Carbon dynamics of the seagrass thalassia testudinum

Carbon dynamics of the seagrass thalassia testudinum

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Carbon dynamics of the seagrass thalassia testudinum
Durako, Michael Joseph
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Tampa, Florida
University of South Florida
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xiii, 161 leaves : ill. ; 29 cm


Subjects / Keywords:
Thalassia testudinum -- Growth ( lcsh )
Thalassia testudinum -- Effect of carbon on ( lcsh )
Dissertations, Academic -- Marine Science -- Masters -- USF ( FTS )


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Thesis (M. S.)--University of South Florida, 1991. Includes bibliographical references (leaves 138-155).

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University of South Florida
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University of South Florida
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All applicable rights reserved by the source institution and holding location.
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028398881 ( ALEPH )
27475852 ( OCLC )
F51-00177 ( USFLDC DOI )
f51.177 ( USFLDC Handle )

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CARBON DYNAMICS OF THE SEAGRASS THALASSIA TESTUDINUM by Michael Joseph Durako A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Marine Science in the University of South Florida April 1991 Co-Major Professor: Kent A. Fanning, Ph.D. Co-Major Professor: Gabriel A. Vargo, Ph.D.


Graduate Council University of South Florida Tampa, Florida CERTIFICATE OF APPROVAL Ph.D Dissertation This is to certify that the Ph.D. Dissertation of Michael Joseph Durako with a major in Marine Science has been approved by the Examining Committee on March 22, 1991 as satisfactory for the dissertation requirement for the Ph. D degree. Examining Committee: -<,e..y 'b'iff.: v-Member: 'Cli'n J'. awes, Ph.D. Member: Larry(P. Solomonson, Ph.D.


ACKNOWLEDGEMENTS The author wishes to thank his major professors Drs. Kent Fanning and Gabe Vargo for their advice and guidance -while our interaction was not extensive, it was critical to my success. I would also like to thank the other members of my committee -Dr. John Paul and Dr. Larry Solomonson for their helpful suggestions, and Dr. Clinton J. Dawes for his longstanding support and friendship. I want to acknowledge and express my appreciation to Dr. Brian Fry who shared some of his ideas on the value and uses of stable isotopes in investigating the carbon metabolism of seagrasses. A special note of thanks goes to Dr. William Sackett for his assistance in obtaining and interpreting the stable carbon isotope data and for his role i n broadening my scientific and personal horizons. Much of the information in Chapters 2 and 3 is a result of research supported by the Coastal Zone Management Program, NOAA, Department of Commerce under Grant No. CM-254 to the Florida Marine R e s earch Institute. Support for the University of Rhode Island, Marine Ecosystems Research Laboratory (MERL) pC02 research was provided by National Science Foundation grant No. NSF-OCE-88-17446. I would like to acknowledge and ii


thank Dr. Ken Hinga and Ms. Cyndi Heil at MERL for logistic and facilities support, Peter Betzer and the providing travel funds. and express my appreciation to Dr. Department of Marine Science for Finally, I would like to thank my wife, Carol, my daughter, Maris, and my son, Corey, for their continued support, encouragement, and love. Thanks guys. iii


TABLE OF CONTENTS LIST OF TABLES LIST OF FIGURES ABSTRACT CHAPTER 1 GENERAL INTRODUCTION CHAPTER 2 INFLUENCE OF CARBON SOURCE ON GROWTH AND STABLE CARBON ISOTOPIC COMPOSITION OF THE SEAGRASS THALASSIA TESTUDINUM Introduction Methods Plant material Experimental o13C Analysis Results 1988 seedling crop 1989 seedling crop Discussion Seedling growth Leaf stable carbon isotope composition CHAPTER 3 EFFECTS OF pC02 ON THE GROWTH AND CARBON ISOTOPIC COMPOSITION OF THE SEAGRASS Thalassia testudinum (HYDROCHARI TACEAE) Introduction Methods Plant material Experimental setup o13C analysis Results Discussion CHAPTER 4 GROWTH RESPONSES OF LIGHT-AND DARK-CULTIVATED AXENIC Thalassia testudinum SEEDLINGS TO ORGANIC CARBON AMENDMENT Introduction Methods iv vi viii xi 1 11 11 13 13 13 15 16 16 20 23 23 27 31 31 34 34 35 38 39 45 56 56 57


