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Mechanistic Basis of Gas Exchange in the Terrestrial Astomatal Acid Metabolism Plant, Chiloschista lunifera (Reichb. f.)...

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PAGE 1

MECHANISTIC BASIS OF GAS EXCHANGE IN THE TERRESTRIAL ASTOMATAL AC ID METABOLISM P LANT Chiloschista lunifera (REICHB.F.) J. J. SM. (ORCHIDACEAE). By 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 2006

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ii ACKNOWLEDGMENTS 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 gui dance 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 appr opriate techniques for measurement of whole plants gas exchange. Additional thanks are extended to Dr. Carlsward for providing me access to her anatomical and phyloge netic 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 Chiloschista 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.

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iii TABLE OF CONTENTS Page ACKNOWLEDGEMENTS.................................................................................................ii TABLE................................................................................................................................iv LIST OF FIGURES.............................................................................................................v ABSTRACT.......................................................................................................................vi CHAPTER 1 INTRODUCTION...................................................................................................1 Terrestrial Astomatal Acid Metabolism (TAAM) ..................................................2 Vegetative Reduction and the Shootless Habit .......................................................3 Photosynthetic Velamentous Roots.........................................................................8 Objectives..............................................................................................................14 2 A MODEL OF GAS EXCHANGE IN PHOTOSYNTHETIC VELAMENTOUS ROOTS...................................................................................15 Introduction...........................................................................................................15 Materials and Methods..........................................................................................17 Plant Material............................................................................................17 Whole Plant Gas-exchange.......................................................................18 Water Vapor Flux Across Isolated Velamen.............................................19 Gas-exchange Model.................................................................................22 Results....................................................................................................................23 Discussion..............................................................................................................29 APPENDIX: GLOSSARY OF SYMBOLS.......................................................................33 LIST OF REFERENCES...................................................................................................35 BIOGRAPHICAL SKETCH.............................................................................................40

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iv TABLE Table Page 2-1 Velamen vapor phase water potentials..................................................................27

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v LIST OF FIGURES Figure Page 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 constant VPD.........................................................................................................28

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vi 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 Chiloschista lunifera (REICHB.F.) J. J. SM. (ORCHIDACEAE). By Jason R. Hupp May 2006 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 biochemistr y, expression and anatomy between various CAM species. While a large body of literature exist s addressing biochemical and expressional variation in CAM, relatively little work has been done addressing the anatomical variants of CAM, particularly Terrestri al 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 ce lls within this structure may act similarly to stomatal guard cells, serving to re gulate gas exchange. However, the few studies that have been conducted to date have focused solely on carbon

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vii and as a result have failed to provide strong evidence for or against a regulatory mechanism. Diel gas exchange patterns for Chiloschista lunifera were indicative of CAM. Transpiration rates tracked those of carbon assimilation, with maximum rate s of both occurring at night. Measurements of water vapor flux across isolated velam en indicated an ability of velamen to absorb water vapor at a vapor pressure deficit (VPD) le ss 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 dec rease in transpiration rates in response to high VPD imply an active means of regulatin g 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 other mechanism.

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1 CHAPTER 1 INTRODUCTION Crassulacean acid metabolism (CAM) represents a versatile and highly plastic photosynthetic pathway, allowing for maximum carbon acquisition under carbon limite d conditions (Cockburn 1985, Cushman 2001, Lttge 2004). Under the traditional view of CAM, carbon uptake is dominated by nocturnal fixation of CO 2 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 CO 2 for Rubisco to act upon (Cockburn 1985, Lttge 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 (C ushman 2001), making a comprehensive and coherent definition of CAM difficult (Lttge 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 fr om deviations in biochemistry (e.g., Bakrim et al. 2001, Chen and Nose 2004, Cockburn 1985, Cushman and Bonhert 1999, Drincovich et al. 2001, Lttge 2000, Taybi et al. 2004) and expression (e.g., Borland and Grifiths 1990, Cockburn 1985, Cockburn 1998,

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2 Cushman 2001, Cushman and Bonhert 1999, Cushman and Borland 2002, Kluge et al. 1997, Lttge 1987, Lttge 2000, Zotz 2002), almost no work has been done addressing the anatomical variants of CAM, particularly Terrestrial Astomotal Aci d Metabolism (TAAM). 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 CO 2 -rich substratum. The photosynthetic organs are covered by a thick waxy cuticle lacking stomata, preventing direct gas exchange with the atmosphere (Cockbur n 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 (Cockburn 1985). The situation for TAAM in the photosynthetic velamentous roots of orchids is quite different then in I. andicola In both cases roots serve as the primary organ of carbon acquisition by TAAM (Cockburn 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 m ay be exposed to long periods of drought between rain events, and therefore must have some

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3 means of regulating transpirational losses to the environment (Benzing and Ott 1981, Benzing et al. 1983) while allowing CO 2 to diffuse in from the surrounding atmosphere. Vegetative Reduction and the Shootless Habit The Orchidaceae is known for its unsurpassed degree of ecological diversity a nd numerous deviations from the typical body plans exhibited by vascular plants (Fig ure 11) (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 pr esent in many epiphytic orchids exhibiting a broad array of vegetative morphologies, the y are most refined in the shootless species where they serve as the sole means of ca rbon 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 (Carlsward 2004). While the forces driving the evolution of shootlessness are not well understood, the most widely cited benefit is the suspected improved water economy associa ted with the habit. By reducing their surface area, these plants presumably decreas e transpirational demand and increase water use efficiency (Benzing and Ott 1981, Benzing et al. 1983, Cockburn 1985, Rolfe 1914).

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4 A B C D E F G Figure 1-1: 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. Chiloschista 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.

