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MECHANISTIC BASIS OF GAS EXCHANGE IN THE TERRESTRIAL
ASTOMATAL ACID METABOLISM PLANT C(/lnl7h/l,0, lunifera
(REICHB.F.) J. J. SM. (ORCHIDACEAE).
JASON R. HUPP
A THESIS PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
MASTER OF SCIENCE
UNIVERSITY OF FLORIDA
I would like to thank my advisor, Dr. Stephen Mulkey, and the members of my
committee, Dr. Karou Kitajima and Dr. Timothy Martin, for their invaluable guidance
during the development of this project.
I would like to thank Dr. Mark Whitten and Dr. Barbra S. Carlsward for providing
access to living specimens of various shootless orchids from the Florida Museum of
Natural History's collection. These plants proved crucial for developing appropriate
techniques for measurement of whole plants gas exchange. Additional thanks are
extended to Dr. Carlsward for providing me access to her anatomical and phylogenetic
studies of the Vandae, and for her conversations with me regarding the physiology and
anatomy of shootless orchids.
I would also like to extend special thanks to Dr. Thomas Sinclair for the central
role he played in the interpretation of my results and the development of the model used
to describe changes in internal conductance within the roots of C/hl/, /ii\1, lunifera.
Without his help only a much less convincing argument could be made for the existence
of a regulatory mechanism over gas exchange in the roots of this plant.
TABLE OF CONTENTS
A CK N OW LED G EM EN TS................................................................................................ ii
T A B LE .. .......................... ............................................................. .................... .... .iv
L IST O F FIG U R E S....................................... ..........................................................v
A B ST R A C T ........................................................................................... ................. ..........vi
1 INTRODUCTION........................ ......... ....... ............................
Terrestrial Astomatal Acid Metabolism (TAAM) ........................... ........... 2
Vegetative Reduction and the Shootless Habit ............................ ........... 3
Photosynthetic Velamentous Roots.....................................................8
O bjectives............................................ ......................................... .................. 14
2 A MODEL OF GAS EXCHANGE IN PHOTOSYNTHETIC
VELAMENTOUS ROOTS................................................. ......................15
Introduction ..................................................................... ......... ........................... 15
M materials and M ethods........................................................ ...................... 17
Plant M aterial................................ ............................................... 17
Whole Plant Gas-exchange........................ .... ....................18
Water Vapor Flux Across Isolated Velamen ...........................................19
G as-exchange M odel............................................... ...................... 22
R esults.. ................................ ............................................... .. .................... .... 23
D iscu ssion ...................................................................... .................................. 2 9
APPENDIX: GLOSSARY OF SYMBOLS....................................................................33
L IST O F R E FE R E N C E S ............................................................................................. ....35
2-1 Velamen vapor phase water potentials........................ ...................... 27
LIST OF FIGURES
1-1 Vegetative morphologies of various orchids..............................................4
2-1 Schematic diagram of the velamen cuvette........................ ...................... 20
2-2 Carbon assimilation and transpiration at constant VPD......................................24
2-3 Net carbon gain, water loss and internal conductance for all vapor
pressure deficits............................................. .......................................... 25
2-4 Transpiration rate response during the first four hours of measurement
at 2.50 kPa VPD ........................ ......................... ......................... 26
2-5 Water vapor flux across isolated velamen tissue.......................................27
2-6 Total plant conductance and internal conductance of water vapor at
con stant V P D ................................................................... ................................28
Abstract of Thesis Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Master of Science
MECHANISTIC BASIS OF GAS EXCHANGE IN THE TERRESTRIAL
ASTOMATAL ACID METABOLISM PLANT Ch',I/i iuia lunifera (REICHB.F.) J. J.
Jason R. Hupp
Chair: Stephen Mulkey
Major Department: Botany
Crassulacean acid metabolism (CAM) represents a versatile and highly plastic
photosynthetic pathway, showing a great degree of variability in biochemistry, expression
and anatomy between various CAM species. While a large body of literature exists
addressing biochemical and expressional variation in CAM, relatively little work has
been done addressing the anatomical variants of CAM, particularly Terrestrial Astomatal
Acid Metabolism (TAAM) and its occurrence in the photosynthetic roots of orchids.
While these TAAM roots lack stomata, they do possess specialized aeration
complexes embedded in the root surface which are thought to provide a diffusive path for
gas exchange. It has been proposed that a pair of differentially thickened cells within this
structure may act similarly to stomatal guard cells, serving to regulate gas exchange.
However, the few studies that have been conducted to date have focused solely on carbon
and as a result have failed to provide strong evidence for or against a regulatory
Diel gas exchange patterns for Clhi ', n\ ,i lunifera were indicative of CAM.
Transpiration rates tracked those of carbon assimilation, with maximum rates of both
occurring at night. Measurements of water vapor flux across isolated velamen indicated
an ability of velamen to absorb water vapor at a vapor pressure deficit (VPD) less than 2
kPa, and likely played a role in the steady decrease observed in transpiration rates with
decreasing VPD. The consistent diurnal variation in transpiration and the rapid decrease
in transpiration rates in response to high VPD imply an active means of regulating gas
exchange in C. lunifera. Further study is necessary to determine if the regulatory
mechanism is based on changes in cortical component aperture, or is the result of some
Crassulacean acid metabolism (CAM) represents a versatile and highly plastic
photosynthetic pathway, allowing for maximum carbon acquisition under carbon limited
conditions (Cockburn 1985, Cushman 2001, LIittge 2004). Under the traditional view of
CAM, carbon uptake is dominated by nocturnal fixation of CO2 by phosphoenyol
pyruvate carboxylase (PEPC) to produce malic acid, which is stored in the vacuole.
Malate is then remobilized behind closed stomata and decarboxylated in the light
providing CO2 for Rubisco to act upon (Cockburn 1985, LIittge 1987, Osmond 1978).
However, because of the great deal of taxonomic diversity among CAM plants (e.g. 16
000 species from 33 families [Cushman 2001, Cushman and Borland 2002, Zotz 2002])
and is undoubtedly polyphyletic origins a vast array of CAM variants exist (Cushman
2001), making a comprehensive and coherent definition of CAM difficult (LIittge 2004).