Results 1987 seedling cultures 1988 seedling cultures Discussion CHAPTER 5 PHOTOSYNTHETIC UTILIZATION OF C02 AND HC03 IN THE 60 59 63 67 SEAGRASS Thalassia testudinum (HYDROCHARITACEAE) 74 Introduction 74 Methods 78 Plant material 78 Incubation media 79 Photosynthesis measurements 83 Results 86 Photosynthesis at variable pH and constant total carbon concentration 86 Photosynthesis at various DIC concentrations with pH maintained close to that of seawater 88 Discussion 99 CHAPTER 6 CARBONIC ANHYDRASE ACTIVITY IN THE MARINE ANGIOSPERM Thalassia testudinum (HYDROCHARITACEAE) 115 Introduction 115 Methods 118 Results and Discussion 120 CHAPTER 7 SUMMARY AND CONCLUSIONS 126 Carbon source and concentration effects 126 The source of carbon for photosynthesis 127 Is carbon limiting to Thalassia testudinum 129 REFERENCES 134 APPENDICES 152 APPENDIX 1 153 APPENDIX 2 155 v


Table 1 2 3 4 5 6 7 8 9 10 LIST OF TABLES Biomass (mg} and resource allocation characteristics in Thalassia testudimum aquarium seedling cultures (means ( s. e.), n=4]. Mean 6C values of the carbonate (DIC} in the synthetic seawater media and Thalassia testudinum seedling leaf tissue, and LlC ( 613C1eat 613Cmd Water quality characteristics of pC02 treatment microcosms. 61 3c values for Thalassia testudinum leaves (mean s. e.) and treatment media TIC. Growth characteristics (mean s.e.) of Thalassia testudinum seedlings cultured for two months in various pC02 treatments. Biomass characteristics (mean s.e.} of Thalassia testudinum seedlings cultured for two months in various pC02 treatments. Biomass (mg dry wt., mean s.e.) characteristics of light-and dark-cultured axenic testudinum seedlings in Von stosch media with varying percent sucrose. Analyses of variance for treatment effects on 1987 axenic testudinum seedling growth. Analyses of variance for treatment effects on 1988 axenic testudinum seedling growth. Mean leaf 613C values ( s. e.) for 1988 axenic T testudinum seedlings after 3 and 6 months in media varying sucrose. vi Page 19 21 36 41 42 44 63 64 68 70


11 12 13 14 15 Calculated free C02 and HCo3 -concentrations (mM) in media with constant total carbon dioxide concentrations (2.2 mM) but varying pH (Park, 1969; Mehrbach et al., 1973; UNESCO, 1987). Calculated free C02 and HC03 -concentrations (mM) in media with varying total carbon dioxide concentrations but relatively constant pH (Park, 1969; Mehrbach et al., 1973; UNESCO, 1987). Comparison of calculated kinetic parameters from linear transformations of the MichaelisMenten equation. Carbonic anhdrase activity expressed as enzyme units (E.U.) per g fresh weight (mean s.e., n=3), mg or mg protein of Thalassia testudinum leaf tissue. Important stresses which limit the distribution and abundance of seagrasses (adapted from Larkum and den Hartog, 1989). vii 83 84 108 126 136