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5 However, little physical evidence supports this theor y. 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 sugge sted 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 d riving 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 ari dity (Benzing and Ott 1981). While CAM has traditionally been thought of as a means to increase carbon gain under water limited conditions (Cockburn 1985, Lttge 1987, Osmond 1978), it is better thought of as a means to increase carbon gain under carbon limited conditions (Cockburn 1985, Cushman 2001, Lttge 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 devel oped among leafy orchids (Benzing et al. 1983). This presumably poses a rather large

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6 resistance to the diffusion of carbon dioxide in to these roots. CAM allows for maximiz ed carbon gain under these conditions by allowing the plant to generate a much gre ater concentration gradient of CO 2 between the root cortex and the surrounding atmosphere, driving the influx of CO 2 in to the plant. In addition remobilization of CO 2 from malate during the light vastly increases the internal CO 2 :O 2 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 (Lttge 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 ove r the rate at which water is pulled in to the velamen. However, by increasing the solute concentr ation 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 i n the velamen and increasing the amount of water that can be pulled into the root following a wetting e vent. 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

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7 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 availabl e to terrestrially rooted vegetation, forcing them to rely on sporadic and short live d 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 sit uation, including litter impounding leaf configurations, phytotelmata, absorptive tric homes, insectivory, myrmecophytism, and the occurrence of mycorrhizae (Benzing 1990, Hie tz et al. 2002, Nadkarni 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 nutri ents to increase fecundity (Benzing et al. 1983).

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8 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 embe dded in a matrix of highly suberised “U” cells that provide a barrier to water movement. T he passage cells contain an extensive membrane network and a greater density of mitochondria than surrounding cells, suggesting some active mechanism for transpor ting 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 com prised 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 matur ity, which occurs within the first few millimeters behind the root tip (Pridgeon 1987). Wa lls of the velamen cells undergo deferential deposition of cellulose fibrils prior t o cell death

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9 resulting in secondary thickenings (Pridgeon 1987). These thickenings maybe heli cal, 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 Ba rthlott 1988). As a result of drying and shrinking of the cell walls of the velamen following c ell death, pores develop ranging in size from 1 m to 50 m in diameter (Pridgeon 1987). These pores originate as small tears and elliptical slits in pit fiel ds 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 siz e of large pores by preventing further elongation (Pridgeon 1987). Species with helical t hickenings, most common in epiphytes (Stern and Carlsward 2004), generally exhibit the larges t pores (Porembski and Barthlott 1988). Across all species the inner most tangential velamen wall shows the greatest density of pores, particularly above passag e 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 inne r most velamen layers and decreases in the outer layers (Pridgeon 1987, Sanford and Adanlawo 1973). I n 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 m 2 g -1 an interesting finding as this is approximately twice that observed for wood and jute and greater than three times that for cotton.

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10 Velamen structure also varies with taxon, but is relatively constant acros s a given species or in many cases a greater taxonomic grouping, particularly at the t ribal 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 ci ted 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 conc erning 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 surroundi ng 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 spec ies 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

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11 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 vela men 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 t o absorb water vapor. V. suavis was able to absorb 15% of its dry weight at a relative humidity (RH) as low as 50% (at 20C), and showed a maximum absorption of nearly 24% of its dry weight at 98% RH (at 20C). These values are much greater than those observed for other cellulosic materials such as wood, jute and cotton (Giles and Agni hotri 1968). Within the cortex of velamentous roots lie thin walled living parenchyma, which may contain chloroplast, and non-living tracheoidal idioblasts resembling wate r 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 poss ible for them function as collapsible water storage cells similar to those found in the pare nchyma 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 a eration complexes spanning the exodermis and velamen (discussed below). Among leafy orchids these intercellular air spaces are within the size range for the interce llular air spaces Modern used here refers to publications post 1900.

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12 found in the leaves of CAM and C 4 species. Shootless taxa possess larger air spaces, but they still remain smaller than those observed for C 3 plants (Benzing et al. 1983). The velamen is especially well developed among shootless orchids, and possesse s specialized aeration complexes which presumably provide a diffusive path for ga s 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 a re 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 ex odermis. 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 cort ical 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 gasexchange 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. 1985).

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13 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 desicca tion 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 aer ation complexes found in the roots of P. lindenii is yet to be determined. In their work with Chiloschista usneoides Cockburn et al. (1985), conclude that it was highly unlikely that the aeration complex could regulate gas exchange (C ockburn 1985). They suggested that the small loss of CO 2 observed during the light in C. usneoides was not the result of an increased diffusive resistance resulting from reducti on in the cortical component aperture, but resulted from the maintenance of internal CO 2 concentration of the plant near atmospheric concentration (Cockburn 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 inacc urate representation of actual internal CO 2 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 velame n layer), in her work with the anatomy of Vandeae, Carlsward (pers. comm.) noted a g reat deal of variation in the distance between the cortical components within a given specie s. The basis of this variation remains undetermined. But it seems likely that it c ould result

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14 from changes in turgor during the process of preparing specimens for anatomi cal study and so maybe analogous to changes in turgor in the living root effecting cortical component aperture. Objectives 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 (Cockburn 1985), the diversity of CAM orchids in tropical forest may mean that TAAM is exhibited by as many as 75% of all C AM 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 ex change (Cockburn 1983, Cockburn 1985, Lttge 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 CO 2 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 resea rch 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.

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15 CHAPTER 2 A MODEL OF GAS EXCHANGE IN PHOTOSYNTHETIC VELAMENTOUS ROOT S Introduction 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 A stomatal Acid Metabolism (TAAM) is unique in that there is a total absence of stomata from t he photosynthetic organs (Cockburn 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 (Cockburn 1985), its occurrence among tropical CAM species maybe quite common as orchids may comprise 75% of all CAM species i n a given tropical forest (Zotz and Ziegler 1997). Diel gas exchange patterns for the photosynthetic roots of orchids are genera lly indicative of CAM, showing predominate carbon assimilation at night (Benzing and Ott 1981, Cockburn 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 negati ve 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

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16 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 bal ance (Benzing and Ott 1981, Cockburn et al. 1985). The magnitude of carbon assimilation in shootless species is generally lower than that of the CAM leaves from other orchi ds (Benzing and Ott 1981) Use of radio labeled 14 CO 2 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 C 3 -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 degre e 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 eff lux of CO 2 in the dark (Cockburn 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 inter nal partial pressures of CO 2 are associated with the decarboxylation of malate during the light by CAM plants (Cockburn 1985, Lttge 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 CO 2 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 photo synthetic 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).