The wealth of variation among CAM plants results from deviations in
biochemistry, expression and anatomy from Osmond's (1978) classical description of
CAM. While a great deal of work has been done regarding CAM variation resulting from
deviations in biochemistry (e.g., Bakrim et al. 2001, Chen and Nose 2004, Cockbum
1985, Cushman and Bonhert 1999, Drincovich et al. 2001, LIittge 2000, Taybi et al.
2004) and expression (e.g., Borland and Grifiths 1990, Cockburn 1985, Cockburn 1998,
Cushman 2001, Cushman and Bonhert 1999, Cushman and Borland 2002, Kluge et al.
1997, Liuttge 1987, Liuttge 2000, Zotz 2002), almost no work has been done addressing
the anatomical variants of CAM, particularly Terrestrial Astomotal Acid Metabolism
Terrestrial Astomotal Acid Metabolism (TAAM)
Terrestrial Astomotal Acid Metabolism is only known from two phylogeneticly
distinct groups of plants, the rare member of the Isoetaceae, Isoetes andicola (= Stylites
andicola) and the photosynthetic velamentous roots of orchids. In both cases stomata are
absent from the photosynthetic organs and carbon fixation is dominated by PEPC
(Cockburn 1985, 1998).
In Isoetes andicola carbon is taken up via an extensive root system from a moist
C02-rich substratum. The photosynthetic organs are covered by a thick waxy cuticle
lacking stomata, preventing direct gas exchange with the atmosphere (Cockbum 1985,
1998). While in general the CAM pathway and an absence of stomata are not unusual in
Isoetes, they are for terrestrial members of the genus (i.e. stomata are absent in aquatic
species), suggesting I. andicola as an intermediate between terrestrial and aquatic forms
The situation for TAAM in the photosynthetic velamentous roots of orchids is
quite different then in L andicola. In both cases roots serve as the primary organ of
carbon acquisition by TAAM (Cockbum 1985, 1998), however the photosynthetic
velamentous roots of orchids exists exposed to the atmosphere in an environment with
sporadically available water resources (Benzing and Ott 1981). These roots may be
exposed to long periods of drought between rain events, and therefore must have some
means of regulating transpirational losses to the environment (Benzing and Ott 1981,
Benzing et al. 1983) while allowing CO2 to diffuse in from the surrounding atmosphere.
Vegetative Reduction and the Shootless Habit
The Orchidaceae is known for its unsurpassed degree of ecological diversity and
numerous deviations from the typical body plans exhibited by vascular plants (Figure 1-
1) (Benzing and Ott 1981). One of the most interesting deviations is the allocation of
photosynthetic carbon acquisition to the roots and the subsequent loss of leaves and
reduction of the stem exhibited by shootless taxa. While photosynthetic roots are present
in many epiphytic orchids exhibiting a broad array of vegetative morphologies, they are
most refined in the shootless species where they serve as the sole means of carbon gain
(Benzing and Ott 1981, Benzing et al. 1983, Carlsward 2004).
The shootless habit is not common within the Orchidaceae (Benzing and Ott
1981) (comprising less than one percent of the family), and is represented in only the
tribe Vandeae (Carlsward 2004, Carlsward et al. 2003). Within the Vandeae
photosynthetic roots and the monopodial growth habit are common though and
presumably provided the framework from which the shootless habit could arise
While the forces driving the evolution of shootlessness are not well understood,
the most widely cited benefit is the suspected improved water economy associated with
the habit. By reducing their surface area, these plants presumably decrease transpirational
demand and increase water use efficiency (Benzing and Ott 1981, Benzing et al. 1983,
Cockburn 1985, Rolfe 1914).
Vegetative morphologies of various orchids. A. Vanilla planifolia a leafy
monopodial species which exhibits both photosynthetic stems and roots.
B. Vanilla barbellata a leafless monopodial species reliant solely on
photosynthetic stems. C. Brassavola nodosa a leafy sympodial species
with stems and roots capable of photosynthesis. D. Cattleya duisosa x Blc
"California girl" a leafy sympodial species with stems and roots capable of
photosynthesis, and stems modified for water storage (psuedobulbs). E.
C/7ll x, l,,\,i lunifera a shootless species reliant solely on photosynthetic
roots for carbon acquisition. F. Close up of B. nodosa showing sympodial
growth habit. G. Phalaenopsis (unnamed hybrid) a leafy monopodial
species with stems and roots capable of photosynthesis.
However, little physical evidence supports this theory. It has been shown that velamen
thickness is positively correlated with habitat aridity (Benzing and Ott 1981, Sanford and
Adanlawo 1973). One would expect that if these species had historically been exposed to
such arid conditions to force the extreme vegetative reduction seen today, as suggested by
some authors (Cockburn 1985, Rolfe 1914), shootless orchids would posses thick
velamen indicative of species originating from arid habitats (Benzing and Ott 1981).
However, shootless taxa tend to posses thin velamen, and in some species, such as
Campylocentrum pachyrrhizum the velamen is almost completely eroded away on mature
roots (Benzing et al. 1983). Detailed habitat information is scanty for most shootless
species, but it is clear from their needs under cultivation that these species are intolerant
of conditions where transpirational demands are high and ambient humidity is low (pers.
obs.). Taken together this suggests that water economy is likely not a primary driving
force behind the origin of shootlessness.
It has been suggested that the CAM based mode of photosynthesis exhibited by
these roots is evidence that these plants had to historically coupe with habitat aridity
(Benzing and Ott 1981). While CAM has traditionally been thought of as a means to
increase carbon gain under water limited conditions (Cockburn 1985, Litttge 1987,
Osmond 1978), it is better thought of as a means to increase carbon gain under carbon
limited conditions (Cockburn 1985, Cushman 2001, LIuttge 2004). Following this line of
reasoning the occurrence of CAM in the photosynthetic roots of those leafy species that
preceded the development of the shootless habit is quite reasonable. The elaborate
ventilation systems seen in many shootless taxa are absent or highly under developed
among leafy orchids (Benzing et al. 1983). This presumably poses a rather large
resistance to the diffusion of carbon dioxide in to these roots. CAM allows for maximized
carbon gain under these conditions by allowing the plant to generate a much greater
concentration gradient of CO2 between the root cortex and the surrounding atmosphere,
driving the influx of CO2 in to the plant. In addition remobilization of CO2 from malate
during the light vastly increases the internal CO2:O2 ratio suppressing costly
photorespiration due to the oxgenase activity of Rubisco (Keeley and Rundel 2003).
