-phosphate (GAP)

dehydrogenase, fructose 1,6-bisphosphatase (FBPase), sedoheptulose

1,7-bisphosphatase (SBPase), and phosphoribulakinase (Buchanan, 1980).

These enzymes are located at positions 1 through 5, respectively, in

Figure 1.1. A number of roles for light in enzyme activation have

been proposed for PCR cycle enzymes. The chloroplast stroma becomes

more alkaline in the light, as compared to the dark, as a result of

proton transport across the thylakoid membranes (Heldt et al., 1973).

The change in pH favors carbon assimilation and is sufficient to

increase CO2 fixation from zero td high rates (Werden et al., 1975).

In exchange for protons moving out of the stroma, Mg2 ions act as

counter-ions and enter the strome thus raising the total Mg2+

concentration (Portis and Heldt, 1976), Portis et al. (1977) have

shown that the light-dependent changes in stromal Mg2 concentration

can control FBPase and SBPase activity. The activation of RuBPCase in

vitro has also been shown to require Mg2+ (Laing and Christeller,

1976; Lorimer et al., 1976). Other mechanisms of light-activation of

PCR cycle enzymes include the ferredoxin/thioredoxin system (Buchanan,

1980) and the light effect mediator (LEM) system (Anderson, 1979a).

These two mechanisms are similar in that both use light energy to

reduce disulfide (oxidized) containing compounds to the sulfhydry1

(reduced) state. In the reduced state they are able to activate

certain enzymes. One difference between the two mechanisms is that

the ferredoxin/thioredoxin system requires a soluble protein factor











whereas the LEM system does not. Very recent evidence from Salvucci

et al. (1985) has shown an apparently different chloroplast protein to

be involved in the activation of RuBPCase. Activation, while

suggested to be catalyzed by the protein, is regulated by the

energization status of the thylakoids (Salvucci et al., 1986b) and is

thus light-dependent. Light effects on some PCR cycle enzymes can

also be mediated by effectors such as ATP and NADPH, both of which are

generated in the light. The relative saturation of the adenylate pool

with phosphate (i.e., ATP levels relative to ADP and AMP levels)

regulates the activity of phosphoribulokinase and 3-phosphaglycerate

(PGA) kinase (Pradet and Raymond, 1983). Both of these enzymes

catalyze reactions requiring ATP (Figure 1.1). Also related to light

are electron transport rates. Dietz et al. (1984) report, however,

that even at high light intensity and saturating CO2, electron

transport rates do not play a direct role in limiting photosynthetic

rates.

Five potential control points associated with the PCR cycle have

been identified by Anderson (1979b) to be possible regulatory sites.

Two of these points are the export of the triose phosphates GAP and

dihydroxyacetone phosphate (DHAP) by the phosphate translocator and

the pathway from fructose 6-phosphate (F6P) to starch. These points

are discussed later. The remaining three points are the enzymes

RuBPCase, FBPase, and SBPase. Dietz and Heber (1984) found even at

high light and CO2, FBPase did not limit photosynthesis. Likewise,

Latzko and Kelly (1979) report all PCR cycle enzymes have been found

to possess activity sufficient to support observed rates of CO2











fixation with the exception of SBPase. Knowles (1985) has suggested

that transketolase may regulate carbon flow through the PCR cycle by

restricting regeneration of RuBP. Evidence from Dietz and Heber

(1984) also indicates that at some point during the regeneration of

RuBP from F6P and triose phosphate, CO2 fixation appears to be limited

under conditions of high CO2 and high light intensity. The activity

of RuBPCase has been suggested to be a limiting factor in

photosynthesis even at high CO2 concentrations (Dietz and Heber,

1984).

Individual reaction rates may be influenced by the accumulation

or depletion of reaction products and substrates. Some enzymes are

also affected by other chloroplast metabolites. These may be

modulated in a positive or negative manner by the binding of a

positive or negative allosteric effector at a site on the enzyme

distinct from the catalytically active site (Robinson and Walker,

1981).


Triose Phosphate Export and Starch Synthesis


Export of triose phosphate from the chloroplast via the phosphate

translocator and the synthesis of starch are processes which utilize

fixed carbon from the PCR cycle (Figure 1.1). The phosphate

translocator is the most powerful of several transport systems

facilitating exchange between the chloroplast and the cytosol (Heber

and Heldt, 1981). It is located at the inner chloroplast envelope

membrane and is capable of transporting triose phosphates (DHAP and

GAP), PGA, and inorganic phosphate (Pi) (Flugge and Heldt, 1984). The











stoichiometry of the transporter is such that export of one molecule

of triose phosphate or import of one molecule of PGA is accompanied by

the counter transport of one Pi (Heber and Heldt, 1981). Thus, the

total amount of phosphate in the stroma is kept constant. The export

of triose phosphate is the mechanism whereby carbon fixed in the

chloroplast can be transported to the cytosol, where it is metabolized

and subsequently translocated to other locations within the plant.

The availability of cytosolic Pi to be transported into the

chloroplast can affect photosynthesis and starch metabolism (Walker

and Sivak, 1986). Triose phosphate can be metabolized to sucrose and

Pi in the cytosal, with Pi becoming available for transport back into

the chloroplast in exchange for additional triose phosphate. Low

rates of sucrose synthesis during photosynthesis may result in

decreased cytosolic Pi for transport and therefore a build-up of some

PCR intermediates in the chloroplast (Huber and Israel, 1982).

Inorganic phosphate is ultimately required for formation of

chloroplastic sugar phosphates. When the rate of CO2 fixation is

greater than the availability of cytosolic Pi for chloroplast import,

triose phosphates will not be formed and PGA may accumulate (Heber and

Heldt, 1981). The subsequent high PGA/Pi ratio in the chloroplast has

been shown to result in starch synthesis (Preiss, 1982). Starch

synthesized in the chloroplast is usually degraded during the

following night period (Heber and Heldt, 1981), The possibility that

photosynthesis may be limited by (1) the inability of the plant to use

sucrose at a rate similar to the rate at which it is produced, or











(2) accumulation of starch in the chloroplast, is discussed in Chapter

II.


Photorespiratory Carbon Oxidation Cycle


Photorespiration may be defined as the oxygen and light-dependent

release of CO2 from certain plants (Somerville and Ogren, 1982). The

rate of photorespiration is often greater than the rate of dark

respiration (Zelitch, 1971). Summarized briefly, RuBP combines with

02 to produce 2-phosphoglycolate (P-GLY) and PGA in a reaction
catalyzed by Rubisco, the same enzyme responsible for catalyzing CO2

fixation in the PCR cycle (Bowes et al., 1971; Ogren and Bowes, 1971).

The P-GLY produced in the photorespiratory carbon oxidation (PCO)

cycle undergoes a series of reactions in the chloroplast, peroxisome

and the mitochondrion where photorespiratory CO2 is released (0gren,

1984; Chollet and Ogren, 1975). Th~e ratio of oxygenase to carboxylase

activity is dependent on the relative concentrations of 02 and CO2'

Rubisco kinetics (Laing et al., 1974) and temperature. Temperature

affects both the kinetics of Rubisco (Jordan and Ogren, 1984) and the

relative solubilities of 02 and CO2 (Jordan and Ogren, 1984; Ku and

Edwards, 1977). Figure 1.2 shows a non-stoichiometric schematic

diagram demonstrating the integration of the PCR and PC0 cycles by the

common enzyme Rubisco and the common substrate RuBP. Besides CO2, NH3

is also released in the PC0 cycle. Keys et al. (1978) have shown that

NH3, like CO2, is released in the mitochondrion during the conversion

of glycine to serine and MH3 is then reassimilated into glutamine in
the cytosal.



























Fig. 1.2. A non-stoichiometric diagram of the integration
of the PCR and PCO cycles in C chloroplasts
(after Lorimer, 1981). The initial reaction in
both cycles is catalyzed by Rubisco and utilizes
RuBP. Triose phosphate represents GAP and DHAP.
P-GLY is 2-phosphoglycolate. Other abbreviations
are as in Fig. 1.1.





















Ru5P RBP OFP-GLY



PCR PCO co,
CYCLE c CYCLE ~




PGA
TRIOSE
PHOSPHATE











Because the PCO cycle results in a loss of CO2 and energy it is

often regarded as a wasteful process. Much research has been aimed at

understanding photorespiration. Although various roles have been

proposed for the PCO cycle, it appears that other than the subsequent

metabolism of any P-GLY produced during RuBP oxygenase activity, there

is no known requirement for photorespiration (0gren, 1984). It has

been suggested that photorespiration is an unavoidable result of both

the nature of the Rubisco active site chemistry and the concentrations

of CO2 and 02 at the active site (Andrews and Lorimer, 1978). Mutants

of Arabidopsis lacking activity of different PCO cycle enzymes have

been found to have inhibited photosynthesis in air and are not viable

(Somerville, 1986). However, under conditions of high CO2 or low 02

normal photosynthesis was observed. This led Somerville and Ogren

(1982) to the conclusion that once carbon enters the PCO cycle it must

continue to be metabolized to prevent photosynthetic inhibition.

Thus, apparently the only way photorespiration can successfully be

reduced is by reducing the oxygenase/carboxylase activity ratio.


RuBP Carboxylase/0xygenase


Introduction


Under saturating light conditions, the amount and degree of

activation of RuBPCase regulates CO2 assimilation (Jensen and Bahr,

1977). This emphasizes the importance of Rubisco (used here Rubisco

refers to the enzyme RuBP carboxylase/oxygenase while RuBPCase and

RuBP oxygenase refer to the carboxylation and oxygenation activities,

respectively). This enzyme represents up to 65% of the total soluble











leaf protein (Ellis, 1979). It is located in the chloroplast stroma

in concentrations of approximately 0.4 to 0.5 mM (Jensen and Bahr,

1977). In higher plants the enzyme is composed of eight large

subunits (containing one active binding site per large subunit) and

eight small subunits whose function is not yet known (Miziorko and

Lorimer, 1983). Thus, the binding site concentration in the

chloroplast is approximately 3 to 4 mM. The prodigious amount of this

enzyme is countered by its slow rate of catalysis. The turnover

number of fumarase (a tricarboxylic acid cycle enzyme) is 50 times

greater than spinach RuBPCase (Seemann and Berry, 1982). Compared to

spinach carbonic anhydrase (Pocker and Miksch, 1978) spinach RuBPCase

is four orders of magnitude slower. Because of its central role in

CO2 assimilation and agricultural productivity, Rubisco has been
previously and is currently the object of intense investigation.


Reactions of Rubisco


The two competitive reactions catalyzed by Rubisco are the

carboxylation and the oxygenation of RuBP (Bowes et al., 1971; Ogren

and Bowes, 1971). As previously described, Oxygenation of RuBP is the

initial step in photorespiration while carboxylation of RuBP initiates

photosynthesis. The ratio of photosynthesis to photorespiration can

be described in terms of enzyme kinetics by the equation of Laing et

al. (1974),

v /vo = VcKoC/VogcO, [1.1]

where vc and vo are the rates of carboxylation and oxygenation, and

Vc, Vo, Kc and Ko are the Vma (theoretical maximum rate of reaction)











and Km (Michaelis constant) values for carboxylation and oxygenation,

respectively. The concentration of CO2 and 02 at the reaction site

are represented by C and 0. At atmospheric conditions of CO2 and 02

and 250C, the ratio of carboxylation/oxygenation is approximately 4/1

(0gren, 1984). In spite of much research to identify factors which

can alter the v /vo ratio, only the substitution of MnZ for Mg2

during the enzyme reaction and temperature have proven effective

(Ellis, 1979). The K (02) is decreased when activation and catalysis
involves Mn2+ rather than Mg2+ (Lorimer, 1981). Temperature has been

found to differentially affect Rubisco kinetics. This was shown using

the substrate specificity factor defined by Jordan and Ogren (1984),

VcKo/VoKc, [1.2]

where the variables are defined as in equation [1.1]. At given

concentrations of CO2 and 02 the specificity factor determines the

relative rates of carboxylation and oxygenation. A high value

indicates a high carboxylase to oxygenase ratio. As temperature

increases Vc, Vo, and Kc increase, however, Ko is not temperature

dependent. The overall effect of the temperature increase is a

decrease in the specificity factor (Jordan and Ogren, 1984). Jordan

and Ogren (1984) found the specificity factor of purified enzyme to

drop to less than one-third of its value as the temperature increased
from 5 to 400C. A similar response was observed by Brooks and

Farquhar (1985) using gas exchange techniques on intact leaves.