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17 (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 shootle ss taxa which lack the stems modified for water storage present among other epiphy tic orchids. 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 sa me 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 det ailed examination of diel gas exchange patterns and through estimations of internal conductance based on a quantitative model describing water vapor flux. Materials and Methods Plant Material Mature greenhouse grown specimens of Chiloschista lunifera (Reichb.f.) J. J. Sm. were obtained from Tropiflora Inc. (Sarasota, Florida). Plants were grow n 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, ensuri ng the roots were the sole means of carbon assimilation and water loss.

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18 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 mols -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 da ys. All fluxes were calculated on a surface area basis, determined by assumin g 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-C OR 2005).

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19 Water Vapor Flux Across Orchid Velamen Water vapor flux across excised pieces of velamen was measured using a LI6400 Portable Photosynthesis System (LI-COR, Inc. Lincoln, Nebraska). Velame n 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 sh eet (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 w ater vapor saturated air space on one side of the velamen via a filter paper wick positi oned 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 co rtical material was removed and the velamen sections were blotted dry prior to mounting in t he cuvette. Excised pieces of velamen were positioned underneath the mounting plate at i ts orifice so that the surface formally facing the root cortex faced the w ater 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 the se gaskets and positioned in the air space under the velamen.

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20 0 1 2 A B Attached to the LI-6400 sensor head along this side, and sealed with rubber O-rings 3 mm square velamen mounting plate orifice Counter sunk holes for mounting to the LI-6400 sensor head Screw holes for connecting upper and lower sections of the cuvette; lower holes threaded 10-32 Closed cell foam rubber gaskets 0.6 mm brass velamen mounting plate Thermocouple inserted here Water reservoir Figure 2-1. Schematic diagram of the velamen cuvette. A. View of chamber f rom 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). Measurement s 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

PAGE 28

21 each decrease in VPD. Measurements were logged every 20 minutes for a total of f ive measurements at each VPD, and the entire experimental procedure was repea ted three times. Flow through the system was held constant at 200 mols -1 and chamber temperature was held constant by maintaining the LI-6400’s block temperature at 27C. It was assumed that the boundary layer conductance for the velamen cuvette, g bvc represented the combined effects of air passing over the plane of the mounting plat e and the unstirred layer of air atop the velamen in the mounting plate orifice (Eq. 2-1) (N obel 1983). g bvc = _D w __P___ 2-1 total r (T a ) Where D w is the diffusivity coefficient of water vapor in air (2.4 x10-4 m 2 s -1 ), and r is the universal gas constant (8.3145 x10 -3 m 3 kPamol -1 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 ms -1 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

PAGE 29

22 to the boundary layer thickness by the layer of unstirred air positioned atop the velam en in the orifice but below the surface of the mounting plate. From VP vel (calculated from Eq. 2-4) an estimation of the vapor phase water potential of the velamen, vel was made based on the natural log of the ratio of VP vel to the saturation vapor pressure of the surrounding air, SVP air Following equation 2-3 vel was calculated and the resulting figure retuned in MPa. vel = _R T a ln _VP vel Eq. 2-3 M SVP air Where R is the universal gas constant expressed in J mol -1 K -1 T a is the air temperature inside the velamen cuvette and M is the molecular weight of water (18.02 g mol -1 ). Gas-exchange Model Measurements of excised velamen indicated a capacity of velamen to absor b water vapor directly from moist air, demonstrating a source/sink role of v elamen for water vapor moving from the root cortex to the atmosphere. A model describing chang es in internal conductance within the root was developed by partitioning water vapor fl ux 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 velame n is at steady state for a particular vapor pressure inside the velamen, transpir ation (E) as determined from the whole plant gas exchange data can be redefined as in equati ons 2-4 and 2-5. E = g bl (VP vel – VP air ) 2-4 = g in t (VP int – VP vel ) 2-5 Where g bl and g int are the boundary layer and internal conductances respectively, VP vel is the vapor pressure inside the velamen, VP int is the vapor pressure inside the root and VP air

PAGE 30

23 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 i nside the cuvette. By rearranging equations 2-4 and 2-5 to solve for the two remaining unknowns, g int and VP vel the model used to estimate g int 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. ____1__________ 2-6 g int = -1 + _(VP int – Vp air )_ g bl E Results Diel gas exchange patterns for both CO 2 and H 2 O were indicative of CAM under all vapor pressure deficits, though the magnitude of these fluxes was quite smal l under the highest VPD (Fig. 2-2). Net carbon assimilation increased in a stepwise manne r 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 re gime plants exhibited the greatest net water loss, showing what appeared to be a lar ge amount of diurnal variation in transpiration (Fig. 2-2B). These apparent spikes in transpirat ion at 10:00, 14:00 and 16:00 corresponded to a rapid decrease in transpiration rate following

PAGE 31

24 B -3.E-06 0.E+00 3.E-06 6.E-06 9.E-06 1.E-05 2.E-05 -3.E-06 0.E+00 3.E-06 6.E-06 9.E-06 1.E-05 2.E-05 -3.E-06 0.E+00 3.E-06 6.E-06 9.E-06 1.E-05 2.E-05 -3.E-06 0.E+00 3.E-06 6.E-06 9.E-06 1.E-05 2.E-05 -3.E-06 0.E+00 3.E-06 6.E-06 9.E-06 1.E-05 2.E-0512:0015:1218:2421:360:484:007:1210:24 -0.04 -0.02 0 0.02 0.04 0.06 0.08 0.1 0.12 -0.04 -0.02 0 0.02 0.04 0.06 0.08 0.1 0.12 -0.04 -0.02 0 0.02 0.04 0.06 0.08 0.1 0.12 -0.04 -0.02 0 0.02 0.04 0.06 0.08 0.1 0.12 -0.04 -0.02 0 0.02 0.04 0.06 0.08 0.1 0.12 12:0015:1218:2421:360:484:007:1210:24 Time Time Assimilation ( mol of CO 2 m 2 s 1 ) Transpiration (mmol of H 2 Om 2 s 1 ) A J I G F E D C H Figure 2-2: Carbon assimilation and transpiration at constant VPD. A. Assimil ation 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 molm -2 s -1 ), dark period shown in gray.