An argument can also be made for a non-carbon acquisition based origin for
CAM in the photosynthetic roots of orchids. CAM allows for very high solute (generally
malate) concentrations to be generated, resulting in a large osmotic potential that can
drive the influx of water into the plant (LIUttge 1987). As rain events are sporadic and
short lived in the epiphytic biotope (Benzing and Ott 1981), the amount of water that can
be scavenged during any given wetting event is of key importance to the health of the
plant. Water uptake by orchid roots is driven by two factors, water movement into the
velamen by capillary action and subsequent movement into the root cortex across an
osmotic gradient (Benzing and Ott 1981, Benzing et al. 1983). As velamen is composed
of non-living cells it is likely that the plant can not exert any direct control over the rate at
which water is pulled in to the velamen. However, by increasing the solute concentration
inside the root, the rate of osmoticly driven uptake from the velamen to the cortex could
be greatly increased. Potentially reducing the residence time for water in the velamen and
increasing the amount of water that can be pulled into the root following a wetting event.
While this would suggest a water economy based origin for CAM in the photosynthetic
root, it doesn't necessitate a water limitation based origin for shootlessness.
An epiparastic mode of nutrition has also been suggested as a driving force
behind the vegetative reduction seen in shootless taxa (Benzing and Ott 1981, Johansson
1977, Ruinen 1953). However, under cultivation these species do best when grown on
non-decomposable substrates such as coarse metal or plastic mesh (Whitten pers.
comm.), suggesting that nutrients scavenged directly from the host (if at all) are not
essential for plant survival. Also the presence of epiphytotic relationships, a possible
means of epiparastism, while not thought to be uncommon among orchids have as far as
this author is aware have not been documented among shootless taxa.
The most recent supposition as to the forces driving the evolution of shootlessness
is the suspected increased nutrient economy of this habit (Benzing and Ott 1981, Benzing
et al. 1983). Nutrient inputs are highly limited in the epiphytic biotope. Occurring high
in the forest canopy, epiphytes are cut off from the lavish nutrient pools available to
terrestrially rooted vegetation, forcing them to rely on sporadic and short lived nutrient
pulses associated with rain events and dust deposition (Nadkarni 1985). As a result they
have developed many, often unusual, strategies to cope with this feast or famine situation,
including litter impounding leaf configurations, phytotelmata, absorptive trichomes,
insectivory, myrmecophytism, and the occurrence of mycorrhizae (Benzing 1990, Hietz
et al. 2002, Nadkami 1985, Nadkarni and Matelson 1991). It has been suggested that by
not investing valuable nutrients in leaves and supporting structures where they would
become permanently fixed, shootless orchids maybe able to reallocate these nutrients to
increase fecundity (Benzing et al. 1983).
Photosynthetic Velamentous Roots
The roots of epiphytic orchids are covered in a spongy layer of hydrophilic cells
called the velamen, which serves to take up water and mineral nutrients from the
environment (Benzing and Ott 1981, Benzing et al. 1982, Benzing et al. 1983, Dycus and
Knudson 1957, Hew et al. 1993, Pridgeon 1987). Upon a wetting event the velamen cells
rapidly engorge with water and dissolved minerals, which are pulled into the root through
highly cytoplasmic "passage cells" in the exodermis. These cells are embedded in a
matrix of highly suberised "U" cells that provide a barrier to water movement. The
passage cells contain an extensive membrane network and a greater density of
mitochondria than surrounding cells, suggesting some active mechanism for transporting
solutes into the root (Benzing et al. 1982, Benzing et al. 1983). The ability of these cells
to move solutes and water into the root is contingent on the length of time that the
adjoining velamina remain engorged with water (Benzing et al. 1982, Benzing et al.
1983), and may be one of the driving forces behind the correlation between velamen
thickness and habitat aridity; As orchids with thicker velamen tend to be found in more
arid habitats (Sanford and Adanlawo 1973). Presumably this thicker velamen would
remain saturated longer and allow for greater nutrient and water uptake.
The tissue layer comprising the velamen is derived from periclinal divisions of
dermatogen resulting in a multiple epidermis (Engard 1944). This layer maybe comprised
of anywhere between one and twenty layers dependent on species (Pridgeon 1987,
Porembski and Barthlott 1988). Velamen cells are non-living and air filled at maturity,
which occurs within the first few millimeters behind the root tip (Pridgeon 1987). Walls
of the velamen cells undergo deferential deposition of cellulose fibrils prior to cell death
resulting in secondary thickenings (Pridgeon 1987). These thickenings maybe helical,
parallel or less often forked in nature (Noel 1974, Pridgeon 1987) and are thought to
stabilize and support the relatively thin walls of velamen cells (Porembski and Barthlott
As a result of drying and shrinking of the cell walls of the velamen following cell
death, pores develop ranging in size from 1 [tm to 50 [tm in diameter (Pridgeon 1987).
These pores originate as small tears and elliptical slits in pit fields within the velamen cell
walls, and rapidly elongate as the velamen shrinks (Noel 1974, Pridgeon 1987). The
secondary wall thickenings supporting the velamen likely act to limit the size of large
pores by preventing further elongation (Pridgeon 1987). Species with helical thickenings,
most common in epiphytes (Stern and Carlsward 2004), generally exhibit the largest
pores (Porembski and Barthlott 1988). Across all species the inner most tangential
velamen wall shows the greatest density of pores, particularly above passage cells in the
exodermis (Porembski and Barthlott 1988).
Composition of velamen cell walls is highly variable among different orchid taxa
(Noel 1974, Pridgeon 1987). All wall layers are cellulosic in nature and exhibit widely
varying degrees of lignin and suberin impregnation (Benzing et al. 1983, Pridgeon 1987).