Activation of RuBP Carboxylase

Prior to becoming catalytically competent, RuBPCase undergoes an

activation process. The proposed model for activation involves CO2
and Mg2+ in the following manner (Lorimer et al., 1976; Laing and

Christeller, 1976),

E + ACO2 _p E ACO2 + M ,:E ACO2 M, [1.3]
(inactive) (inactive) (active)

where E is enzyme, ACO2 is activator CO2 (distinct from substrate

CO2), and M is a divalent metal cation, usually Mg2+ The formation
of the E C form (E CO2) is slow while formation of the E C M

form (E ACO2 M) is rapid. In intact chloroplasts activation has

been shown to depend an light and CO2 (Bahr and Jensen, 1978).
Activation and catalysis are separate phases in the RuBPCase reaction.

Lorimer et al. (1977) have described methods for the activation of the

enzyme in vitro. Activation of Rubisco is necessary for both

carboxylase and oxygenase activities (Lorimer, 1981). Inhibition of

RuBPCase by substrate RuBP (Jordan and Chollet, 1983; Laing and

Christeller, 1976) and by HCO3- (Machler and Nosberger, 1980) have

been reported in in vitro studies. In the light and in air-CO2

levels, RuBPCase (in vivo) is typically activated to a substantial

degree (Perchorowicz et al., 1981). Herein lies an enigma in that

conditions believed to exist in the stroma in the light (5 to 10 pM

CO2, 5 to 10 mM Mg2+ and pH 8.0) are not sufficient to activate
RuBPCase in vitro (Miziorko and Lorimer, 1983). A number of

metabolites have been shown to affect RuBPCase activation and/or

activity. This group of metabolites has been reported to include











NADPH, 6-phosphogluconate, ribose 5-phosphate, 3-phosphoglycerate,

fructose 1,6-bisphosphate and several other compounds (Jordan et al.,

1983; Badger and Lorimer, 1981; Hatch and Jensen, 1980; Lorimer et al.,

1978; Challet and Anderson, 1976; Chu and Bassham, 1975). These

effectors were suggested to act at allosteric regulatory sites (Chu and

Bassham, 1975) but more recent evidence indicates that the effectors

bind competitively at the same active site as does RuBP (Jordan et al.,

1983; Badger and Lorimer, 1981; McCurry et al., 1981). It has been

suggested that the concentration of these effectors in the stroma

(Lorimer et al., 1978) and the magnitude of their induced responses

(Akazawa, 1979) are inadequate to be physiologically important in vivo.

Somerville et al. (1982) have identified a mutant of Arabidopsis

thaliana in which RuBPCase is present in a nonactivatable form in vivo.

This implies that a factor necessary for activation is absent in the

mutant. Recently, Salvucci et al. (1985) have discovered two

polypeptides missing from the same Arabidopsis mutant and have linked

these polypeptides to a soluble chloroplast enzyme designated Rubisco

activase. These data suggest activase may be involved in light-

activation of RuBPCase in vivo and that activation is a catalyzed and

not a spontaneous process (Salvucci et al., 1986a).

An additional regulatory aspect of light on RuBPCase was

discovered independently by Vu et al. (1983), Mcnermitt et al. (1983),

and Ku et al. (1982). They found crude extracts of RuBPCase from

leaves collected in the dark to be less catalytically active than from

leaves collected in the light. This light/dark modulation has been

found in a number of different species from different photosynthetic











categories (Vu et al., 1984a). Restoration of catalytic ability by

ammonium sulfate fractionation of the crude extract of dark collected

leaves indicated the potential involvement of an inhibitory compound

(Vu et al., 1984b). Subsequent work by Seemann et al. (1985) and

Servaites (1985) have shown the inhibitor to be a phosphorylated

compound which binds to the active site of RuBPCase. Berry et al,

(1986) have identified the inhibitor as carboxyarabinitol 1-phosphate.


Non-Catalytic Roles of Rubisco


Due to its high concentration in the chloroplast, Rubisco has been

suggested to function as a storage protein (Huffaker and Miller, 1978;

Huffaker and Peterson, 1974). It also has been suggested to be a major

source of protein for animals for the same reason (Huffaker and

Peterson, 1974). Another function, that of a metabolite buffer, has

been proposed by Ashton (1982). The ability of compounds such as

fructose 1,6-bisphosphate (FBP) to bind to Rubisco and the relative

concentrations of FBP and Rubisco binding sites imply that greater than

98% of the FBP could theoretically be bound to Rubisco in illuminated

chloroplasts (Ashton, 1982). The physiological significance of this

effect is apparently speculative.


Experimental Approach


The CO2 in the atmosphere surrounding a leaf, or other

photosynthetic organ, is the source of carbon for terrestrial

photosynthesis. Manipulation of the CO2 concentration and observation

of the resulting photosynthetic responses provide insight into the











control and mechanism of CO2 fixation. This approach has been carried

out by a number of investigators (see reviews by Kimball, 1983; Lemon,

1983; Strain and Cure, 1985), not only to learn more about

photosynthesis but to study the effects of CO2 supply on yield and

what effect rising atmospheric levels of CO2 might have on vegetation.

In the experiments reported in the following chapters, soybeans were

grown in outdoor, naturally sunlit, controlled environment chambers,

in which CO2 concentration and dry bulb and dew point temperatures

were controlled to pre-selected values. Gas exchange techniques were

used to measure leaf and canopy photosynthetic rate response to

different CO2 concentrations. Leaf tissue samples were collected for

analysis of RuBP and RuBPCase, the substrate and enzyme involved in

CO2 fixation.
The purpose of the experiments described in the following

chapters was to examine the effects of CO2, both in the short-term and

the long-term, on the physiology and biochemistry of photosynthesis in

soybean. It was hypothesized that long-term exposure (exposure during

growth) to different CO2 concentrations could result in a change in

the capacity for photosynthesis in soybean. To examine this

hypothesis, specific objectives were:

(1) to determine the leaflet photosynthetic rate response to CO2

for soybeans grown at atmospheric and twice-atmospheric CO2

concentrations,

(2) to examine the effects of CO2 concentration (during short-
and long-term exposures) on RuBP levels,











(3) to examine the effects of CO2 concentration (during short-

and long-term exposures) on RuBPCase activity,

(4) to determine the effects of growth in subatmospheric and

superatmospheric concentrations of CO2 on kinetics of RuBPCase,

(5) to examine the effects of growth air temperature on RuBP

levels and RuBPCase activity, and

(6) to determine if either the RuBP level or RuBPCase activity

may be limiting to photosynthesis under high quantum flux density and

various CO2 concentrations.

In Chapter II, experiments are described in which soybeans were

grown at atmospheric and twice-atmospheric concentrations of CO2

Short-term exposures (1 br) to various CO2 concentrations allowed

leaflet photosynthetic rate response to CO2 to be measured as well as

RuBP levels and RuBPCase activities. In Chapter III, the effects of

growth in various subatmospheric and superatmospheric concentrations

of CO2 on canopy photosynthetic rates are described. The effects of

CO2 concentration on levels of RuBP and the activity and kinetics of
RuBPCase were also determined. The effects of three different

day/night air temperature regimes on canopy photosynthesis, RuBP

levels, and RuBPCase activity of soybean grown at atmospheric and

twice-atmospheric CO2 concentrations were investigated and are
discussed in Chapter IV. In Appendix A, the photosynthetic rate

response to light for leaflets and canopies is discussed. The effect

of leaf tissue sample size on the in vitro assay of RuBPCase activity

is discussed in Appendix B. Parameters from linear regression

analyses are tabulated in Appendix C.











The long-range goal of research such as described herein is to

reach a greater understanding of the fundamental process of

photosynthesis. This knowledge may hopefully contribute to

improvements in agricultural productivity.













CHAPTER II
TH7E EFFECTS OF SHORT-TERM EXPOSURES TO CO2 ON LEAF PHOTOSYNTHETIC
RATE, RuBP CARBOXYLASE ACTIVITY 2AND RuBP LEVEL


Introduction


That present day concentrations of atmospheric CO2 are limiting

to photosynthesis in Cg plants is widely recognized (Pearcy and

Bjorkman, 1983). It is well documented that photosynthetic rates

increase when C3 plants are exposed to higher than normal CO2

concentrations (Tolbert and Zelitch, 1983; Osmond et al., 1980; Allen,

1979). The increase in CO2 not only provides more substrate for
carbon assimilation, but also alters the photosynthetic/

photorespiration ratio by reducing photorespiration (0gren, 1984).

Investigations into the effects of CO2 on photosynthesis have

proceeded in several directions including long-term and short-term

exposures of plants to various CO2 concentrations. Often times long-
term exposure involves growing plants from seed to maturity at

elevated CO2 concentrations. Experiments of this type, in which

plants were grown at both atmospheric and elevated CO2 concentrations,

have yielded mixed results when leaf photosynthetic rates were

measured at the respective growth CO2 concentrations. In some

experiments, plants grown at atmospheric CO2 bad greater

photosynthetic rates than high CO2 grown plants (Peet et al., 1986;
von Caemmerer and Farquhar, 1984; Hofstra and Hesketh, 1975). In

other experiments the reverse was found, leaf photosynthetic rates











were greater in high CO2 grown plants when both were measured at their

growth CO2 concentration (Peet et al., 1986; Havelka et al., 1984;
Huber et al., 1984; Downton et al., 1980; Wong, 1979; Ho, 1977).

In other experiments in which various C3 species were grown

either from seed or for long periods of time at different CO2

concentrations, photosynthesis was measured over a range of CO2

concentrations. Results from these experiments suggest leaf

photosynthetic rate responses appear to fit into one of three

categories. These categories may in fact represent a continuum of

possible responses that depend on species, growth conditions, stage of

growth, and other factors, including experimental conditions. These

categories may be described as follows: (1) leaf photosynthetic rates

are greater in plants grown at higher rather than lower CO2 when

measured at all CO2 concentrations, (2) leaf photosynthetic rates are

greater in plants grown at lower rather than higher CO2 when measured

at all CO2 concentrations, and (3) leaf photosynthetic rates are

greater in plants grown at lower CO2 when measured at low CO2 but

higher in plants grown in high CO2 when measured at high CO .
Hicklenton and Jolliffe (1980a), working with young tomato plants,

found leaf photosynthetic rates, on a fresh weight basis, to be greater

in plants grown at 1000 pl CO2 1- than those grown at 300 pl CO2 1-

when measured over a range of CO2 concentrations. With older plants

the difference in photosynthetic rate response of the leaves was less.

Plants grown at 5000 pl CO2 1-1, however, were always found to have

leaf photosynthetic rates lower than 300 pl CO2 1-1 grown tomato

plants. Mlauney et al. (1979) grew soybeans at 330 and 630 pl CO2 1-










and found that when leaf photosynthetic rates were measured at the

lower CO2 concentration the rates were the same but at high CO2

concentration the rates were greater in the 630 pl CO2 1- grown

plants. The majority of the data in the literature shows leaf

photosynthetic rates, when expressed on an area basis, to be greater

in plants grown at lower rather than higher CO2 when measured over a

range of CO2 concentrations. This type of relationship has been

reported for experiments run under a variety of environmental

conditions with various species such as cotton (Delucia et al., 1985;

Mauney et al., 1979), Phaseolus vulgaris (Ehret and Jolliffe, 1985;

von Caemmerer and Farquhar, 1984), sunflower (Mauney et al., 1979),

tomato (Ho, 1977), Nerium 01eander and Larrea divaricata (Downton et

al., 1980), and waterbyacinth (Spencer and Bowes, 1986). Plants in

which the relative rates of leaf photosynthesis shift between low and

high CO2 grown plants, depending on the CO2 concentration during

measurement, make up the third response category. Examples of this

type of response have been reported with cotton (Wong, 1979), grape

(Kriedemann et al., 1976), and Amorphophallus konjac (Imai and

Coleman, 1983).