Figure 1 2 3 4 5 6 7 8 LIST OF FIGURES Mean leaf areas of testudinum seedling cultures in six sediment treatments (n=4, s.e. not shown for clarity). &C values of 1988 testudinum seedlings leaf tissues cultured in six sediment treatments. Leaf area (mean s.e.) of 1989 testudinum seedlings after 110 and 195 days in culture in three sediment treatments. Root and shoot biomass of 1989 T testudinum seedlings after 110 and 195 days in culture in three sediment treatments. &13c values (mean s. e.) of 1989 testudinum seedlings after 110 and 195 days in culture in three sediment treatments. Thalassia testudinum seedling leaf o13C (closed symbols) and fractionation (.113C, open symbols) as a function of pC02 (mean s.e.). Growth characteristics (mean s.e.) of 1987 light-and dark-cultured axenic T testudinum seedlings in media with 0% (open circles), 1% (closed circles), and 3% (triangles) sucrose. Growth charactersistics (mean s.e.) of 1988 light-and dark-cultured axenic T testudinum seedlings in media with 0% (open circles), 1% (closed circles), and 3% (triangles) sucrose. viii Page 17 22 24 25 26 45 62 67


9 10 11 12 13 14 15 16 17 Photosynthetic rates of T testudinum leaf segments as a function of pH in June, 1989. Lineweaver-Burk plots of T testudinum photosynthesis versus C02 (top) and HC03 (bottom) in June, 1989. Photosynthetic rates of T testudinum leaf segments at pH 7.80, 8.21, and 8.61 as a function of DIC concentration in July, 1989. Lineweaver-Burk plot of T testudinum photosynthesis as a function of DIC concentration in July, 1989. Photosynthetic rates of T testudinum leaf segments at pH 7.80, 8.21, and 8.61 as a function of C02 (top) and HC03 (bottom) in July, 1989. Lineweaver-Burk plots of T. testudinum photosynthesis versus C02 (top) and HCo3 -(bottom) in July, 1989. Photosynthetic rates of T. testudinum leaf segments at pH 7.80, 8.21, and 8.61 as a function of DIC concentration in September, 1989. Lineweaver-Burk plot of T testudinum photosynthesis as a function of DIC concentration in September, 1989. Photosynthetic rates of T testudinum leaf segments at pH 7.80, 8.21, and 8.61 as a function of C02 (top) and HCo3 -(bottom) in September, 1989. ix 89 91 92 93 94 96 97 98 99


18 19 20 21 22 23 24 25 Lineweaver-Burk plots of testudinum photosynthesis versus C02 (top) and HC03 (bottom) in September, 1989. Photosynthetic rates of testudinum segments as a function of DIC concentration in December, 1989. Lineweaver-Burk plot of testudinum photosynthesis as a function of DIC concentration in December, 1989. Photosynthetic rates of testudinum leaf segments at pH 7.80, 8.21, and 8.61 as a function of C02 (top) and HC03 -(bottom) in December, 1989. Lineweaver-Burk plots of testudinum photosynthesis versus C02 (top) and HCo3 -(bottom) in December, 1989. Photosynthetic rates of testudinum leaf segments at relatively constant C02 and increasing HC03-. Linearity of carbonic anhydrase assay over a range of standard volumes. Leaf o13C values (mean s.e.) testudinum short-shoots subjected to in situ light reduction at one and three months posttreatment. X 100 102 103 104 105 111 124 134


CARBON DYNAMICS OF THE SEAGRASS Thalassia testudinum by Michael Joseph Durako An Abstract Of a dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Marine Science in the University of South Florida April 1991 Co-Major Professor: Kent A Fanning, Ph.D. Co-Major Professor: Gabriel A. Vargo, Ph.D. xi


The effects of dissolved inorganic and organic carbon sources on stable carbon isotope composition, growth, and photosynthetic characteristics of the seagrass Thalassia testudinum Banks ex Konig were investigated. Media co2 enrichment resulted in a temporal decrease in leaf 613c which, after nine months in culture, was the lowest ever reported for a higher plant (-57.1 /00). Isotopically distinct sediment organic carbon sources also affected the isotopic composition of seedling leaves. Increasing pco2 elicited few growth responses, except at the highest level 3300 but isotopic fractionation increased as a function of pC02 ranging from -8.6 to -21.0 /00 Because of inter-microcosm pH variability, seedlings in the ambient pco2 treatment were exposed to pC02 levels which were about 15% higher than seedlings in the 2-times ambient pco2 treatment, coincident with dissolved inorganic carbon levels that were about 24% lower. Comparatively larger isotopic fractionations exhibited by the control seedlings indicate a greater importance for free-co2 and a correspondingly lower relative importance for HC03 -as the initial substrate for carbon fixation. Growth of axenic T testudinum seedlings was enhanced by the addition of sucrose to the media, indicating possible heterotrophic carbon assimilation. This was confirmed through the use of a natural-abundance isotopic tracer (sucrose derived from the Sugar Beet, = -24.1 Similarities in quantitative growth characteristics between xii