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25 0 0.001 0.002 0.003 0.004 0.005 0.006 0.007 0.008 0.009 0.01 0 0.01 0.02 0.03 0.04 0.05 0.06 Net carbon gain (g of Cm -2 ) Net water loss (g of H 2 Om -2 ) 0.0E+00 5.0E-06 1.0E-05 1.5E-05 2.0E-05 2.5E-05 3.0E-05 3.5E-05 4.0E-05 4.5E-050.701.201.702.202.70 Mean gint A B C VPD ( kPa) Figure 2-3: Net carbon gain, water loss and internal conductance for all vapor pre ssure deficits. Net carbon gain (A) and net water loss averaged for all plants (B) integrated for day (open circles) and night (closed circles). C. Inter nal conductance averaged for day (open circles) and night (closed circles). All values are shown plus or minus standard error.

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26 -0.60 -0.50 -0.40 -0.30 -0.20 -0.10 0.00 0306090120150180210240 Change in transpiration rate (E t=x /E t =0 1) Time (min) 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 ze ro 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 exhibit ed 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 l eading to an almost neutral water balance. Figure 2-4: Transpiration rate response during the first four hours of measure ment 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.

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27 -6 -5 -4 -3 -2 -1 0 1 2 3 0.701.201.702.202.70VPD (kPa) Traspiration ( molm 2 s 1 ) 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 isola ted velamen mounted in the velamen cuvette under all VPDs (Table 2-1). Figure 2-5: Water vapor flux across isolated velamen tissue. The units of transpi ration have been converted to molm -2 s -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. VPD Velamen cuvette Whole plant __Mean__ _Std. Error_ __Mean__ _Std. Error_ 2.50 -163.52 1.28 -176.66 15.37 2.12 -139.88 0.95 -136.19 1.74 1.78 -107.82 0.75 -105.46 4.34 1.43 -91.73 0.88 -92.19 5.52 1.07 -68.52 2.27 -66.12 3.28

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28 A B 0 0.0001 0.0002 0.0003 0.0004 0.0005 0.0006 0.0007 0.0E+00 5.0E-06 1.0E-05 1.5E-05 2.0E-05 2.5E-05 3.0E-05 3.5E-05 4.0E-05 0.0E+00 5.0E-06 1.0E-05 1.5E-05 2.0E-05 2.5E-05 3.0E-05 3.5E-05 4.0E-05 0.0E+00 5.0E-06 1.0E-05 1.5E-05 2.0E-05 2.5E-05 3.0E-05 3.5E-05 4.0E-05 0.0E+00 5.0E-06 1.0E-05 1.5E-05 2.0E-05 2.5E-05 3.0E-05 3.5E-05 4.0E-05 0 0.0001 0.0002 0.0003 0.0004 0.0005 0.0006 0.0007 0 0.0001 0.0002 0.0003 0.0004 0.0005 0.0006 0.0007 0 0.0001 0.0002 0.0003 0.0004 0.0005 0.0006 0.0007 0 0.0001 0.0002 0.0003 0.0004 0.0005 0.0006 0.000712:0015:1218:2421:360:484:007:1210:24 Time Time Internal conductance (mmolm 2 s 1 ) Total conductance (mmolm 2 s 1 ) 0.0E+00 5.0E-06 1.0E-05 1.5E-05 2.0E-05 2.5E-05 3.0E-05 3.5E-05 4.0E-0512:0015:1218:2421:360:484:007:1210:24 J I G F E D C H Figure 2-6: 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 molm -2 s -1 ), dark period shown in gray.

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29 Discussion 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, Chiloschista 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 CO 2 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 drivi ng 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 ex change. Without such a mechanism it seems highly unlikely that any change in diel transpi ration 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 dur ing the day do to the energy contributed to the system by light striking the plant.

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30 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 f or measurement directly from the greenhouse, moving from the relatively moist c ool 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 transpir ation rates, this decline in transpiration rate can not be explained by changes in the forces drivi ng 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 vela men 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 2 4 hour time period, leading to a near neutral water balance. However, it remains to be seen i f 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.

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31 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 (B enzing 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 regulatin g 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 CO 2 evolution during the day and the decrease in transpiration rates following the drasti c increase in VPD at the start of measurement are all indicative of some reg ulatory mechanism over gas exchange. The variation in transpiration throughout the day and the observation that transpiration rates tracked those of assimilation suggest a s tomata 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 regula tory mechanism exists it does not provide tight control over gas exchange. Estimati ons of vapor phase water potential of the velamen are very negative at these VPDs, s uggesting

PAGE 39

32 the velamen tissue is dry. It therefore seems highly probable that the mechani sm 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 allow s C. lunifera to exert control over fluxes of CO 2 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 investiga tion will reveal that the observed changes in transpiration rates throughout the day cor respond to changes in cortical component aperture.

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33 GLOSSARY OF SYMBOLS Symbol Description Units D Mounting plate orifice depth m D w Diffusivity coefficient of water in air (2.4 x 10 -4 ) m 2 s -1 E Transpiration rate mmolm -2 s -1 g bl Boundary layer conductance of water vapor mmolm -2 s -1 g bvc Boundary layer conductance for velamen mounted in the velamen cuvette mmolm -2 s -1 L Characteristic dimension m M Molecular weight of water (18.02) gmol -1 P Atmospheric pressure kPa r Universal gas constant (8.3145 x10 -3 ) m 3 kPamol -1 K -1 R Universal gas constant (8.3145) Jmol -1 K -1 r bl Boundary layer resistance to water vapor flux r exo Resistance to water vapor flux due to the exodermis r pnue Pneumathode resistance to water vapor flux T a Air temperature inside the cuvette K T r Root temperature C V Wind speed ms -1 VP air Water vapor pressure of air kPa VP int Water vapor pressure inside the root kPa

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34 34 VP vel Water vapor pressure of velamen tissue kPa total Total boundary layer thickness for velamen mounted in the velamen cuvette m vel Vapor phase water potential of the velamen MPa