The different responses to CO2 may be explained in part by the

species chosen. However, the species alone cannot account for all of

the variation in photosynthetic rates since some species demonstrated

more than one type of response. For example, Ho (1977) and Hicklenton

and Jolliffe (1980a) both worked with tomato but observed different

types of photosynthetic behavior. While their experiments were

similar in regard to CO2 concentrations, differences existed in plant










age, growth photoperiod, growth temperature, and whether the plants

were grown from seed or were transferred to a particular CO2

concentration at an early age. In addition, Mauney et al. (1979)

obtained different results working with the same species under

apparently similar experimental conditions in two consecutive years.

Measurement protocol as well as growth and measurement conditions, and

possibly other factors, apparently influence leaf photosynthetic rate

response to CO2 (Woo and Wong, 1983).

There are numerous reports where long-term growth in high CO2

resulted in declining leaf photosynthetic rates, which ultimately

became lower than rates of plants maintained at atmospheric CO2

concentrations (Kramer, 1981). This reduction in photosynthetic rate

has sometimes been shown to be reversible when plants are switched

from high to low CO2 conditions (Sasek et al., 1985; Kriedemann and

Wong, 1984). Sasek et al. (1985) suggest that feedback inhibition of

photosynthesis by starch accumulation is responsible for these types

of observations, but according to Raven (1981), there is little

evidence for feedback inhibition of photosynthetic rates by

photosynthetic product accumulation. Growing plants in air enriched

with CO2 has often been shown to increase the amount of starch present

in the leaf (Cave et al., 1981; Mauney et al., 1979; Hofstra and

H~esketh, 1975; Madsen, 1968). These high starch levels have sometimes

been linked to chloroplast disruption (Cave et al., 1981; Carmi and

Shomer, 1979). Neales and Incol1 (1968) have reviewed reports that

suggest chloroplast disruption may include reduction of light incident

to the grana and interference with CO2! diffusion inside the leaf. The










relationship between high starch levels and changes in leaf

photosynthetic rates is equivocal. There are a number of examples

where high levels of starch have been correlated to reduced

photosynthetic rates (Delucia et al., 1985; Sasek et al., 1985; Azcon-

Bieto, 1983; Mauney et al., 1979; Nafziger and Koller, 1976; Hofstra

and Hesketh, 1975), and a number of examples where starch was not

observed to affect photosynthetic rates (Potter and Breen, 1980; Carmi

and Shamer, 1979; Mauney et al., 1979; Little and Loach, 1973). In

fact, Mauney et al. (1979) and Little and Loach (1973) showed positive

correlations between starch levels and leaf photosynthetic rates. It

has been suggested (Milford and Pearman, 1975) that starch may not

inhibit photosynthesis until a threshold level, which is not normally

attained under field conditions, is reached. Accumulation of starch

in the leaf may be related to, among other things, the assimilate

demand of the plant. The role of assimilate demand in leaf

photosynthesis has been reviewed by Neales and Incoll (1968) and

Geiger (1976). The possible mechanisms involved have been discussed

by Herold (1980). Most of the data in the literature suggest high

assimilate demand results in high photosynthetic rates (Geiger, 1976).

King et al. (1967), however, have reviewed several reports showing

both positive and negative influences on photosynthetic rates.

Positive correlations between leaf photosynthetic rates and increased

assimilate demand have been demonstrated in a variety of depodding and

leaf shading experiments (Wlittenbach, 1983; Clough et al., 1981;

Mondal et al., 1978; Thorne and Koller, 1974; King et al., 1967).










In addition to the above mentioned effects on photosynthesis, age

or the developmental stage of a plant may influence CO2 assimilation

rates. The podfilling stage in soybeans can be a period of high

photosynthetic activity (Enos et al., 1982; Hesketh et al., 1981;

Woodward and Rawson, 1978; Dornhoff and Shibles, 1970), however,

Sinclair (1980) has pointed out that there are substantial

differences, among cultivars, in the ability to maintain high

photosynthetic rates late in the season. Differences in RuBPCase

activity in soybean have been noted between expanding and mature

leaves (Vu et al., 1983). Changes in the relative photosynthetic rate

responses to CO2 in atmospheric concentrations and high CO2 grown

leaves have been shown to occur as plants become older (Peet et al.,

1986; Ehret and Jolliffe, 1985; Hicklenton and Jolliffe, 1980a).

Baysdorfer and Bassham (1985) have found that as alfalfa progressed

from seedling to mature crop, photosynthesis shifted from being

source-limited to sink-limited.

Different leaf photosynthetic rate responses to CO2 have been

obtained with a variety of species and under wide ranging

environmental and experimental conditions,which may account for much

of the variation in results. Additionally, the diversity of

interpretation of the results implies that regulation of

photosynthesis is not, as yet, well understood. A confounding

possibility is the suggestion (Maggs, 1964) that leaves usually

operate below their maximum level.

The objectives of this study were to measure leaflet

photosynthetic rate response to CO2 for soybean grown at atmospheric











and twice atmospheric concentrations of CO2. In addition, the effects

of the two CO2 growth treatments and short-term response to a range of

CO2 concentrations on the activity of RuBPCase and the level of RuBP

were investigated. The photosynthetic rate response to CO2 and the

response of RuBPCase and RuBP to CO2 were examined to determine what

role the biochemical parameters may have in regulating leaflet

photosynthesis under conditions of high quantum flux density and

various concentrations of CO2.


Materials and Methods


Plant Material and Growth Conditions


Soybeans (Glycine max L. Merr. cv Bragg) were planted in outdoor,

computer-managed, environmentally controlled plant growth chambers

located at the University of Florida's Irrigation Research and

Education Park, in Gainesville, on 30 Aug. 1983. The upper part of

each growth chamber was constructed of clear acrylic and polyester

film, allowing the plants to receive 88% of the natural solar

irradiation. The chamber tops measured 2 m by 1 m in cross section by

1.5 m in height. The lower steel part of the chamber was of the same

cross section and 1 m in depth. It was filled with a reconstructed

Arredondo fine sand profile, which was sealed from the upper aerial

part following seedling emergence to prevent the mixing of the soil

and aerial atmospheres. The dry bulb temperature of the chamber

atmosphere was controlled to 310C during the day and to 230C at night.

The dewpoint temperature was controlled to 16oC. The CO2

concentration of the chamber atmosphere was controlled, from the date











of planting until final harvest, to either 330 pl CO2 11 or 660 #1

CO2 1^1. A general description of growth chamber operation may be
found in Jones et al. (1985b), while Jones et al. (1984b) provide a

detailed description of the growth chamber design and the computer

control system.

For the experiments described here, two of six plant growth

chambers were used. Within each of these plant growth chambers were

placed two leaf chambers, each capable of accommodating one fully

expanded soybean leaflet. The leaf chambers were constructed of an

acrylic frame covered with a clear polyester film which transmitted

88% of the incident solar radiation. The internal volume of each leaf

chamber was 0.375 liters. Chilled water, circulating through the

chamber frame, maintained the temperature of the air in the leaf

chamber close to the air temperature in the plant chamber. The leaf

chambers were controlled by a computer system similar to but separate

from the system controlling the plant chambers.

The origin of the air circulating through the leaf chamber system

was the respective plant chamber. Air was circulated, by diaphragm

pumps, from the plant chamber through homogenizing containers and then

through the leaf chamber system. The leaf chamber system consisted of

two IR gas analyzers (Beckman, model 865), two dewpoint hygrometers

(General Eastern, model 1100 DP), one thermocouple (0.25 mm diameter)

placed in each leaf chamber to monitor air temperature and three

thermocouples (0.076 mm diameter) wired in parallel and placed in

contact with the abaxial leaflet surface to monitor leaflet

temperature. Each IR gas analyzer and hygrometer was dedicated to two










two leaf chambers. Air lines were heated and insulated to help

prevent condensation. Air flow rates through the leaf chambers were

between 0.318 and 0.468 m3 per hour (5.3 and 7.8 liters per minute).

The dry bulb and dewpoint temperatures and the CO2 concentration in

the leaf chambers were similar to conditions in the respective plant

chambers.

The plants completed germination approximately 4 days after

planting (4 DAP). On October 18, 49 DAP, the plants were thinned to a

density of 30 plants per m2. Throughout the experiments, shadecloth

(approximately 50% shading) was attached to the outside of the plant

chamber at a height equal to the top of the canopy to approximate a

closed canopy and reduce the solar irradiance on the sides of the

canopy.


CO2 Concentration Experiments

A series of short-term experiments were performed from October 25

to October 30 (56 to 61 DAP), during which time all plants were at the

beginning seed or R5 stage of development (Fehr and Caviness, 1977).

During this period the CO2 concentrations in the plant chamber, and

thus also in the leaf chamber, were controlled to various levels

different than the normal CO2 growth concentrations. These additional

CO2 concentrations (110, 220, 330, 550, 660, and 880 pl CO2 1-1) were
imposed at midday and were maintained for approximately 1 hour.

During these exposure times photosynthetic rate data were collected,

and immediately following these measurements leaf tissue samples were

rapidly collected for subsequent laboratory analysis. Supplementary










CO2 concentrations (160, 440, and 990 lil CO2 1 1) were imposed in the

plant and leaf chambers after plant tissue sampling to expand the CO2

range over which photosynthetic rate measurements were collected. In

all cases, when the CO2 was changed from one concentration to another,

steady state conditions were allowed to return inside the plant and

leaf chamber prior to collecting data for analysis. This always

represented a period of not less than 10 minutes. All data collected

during these CO2 experiments were obtained between 1100 and 1430

Eastern Standard Time (EST). During each day this was a cloud-free

high irradiance period when the quantum flux density (400 to 700 nm)

was measured to be at least 1000 Clmol quanta m2s- at the leaf

level, which in these experiments was saturating for leaflet

photosynthesis. Quantum flux density was measured with a quantum

sensor (Li-Cor, model LI-190S) and corrections were made for the

transmittance through the plant and leaf chambers.


Leaf Photosynthesis Measurements


The leaf chamber system was used to collect leaf gas exchange

data, at 5-minute intervals, continuously during the photosynthesis

experiments. Measurements of CO2 concentration and dewpoint

temperatures were made on air entering and leaving the leaf chambers.
In addition, measurements were made of the dry bulb temperature of the

air inside the leaf chambers, the leaflet temperature, and the air

flow rate. The net photosynthetic rate (Pn) of the leaflet was

calculated using the following equation from Gaastra (1959),










C. Cou
Pn = n ou flow rate, [2.1]


where Cin and Cou are the CO2 concentrations of the air stream

entering and leaving the leaf chamber, respectively, A is the area of

the leaflet, and flow rate is the rate of the air-stream flowing

through the leaf chamber system.

The concentration of CO2 in the air in the leaf intercellular

space (Ci), was calculated based on the method of Farquhar and Sharkey

(1982),

Ci = Ca (Pn Eleaf 1.6), [2.2]


where Ca is the CO2 concentration of the air ambient to the leaflet,

Rleaf is the total leaf resistance to diffusion of water vapor and 1.6
is the ratio of the binary diffusivities of water vapor/air and

002/air (Farquhar and Sharkey, 1982). The product of Rleaf *16i
the leaf resistance to diffusion of CO2' This method of estimating Ci

was found by Sharkey et al. (1982) to be in close agreement with

measured values of the intercellular concentration of CO2. The

calculation of Rleaf Was based on the equations of Neumann and

Thurtell (1972), using measured values of dewpoint and dry bulb air

temperatures, air flow rates, and leaflet area. Photosynthetic rates

for leaflets grown at each CO2 concentration are the pooled values
from two leaflets.