In multilayered velamen lignification is greatest in the walls of the inner most velamen
layers and decreases in the outer layers (Pridgeon 1987, Sanford and Adanlawo 1973). In
a study of velamen properties Giles and Agnihotri (1968) determined the velamen of
Vanda suavis to be comprised of 51.5 % cellulose with a specific surface area of 425
m2 g-1, an interesting finding as this is approximately twice that observed for wood and
jute and greater than three times that for cotton.
Velamen structure also varies with taxon, but is relatively constant across a given
species or in many cases a greater taxonomic grouping, particularly at the tribal and
subtribal levels (Pirdgeon 1987, Porembski and Barthlott 1988). Porembski and Barthlott
(1988) identified ten distinct, and one unspecified, velamen types in an exhaustive survey
of 344 species from 262 genera, based on properties of the cell wall, number and
stratification of the velamen layers, and tilosomes, in addition to other morphological
characters of the root.
Velamen physiology as it relates to gas fluxes in and out of the root has been
generally ignored since the discovery of velamen in the mid 1800s. Speculation as to
velamen function, following in the first few decades after its discovery, often cited a role
of velamen in condensing water vapor, though little or no experimental evidence
supported this conclusion (see Pridgeon 1987 for review). As a result this view quickly
fell out of favor and the vast majority of subsequent physiological research concerning
velamen function has focused on its now well established role in water and nutrient
uptake (see Pridgeon 1987 for review).
Velamen is often cited as means to resist desiccation under the arid conditions
associated with epiphytism, either by increasing the boundary layer surrounding the root
(Pridgeon 1987) or by some undocumented resistive property of the tissue. While
velamen thickness has been shown to correlate with habitat aridity (Sanford and
Adanlawo 1973), investigations into the desiccation resistance of various orchid species
has not shown a similar relationship (Benzing et al. 1983). Benzing et al. (1983) found
that among the ten orchid taxa studied Polyradicion lindenii (= Dendrophylax lindenii see
Carlsward et al. 2003) showed the greatest resistance to desiccation, though this species
has only a moderate surface to volume ratio and thin velamen, two to four cells thick
(Benzing et al. 1983, Carlsward 2004). Similarly structured roots of other species with
thicker velamen layers desiccated much faster, suggesting that the velamen layer may not
play a significant role in desiccation resistance (Benzing et al. 1983).
The author is aware of only one investigation in the modern literature* examining
the ability of velamen to absorb water vapor. Giles and Agnihotri (1968) developed
absorption isotherms for the velamen of both Philodendron gigantum (Aeraceae) and
Vanda suavis (Orchidaceae). The velamen of both species, though from quite different
taxonomic backgrounds, showed similar chemical composition and an amazing ability to
absorb water vapor. V. suavis was able to absorb 15% of its dry weight at a relative
humidity (RH) as low as 50% (at 200C), and showed a maximum absorption of nearly
24% of its dry weight at 98% RH (at 200C). These values are much greater than those
observed for other cellulosic materials such as wood, jute and cotton (Giles and Agnihotri
Within the cortex of velamentous roots lie thin walled living parenchyma, which
may contain chloroplast, and non-living tracheoidal idioblasts resembling water
conducting cells (Carlsward 2004, Benzing et al. 1983, Solereder and Meyer 1930). The
cell walls of these tracheoidal idioblasts lack suberin and lignin, making it possible for
them function as collapsible water storage cells similar to those found in the parenchyma
of many succulents. In addition the cortex of photosynthetic orchid roots also contain a
network of gas filled passages, which approach or are connected to specialized aeration
complexes spanning the exodermis and velamen (discussed below). Among leafy orchids
these intercellular air spaces are within the size range for the intercellular air spaces
* Modem used here refers to publications post 1900.
found in the leaves of CAM and C4 species. Shootless taxa possess larger air spaces, but
they still remain smaller than those observed for C3 plants (Benzing et al. 1983).
The velamen is especially well developed among shootless orchids, and possesses
specialized aeration complexes which presumably provide a diffusive path for gas
exchange (Benzing and Ott 1981, Benzing et al. 1983, Cockburn 1985, Cockburn et al.
1985). Though these aeration complexes are not restricted to shootless taxa, they are most
refined in these species. Embedded within the velamen of these roots are areas of highly
suberised cells, the pneumathode, which remain void even when the velamen is fully
engorged with water (Benzing and Ott 1981, Benzing et al. 1983, Dycus & Knudson
1957, Pridgeon 1987). These regions often sit atop a thin walled aeration cell, which in
turn sits atop two differentially thickened cortical components in the root's exodermis.
These cortical components potentially act as guard cells and may provide a means of
regulating gas exchange (Benzing and Ott 1981, Benzing et al. 1983).
Benzing et al. (1983) proposed two methods by which the aeration complex could
regulate gas exchange. They suggested that (1) by expansion and contraction of the two
cortical components, that they could function analogously to stomatal guard cells and
could provide a homiohydrous mode of regulating gas exchange. Alternatively, (2) as the
roots swells or contracts in relation to the overall root water status the two cortical
components may be forced together or pulled apart, providing a poikilohydrous mode of
regulation (Benzing et al. 1983). However, data from the few studies looking at gas-
exchange in photosynthetic orchid roots has not provided strong evidence for or against
an aeration complex based regulatory mechanism (Benzing et al. 1983, Cockburn et al.
It seems quite likely that some regulatory mechanism exists, though data
supporting this are inconclusive. In their work with P. lindenii Benzing et al. (1983)
demonstrated that this species showed the greatest resistance to desiccation and that this
could not be attributed to properties of the velamen. Similarly structured roots of a leafy
species with a thicker velamen layer desiccated nearly twice as fast, suggesting that P.
lindenii had at least some control over water loss (Benzing et al. 1983). Whether or not
this increased desiccation resistance is the result of the more refined aeration complexes
found in the roots of P. lindenii is yet to be determined.
In their work with C'hli\, ihl usneoides Cockbum et al. (1985), conclude that it
was highly unlikely that the aeration complex could regulate gas exchange (Cockburn
1985). They suggested that the small loss of CO2 observed during the light in C.
usneoides was not the result of an increased diffusive resistance resulting from reduction
in the cortical component aperture, but resulted from the maintenance of internal CO2
concentration of the plant near atmospheric concentration (Cockbum et al. 1985).