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35 LIST OF REFERENCES Bakrim N, J Brulfert, J Vidal and R Chollet. 2001. Phosphoenolpyruvate carboxyl kinase is controlled by a similar signaling cascade in CAM and C 4 plants. Biochemical and Biophysical Research Communications 286 : 1158-1162 Benzing DH. 1990. Vascular epiphytes Cambridge, Massachusetts: Cambridge University Press. Benzing DH and DW Ott. 1981. Vegetative Reduction in Epiphytic Bromeliaceae and Orchidaceae: Its Origin and Significance. Biotropica 13 : 131-140 Benzing DH, DW Ott and WE Friedman. 1982. Roots of Sobralia macrantha (Orchidaceae): Structure and Function of the Velamen-Exodermis Complex. American Journal of Botany 69 : 608-614 Benzing DH, WE Friedman, G Peterson and A Renfrow. 1983. Shootlessness, Velamentous Roots, and the Pre-Eminence of Orchidaceae in the Epiphytic Biotope. American Journal of Botany 70 : 121-133 Borland AM and H Griffiths. 1990. The regulation of CAM and respiratory recycling by water supply and light regime in the C 3 -CAM intermediate Sedum telephium Functional Ecology 4 : 33-39 Carlsward BS. 2004. Molecular systematics and anatomy of Vandeae (Orchidaceae): t he evolution of monopodial leaflessness. Doctoral dissertation. University of Florida, Gainesville, Florida. Carlsward BS, WM Whitten and NH Williams. 2003. Molecular phylogenetics of neotropical leafless Angraecinae (Orchidaceae): Reevaluation of generic concepts. International Journal of Plant Science 164 : 43-51 Chen L and A Nose. 2004. Day-night changes in energy-rich compounds in crassulacean acid metabolism (CAM) species utilizing hexose and starch. Annals of Botany 94 : 449-455 Cockburn W. 1983. Stomatal mechanism as the basis of the evolution of CAM and C4 photosynthesis. Plant, Cell and Environment 6 : 275-279 Cockburn W. 1985. Tansley Review No 1. Variation in Photosynthetic Acid Metabolism in Vascular Plants: Cam and Related Phenomena. New Phytologist 101 : 3-24

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36 Cockburn W. 1998. Rapid-cycling CAM; an hypothetical variant of photosynthetic metabolism. Plant, Cell and Environment 21 : 845-848 Cockburn W, GJ Goh and PN Avadhani. 1985. Photosynthetic carbon assimilation in a shootless orchid, Chiloschista usneoides (DON) LDL. Plant Physiology 77 : 8386 Cushman JC. 2001. Crassulacean acid metabolism. a plastic photosynthetic adaptation to arid environments. Plant Physiology 127 : 1439-1448 Cushman JC and HJ Bonhert. 1999. Crassulacean acid metabolism: Molecular genetics. Annual Review of Plant Physiology 50 : 305-332 Cushman JC and AM Borland. 2002. Induction of Crassulacean acid metabolism by water limitation. Plant, Cell and Environment 25 : 295-310 Drincovich MF, P Casati and CS Andreo. 2001. NADP-malic enzyme from different plants: a ubiquitous enzyme involved in different metabolic pathways. Federation of European Biochemical Societies Letters 490 : 1-6 Dycus A M and L Knudson. 1957. The role of the velamen of the aerial roots of orchids. Botanical Gazette 119 : 78-87 Engard CJ. 1944. Morphological identity of the velamen and exodermis in orchids. Botanical Gazette 105 : 457-462 Erickson LC. 1957. Respiration and photosynthesis in Cattleya roots. American Orchid Society Bulletin 26 : 401-402 Giles CH and VG Agnihorti. 1968. Water vapor adsorption by aerial roots: a new type of high specific surface cellulose. Chemistry and Industry 35 : 1192-1194 Goh CJ, J Arditti and PN Avadhani. 1983. Carbon fixation in orchid aerial roots. New Phytologist 95 : 367-374 Hew CS. 1987. Respiration in orchids. In: Arditti J, ed. Orchid biology. Reviews and perspectives IV Ithaca, New York: Cornell University Press. 227-259 Hew CS, LY Lim and CM Low. 1993. Nitrogen uptake by tropical orchids. Environmental and Experimental Botany 33 : 273-281 Hew CS, YW Ng, SC Wong, HH Yeoh and KK Ho. 1984. Carbon dioxide fixation in orchid aerial roots. Physiologia Plantarum 60 : 154-158

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37 Hew CS, QS Ye and RC Pan. 1991. Relation of respiration to CO 2 fixation by Aranda orchid roots. Environmental and Experimental Botany 31 : 327-331 Hietz P, W Wanek, R Wania, and N Nadkarni. 2002. Nitrogen-15 natural abundance in a montane cloud forest canopy as an indicator of nitrogen cycling and epiphyte nutrition. Oecologia 131 : 350-355 Johansson DR. 1977. Epiphytic orchids as parasites of their host trees. Bulletin of the American Orchid Society 46 : 703-707 Keeley JE and PW Rundel. 2003. Evolution of CAM and C4 carbon-concentrating mechanisms. International Journal of Plant Science 164 : 555-577 Kluge M, B Vinson and H Ziegler. 1997. Ecophysiological studies on orchids of Madagascar: incidence and plasticity of crassulacean acid metabolism in s pecies of the genus Agraecum Bory. Plant Ecology 135 : 47-57 Lambers H, FS Chapin and TL Pons. 1998. Plant physiological ecology New York, New York: Springer-Verlag New York, Inc. LI-COR Biosciences. 2005. Using the LI-6400 portable photosynthesis system; Version 5.3. LI-COR, Inc. Lincoln, Nebraska Lttge U. 1987. Tansley review no. 10 Carbon dioxide and water demand: Crassulacean acid metabolism (CAM), a versatile ecological adaptation exemplify ing the need for integration in ecophysiological work. New Phytologist 106 : 593-629 Lttge U. 2000. The tonoplast functioning as the master switch for circadian regulation of crassulacean acid metabolism. Planta 211 : 761-769 Lttge U. 2004. Ecophysiology of crassulacean acid metabolism (CAM). Annals of Botany 93 : 629-652 Meidner H. 1987. The humidity response of stomata and its measurement. Journal of Experimental Botany. 38 : 877-882 Nadkarni NM. 1985. Roots that go out on a limb. Natural History 94 : 42-49 Nadkarni NM and TJ Matelson. 1991. Fine litter dynamics within the tree canopy of a tropical cloud forest. Ecology 72 : 2071-2082 Nobel PS. 1983. Biophysical plant physiology and ecology San Francisco, California: WH Freeman and Company.