Plant Sampling Procedure


Leaf tissue samples were collected via access doors located on

the rear (north side) of the plant chambers. Inside each door was










positioned a curtain of polyester film that reduced disturbance of the

atmosphere within the plant chamber during plant tissue sampling.

This procedure was found to result in small and only brief

disturbances of the atmosphere during sampling events. The plant

tissue collected was from the upper canopy and consisted of 20 to 25

fully expanded, non-shaded, and visibly healthy leaflets. These

leaflets were selected in part based on visual similarity to the

leaflets used in the leaf chambers for photosynthetic rate

measurements. Leaflet lamina were removed at the petiolule and

immediately plunged into liquid N2. This process was completed in

approximately 1 second. The leaf tissue was then ground in a liquid

N2 chilled mortar and the resulting leaf powder was stored in a

container in liquid N'2. The leaf tissue was kept at liquid N2

temperature from the time of harvesting until laboratory analysis

which occurred at a later date. Vu et al. (1984a) have shown this

method to preserve enzymatic activity for prolonged periods of time.


RuBP Carboxylase Assay


A quantity of frozen leaf powder (100 to 170 mg dry weight) was

removed from liquid N2 storage and placed in a pre-chilled Ten Broeck

tissue homogenizer. Added to the leaf powder was 10 ml of extraction

buffer consisting of 50 mMI Tris (pH 8.5), 5mM DTT, 0.1 mM EDTA, and

1.5% (w/v) PVP-40. The leaf tissue was homogenized for approximately

60 seconds at 00C, at which point an aliquot of the homogenate was

reserved for chlorophyll determination, and the remainder was

centrifuged at 12,000 g for 3 minutes. The supernatant of the crude










extract was either assayed immediately or else following a 5-minute

activation period at 300C in 10 mM NaHCO3 and 10 mM MgC12. Assays
were carried out in triplicate at 300C, with continuous shaking (125

strokes min-1), in 5-m1 glass vials with screw-on septum caps. The

assay buffer consisted of 50 mM Tris (pH 8.5), 5 mM DTT, 0.1 mM EDTA,

10 mM MgC12, 0.5 mM RuBP, and 20 mMl NaHIi4CO2 (7.54 GBq mol-1). The

sealed vials were purged with N2 for 10 minutes prior to the addition

of the Tris buffer and the NaH14CO2. The total assay volume was 1 mL.

Assays of enzyme activity were initiated with the injection, through

the septum cap, of 0.1 ml of either nonactivated or HCO3 /Mg2+

activated crude extract to determine initial or total activity,

respectively (Perchorowicz et al., 1981). Assays were terminated

after 45 seconds with the injection of 0.1 ml of 6N HC1. A 0.9 ml

aliquot of the assay mixture was transferred to a 20-m1 glass

scintillation vial which was placed on a warm heating plate under an

air-stream, and remained there until the contents were dried. W~hen

dry, 2.5 ml of water and 5 ml of scintillation cocktail were added to

the vials and acid-stable 14C products were determined by liquid

scintillation spectrometry.


RuBP Determination


The determination of RuBP was based on the method of Latzko and

Gibbs (1974) with modifications by Vu et al. (1983). A quantity of

frozen leaf powder (85 to 150 mg dry weight) was removed from liquid

N2 storage and placed in a pre-chilled Ten Broeck tissue homogenizer.
Added to the leaf powder was 10 m1 of 0.5N HC1 at 00C. The leaf










tissue was homogenized for approximately 60 seconds at 00C, an aliquot

was reserved for pheophytin determination and the remainder was then

centrifuged at 12,000 g for 5 minutes. To 5 ml of the supernatant was

added 0.75 ml 2M Tris base and 0.44 ml 4N ROH. The neutralized

supernatant (pH 8.3) was then stored on ice. Assays were carried out

in triplicate in 5-m1 glass vials with screw-on septum caps at 300C

with continuous shaking (125 strokes min-1). The assay buffer

consisted of 50 mM Tris (pH 8.5), 5 mM DTT, 10 mM MgC12, 20 nmF

NaH14CO2 (7.541 GBq mol-1), and 0.5 ml of the neutralized leaf extract

supernatant. The total assay volume was 1 ml. The RuBP determination

was initiated with the injection of 0.1 ml of activated crystallized

RuBPCase from tobacco (equivalent to approximately 55 pg protein).

The tobacco enzyme had been prepared previously according to the

method of Kung et al. (1980), and was reactivated by dissolving the

enzyme in 50 mM Tris (pH 8.5), 10 mM MgC12, 10 mM NaHCO2 and 100 mM
NaC1 and incubating for 25 minutes at 50aC (Kung et al., 1980). After

60 minutes the assay was terminated with the injection of 0.1 ml 6N

HC1. An aliquot (0.9 ml) of the assay mixture was transferred to a

20-m1 glass scintillation vial which was dried on a warm heating plate

under an air-stream. When dried, 2.5 ml water and 5 ml scintillation

cocktail were added to each vial and acid-stable 14C products were

determined by liquid scintillation spectrometry.


Chlorophyll, Protein, and Specific Leaf Weight Determinations


Chlorophyll determinations were performed on sample aliquots

reserved during the RuBPCase assays. Chlorophyll was extracted in 80%










acetone and calculations were by the method of Arnon (1949). The

chlorophyll in sample aliquots reserved during RuBP determinations was

converted to pheophytin during extraction with acid, therefore the

original chlorophyll concentration was determined using the method of

Vernon (1960). In addition, chlorophyll was determined in leaf disks

of known surface area, collected and assayed at the same time that

leaf tissue was collected for RuBPCase and RuBP assays. Soluble

protein determinations were performed on aliquots of the same

supernatant from the crude extracts used to initiate the RuBPCase

assays. The dye binding spectrophotometric method of Bradford (1976)

was used. Protein standards were prepared from crystallized and

lyophilized BSA (bovine serum albumin) dissolved in the same buffer

used in extraction of RuBPCase from leaf tissue. Specific leaf weight

(SLW) was determined by drying, freshly harvested leaves of known

surface area, collected 49 DAP from the unshaded upper canopy, to

constant weight in a 7000 oven.


Pod Load and Leaf Area Measurements


On October 18, 1983 (49 DAP), 12 plants were removed from each

chamber for determination of pod weight (grams dry weight) and leaf

area. To measure pod weight, all viable pods were removed from the

plants and dried to constant weight in a 70"C oven. To determine leaf

area, all green leaves were removed from the plants and the surface

area (one side of each leaf) was measured with an area meter (L~ambda,

model LI 3000).











Analysis of Statistical Significance


To determine the statistical significance of experimental

results, simple linear regressions were performed using the short-term

CO2 concentrations to which plants were exposed as regressor.

Comparisons of slopes and intercepts between CO2 growth treatments,

and comparison of slopes to zero, were used as tests to determine if

there were significant differences between treatments and also if

there were significant responses to the various short-term CO2

concentrations. In addition to simple linear regression, a quadratic

regression was also performed on the RuBP data. Both types of

regressions gave very similar results regarding the significance of

RuBP response to CO2. In cases where data were collected following

growth at the two CO2 treatments (but prior to exposure to the various

short-term CO2 concentrations) t-tests were used to determine the

significance of the growth CO2 treatments on certain plant

characteristics. In all cases, all tests of significance were made at

the 5% level unless otherwise noted. Regression parameters are

tabulated in Appendix C.


Results


Response of Leaf Characteristics to CO,

Soybean plants were grown from seed at atmospheric and twice

atmospheric CO2 concentrations. As shown in Table 2.1, specific leaf

weight increased significantly at elevated CO2. Chlorophyll and total

soluble protein (expressed on a leaf area basis) were not

significantly different in the two CO2 treatments. Pod weight, leaf











Table 2.1. Effects of growth CO2coenrioonla
characteristics. Specific leaf weight determined on
samples collected 49 DAP. All other samples collected
56 to 60 DAP. Mean values + SD are presented.



Growth CO2 concentration

pl CO2 11

-- 330 - 660 --

Specific leaf weight
g dr wt.m-217.00 + 0.10 23.70 + 0.04


Chlorophyll2
-2 0.475 f 0.005 0.520 + 0.017
gm


Total soluble protein
-2 4.03 c 0.04 4.26 r 0.14
gm


Protein/Chlorophyll
ratio 8.5 8.2


1
2t = 67.03, df = 2
3t = 3.39, df = 2
t = 2.06, df = 2


Ho:M660-u330 = 0
Ho:p660-u330 = 0
Ho:u660-u330 = 0


rejected at 5% level.
not rejected at 5% level.
not rejected at 5% level.











area per plant, and the ratio of pods to leaf area all increased with

CO2 (Table 2.2), however, the differences in pod weight and leaf area
were not significant. These morphological and biochemical differences

reflect the effects of increased C02 concentration which also affects

leaf photosynthetic rate response.


Leaf Photosynthetic Rate


The effects of C02 concentration on leaf photosynthesis were

examined following long-term and short-term exposures to different CO2

concentrations. Intercellular CO2 concentrations (Ci) were calculated

as the CO2 concentration ambient to the leaf (Ca) was varied from 80

to 1000 ill CO2 1-1. In Figure 2.1, Ci is plotted against Ca for

leaves grown at both CO2 concentrations. Linear regression analysis

of the data yields slopes, and hence Ci/Ca ratios, of 0.72 (r=0.985)

and 0.55 (r=0.965), respectively, for the 330 and 660 pl CO2 1-1 grown

leaves. The difference in the Ci/Ca ratio was found to be

significant. Because the Ci/Ca ratio was lower in high CO2 grown

leaves, the Ci calculated at any ambient CO2 concentration was greater

in leaves grown at 330 ul CO2 1-

Leaf photosynthetic rates were greater in high CO2 grown plants

at all CO2 concentrations in which they were measured (Figure 2.2).

When plotted against Ci, plants grown at high CO2 had greater maximum

leaf photosynthetic rates. Plotting leaf photosynthetic rate against

Ci allows evaluation of the CO2 assimilation rate response to CO2

concentration independent of stomatal influences. Each point in

Figure 2.2 represents one photosynthetic rate measurement made at a











Table 2.2. Effects of growth CO2 concentration on pod weight and
total green leaf area per plant on samples collected 49
DAP. Mean value + SD are presented for leaf area. Pod
weight represents total dry weight of pods divided by the
number of plants.



Growth CO2 concentration

pl CO2 1-1


---660 - --660/330 ---


----- 330 ---


0.084 + 0.023


Pod weight

g dry wt. plant-1


0.125 +_ 0.052


1.48


Leaf area2

m2 plant-1


0.1475 + 0.0546


0.1855 + 0.0432




0.674


Pod/Leaf area

gm


0.569


1.18


1
2t = 1.23, df = 4
t = 1.89, df = 32


Ho:W660-9330 = 0
Ho:W660-3330 = 0


not rejected at 5% level.
not rejected at 5% level.




























Fig 2.1, Intercellular CO2 concentration versus ambient
CO concentration for leaves grown at two
CO2 cof centrations. In leaves grown at 330 ul
CO 1~ (+), Ci/Ca = 0172 (r = 0.985). In leaves
grown at 660 pl CO2 1 (a), Ci/Ca = 0.55 (r =
0.965).
























1UUU

GROWTH COg CONCENTRATION
o + 330 atCO2I`
~ 8001 660 AlCO2I~





Zo ++









8 200 400 600 80 10888
CO, CONCENTRATION (41I C02 I 1)


























Fig. 2.2. Leaf photosynthetic rate versus intercellular CO2
co centration for leaves ~rown at 330 pl CO2
1 (A) and 660 pl CO2 1 (B). Each data point
represents one measurement made at 5 minute
intervals. The solid curves were generated by
non-linear regression analysis of the data. The
regression model was P=Pmax*Ci/(KCi+Ci)+R; where
P is leaf net photosynthetic rate, Pmax is the
maximum value of P-R, Ci is intercellular CO2
KCi is the Michaelis constant for Ci and R is the
estima ed respiration rate at Ci=0. For 330 pl
CO, 1 grown leaves: Pma =55.5, KCi=206 and R=
-11.0. For 660 pl CO2 1 grown leaves:
Pmax=96.1, KCi-22 and R=-13.8. Pmax andlR are
in pmol CO2 m s and KCi is in pl CO3 1 .o6
Photosynthtic measurements were made 5 o6
DAP.

