Samples taken from the intercellular air spaces of these roots by the authors were
admittedly contaminated by air from outside the root, and may have been an inaccurate
representation of actual internal CO2 concentrations.
While no direct observation of cortical component aperture over any time course
has been made to date (presumably because of obstruction by the overlying velamen
layer), in her work with the anatomy of Vandeae, Carlsward (pers. comm.) noted a great
deal of variation in the distance between the cortical components within a given species.
The basis of this variation remains undetermined. But it seems likely that it could result
from changes in turgor during the process of preparing specimens for anatomical study
and so maybe analogous to changes in turgor in the living root effecting cortical
Terrestrial Astomatal Acid Metabolism represents a unique and poorly understood
anatomical variation on the tradition CAM pathway. While the occurrence of TAAM is
limited to only two plant groups (Cockbum 1985), the diversity of CAM orchids in
tropical forest may mean that TAAM is exhibited by as many as 75% of all CAM species
in a particular area (Zotz and Ziegler 1997). Given that much of our understanding of
CAM physiology stresses the importance of stomata in regulating gas exchange
(Cockburn 1983, Cockburn 1985, LIUttge 1987), TAAM represents a potentially large gap
in our understanding of CAM. It is difficult to imagine how a plant could maintain the
associated high internal CO2 concentrations of a CAM pathway, or how these plants
could mediate water losses in the water limited environment of the forest canopy without
some mechanism to regulate gas exchange. The principal goals of my research were to
develop a model describing gas exchange for photosynthetic velamentous orchid roots
exhibiting TAAM and to provide evidence either for or against an aeration complex
based regulatory mechanism.
A MODEL OF GAS EXCHANGE IN PHOTOSYNTHETIC VELAMENTOUS ROOTS
The photosynthetic roots of epiphytic Orchids exhibit an unusual and poorly
understood variation of Crassulacean Acid Metabolism (CAM). While thought to follow
the same general biochemistry as other CAM variations, Terrestrial Astomatal Acid
Metabolism (TAAM) is unique in that there is a total absence of stomata from the
photosynthetic organs (Cockbum 1985). While TAAM is known to occur in only two
phylogeneticly distinct plant groups, the rare member of the Isoetaceae Isoetes andicola
and the photosynthetic roots of orchids (Cockbum 1985), its occurrence among tropical
CAM species maybe quite common as orchids may comprise 75% of all CAM species in
a given tropical forest (Zotz and Ziegler 1997).
Diel gas exchange patterns for the photosynthetic roots of orchids are generally
indicative of CAM, showing predominate carbon assimilation at night (Benzing and Ott
1981, Cockbum et al. 1985, Hew 1987, Hew et al. 1984, Hew et al. 1991). For most taxa
possessing photosynthetic leaves and/or stems carbon assimilation in the roots is
generally less than losses due to respiration, resulting in an apparently negative carbon
balance for these roots (Dycus and Knudson 1957, Erickson 1957, Hew et al. 1984,
Kwok-ki et al. 1983). In shootless species, where photosynthetic roots represent the sole
means of carbon acquisition for the majority of the plant's life* nocturnal carbon
assimilation is significantly large enough to result in a net positive carbon balance
(Benzing and Ott 1981, Cockbum et al. 1985). The magnitude of carbon assimilation in
shootless species is generally lower than that of the CAM leaves from other orchids
(Benzing and Ott 1981)
Use of radio labeled 14CO2 has demonstrated that photosynthetic roots are able to
assimilate carbon from the surrounding atmosphere both day and night (Benzing and Ott
1981, Goh et al. 1983, Hew et al. 1984), suggesting a possible C3-CAM mode of carbon
uptake. The magnitude of diel fluctuations in titratable acidity seem to indicate that for
many species potential carbon loss through respiration is mediated by some degree of
CAM-cycling (Hew et al. 1984). CAM-cycling is further supported by the identification
of malate as the product of nocturnal assimilation in species which showed net efflux of
CO2 in the dark (Cockbum et al. 1985, Hew et al. 1984). This may be evidence of a
highly plastic application of the CAM machinery in the photosynthetic roots of orchids.
The presence of a CAM based mode of carbon acquisition and the absence of
stomata exhibited by these roots raises some interesting questions. High internal partial
pressures of CO2 are associated with the decarboxylation of malate during the light by
CAM plants (Cockbum 1985, LIittge 1987), yet for several of the photosynthetic roots
examined to date net assimilation rates are near zero throughout much of the light
(Corckburn et al. 1985, Benzing and Ott 1981). How then do these orchids prevent the
loss of CO2 during the light period? In addition many of these orchids live high in forest
canopy where water availability is sporadic and evaporative demand may be quite high
*Like many orchids these species often posses photosynthetic inflorescences and fruit, capable of off
setting much of their carbon costs (Zotz et al. 2003). In addition young plants of some shootless orchids
may possess small green leaves which are lost with age (Cralsward 2004).
(Benzing 1990), yet they often possess a multitude of aerial roots exposed to the
surrounding atmosphere. How then do these plants mediate water loss under these
seemingly water demanding conditions? A question even more perplexing for shootless
taxa which lack the stems modified for water storage present among other epiphytic
It has been proposed that specialized aeration complexes present in many orchid
roots may provide a regulatory mechanism over gas exchange (Benzing et al. 1983),
though experimental evidence supporting or refuting this are scanty (Benzing et al. 1983,
Cockburn et al. 1985). These structures which span the exodermis-velamen complex
contain two differential thickened cortical cells which may act in much the same manner
as stomatal guard cells (Benzing et al. 1983). It is the purpose of this report to provide
evidence either for or against the presence of a regulatory mechanism based on detailed
examination of diel gas exchange patterns and through estimations of internal
conductance based on a quantitative model describing water vapor flux.
Materials and Methods
Mature greenhouse grown specimens of C/hlI\, hl\ia lunifera (Reichb.f.) J. J. Sm.
were obtained from Tropiflora Inc. (Sarasota, Florida). Plants were grown on a course
plastic mesh and maintained in the Botany Greenhouse (Department of Botany,
University of Florida, Gainesville, Florida) for at least four months prior to use in gas
exchange studies. Plants were non-reproductive at the time of measurement, ensuring the
roots were the sole means of carbon assimilation and water loss.