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38 Kwok-Ki H, Y Hock-Hin and H Choy-Sin. 1983. The presence of photosynthetic machinery in aerial roots of leafy orchids. Plant and Cell Physiology 24 : 13171321 Noel ARA. 1974. Aspects of cell wall structure and the development of the velamen in Ansellia gigantea Reichb. f. Annals of Botany 38 : 495-504 Osmond CB. 1978. Crassulacean acid metabolism: a curiosity in context. Annual Review of Plant Physiology 29 : 379-414 Porembski S and W Barthlott. 1988. Velamen radicum micromorphology and classification of Orchidaceae. Nordic Journal of Botany 8: 117-137 Pridgeon AM. 1987. The velamen and exodermis of orchid roots. In: Arditti J, ed. Orchid biology. Reviews and perspectives IV Ithaca, New York: Cornell University Press. 139-192 Rolfe R. 1914. Leafless orchids. Orchid Review 22 : 73-75 Ruinen J. 1953. Epiphytosis. A second view on epiphytism. Annals of Bogor 1 : 101-157 Sanford WW and I Adanlawo. 1973. Velamen and exodermis characteristics of West African epiphytic orchids in relation to taxonomic grouping and habitat toleranc e. Journal of the Linnaean Society of Botany 66 : 307-321 Schulze ED, NC Turner, T Gollan and KA Shackel. 1987. Stomotal response to air humidity and to soil drought. In: Zieger J, GD Farquhar and IR Cowan, ed. Stomatal function Stanford, California: Stanford University Press. 311-321 Solereder H and FJ Meyer. 1930. Systematic anatomy of the monocotyledons. VI. Microspermae. Jerusalem, Israel: Israel Program for Scientific Translations Stern WL and B Carslward. 2004. Vegetative constants in the anatomy of epiphytic orchids. Orchid Review 112 : 119-122 Taybi T, HG Nimmo and AM Borland. 2004. Expression of phosphoenolpyruvate carboxylase and phosphoenolpyruvate carboxyl kinase genes. Implications for genotypic capacity and phenotypic plasticity in the expression of crassulac ean acid metabolism. Plant Physiology 135 : 587-598 Zotz G. 2002. Categories and CAM – blurring the divisions, increasing understanding? New Phytologist 156 : 1-8 Zotz G, B Vollrath and G Schmidt. 2003. Carbon relations of fruits of epiphytic orchids. Flora 198 : 98-105

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39 Zotz G and H Ziegler. 1997. The occurrence of crassulacean acid metabolism among vascular epiphytes from Central Panama. New Phytologist 137 : 223-229

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40 BIOGRAPHICAL SKETCH 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 degr ee 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, F lorida, and began work on a master’s degree at the University of Florida under the super vision of Stephen S. Mulkey.


Permanent Link: http://ufdc.ufl.edu/UFE0014180/00001

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Title: Mechanistic Basis of Gas Exchange in the Terrestrial Astomatal Acid Metabolism Plant, Chiloschista lunifera (Reichb. f.) J. J. Sm. (Orchidaceae)
Physical Description: Mixed Material
Copyright Date: 2008

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Source Institution: University of Florida
Holding Location: University of Florida
Rights Management: All rights reserved by the source institution and holding location.
System ID: UFE0014180:00001

Permanent Link: http://ufdc.ufl.edu/UFE0014180/00001

Material Information

Title: Mechanistic Basis of Gas Exchange in the Terrestrial Astomatal Acid Metabolism Plant, Chiloschista lunifera (Reichb. f.) J. J. Sm. (Orchidaceae)
Physical Description: Mixed Material
Copyright Date: 2008

Record Information

Source Institution: University of Florida
Holding Location: University of Florida
Rights Management: All rights reserved by the source institution and holding location.
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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).












By

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


2006















ACKNOWLEDGMENTS

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

Page

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

CHAPTER

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

BIOGRAPHICAL SKETCH...........................................................................................40















TABLE

Table Page

2-1 Velamen vapor phase water potentials........................ ...................... 27















LIST OF FIGURES


Figure Page

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.
SM. (ORCHIDACEAE).

By

Jason R. Hupp

May 2006

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

mechanism.

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

other mechanism.















CHAPTER 1
INTRODUCTION

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

(TAAM).

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

(Cockbum 1985).

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

(Carlsward 2004).

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.


Figure 1-1:









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

1988).

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

1968).

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.

1985).









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

component aperture.

Objectives

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.















CHAPTER 2
A MODEL OF GAS EXCHANGE IN PHOTOSYNTHETIC VELAMENTOUS ROOTS

Introduction

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

orchids.

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

Plant Material

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

2005).









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
0 1


3 mm square velamen
mounting plate orifice

Counter sunk holes for
mounting to the LI-6400
sensor head

Screw holes for connecting
upper and lower sections of
the cuvette; lower holes
threaded 10-32

Closed cell foam rubber
gaskets

0.6 mm brass velamen
mounting plate


Thermocouple inserted here


Water reservoir


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


A


Figure 2-1.









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

1983).

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
M SVPair

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).

Gas-exchange Model

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

and 2-5.

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.

1 2-6
giant = :-l + (VPi.nt-Vairp)
gbl E

Results

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
















Ol
012
0 1
008
006
0 04

0 02
0
002
0 04


012
o:Q
008
006
004 ri.
0 02

002
004


012
o:
008
006


002

002
004


012 0

002
006
004
002







0 12


0 04
0 02



004


2 E 05

1E 05
9 E 06

6 E 06

3 E 06
0 E+00

3 E 06



2E05 (

1E 05

9 E 06

6E06
3 E06 6kIP-

0 E+00

-3 E06


0


2E05
2 E 05






1E-05
9 E 06

6 E 06

3 E-06
0 E+00

3 E-06



2E05

1E 05

9 E-06

6 E 06

3 E 06 6-------------

0 E+00
3 E06


1200 15 12 1824 2136 048 400 7 12 1024 1200 1512 1824 2136 048 400 712 1024
Time Time

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.
