GROWTH CO, CONCENTRATION A







4 ++


80-


60-


40-




201


I


I I


) 200 400 600 800
INTERCELLULAR CO2! CONCENTRATION (pl CO, I`')










5-minute interval, and are the pooled values from two leaflets at each

CO2 treatment. The highest rate measured for a leaf grown at 330 pl

CO2 1- was 41 umol CO2! m- s-1, and for a 660 pl CO2 1- grown leaf
69 pmol CO2 -2 s- (Figure 2.2, A and B). At low Ci, high CO2 grown

leaves showed greater rate response to increases in CO2. The solid

curves in Figure 2.2 (A and B) were generated by non-linear regression

analysis of the data points.

The data in Figure 2.2 were divided into 10 discrete groups based

on CO2 concentration, and the mean Ci and mean leaf photosynthetic

rate were calculated. The Ci values in each group varied less than 5%

from the mean. These means are plotted in Figure 2.3 (A). Comparison

of Figure 2.2 (A and B) with Figure 2.3 (A), indicates that plotting

the means of the data did not affect the relationship between

photosynthetic rates nor the relationship between photosynthetic rate

and Ci. Since there was a difference in SLW between leaves grown at

the two CO2 concentrations, mean leaf photosynthetic rates were also

calculated based on dried leaf weight and are plotted against Ci in

Figure 2.3 (B). The difference in photosynthetic rates between high

CO2 and atmospheric CO2 concentration grown leaves was less when

expressed on a dry weight basis, particularly at lower CO2

concentrations. However, leaf photosynthetic rates were still greater

in the high CO2 grown leaves at all CO2 concentrations, suggesting

that the increase in SLW in the high CO2 grown leaves did not account

for all of the increase in leaf photosynthetic rate. Arrows in Figure

2.3 (A and B) indicate the mean photosynthetic rates obtained when

measured at the respective ambient growth CO2 concentrations. The



























Fig. 2.3. Mean leaf photosynthetic rate versus mean
intercellular 000 cone ntration for leaves _1
grown at 330 pl 602 1 (*) and 660 ill CO2 1
(A). Photosynthetic rates are expressed on a
leaf area basis (A) and a leaf dry weight basis
(B). Data are from Figure 2.2. Arrows indicate
mean photosynthetic rates measured at the
respective ambient CO2 growth concentrations.
Vertical lines represent + SD.











photosynthetic rates of leaves grown and measured at 660 pl CO2 1-

were greater than in leaves grown and measured at 330 #1 CO2 11


RuBP Carboxylase Activity


Assays of RuBPCase activity were performed on leaf tissue sampled

from plants at their growth CO2 concentration and also following

short-term exposure to a range of CO2 concentrations. Both initial

(nonactivated) and total (HCO3 /Mg2+ activated) activities were

assayed in samples (collected under high light conditions) that were

extracted without added Mg2+, The results of these assays are shown

in Figure 2.4 (A and B). Each data point is the mean of triplicate

assays. Enzyme activity in Figure 2.4 is expressed on a leaf area

basis so a more meaningful comparison can be made with leaf

photosynthetic rates. Figure 2.4 (A) shows that initial activity of

RuBPCase did not significantly respond to short-term exposure to

different CO2 concentrations. There was no significant difference

between the two growth CO2 concentrations. Total activity was also

independent of short-term CO2 concentrations [Figure 2.4 (B)]. It

also did not significantly respond to increases in CO2. The catalytic

rates were quite similar (not significantly different) between the two

growth CO2 concentrations whether measured as initial or total enzyme

activity at all CO2 concentrations. On a leaf area basis there was
less than a 5% difference between the activities (both initial and

total) of RuBPCase when sampled at the respective growth CO2

concentrations. Initial and total enzyme activities were also

calculated on a chlorophyll basis and these data are presented in
























Fig. 2.4. Leaf RuBPCase activity versus CO, concentration
for samples collected following I hour exposures
to six different CO2 icnrtos Plantilwere
gron a 30 p C2 1 (e) or 660 pl CO,
(A). Both initial activity (A) and total
activity (B) were assayed. Mean values of
triplicate assays are presented.


























INITIAL ACTIVITY

GROWTH COp CONCENTRATION
4 330 p.I CO2 I`
A 660 pl CO, I'

















TOTAL ACTIVITY

GROWTH CO2 CONCENTRATION
S330 Aul CO2 ~
A 660 st CO2 `


120 -




;m80-




40 -


O


--
o 160-


-

S120-




m 80-


1000


O 2d00


400 600 800


CONCENTRATION (p.1 CO, 1' )










Table 2.3. Due to the difference in the amount of chlorophyll per

unit leaf area, the relative enzyme activities shift somewhat when

expressed on a different basis. When expressed on a chlorophyll basis,

leaves grown and sampled at 330 pl CO2 11 had initial and total

activities 10 and 13% greater than leaves grown and sampled at 660 Cll

CO2-1. However, the response to CO2 of both initial and total
RuBPCase activities was not significantly different between the two

growth CO2 treatments. The activation state of RuBPCase invivo may
be estimated by initial activity/total activity # 100%. As would be

expected based on the independence of initial and total enzyme

activities from CO2 concentration (Figure 2.4), the activation was

also independent of CO2 (Figure 2.5). Th~e response of activation to

CO2 concentration was insignificant (at the 1% level) for both CO2
treatments. There was no significant effect of exposure to different

short-term CO2 concentrations or to long-term growth CO2 concentration
on activation.


RuBP Levels


Steady state RuBP levels were measured in the same tissue samples

collected for RuBP carboxylase assays. Samples were collected at

growth CO2 concentrations and also following the short-term exposures

to the various 002 concentrations. RuBP data are reported on a leaf
area basis in Figure 2.6. Each data point represents the mean of

triplicate assays. There was a significant response of RuBP levels to

CO2 concentration. In both growth CO2 treatments, below a CO2
concentration of 330 ul CO2 1-1, RuBP levels increased as CO2











Table 2.3. Effects of two growth CO2 cnetain nRB~s
activity in leaves collected following 1-hour exposures
to six different CO, concentrations. Both initial and
total enzyme activity were assayed. Mean values of
triplicate assays + SD are presented.



RuBP carbosylase activity


Ambient CO2

Concentration

plC -1

110

220

330

550

660

880


330 pul CO21-1


660 ul CO2

mg Chl- hr-

Initial

651 &26

627 & 18

847 f 9

614 & 18

720 1 19

624 & 18


ilmol CO2

Total

731 & 53

880 & 6

890 & 24

771 & 29

800 & 16

809 & 17


Initial

700 1 13

803 1 20

803 & 28

665 & 5

706 1 6

715 &7


Total

726 & 10

793 & 5

903 A 14

735 & 32

771 18

757 I 6


























Fig. 2.5. Activation status of RuBPCase versus CO
concentration for leaves grown at 330 ii CO2 1-
(*) or 660 pl CO2 1~ (r). Mean values of
triplicate assays are presented. Percent
activation calculated from data in Figure 2.4.























S80


F o




> 40
O

1 20


CO, CONCENTRATION (.i CO2 I ')



























Fig. 2.6. Leaf RuBP levels versus CO2 concentration in
samples collected following 1-hour exposures to
six different CO, cone ntrations. Leaves we e
grown at 330 ul CO2 1~ () or 660 pl CO21
(r) Mean values of triplicate as says are
presented.

















100


80-


S60-


S40-

GROWTH COe CONCENTRATION
201 330 41 COe I''
A660 41I CO, I-'

O 200 400 600 800 1000
CO, CONCENTRATION (yl C02 l"')










decreased. Above this concentration RuBP was rather insensitive to

CO2. The levels of RuBP were higher in leaves grown at high CO2

regardless of the different short-term CO2 concentrations. The RuBP

levels showed significant responses to both the short-term CO2

concentrations and to growth CO2 treatment. Due to the difference in

chlorophyll content, RuBP levels were also calculated on a chlorophyll

basis. The concentration of RuBP in the chloroplast stroma was

calculated assuming RuBP is present only in the chloroplast (Heber,

1974) and that the stromal volume is equivalent to 25 pl mg

chlorophyll-1 (Sicher and Jensen, 1979). These data are shown in

Table 2.4. As was the case on an area basis, the RuBP levels on a

chlorophyll basis were significantly higher in the high CO2 grown

leaves. The RuBP level decreased significantly with increasing CO2

concentration when expressed on either a chloropyll basis or as the

stromal concentration of RuBP.


Discussion


Soybean leaflet photosynthetic rates increased with increasing

CO2 concentration in plants grown at both atmospheric and twice

atmospheric CO2 concentrations. There are relatively few examples of

high CO2 grown plants having greater leaf photosynthetic rates than

atmospheric CO2 grown plants, when both are measured over the same

range of CO2 concentration. However, at all CO2 concentrations in

which photosynthesis was measured, rates were greater in leaflets

grown at the higher CO2 concentration (Figures 2.2 and 2.3). Thus,
these results agree with those of Hlicklenton and Jolliffe (1980a) and





Tabl 2.. Efecs o grwthCOconcentration on RuBP levels in
Tale24.Efet o rotleaves collected following 1-hour exposures to six
different CO2 concentrations. Levels of RuPB are
expressed both on a chlorophyll basis and a chloroplast
concentration basis, Mean values of triplicate assays
+ SD are presented.


RuBP


Ambient
CO
Concen rat on
ill CO2 1

110

220

330

550

660

880


Growth CO, Conce ntraton

-1660 330
mgChl


330
nmol

141 & 2

128 1

108 f 5

115 + 1

109 +1

113 + 1


158 &

186 &

106 +

103 &

135 &

123 +


+ 0.08

+ 0.04

f 0.20

f 0.04

+ 0.04

+ 0.04











are similar to the results of Mauney et al. (1979) with soybean. The

implication of this type of relationship between leaf photosynthesis

and CO2 with regard to control of leaf photosynthetic rate is

discussed below.

Intercellular CO2 concentrations were calculated and leaflet

photosynthetic rates were then plotted against Ci. Figure 2.1 shows

the relationship between Ci and Ca to be linear and therefore the

ratio of Ci/Ca was found to be constant across a range of CO2

concentrations from 80 to 1000 pl CO2 1-1. Whereas Coudriaan and van

Laar (1978) found Ci/Ca to be constant in Phaseolus vulgaris only when

Ca was below and not above 300 pl CO2 1-1, the results reported here

are consistent with those of most other researchers (Spencer and

Bowes, 1986; Sharkey et al., 1982; Wong et al., 1979). While the

Ci/Ca ratios were constant at all CO2 concentrations, the ratio was

significantly lower (by 23%) in plants grown at higher CO2. This
could be due to the higher photosynthetic rates or differential

stomatal response in the high CO2 grown leaves. Either factor might

lower the Ci. However, another factor may be responsible for the

Ci/Ca ratio difference. Growth of soybean at elevated CO2

concentrations can result in thicker leaves with more palisade cells

per unit leaf area (J.C.V. Vu, personal communication; Thomas and

Harvey, 1983), and therefore an increased mesophyll cell surface

area/external leaf surface area ratio. An increased internal surface

area would permit greater uptake of CO2 from the leaf intercellular

air spaces and result in a lower Ci value. Nobel et al. (1975) and

Nobel (1980) have discussed the influence of several environmental











variables, other than CO2, on the internal to external surface area

ratio. The effects of increased mesophyll cell surface area on leaf

photosynthesis are discussed below. Wong et al. (1985) and Spencer

and Bowes (1986) did not find a difference in Ci/Ca ratios with

different growth CO2 concentrations.