Whole Plant Gas-exchange
Diel gas exchange measurements were performed in the lab. Measurements were
made on a whole plant basis using open-system analysis with a LI-6400 Portable
Photosynthesis System (LI-COR, Inc. Lincoln, Nebraska) fitted with a custom cuvette
(20 cm L x 9 cm W x 4cm H), constructed of Polycast acrylic sheet (SPARTECH
Corporation, Stamford, Connecticut). Measurements were made over a 24 hour period,
with 12 hours of light and 12 hours of darkness, at a constant vapor pressure deficit
(VPD) supplied by a LI-610 Portable Dew Point Generator (LI-COR, Inc. Lincoln,
Nebraska). Light was supplied by a 50 W halogen lamp mounted 35 cm above the
chamber in a darkened room. Flow through the system was held constant at 200 [tmol-s-1,
and chamber temperature was held constant by maintaining the block temperature at
27C. To account for absorption and desorption of water by the acrylic body of the
cuvette, a one hour equilibrium period was allowed prior to measurement. Measurements
were made at a constant VPD of 2.50, 2.14, 1.78, 1.43 and 1.07 kPa for each 24 hour
period, starting at the highest VPD and decreasing stepwise over a period of five days.
All fluxes were calculated on a surface area basis, determined by assuming
regular geometry and treating roots as cylinders of constant diameter. Root diameter was
determined on a per plant basis as the average diameter of ten roots.
Net assimilation, transpiration and total root conductance were calculated using
the standard equations used by the LI-6400 Portable Photosynthesis System (LI-COR
Water Vapor Flux Across Orchid Velamen
Water vapor flux across excised pieces of velamen was measured using a LI-6400
Portable Photosynthesis System (LI-COR, Inc. Lincoln, Nebraska). Velamen was
mounted in a custom cuvette (Fig. 2-1) functionally similar to the porometer chamber
described by Meidner (1986), and attached to the LI-6400 sensor head (LI-COR, Inc.
Lincoln, Nebraska). The cuvette was designed to replace the lower section of the standard
2X3 Leaf Chamber (LI-COR, Inc. Lincoln, Nebraska). Polycast acrylic sheet
(SPARTECH Corporation, Stamford, Connecticut) was used to construct the body of the
cuvette. The mounting plate used to hold the velamen in position was constructed from a
0.6 mm thick brass plate, with a 3 mm square orifice machined at the center of the
chamber. A water reservoir machined into the lower half of the cuvette provided a water
vapor saturated air space on one side of the velamen via a filter paper wick positioned
just below the velamen.
Velamen was carefully peeled from healthy living roots at positions well back
from the actively growing root tip, using a sharp razor blade. Any remaining cortical
material was removed and the velamen sections were blotted dry prior to mounting in the
cuvette. Excised pieces of velamen were positioned underneath the mounting plate at its
orifice so that the surface formally facing the root cortex faced the water reservoir. The
velamen was sealed to the lower half of the cuvette via two closed cell foam rubber
gaskets. A type E Chromel-Constantan thermocouple (Omega Engineering, Inc.
Stamford, Connecticut) connected to the LI-6400 sensor head was inserted between these
gaskets and positioned in the air space under the velamen.
Attached to the LI-6400 sensor head along
this side, and sealed with rubber O-rings
0 1 I
3 mm square velamen
mounting plate orifice
Counter sunk holes for
mounting to the LI-6400
Screw holes for connecting
upper and lower sections of
the cuvette; lower holes
Closed cell foam rubber
0.6 mm brass velamen
Thermocouple inserted here
Schematic diagram of the velamen cuvette. A. View of chamber from
above. B. Cut-away view of chamber along axis drawn through A (heavy
dashed line), as viewed from the side. Hidden lines represented as dashed
lines. Scale is given in centimeters.
A constant vapor pressure deficit (VPD) was supplied to the cuvette by a LI-610
Portable Dew Point Generator (LI-COR, Inc. Lincoln, Nebraska). Measurements were
made on the same piece of velamen at a constant VPD of 2.50, 2.14, 1.78, 1.43 and 1.07
kPa, with a one hour equilibrium period prior to beginning measurement and between
each decrease in VPD. Measurements were logged every 20 minutes for a total of five
measurements at each VPD, and the entire experimental procedure was repeated three
times. Flow through the system was held constant at 200 [tmol-s-1, and chamber
temperature was held constant by maintaining the LI-6400's block temperature at 270C.
It was assumed that the boundary layer conductance for the velamen cuvette, gbvc,
represented the combined effects of air passing over the plane of the mounting plate and
the unstirred layer of air atop the velamen in the mounting plate orifice (Eq. 2-1) (Nobel
gbvc= Dw_ P 2-1
total r (Ta)
Where Dw is the diffusivity coefficient of water vapor in air (2.4 x10-4 m2.-1), and r is
the universal gas constant (8.3145 x10-3 m3 kPa-mol --K 1). The total boundary layer
thickness for the velamen cuvette, total, is calculated from equation 2-2 and expressed in
meters (Nobel 1983).
total = 4.0 /(L/V) + D 2-2
Where L is the characteristic dimension in the direction of air flow and D is the depth of
the mounting plate orifice, both expressed in meters. V is the wind speed inside the
chamber, expressed in m-s Since velamen mounted in the cuvette is positioned below
the bottom plane of the chamber and parallel to the direction of air flow, the entire
bottom surface of the chamber contributes to the thickness of the boundary layer, not just
the area of exposed velamen at the orifice. So L is equal to 0.02 m, as this is the length of
the chamber in the direction of air flow. In addition a contribution 0.0006 m (D) is made
to the boundary layer thickness by the layer of unstirred air positioned atop the velamen
in the orifice but below the surface of the mounting plate.
From VPvel (calculated from Eq. 2-4) an estimation of the vapor phase water
potential of the velamen, Yvel, was made based on the natural log of the ratio of VPvei to
the saturation vapor pressure of the surrounding air, SVPair. Following equation 2-3 Yvel
was calculated and the resulting figure retuned in MPa.
Yvel = R Ta. In VPvel_ Eq. 2-3
Where R is the universal gas constant expressed in J'mol-"K-1, Ta is the air temperature
inside the velamen cuvette and M is the molecular weight of water (18.02 g'mol 1).