'h


si,
co

Z


c~~
c~,~'
30 E
O
~1
6,
u o


0.06

0.05

0.04

0.03

0.02

0.01


0.01
0.009
0.008
0.007
0.006
0.005
0.004
0.003
0.002
0.001
0


Q


4.5E-05
4.0E-05
3.5E-05
3.0E-05
2.5E-05
2.0E-05
1.5E-05
1.0E-05
5.0E-06
0.OE+00
0.70


1.70

VPD (kPa)


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.


0.00


-0.10


-0.20


-0.30


-0.40


-0.50


Figure 2-4:


-0.60 ...
0 30 60 90 120 150 180 210 240
Time (min)

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).


3,


VPD (kPa)


Figure 2-5:


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.


Velamen cuvette


Mean

-163.52
-139.88
-107.82
-91.73
-68.52


Whole plant


Std. Error

1.28
0.95
0.75
0.88


Mean

-176.66
-136.19
-105.46
-92.19


2.27 -66.12


Std. Error

15.37
1.74
4.34
5.52
3.28


VPD


2.50
2.12
1.78
1.43
1.07

























000
o oo






ooo



o ooo
0o c0
0000
o 000
0000
0 000
o co000



o co000
o co000
o coo
o oo,
o coo


o ooo




oooo





1200 1512 1824 2136 048 400 12 1024
Time
Time


Figure 2-6:


0


0
0''
r-e
0
0)
a *
^..

..


0
U


0/-~




f9O


1200 1512 1824 2136 048 400 712 1024

Time


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
in gray.










Discussion

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.


















Symbol

D

Dw

E

gbl

gbvc


L

M

P

r

R

rbl
rh

rexo

rpnue

Ta

Tr

V

VPair

VPint


APPENDIX
GLOSSARY OF SYMBOLS

Description

Mounting plate orifice depth

Diffusivity coefficient of water in air (2.4 x 10-4)

Transpiration rate

Boundary layer conductance of water vapor

Boundary layer conductance for velamen mounted in the
velamen cuvette

Characteristic dimension

Molecular weight of water (18.02)

Atmospheric pressure

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

Root temperature

Wind speed

Water vapor pressure of air

Water vapor pressure inside the root


Units

m

m2 -1


mmol-m-2s-1

mmol-m-2s-1

mmol-m-2s-1


m

g-mol-1

kPa

m3 kPa-mol1-K-1

J-mol 1-K-1








K

C

m-s1

kPa

kPa









VPvei Water vapor pressure of velamen tissue kPa

total Total boundary layer thickness for velamen mounted in the m
velamen cuvette

've1 Vapor phase water potential of the velamen MPa















LIST OF REFERENCES


Bakrim N, J Brulfert, J Vidal and R Chollet. 2001. Phosphoenolpyruvate carboxyl
kinase is controlled by a similar signaling cascade in CAM and C4 plants.
Biochemical and Biophysical Research Communications 286: 1158-1162

Benzing DH. 1990. Vascular epiphytes. Cambridge, Massachusetts: Cambridge
University Press.

Benzing DH and DW Ott. 1981. Vegetative Reduction in Epiphytic Bromeliaceae and
Orchidaceae: Its Origin and Significance. Biotropica 13: 131-140

Benzing DH, DW Ott and WE Friedman. 1982. Roots of Sobralia macrantha
(Orchidaceae): Structure and Function of the Velamen-Exodermis Complex.
American Journal of Botany 69: 608-614

Benzing DH, WE Friedman, G Peterson and A Renfrow. 1983. Shootlessness,
Velamentous Roots, and the Pre-Eminence of Orchidaceae in the Epiphytic
Biotope. American Journal of Botany 70: 121-133

Borland AM and H Griffiths. 1990. The regulation of CAM and respiratory recycling
by water supply and light regime in the C3-CAM intermediate Sedum telephium.
Functional Ecology 4: 33-39

Carlsward BS. 2004. Molecular systematics and anatomy of Vandeae (Orchidaceae): the
evolution of monopodial leaflessness. Doctoral dissertation. University of Florida,
Gainesville, Florida.

Carlsward BS, WM Whitten and NH Williams. 2003. Molecular phylogenetics of
neotropical leafless Angraecinae (Orchidaceae): Reevaluation of generic
concepts. International Journal of Plant Science 164: 43-51

Chen L and A Nose. 2004. Day-night changes in energy-rich compounds in crassulacean
acid metabolism (CAM) species utilizing hexose and starch. Annals of Botany 94:
449-455

Cockburn W. 1983. Stomatal mechanism as the basis of the evolution of CAM and C4
photosynthesis. Plant, Cell and Environment 6: 275-279

Cockburn W. 1985. Tansley Review No 1. Variation in Photosynthetic Acid Metabolism
in Vascular Plants: Cam and Related Phenomena. New Phytologist 101: 3-24











Cockburn W. 1998. Rapid-cycling CAM; an hypothetical variant of photosynthetic
metabolism. Plant, Cell and Environment 21: 845-848

Cockburn W, GJ Goh and PN Avadhani. 1985. Photosynthetic carbon assimilation in
a shootless orchid, (C'uh/,,, ui, usneoides (DON) LDL. Plant Physiology 77: 83-
86

Cushman JC. 2001. Crassulacean acid metabolism, a plastic photosynthetic adaptation
to arid environments. Plant Physiology 127: 1439-1448

Cushman JC and HJ Bonhert. 1999. Crassulacean acid metabolism: Molecular
genetics. Annual Review of Plant Physiology 50: 305-332

Cushman JC and AM Borland. 2002. Induction of Crassulacean acid metabolism by
water limitation. Plant, Cell and Environment 25: 295-310

Drincovich MF, P Casati and CS Andreo. 2001. NADP-malic enzyme from different
plants: a ubiquitous enzyme involved in different metabolic pathways. Federation
of European Biochemical Societies Letters 490: 1-6