In plants grown at both atmospheric and elevated CO2, RuBPCase
activity (on a chlorophyll basis) was not significantly greater in the

leaves grown and sampled at the lower rather than the higher CO2

concentration (Table 2.3). An apparently significant effect of CO2

has been reported in the literature for a variety of C3 plants

including cotton (Wong, 1979), Nerium 01eander, and Atriplex

triangiularis (Downton et al., 1980), Phaseolus (von Caemmerer and

Farquhar, 1984; Porter and Grodzinski, 1984), soybean (Vu et al.,

1983), tomato (Hicklenton and Jolliffe, 1980a), and waterbyacinth

(Spencer and Bowes, 1986). When RuPBCase activity is expressed on a

leaf area basis (Figure 2.4) there is also no significant difference

between CO2 treatments in the enzyme response to CO2 concentration.

In plants that were grown at a particular CO2 concentration and then

exposed for short periods of time to concentrations of CO2 ranging

from 110 to 880 pl CO2 1-1, prior to sampling leaves, there was no

significant effect of the short-term exposures on initial or total

enzyme activity (Figure 2.4). The independence of initial activity

from short-term exposure to CO2 in the light has also been reported in

Arabidopsis (Salvucci et al., 1986a) anid white clover (Schnyder et

al., 1984). When the CO2 concentration was raised to 5000 pl CO2 11

Schnyder et al. (1984), however, found a 50% decrease in activity











compared to the activity at the CO2 compensation point. The percent
activation of RuBPCase, an estimation of the in vivo enzyme activation

status, like the initial and total activities was essentially not

affected by CO2 (Figure 2.5). Perchorowicz and Jensen (1983) and

Schnyder et al. (1984) reported similar results with wheat and white

clover, respectively. Although CO2 is necessary in the activation of

RuBPCase (Bahr and Jensen, 1978; Lorimer et al., 1976), there was no

indication that even at CO2 concentrations as low as 110 pl CO2 1-

(and corresponding Ci value of 60 to 75 pl CO2 1-1) the enzyme
suffered a significant decrease in activation. This indicates that a

high CO2 concentration inside the leaf is not required for a high
level of RuBPCase activation at high light intensity.

Unlike the apparent lack of effect of CO2 concentration on
RuBPCase activity in vitro, steady state RuBP levels were found to

respond to CO2. Plants grown at both CO2 concentrations had the

highest levels of RuBP following exposure to low CO2 concentrations.

The RuBP levels declined as CO2 increased (Figure 2.6). Work by other

researchers has yielded similar results (Badger et al., 1984; Dietz

and Heber, 1984; Mott et al., 1984; Collatz, 1978). The results of

Dietz and Heber (1986) indicated approximately two times the

concentration of CO2 was required with spinach, as compared with the

soybean data in Figure 2.6, prior to the onset of the decline in RuBP.

Hitz and Stewart (1980) did not find changes in RuBP levels in soybean

during steady state photosynthesis in 21% 02 and CO2 concentrations

ranging from 50 to 500 pl CO2 1-1. Levels of RuBP decreased as leaf

photosynthetic rate increased with increasing CO2 regardless of growth










at 330 or 660 pl CO2 11 (Figure 2.6). The lower levels of gRuP (as

CO2 concentration was increased) are presumably a result of greater
consumption due to higher photosynthetic rates associated with the

increased CO2 concentration. Although both photosynthesis and RuBP

levels were greater in leaves grown at high CO2, the turnover time for

the pool of RuBP was about the same for leaves grown at either 002

concentration when calculated at both low and high ambient CO2 (110

and 880 pl1 CO2 1-1). This suggests coordination between leaf

photosynthetic rate and RuBP levels. Turnover times were calculated

based on the rate of photorespiration being 15% of the rate of

photosynthesis (Canvin, 1979), one mole RuBP consumed per mole CO2

assimilated (Bassham, 1979), and two moles RuBP consumed per mole CO2

released during photorespiration (Ogren, 1984). This stoichometry,

the leaf photosynthetic rates, and the measured steady state levels of

RuBP yielded turnover times of 11.5 and 10.8 seconds for leaves grown

at 330 and 660 ill CO2 1-1, respectively, when measured at 110 pl

CO2 1-1 and 1.1 and 0.8 seconds when measured at 880 ul CO2 1-1 The
RuBP concentrations (Table 2.4) were always greater than the estimated

RuBPCase binding site concentration for RuBP of 3 to 4 mN (Jensen and

Bahr, 1977), indicating that RuBP was probably at saturating

concentrations. The similarity of turnover times and the

concentration of RuBP greater than the estimated binding site

concentration, suggest that RuBP was probably not limiting leaf

photosynthetic rates in these experiments.

Initial RuBPCase activity [Figure 2.6 (A)] was greater at all CO2

concentrations than the leaf photosynthetic rate [Figure 2.3 (A)] when










both were expressed on a leaf area basis. Results of this nature have

previously been reported (Bjorkman, 1981; Singh et al., 1974). There

are a number of reasons why leaf photosynthetic rate measured in situ

would be less than RuBPCase activity measured in vitro. The enzyme

assays are performed under saturating inorganic carbon concentrations

which not only provides more CO2 than is normally available within the

leaf chloroplast in the field, but also essentially eliminates the

competitive oxygenase reaction. The effects of dark respiration are

not measured in the enzyme assay. Also, extraction of the enzyme from

its intrachloroplastic location prior to assay will presumably remove

metabolic regulation that may normally function in the intact

photosynthetic cell. Furthermore, the assay procedure used to

determine RuBPCase activity measures both the E-C and E-C-M forms of

the enzyme while in the intact leaf only the E-C-M form will be active

(Seftor et al., 1986). If the E-C form is present in significant

quantities the in vitro enzyme assay will tend to overestimate the

active species of RuBPCase in vivo.

Farquhar et al. (1980) have proposed a model suggesting leaf

photosynthetic rate is limited by RuBPCase at low Ci and by RuBP

regeneration at high Ci. Results supporting this model have been

reported by von Caemmerer and Farquhar (1981), while Makino et al.

(1985) have indicated their results suggest RuBPCase was always

limiting to leaf photosynthesis. Results reported here show no

significant effect of Ci on RuBPCase activity and suggest that RuBP

levels were probably saturating for RuBPCase binding sites at all Ci











values. These data, therefore, do not appear to support the model of

Farquhar et al. (1980).

Since the leaflet photosynthetic rates were greater in leaves

grown at twice the atmospheric concentration of CO2, yet the

difference in RuBPCase activity between the two CO2 growth treatments

were not significant, and RuBP appeared to be at saturating levels,

three possibilities are suggested which may account for the greater

leaflet photosynthetic rates of the high CO2 grown plants. First, as

already described, growth at elevated CO2 concentration can result in

an increase in the mesophyll cell surface area/1eaf surface area

ratio. Nobel et al. (1975) have shown an increase in this ratio to

result in higher photosynthetic rates. This may have occurred in the

high CO2 treatment. Second, leaflet photosynthetic rates were

measured during the pod filling stage, and plants grown at high CO2

had a greater pod weight per plant and per unit leaf area. Long-term

growth in high CO2 has been shown to increase the number of fruit per

plant in several cases (Havelka et al., 1984; Baker and Enoch, 1983;

Cooper and Brun, 1967), and these increases represent an increase in

assimilate demand. An increase in assimilate demand has often been

associated with increased photosynthesis (Gifford and Evans, 1981;

Geiger, 1976; King et al., 1967). Plants grown at high CO2 had

greater leaf photosynthetic rates as well as greater pod weights per

plant. Enos et al. (1982) have also reported higher photosynthetic

rates in soybean plants with heavier pods. Third, the CO2-saturated

RuBPCase activity in vitro may not be an accurate representation of

activity in vivo. There may be differential regulation of RuBPCase in










vivo in soybean grown at different CO2 concentrations, however, no

evidence of this was observed.

An additional factor needs to be addressed with regard to

photosynthetic rates; the effects of leaf starch. Although starch was

not measured quantitatively in these experiments, visual estimations

of relative starch levels performed prior to enzyme assays indicated

that leaves grown at high CO2 contained more starch. In previous

studies, where starch was measured quantitatively, it was found to be

higher in soybean leaves grown at elevated CO2 concentrations (Allen

et al., 1983). In the experiments reported here the results are in

agreement with those in the literature that indicate no evidence of

photosynthetic rate inhibition by starch accumulation at high CO2'

Based on the results presented here from soybean, it is shown

that growth at twice the atmospheric concentration of CO2 can result

in an enhanced capacity for leaflet photosynthesis. Since the

response of RuBPCase activities was not significantly different with

growth CO2 treatment and the levels of RuBP appeared to be saturating

with regard to RuBPCase binding sites, the role of either in the

enhanced photosynthetic capacity remains unsupported. The increased

photosynthetic capacity following growth in elevated CO2 may be due to

either an increase in the internal/external leaf area ratio or greater

assimilate demand or a combination of both,













CHAPTER III
RESPONSE OF PHOTOSYNTHETIC BIOCHEMISTRY AND PHYSIOLOGY TO LONG-TERM
EXPOSURE TO SUBATM1OSPHERIC AND SUPERATMOSPHERIC CO2 CONCENTRATIONS


Introduction


Much of the interest in the effects of CO2 on vegetation is based

on the fact that the atmospheric concentration of 002 has been
increasing for the last century (Baes et al., 1977). Research has

focused on predicting how this continuing trend will affect future

crop yields and water use. In addition to learning the answers to

these questions, experiments with 002 concentrations can enhance our

comprehension of plant processes such as photosynthesis. Since the

response of plants to CO2 is largely mediated by the photosynthetic

process, understanding the effects of CO2 on photosynthesis is

paramount to understanding the effects on whole crop responses.

Almost all of the research on the long-term effects of CO2 on

plants has involved exposing plants to elevated concentrations of CO2

(Lemon, 1983; Kramer, 1981). It appears that long-term research on

plants grown at reduced rather than elevated CO2 concentrations has

previously just involved plants native to high altitudes where they

normally grow at CO2 partial pressures below those at or near sea-
level (Mooney et al., 1966; Billings et al., 1961). Long-term

exposure to elevated CO2 results in a number of changes in plant
characteristics. Leaf area on a whole plant basis has been shown to

increase with CO2 (Jones et al., 1984a; O'Leary and Knecht, 1981;










Cooper and Brun, 1967). Stomatal density stomataa mm-2) increased,

although not significantly, in soybean grown at high CO2 (Thomas and

Harvey, 1983). In Phaseolus fewer stomates were found on the abaxial

surface of leaves grown at high CO2, but the leaves were larger and

thus the overall result was more stomates per leaf (0'Leary and

Knecht, 1981). Increases in specific leaf weight (SLW) following

growth at elevated CO2 have been reported in tomato (Madsen, 1968),
Nerium oleander (Downton et al., 1980), Phaseolus (Jolliffe and Ehret,

1985), and soybean (Havelka et al., 1984; Jones et al., 1984a; Thomas

and Harvey, 1983; Hofstra and Hesketh, 1975). Chlorophyll content of

leaves has been shown to either increase (Downton et al., 1980),

decrease (von Caemmerer and Farquhar, 1984), or stay the same (Havelka

et al., 1984) in plants grown at elevated CO2. Other cytological

responses to long-term high CO2 exposure include increased cell water
content (Madsen, 1968), and changes in cell volume (Gates et al.,

1983; Madsen, 1968). In soybean, the presence of a third layer of

palisade cells not found in plants grown at atmospheric CO2

concentration was observed in high CO2 grown plants (Thomas and

Harvey, 1983). Carbon dioxide concentration has been shown to affect

the concentration of proteins as well as enzyme activities. In

soybean grown at elevated CO2 seed protein was found to decrease as

CO2 increased (Rogers et al., 1984), but in another study there was no
effect of CO2 on pod nitrogen levels (Hardy and Havelka, 1976). The

response of total soluble leaf protein to CO2 varies. It has been
shown to increase (Dow~nton et al., 1980), decrease (W'ong, 1979), and

not change (Havelka et al., 1984; Porter and Gradzinski, 1984) with










long-term exposure to elevated CO2. Most reports indicate that growth

at high CO2 results in reduced activity of RuBPCase when compared to

plants grown at atmospheric CO2 concentrations, when activity is

expressed on either a chlorophyll basis (Spencer and Bowes, 1986; Vu et

al., 1983; Downton et al., 1980) or a leaf area basis (von Caemmerer

and Farquhar, 1984; Wong, 1979). However, Fair et al. (1973) have

reported higher activity, when expressed on a fresh weight basis, in

young barley plants grown at 10,000 to 50,000 pl CO2 1-1 The
difference in activity became less as the plants aged. The proportion

of leaf soluble protein composed of RuBPCase (mg RuBPCase/g soluble

protein) decreased 22% in Nerium 01eander when the growth CO2

concentration was increased from 330 to 660 Irl CO2 1-1 (Downton et al.,

1980). The effects of CO2 on a variety of other enzymes have also been

reported. Carbonic anhydrase activity increased in oat when grown at

80 #1 CO2 1- and decreased when~ grown at 600 pl CO2 11 (Cervigni et

al., 1971). In Phaseolus, carbonic anhydrase activity decreased

following growth at 1200 pl CO2 1-1 (Porter and Gradzinski, 1984).