Measurements of excised velamen indicated a capacity of velamen to absorb
water vapor directly from moist air, demonstrating a source/sink role of velamen for
water vapor moving from the root cortex to the atmosphere. A model describing changes
in internal conductance within the root was developed by partitioning water vapor flux to
include movement of water into the velamen from both the atmosphere and the root
cortex. By assuming the rate of water vapor adsorption and desorption by the velamen is
at steady state for a particular vapor pressure inside the velamen, transpiration (E) as
determined from the whole plant gas exchange data can be redefined as in equations 2-4
E = gbl (VPvel VPair) 2-4
= giant (VPint VPve) 2-5
Where gbl and git are the boundary layer and internal conductances respectively, VPvel is
the vapor pressure inside the velamen, VPint is the vapor pressure inside the root and VPair
is the vapor pressure of the air surrounding the root. The boundary layer conductance was
calculated by assuming regular geometry and treating roots as cylinders oriented
perpendicular to the direction of air flow (Nobel 1983). The internal vapor pressure was
assumed to be at saturation and was calculated based on measurement of root internal
temperature by a type E Chromel-Constantan thermocouple (Omega Engineering, Inc.
Stamford, Connecticut) connected to the LI-6400 sensor head and inserted into the root
cortex. The vapor pressure of the air surrounding the root was calculated based on the
concentration of water vapor in the sample cell of LI-6400 and the air temperature inside
the cuvette. By rearranging equations 2-4 and 2-5 to solve for the two remaining
unknowns, giant and VPvei, the model used to estimate gi,t results (Eq. 2-6). This model
was used to estimate changes in internal conductance exhibited by C. lunifera throughout
the course of each diel gas exchange measurement.
giant = :-l + (VPi.nt-Vairp)
Diel gas exchange patterns for both C02 and H20 were indicative of CAM under
all vapor pressure deficits, though the magnitude of these fluxes was quite small under
the highest VPD (Fig. 2-2). Net carbon assimilation increased in a stepwise manner as
VPD decreased, showing the greatest net carbon gain under the lowest VPD (Fig. 2-3A).
Patterns of water vapor flux were most interesting. Under the highest VPD regime
plants exhibited the greatest net water loss, showing what appeared to be a large amount
of diural variation in transpiration (Fig. 2-2B). These apparent spikes in transpiration at
10:00, 14:00 and 16:00 corresponded to a rapid decrease in transpiration rate following
2 E 05
9 E 06
6 E 06
3 E 06
3 E 06
9 E 06
3 E06 6kIP-
2 E 05
9 E 06
6 E 06
6 E 06
3 E 06 6-------------
1200 15 12 1824 2136 048 400 7 12 1024 1200 1512 1824 2136 048 400 712 1024
Figure 2-2: Carbon assimilation and transpiration at constant VPD. A. Assimilation at
2.50 kPa. B. Transpiration at 2.50 kPa. C. Assimilation at 2.14 kPa. D.
Transpiration at 2.14 kPa. E. Assimilation at 1.78 kPa. F. Transpiration at
1.78 kPa. G. Assimilation at 1.43 kPa. H. Transpiration at 1.43 kPa. I.
Assimilation at 1.07 kPa. J. Transpiration at 1.07 kPa. Light period shown
in white (-200 kmolm-2.s'1), dark period shown in gray.
Figure 2-3: Net carbon gain, water loss and internal conductance for all vapor pressure
deficits. Net carbon gain (A) and net water loss averaged for all plants (B)
integrated for day (open circles) and night (closed circles). C. Internal
conductance averaged for day (open circles) and night (closed circles). All
values are shown plus or minus standard error.
the start of each new diel gas exchange measurement (Fig. 2-4). Net water loss decreased
as VPD decreased, with afternoon transpiration rates gradually approaching zero with
each drop in VPD (Fig. 2-3B). Measurements of excised velamen indicated water uptake
by this tissue layer at VPDs less than 2 kPa (Fig. 2-5), and this likely played a role in the
gradual decrease in transpiration rates. Under the lowest VPD plants exhibited negative
transpiration rates for a period of almost 10 hours. This period of water gain by the plants
was almost enough to completely counter transpirational losses during the night leading
to an almost neutral water balance.
0 30 60 90 120 150 180 210 240
Transpiration rate response during the first four hours of measurement at
2.50 kPa VPD. Though the rate of decrease was slightly different between
trials, transpiration showed an initial rapid drop off following the start of
each set of measurements. Values are shown plus or minus standard error.
Estimations of total root conductance and internal conductance were indicative of
CAM under all but the highest VPD (Fig. 2-6) and similar to transpiration, conductance
was lower during the day than at night (Fig. 2-3C). The vapor phase water potentials for
velamen sheathing the intact root were similar to those determined for isolated velamen
mounted in the velamen cuvette under all VPDs (Table 2-1).
Water vapor flux across isolated velamen tissue. The units of transpiration
have been converted to tmol-m-2.-1 for convenience as flux rates are
small. Error bars represent standard error.
Table 2-1: Velamen vapor phase water potential. 'vel (MPa) was estimated from
measurements of isolated velamen mounted in the velamen cuvette and for
intact velamen based on whole plant gas exchange data.
1200 1512 1824 2136 048 400 12 1024
1200 1512 1824 2136 048 400 712 1024
Total plant conductance and internal conductance of water vapor at
constant VPD. A. Total conductance at 2.50 kPa. B. Internal conductance
at 2.50 kPa. C. Total conductance at 2.14 kPa. D. Internal conductance at
2.14 kPa. E. Total conductance at 1.78 kPa. F. Internal conductance at
1.78 kPa. G. Total conductance at 1.43 kPa. H. Internal conductance at
1.43 kPa. I. Total conductance at 1.07 kPa. J. Internal conductance at 1.07
kPa. Light period shown in white (-200 tmol-m-2.s-1), dark period shown
The rates of carbon assimilation in photosynthetic orchid roots have been
demonstrated to be quite low, similar to those rates observed in the present work
(Benzing and Ott 1981, Cockburn et al. 1985, Dycus and Knudson 1957, Erickson 1957,
Hew et al. 1984, Kwok-ki et al. 1983). Consistent with the results reported for other
shootless species, C/I "I,, In\ii lunifera exhibited a strong CAM signal at night and
showed little or no carbon efflux during the day (Cockburn et al. 1985). While C. lunifera
showed diel gas exchange patterns for CO2 similar to those reported for other shootless
orchids, these results and those published by other authors (Benzing and Ott 1981,
Cockburn et al. 1985) by themselves shed little light on a possible regulatory mechanism
over gas exchange.