Dycus A M and L Knudson. 1957. The role of the velamen of the aerial roots of
orchids. Botanical Gazette 119: 78-87

Engard CJ. 1944. Morphological identity of the velamen and exodermis in orchids.
Botanical Gazette 105: 457-462

Erickson LC. 1957. Respiration and photosynthesis in Cattleya roots. American Orchid
Society Bulletin 26: 401-402

Giles CH and VG Agnihorti. 1968. Water vapor adsorption by aerial roots: a new type
of high specific surface cellulose. Chemistry and Industry 35: 1192-1194

Goh CJ, J Arditti and PN Avadhani. 1983. Carbon fixation in orchid aerial roots. New
Phytologist 95: 367-374

Hew CS. 1987. Respiration in orchids. In: Arditti J, ed. Orchid biology. Reviews and
perspectives IV. Ithaca, New York: Cornell University Press. 227-259

Hew CS, LY Lim and CM Low. 1993. Nitrogen uptake by tropical orchids.
Environmental and Experimental Botany 33: 273-281

Hew CS, YW Ng, SC Wong, HH Yeoh and KK Ho. 1984. Carbon dioxide fixation in
orchid aerial roots. Physiologia Plantarum 60: 154-158









Hew CS, QS Ye and RC Pan. 1991. Relation of respiration to CO2 fixation by Aranda
orchid roots. Environmental and Experimental Botany 31: 327-331

Hietz P, W Wanek, R Wania, and N Nadkarni. 2002. Nitrogen-15 natural abundance
in a montane cloud forest canopy as an indicator of nitrogen cycling and epiphyte
nutrition. Oecologia 131: 350-355

Johansson DR. 1977. Epiphytic orchids as parasites of their host trees. Bulletin of the
American Orchid Society 46: 703-707

Keeley JE and PW Rundel. 2003. Evolution of CAM and C4 carbon-concentrating
mechanisms. International Journal of Plant Science 164: 555-577

Kluge M, B Vinson and H Ziegler. 1997. Ecophysiological studies on orchids of
Madagascar: incidence and plasticity of crassulacean acid metabolism in species
of the genus Agraecum Bory. Plant Ecology 135: 47-57

Lambers H, FS Chapin and TL Pons. 1998. Plant physiological ecology. New York,
New York: Springer-Verlag New York, Inc.

LI-COR Biosciences. 2005. Using the LI-6400 portable photosynthesis system; Version
5.3. LI-COR, Inc. Lincoln, Nebraska

Liittge U. 1987. Tansley review no. 10 Carbon dioxide and water demand: Crassulacean
acid metabolism (CAM), a versatile ecological adaptation exemplifying the need
for integration in ecophysiological work. New Phytologist 106: 593-629

Liittge U. 2000. The tonoplast functioning as the master switch for circadian regulation
of crassulacean acid metabolism. Planta 211: 761-769

Liittge U. 2004. Ecophysiology of crassulacean acid metabolism (CAM). Annals of
Botany 93: 629-652

Meidner H. 1987. The humidity response of stomata and its measurement. Journal of
Experimental Botany. 38: 877-882

Nadkarni NM. 1985. Roots that go out on a limb. Natural History 94: 42-49

Nadkarni NM and TJ Matelson. 1991. Fine litter dynamics within the tree canopy of a
tropical cloud forest. Ecology 72: 2071-2082

Nobel PS. 1983. Biophysical plant physiology and ecology. San Francisco, California:
WH Freeman and Company.









Kwok-Ki H, Y Hock-Hin and H Choy-Sin. 1983. The presence of photosynthetic
machinery in aerial roots of leafy orchids. Plant and Cell Physiology 24: 1317-
1321

Noel ARA. 1974. Aspects of cell wall structure and the development of the velamen in
Ansellia gigantea Reichb. f. Annals of Botany 38: 495-504

Osmond CB. 1978. Crassulacean acid metabolism: a curiosity in context. Annual
Review of Plant Physiology 29: 379-414

Porembski S and W Barthlott. 1988. Velamen radicum micromorphology and
classification of Orchidaceae. Nordic Journal of Botany 8: 117-137

Pridgeon AM. 1987. The velamen and exodermis of orchid roots. In: Arditti J, ed.
Orchid biology. Reviews and perspectives IV. Ithaca, New York: Cornell
University Press. 139-192

Rolfe R. 1914. Leafless orchids. Orchid Review 22: 73-75

Ruinen J. 1953. Epiphytosis. A second view on epiphytism. Annals of Bogor 1: 101-157

Sanford WW and I Adanlawo. 1973. Velamen and exodermis characteristics of West
African epiphytic orchids in relation to taxonomic grouping and habitat tolerance.
Journal of the Linnaean Society of Botany 66: 307-321

Schulze ED, NC Turner, T Gollan and KA Shackel. 1987. Stomotal response to air
humidity and to soil drought. In: Zieger J, GD Farquhar and IR Cowan, ed.
Stomatal function. Stanford, California: Stanford University Press. 311-321

Solereder H and FJ Meyer. 1930. Systematic anatomy of the monocotyledons. VI.
Microspermae. Jerusalem, Israel: Israel Program for Scientific Translations.

Stern WL and B Carslward. 2004. Vegetative constants in the anatomy of epiphytic
orchids. Orchid Review 112: 119-122

Taybi T, HG Nimmo and AM Borland. 2004. Expression of phosphoenolpyruvate
carboxylase and phosphoenolpyruvate carboxyl kinase genes. Implications for
genotypic capacity and phenotypic plasticity in the expression of crassulacean
acid metabolism. Plant Physiology 135: 587-598

Zotz G. 2002. Categories and CAM blurring the divisions, increasing understanding?
New Phytologist 156: 1-8

Zotz G, B Vollrath and G Schmidt. 2003. Carbon relations of fruits of epiphytic
orchids. Flora 198: 98-105






39


Zotz G and H Ziegler. 1997. The occurrence of crassulacean acid metabolism among
vascular epiphytes from Central Panama. New Phytologist 137: 223-229















BIOGRAPHICAL SKETCH

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.