Phosphoenolpyruvate carboxylase activity decreased when waterhyacinth

was grown at 600 el CO2 1-1 (Spencer and Bowres, 1986), as did nitrate

reductase in barley grown at 10,000 to 50,000 pl CO2 1-1 (Fair et al.,

1973). There was no difference in fructose 1, 6-biphosphatase

activity in Nerium oleander grown at atmospheric and twice

atmospheric CO2 concentrations (Downton et al., 1980). Glycolate

oxidase activity decreased when grown at high CO2 in both Phaseolus

(Porter and Grodzinski, 1984) and barley (Fair et al., 1973),

but in tomato no well-defined response to CO2 was apparent









(Hicklenton and Jolliffe, 1980a). Catalase activity was lower in

barley grown at high CO2 (Fair et al., 1973). There were no

significant differences in sucrose phosphate synthase activity in

soybeans grown at atmospheric or elevated CO2 (Huber et al., 1984) or

in soybean proteolytic enzyme activity (Havelka et al., 1984).

Whether the differences in the activities of these enzymes from plants

exposed to various CO2 treatments are always significant is not clear.

The physiological significance of the responses to CO2 of all of these
enzymes is not always evident.

There are reports of plant damage, sometimes extreme, as a result

of growth at high concentrations of CO2. Accumulation of starch in

plants grown at 1000 pl CO2 1- was found to cause chloroplast
disruption (Cave et al., 1981). Chlorosis occurred in Phaseous grown

at 1400 pl CO2 1-1 (Ehret and Jolliffe, 1985) and in tomato (Thomas

and Hill, 1949). Thomas and Hill (1949) also reported the appearance

of necrotic areas on tomato leaves at high CO2. Brown and Escombe

(1902) reported a variety of disorders in plants grown at 1100 pl

CO21 1-. These included loss of leaves, reduced number of flowers and
lack of fruit formation. According to Ehret and Jolliffe (1985), it

has been suggested that the injuries reported by Brown and Escombe

(1902) may have been due to the impurities in the air in the enclosed

greenhouse. While ethylene contamination of compressed CO2 cylinders
was demonstrated by Morrison and Gifford (1984), presumably most

reports of plant injury are not the result of tainted air. There are

numerous examples of plants exposed to high CO2 with no injurious

effects, including exposure for 14 days to CO2 as high as 50,000 pl










CO2 1-1 (Hicklenton and Jolliffe, 1980b), suggesting that exposure to

high CO2 per se is not damaging to all plants.

Whole canopy photosynthetic rate responses to CO2 of canopies

grown at atmospheric and elevated CO2 have been reported for soybeans

by Acock et al. (1985) and Jones et al. (198Ga). In both cases

soybeans were grown in outdoor sunlit chambers for an entire season.

Both Acock et al. (1985) and Jones et al. (1984a) showed greater

photosynthetic rates, at all levels of solar irradiance, in canopies

grown at elevated CO2 when compared to canopies grown at 330 pl

CO2 1-1 Jones et al. (1984a) reported maximum canopy photosynthetic

rates, measured at the respective growth CO2 concentration and

approximately 1900 pmol quanta m2 s-1, were 50% greater in the canopy

grown at 800 pl CO2 1- compared to the 330 pl CO2 1- grown canopy.

In Chapter II a study was described in which soybeans were grown

continuously from seed at atmospheric and twice atmospheric

concentrations of CO2 to investigate the effects on photosynthesis.

In the study presented here, the range of growth CO2 concentrations

was expanded. Soybeans were grown at three subatmospheric,

atmospheric, and two superatmospheric concentrations of CO2. The

objective of this study was to investigate the effects of long-term

growth in various concentrations of CO2, ranging from subatmospheric
to superatmospheric levels, on soybean. Specific objectives were to

determine the effects on the activity and kinetics of RuBPCase and on

the levels of RuBP. In addition, the effects of CO2 growth

concentration on several plant characteristics and on canopy

photosynthesis were investigated.











Materials and Methods


Plant Material and Growth Conditions


Soybeans (Glycine max L. Merr. cv Bragg) were planted in six

outdoor environmentally controlled plant growth chambers (described in

Chapter II) on 14 Sept. 1984. The CO2 concentration was controlled to

160, 220, 280, 330, 660, or 990 pl CO2 1- in each chamber from the

date of planting until harvest. The chamber dry bulb and dewpoint

temperatures were controlled to 31 and 160C, respectively. The

chambers received natural solar irradiation. The quantum flux density

(400-700 nm) values reported here are measurements made at the upper

canopy level (the chambers transmit 88% of the incoming solar

radiation). These values were integrated over 5 min intervals from

data collected every 20 s.

Photosynthetic rate measurements and collection of all plant

material for analysis were made on 18 October (34 DAP). At this time

the plants had not yet started reproductive development and had been

thinned to a density of 30 plants per m2. The canopies were at the V7

to V8 stage of development (Febr and Caviness, 1977). Leaf tissue for

biochemical analysis was collected and stored in liquid N2 as

previously described. For each canopy, leaf area index (LAI) was

estimated from the measured leaf area of four plants harvested from

each chamber on 18 October.










Canopy Photosynthesis Measurements


Measurements of net photosynthetic rate of whole canopies were

made based on a whole chamber carbon mass balance which was corrected

for leakage of CO2 from the system (Jones et al., 1985b). The desired

CO2 concentration in a chamber was maintained by injecting pure CO2

into the chamber to replace the CO2 assimilated by the canopy. The

CO2 injections were based on light response algorithms determined for
each canopy. The algorithms were updated as the canopies developed.

Corrections for drift in this procedure were made every 5 min by

making chamber CO2 concentration measurements with an IR gas analyzer

(Jones et al., 1984a).

Canopy photosynthetic rate response to light was measured as the

solar irradiation varied throughout the day. Measurements were made

every 5 min over a 10.5 h period (0750 to 1800 EST) which was cloud

free. During this time period, irradiance at the upper canopy level

varied from 145 Llmol quanta m2s- in the morning, to a midday

maximum of 1370 umol quanta m-2s1 to 15 Llmol quanta m-s1 in the

evening.


RuBP Carboxylase Assay and RuBP Determination


The methods for sampling leaf tissue and for the assay of

RuBPCase activity and the determination of RuBP levels were the same

as those described in Chapter II. For the determination of the

M~ichaelis constant, K (CO2), and Vmax of RuBPCase, the assay

procedures were modified and are described in the following section.











Determination of Apparent K (CO2)and V _x


Assays of RuBPCase activity were performed to determine the

Michaelis constant, K (CO2), and Vmax. The K (CO2) reported here is

actually the apparent K (CO2) as assays were performed on a crude

extract from the leaf rather than the purified enzyme. The assay

procedures were similar to those described in Chapter II with some

modification and are described as follows. A quantity of frozen leaf

powder (70 to 150 mg dry weight) was removed from liquid N2 storage

and placed in a pre-chilled Ten Broeck tissue homogenizer. Added to

the leaf powder was 5 ml of extraction buffer consisting of 100 mM

Tris-HC1 (pH 8.0), 5 mM DTT, 10 mM isoascorbate, 5 mM MlgC12, and 1.5%

(w/v) PVP-40. The leaf tissue was homogenized for approximately 60 s

at 00C. An aliquot of the homogenate was reserved for chlorophyll

determination, and the remainder was centrifuged at 12,000 g for 3

min. Following centrifugation the supernatant was activated and used

to initiate the assays (described later) and the pellet was discarded.

The buffer media used in the assay consisted of 50 mMl Tris-HC1, 5 mM

DTT, 5 mMl MgC12, and 10 mM isoascorbate. The media was prepared CO2

free by purging at pH 3.1 for 15 min with N2 then raising the pH to

8.0 with CO2-free Na0H solution. To a 5-m1 assay vial was added the

CO2-free buffer media, 0.5 mM RuBP, and 54 Wilbur-Anderson units of
carbonic anhydrase (CA) (from bovine erythrocytes). The carbonic

anhydrase was added to prevent depletion of CO2 during the assay,

particularly at the lower concentrations of HCO3- (Bird et al., 1980).

The vials were capped and then purged with N2 for 10 min. Through the

cap septum NaH14CO2 (7.54 GBq/mol) was added in eight different final









concentrations ranging from 0.25 to 10 mM. The consumption of

substrate H14CO3- was always less than 20% and usually less than 10%
during each of the assays. The assays were initiated by the injection

of activated supernatant from the homogenized crude extract. The

supernatant was activated at 00C for 45 min in 50 mM Tris (pH 8.0), 5

mMI DTT, 10 mM isoascorbate, 5 mM MgC12, and 10 mM N'aH1CO3. Following

activation, the supernatant was kept at 00C while the assays were

being performed. The injection of 25 pl of activated supernatant

carried over 0.25 mM H14CO3 into the assay vials and this quantity
was taken into consideration when the final H14CO3- concentration

calculations were made. Assays were performed in triplicate, at 300C,

with continuous shaking (125 strokes min-1), in a total volume of 1

m1. The assays were terminated after 45 s with 6N formic acid in

methanol. An aliquot (0.4 ml) of the assay mixture was then

transferred to a 5-m1 plastic scintillation vial which was placed

under an air-stream until all remaining 14C not fixed into acid-stable

products was driven off. This required leaving the vials in the air-

stream overnight. To the approximately 0.4 ml remaining in each vial

was added 4 ml of scintillation cocktail. Acid-stable 14C products

were determined by liquid scintillation spectrometry.

Since CO2 is the form of inorganic carbon used as a substrate by
RuBPCase (Cooper et al., 1969), it was necessary to calculate the

concentration of dissolved CO2 in the assay mixture based on the added

quantities of H14CO3 At the assay temperature of 300C the

solubility coefficient of CO2 (a) in water is 0.665 ml ml1 (Umbreit
et al., 1972) and the pK' of carbonic acid is 6.327 (Harned and










Bonner, 1945). Using these values, the gas space volume above the

liquid in the assay vial, and the Henderson-Hasselbach equation, the

partitioning of inorganic carbon between dissolved CO2, CO2 in the gas

space and bicarbonate was calculated (0gren and Hunt, 1978). No

corrections were made for the effect of ionic strength on a or pK',

While the effect of salts on a appears to be minor in the

concentration range encountered in these assays (Umbreit et al., 1972)

the effect on pK' is more substantial (Harned and Bonner, 1945).