Diel changes in transpiration rates tracked that of assimilation, showing maximum
transpiration rates at night when transpirational demands were presumably the lowest.
This change in transpiration could not be attributed to any change in the forces driving
water vapor flux from the roots of C. lunifera as measurements were made under constant
temperature and VPD, nor could they be explained by evaporation of water absorbed by
the velamen. Estimations of internal conductance tracked transpiration and provide
evidence that some mechanism inside the plant must exist to regulate gas exchange.
Without such a mechanism it seems highly unlikely that any change in diel transpiration
patterns would be observed in C. lunifera under these measurement conditions, or if
transpiration rates were to show diel variation, maximum rates would be expect during
the day do to the energy contributed to the system by light striking the plant.
In addition to diel variation in transpiration, C. lunifera showed an immediate
response to a sudden large scale change in VPD. Plants were placed in the cuvette for
measurement directly from the greenhouse, moving from the relatively moist cool
conditions of the greenhouse to the much drier conditions used for the first set of diel gas
exchange measurements (VPD of 2.50 kPa). Plants exhibited a fairly rapid decline in
transpiration rate, with transpiration rates on average reduced by 40% within 3.5 hours
after the start of measurement. As with the diel variation observed in transpiration rates,
this decline in transpiration rate can not be explained by changes in the forces driving
water vapor flux from the roots of C. lunifera, nor by water adsorption or desorption by
the velamen. Similar responses have been observed for other plant organs in response to
decreased humidity during gas exchange measurements and have been ascribed to
changes in stomatal conductance (Schulze et al. 1987). It seems likely that this is
evidence that some mechanism exists within the roots of C. lunifera that allows the plant
to regulate gas exchange.
The overall drop in transpiration with each decrease in VPD, and the eventual ten
hours of apparent water vapor uptake by the plants is likely due to the ability of velamen
to adsorb water vapor from the surrounding air (Giles and Agnihotri 1968). The
magnitude of water vapor fluxes into the velamen and out of the root are both small, and
at the lowest VPD these fluxes were nearly equal when integrated over a 24 hour time
period, leading to a near neutral water balance. However, it remains to be seen if the
water vapor absorbed by the velamen remains bound there or is able to be pulled into the
root cortex and used by the plant.
The anatomy of roots from many shootless taxa has been well defined (Benzing et
al. 1983, Carlsward 2004). Within these roots specialized aeration complexes exist
spanning the exodermis and velamen, which presumably provide a pathway for diffusive
gas exchange (Benzing et al. 1983, Cockburn 1985). These complexes are comprised of a
highly suberised region in the velamen, the pneumathode, which remains void even when
other velamen cells are fully saturated (Benzing and Ott 1981, Benzing et al. 1983,
Dycus & Knudson 1957, Pridgeon 1987), an eroded aeration cell spanning the exodermis,
and two differential thickened cortical cells subtending the aeration cell (Benzing and Ott
1981, Benzing et al. 1983, Carlsward 2004). It has been proposed that these cortical cells
may act analogously to stomatal guard cells to provide a means of regulating gas
exchange in orchid roots (Benzing et al. 1983). These structures are present in the roots
of C. lunifera (Carlsward 2004), and represent the most likely means by which C.
lunifera could regulate gas exchange. Diel transpiration patterns, the lack of CO2
evolution during the day and the decrease in transpiration rates following the drastic
increase in VPD at the start of measurement are all indicative of some regulatory
mechanism over gas exchange. The variation in transpiration throughout the day and the
observation that transpiration rates tracked those of assimilation suggest a stomata like
mechanism under active control of the plant.
The overall higher transpiration rates at the higher VPDs may be the result of
desorption of water vapor from the velamen following movement from the moist
greenhouse to the much drier cuvette, or they maybe evidence that while a regulatory
mechanism exists it does not provide tight control over gas exchange. Estimations of
vapor phase water potential of the velamen are very negative at these VPDs, suggesting
the velamen tissue is dry. It therefore seems highly probable that the mechanism used to
regulate gas exchange in C. lunifera is crude and is not capable of completely closing off
the root cortex from the surrounding atmosphere.
It is clear from the data that a regulatory mechanism exists that allows C. lunifera
to exert control over fluxes of C02 and water vapor in and out of its roots. While the
mechanism does not appear to be as refined as that seen in other plants (e.g. stomata), it
does appear to be under active control of the plant. It is likely that further investigation
will reveal that the observed changes in transpiration rates throughout the day correspond
to changes in cortical component aperture.
GLOSSARY OF SYMBOLS
Mounting plate orifice depth
Diffusivity coefficient of water in air (2.4 x 10-4)
Boundary layer conductance of water vapor
Boundary layer conductance for velamen mounted in the
Molecular weight of water (18.02)
Universal gas constant (8.3145 xl0-3)
Universal gas constant (8.3145)
Boundary layer resistance to water vapor flux
Resistance to water vapor flux due to the exodermis
Pneumathode resistance to water vapor flux
Air temperature inside the cuvette
Water vapor pressure of air
Water vapor pressure inside the root
VPvei Water vapor pressure of velamen tissue kPa
total Total boundary layer thickness for velamen mounted in the m
've1 Vapor phase water potential of the velamen MPa
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Jason Robert Hupp was born in Lexington, North Carolina, on October 15, 1980.
He graduated from Mooresville Senior High School, in 1998. The following spring he
enrolled at Catawba College, in Salisbury, North Carolina, and began working under the
guidance of Michael J. Baranski. Jason received his Bachelor of Science degree in 2003,
after completing the requirements for both a major in biology and in environmental
science, and a minor in chemistry. In the fall of 2003 he relocated to Gainesville, Florida,
and began work on a master's degree at the University of Florida under the supervision of
Stephen S. Mulkey.