However, since all assays had essentially the same salt concentration

the relative effects on the kinetic values are insignificant. The

concentration of H14CO3- in each vial was corrected for the

consumption of H14CO3~ during the assay. This required the assumption

that the velocity of the reaction catalyzed by RuBPCase was constant

during the 45 s assay. The corrected substrate concentrations and the

reaction velocities were used to calculate R (CO2) and Vmax values

using Lineweaver-Burke plots and the least squares method (Cleland,

1979). These kinetic values were also calculated using Eadie-Hofstee

plots (data not shown) and were found to be very similar to the values

presented here.


Estimation of Dissolved Free CO2 at the Cell Wall


The dissolved free CO2 at the cell wall of the mesophyll tissue

was assumed to be in equilibrium with the CO2 in the air in the leaf

intercellular spaces. Data from Figure 2.1 (Chapter II) yields a

value of 0.72 for the ratio of the concentrations of intercellular to

ambient CO2, Ci/Ca, for plants grown at 330 #1 CO2 1-1 The Ci/Ca









ratio for plants grown at 660 pl CO2 1- differed from 330 pl CO2 11

grown plants by 23% (Figure 2.1). Since Ci/Ca ratios were not

determined for all the growth CO2 concentrations used in this study,

and the exact nature of the relationship between the Ci/Ca ratio and

growth CO2 concentration is not known, the value for 330 Ul CO2 1-1

grown leaves, Ci/Ca = 0.72, was used for all calculations. Other

assumptions included an atmospheric pressure of 760 mm Hg and a

solubility coefficient, a, for CO2 in water of 0.665 ml ml-1 All

calculations were based on a temperature of 300C. The calculation of

free CO2 dissolved in the cell wall was by the method of Umbreit et

al., 1972),
Pwa t Ci' 1000
CO2 [3.1]
760 't V 100

where CO2 is in units of moles liter-1 (M, molar concentration), the

term P/760 converts atmospheric pressure to standard conditions, Ci'

is the intercellular CO2 concentration in percent (v/v), the term

1000/V converts a from ml ml-1 to moles liter-1, and 100 converts

percent CO2 to pCO2 (partial pressure of CO2 in mm Hg).

Chlorophyll, Protein, and Specific Leaf Weight Determinations


The measurements of chlorophyll, total soluble leaf protein, and

specific leaf weight (SLW) w~ere made using the same methods described

in Chapter II, with the exception that leaves for the SLW

determination were collected from either nodes 5 and 6 or 6 and 7. In

each canopy leaves from these nodes represented tw;o of the most

recently fully-expanded leaves in the upper canopy. All plant










material used for these measurements was collected on 18 Oct. 1984 (34

DAP).


Analysis of Statistical Significance


Simple linear and quadratic regression analyses were performed to

determine the statistical significance (at the 5% level) of

experimental results. In this chapter, the CO2 concentration during

growth was used as regressor. The methods used are described further

in Chapter II. Regression parameters are tabulated in Appendix C.


Results


Response of Plant Characteristics to CO,


Continuous exposure during growth of soybeans to a range of CO2

from 160 to 990 pl CO2 1- resulted in changes in leaf and canopy

characteristics. There was a significant, almost linear increase in

SLW with increasing CO2 (Table 3.1). The plants grown at the highest

CO2 concentration had leaves with SLW 50% greater than those grown at
the lowest concentration. Below atmospheric concentration of CO2 (330

pl1 CO2 1-) there was a minor response of SLW to CO2. The greatest

response occurred as CO2 was increased from 330 to 990 01 CO2 -1
The LAI increased two-fold as CO2 increased from 160 to 990 pl CO2 11

(Table 3.1). The LAI generally increased with increasing CO2, showing

a significant response to CO2 concentration. The LAI values were

similar for canopies grown at 160 and 220 pl CO2 1 1, and although

higher, similar for canopies grown at 280, 330, and 660 #1 CO2 11










Table 3.1. Effect of growth CO2onettinnSLLI
chloophll, nd ota leaf soluble protein. The SLW was
calculated based on fully-expanded leaves collected from
the upper canopy level. Canopy LAI was estimated from
the total leaf area of four representative plants. All
measurements were made on leaf samples collected 18
October (34 DAP) when plants were in the V7 to V8
vegetative stage.



Growth CO2 Specific Leaf Leaf Area Total Soluble
Concentration Weight Index Chlorophyll Protein

-1 -2 m2 m-2 -2 -2
pl CO2 1 g dry wt. m g 2 m g m


160 20.3 + 2.1a 1.63 + 0.11 0.204 + 0.001 2.53 + 0.01

220 20.9 + 1.7 1.61 + 0.03 0.261 + 0.002 3.23 + 0.02

280 21.4 + 2.4 2.40 + 0.42 0.248 + 0.002 2.58 + 0.02

330 21.4 + 2.4 2.54 + 0.20 0.214 + 0.001 2.31 + 0.01

660 26.6 + 5.2 2.40 + 0.42 0.205 + 0.004 2.28 + 0.04

990 30.5 f 5.2 3.25 + 0.33 0.234 + 0.001 2.29 + 0.01



aMean values + SD.









The canopy grown at the highest CO2 concentration had an LAI at least

28% greater than each of the other canopies.

On a leaf area basis, the chlorophyll and leaf soluble protein

levels showed similar responses to CO2 (Table 3.1). The general trend

was a decrease in value with increasing CO2, but the response to CO2

of both chlorophyll and soluble protein was not significant. Because

of the variation in SLWJ, chlorophyll and soluble protein are also

expressed on a dry weight basis in Table 3.2. When expressed on this

basis, the response of chlorophyll and soluble protein to CO2 is

significant. On a dry weight basis the levels of both chlorophyll and

soluble protein in the 330 ill CO2 1-1 grown leaves were approximately

midway between the highest and lowest values, found in the 220 and 990

ill CO2 1-1 grown canopies, respectively. Soluble protein on a dry

weight basis decreased 50% as CO2 was increased from 220 to 990 #1

CO2 1-1. While the direction of responses to CO2 was similar for both
chlorophyll and soluble protein, the magnitude of these responses

varied. This is shown in Figure 3.1 where the protein/chlorophyll

ratio is plotted against CO2 concentration. The ratio is highest at

low CO2. The response to CO2 of the protein/chlorophyll ratio was

found to be significant.


Canopy Photosynthetic Rate

Canopy photosynthetic rate responses to sunlight for plants grown

at each of the six CO2 concentrations are shown in Figure 3.2. Data

points represent measurements made at 5 min intervals as solar

irradiance varied throughout the day. When canopy photosynthetic










Table 3.2. Effect of growth CO2 concentration on chlorophyll and
total leaf soluble protein expressed on a dry weight
basis. Values are calculated from data in Table 3.1.



Growth CO2 Total Soluble
Concentration Chlorophyll Protein

el CO2 -1 mg (g dry wt.)-1 mg (g dry vt.)-1


160 10,05 + 0.05a 124.7 + 0.6

220 12.49 + 0.09 154.5 + 1.1

280 11.59 + 0.09 120.5 c 0.9

330 10.00 + 0.05 107.9 + 0.5

660 7.71 + 0.15 85.7 + 1.7

990 7.67 + 0.03 75.1 + 0.3



aMean values + SD.
































Fig. 3.1. The soluble protein/chlorophyll ratio versus
groth O2concentration. Data were calculated
from the mean values in Table 3.1. Vertical
lines through data points represent f SD.

























Fig. 3.2. A-F. Canopy net photosynthesis (on a land area
basis) versus solar irradiance for canopies grown
atl6 different CO ccetain.A) 1 0 pl CO,1
1 ,B) 2 0 l CO 1~ C) 2 0 pl CO, 1 ,D)3
pul C~O2 E) 66~ pl C 1,F) 99 # C2
Each nata point represents a mreasurement maae at
a 5 min interval. Data were collected over a
10.5 b period (0750-1800 EST) on October 18 (34
DAP). Maximum solar irradiance occurred at
midday when quantum flux density ras-1
approximately 1370 pumol quanta m s.Light
levels are values for the upper canopy surface.
Growth chambers transmit 88% of incoming solar
irradiance. The canopy LAI's varied two-fold
across the CO2 concentration range.

















60



40-









S20



S60

z
1j 40-



S20 -



60


D

330 MI CO, 1-




L .


160 vi C02 1-1







*.*.. :aaa.~~


660 ~1CO, I'





Fe'





990 al1 CO, F


B

220 pl COe I '


C -. 1~C. C~~

"ieo I co T 2_~r ___Zr


500 1000 O 500 1000

QUANTUM FLUX DENSITY ( Amol quanta m2 s )


1500









rates were measured (at 34 DAP) canopies grown at 160 and 220 pl

002 11 were light saturated at light levels lower than 1000 umol
quanta m2s1 (Figure 3.2 A and B). The canopy grown at 280 ul

CO2 1- (Figure 3.2 C) did not appear to light saturate at midday
light levels of 1370 umol quanta m-2 s-1, but did not respond with

increasing photosynthetic rates as high as the 330 nl CO2 1-1 grown

plants (Figure 3.2 D). The photosynthetic rate response increased

continuously with increasing irradiance in plants grown at 330, 660,

and 990 pl CO2 11 (Figure 3.2 D, E, and F). At two and three times

atmospheric CO2 concentration the photosynthetic rate response to

light was clearly still increasing, even at maximum midday irradiance,

showing no indication of light saturation. Based on the visually

estimated intercept of response curves in Figure 3.2, the canopy light

compensation points did not appear to be strongly CO2 dependent.

Compensation points for each canopy were in the range of 50 to 150
-2 -1
pmol quanta m s.

The maximum photosynthetic rates of the canopies are plotted

against growth CO2 concentration in Figure 3.3. Each data point is

the mean of between 7 to 10 measurements made at the growth CO2

concentration at midday when irradiance inside the chambers was at its

peak of 1250-1370 pmol quanta m- s 1. The maximum rates were greater

as the CO2 concentration during growth increased. The slope of the

response is steeper at the lower CO2 concentrations. Because the

total leaf area of a canopy varied by two-fold over the range of CO2

concentrations, the canopy photosynthetic rates in Figures 3.2 and 3.3

are a reflection, in part, of the differences in LAL.




























Fig. 3.3. Maximum canopy net photosynthetic rate versus
growth CO2 concentration, Photosynthesis is on a
land area basis. Each data point is the mean of
7-10 measurements made at midday when the2quaptum
flux density was 1250-1370 pmol quanta m s.
Data are from Fig 3.2. Vertical lines through
data points represent f SD.










RuBP Carboxylase Activity and RuBP Levels


The RuBPCase activity was assayed from fully-expanded leaves

collected from the upper part of each canopy. The means of triplicate

assays are plotted in Figure 3.4 (A). Both initial and total

activities decreased significantly as the CO2 concentration increased,

with the highest activities occurring at the lowest CO2. The initial

activity decreased by 28% as CO2 increased from 160 to 990 pl CO2 11

while the total activity decreased by 23% over the same CO2 range.

The activation of RuBPCase was calculated from data in Figure 3.4 (A)

and was found to be quite high, particularly at low CO2 [Figure 3.6

(B)]. Activation did show a significant but not a great response to

CO2, however, the highest activation (greater than 95%) occurred at
the lower CO2 concentrations. Above atmospheric concentrations of CO2

there was not much activation response to CO2. The initial and total

RuBPCase activities tended to parallel each other regardless of CO2
concentration.

The RuBP levels were determined in a subset of the same leaf

samples used for RuBPCase assays. The means of triplicate assays are

shown in Figure 3.5. The level of RuBP decreased significantly as CO2

increased, however, at CO2 concentrations greater than 660 ul CO2 1-

the measured levels of RuBP did not appear to respond strongly to CO2'

The RuBP at 660 ul CO2 1- was only 30% of the level at 160 il1 CO2 1-

Assuming that RuBP is present only in the chloroplast (Heber, 1974),

and that the stromal volume of the chloroplast is 25 pl mg Chl-1

(Sicher and Jensen, 1979), chloroplast concentrations of RuBP can be




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