light-and dark-cultured axenic seedlings reflected the -significant contribution of stored reserves to early growth; differences in plastochrone intervals and root production indicated that these characteristics may be under photomorphogenic control. Photosynthetic measurements of leaf segments indicated that ,rr. testudinum utilizes both free-co2 and HCO3 in photosynthesis. Kinetic data revealed a very high affinity for co2 with K5(C02 ) values ranging from 3 to 18 compared to K5(HC03-) values ranging from 1.2 to 7.2 mM. Leaf-associated carbonic anhydrase was detected suggesting that Hco3 -may be converted to C02 at the leaf surface rather than being directly assimilated. The results of this investigation indicate that the degree to which carbon is limiting to ,'r. testudinum varies according to the level of organization (i.e. tissue versus whole plant) being considered. Abstract approved: Co-Major Pro.teJ;>sor: Kent A. Fa:ti?fing, Ph. D. Co-Major Professor: xiii


CHAPTER 1 GENERAL INTRODUCTION 1 seagrasses are aquatic angiosperms that live and complete their life cycles totally submerged in a saline-to-brackish medium (Thayer et al., 1975). They are unique in that they are the only group of angiosperms that have totally returned to the sea (den Hartog, 1970). Based on fossil evidence, this probably occurred about one hundred million years ago during the cretaceous Period (den Hartog, 1970). Thus, seagrasses diverged from the mainstream of angiosperms at an early stage since fossil evidence indicates that angiosperms arose in the late Jurassic or early cretaceous Period (Larkum and den Hartog, 1989). The change to a submerged growth habit has resulted in a number of distinctive morphological and anatomical changes in seagrasses, such as the loss of stomata, the development of gas lacunae, and modification in reproductive structures to accomodate hydrophilous pollination. In addition, seagrasses have developed physiological and biochemical adaptations that allow them to (1) access sediment and water column nutrients (McRoy and McMillan, 1977; Iizumi and Hattori, 1982; Thursby and Harlin, 1982; Brix and Lyngby, 1985), (2) tolerate hypoxicfanoxic sediments (Penhale and Wetzel, 1983; Thursby, 1984b; Smith et al., 1984, 1988; Pulich, 1989), and (3)


2 efficiently photosynthesize at relatively low light levels (Williams and McRoy, 1982; Dennison, and Alberte, 1985, 1987; Dennison, 1987; Dawes and Tomasko, 1988). At present there is world-wide recognition of about fifty eight seagrass species belonging to twelve genera, four families, and two orders (Kuo and McComb, 1989). Although not true grasses, their growth habit and ribbon-like leaves result in meadows or beds resembling terrestrial grasslands. This complex habitat structure, in an otherwise relatively featureless environment, provides substrate and protection for large populations of invertebrates and serves as a nursery and feeding ground for numerous fishes, many with food and commercial value (see Zieman, 1982; Phillips, 1984; Thayer et al., 1984; Zieman and Zieman, 1989). The ability of seagrasses to exert a major influence on coastal marine systems is due, in large part, to their rapid growth and high net producti vi ties, which rival those of cultivated agricultural crops (0.4-16 g c m 2 day for Florida seagrasses versus 0. 9-4. 7 g C m 2 day for wheat, corn, rice, and sugar cane, see Durako, 1988). This primary production supplies carbon to herbivores and to a complex detritus-based food web (Zieman et al., 1979; Kikuchi, 1980; Ogden, 1980; Klumpp et al. 1989) Alternatively, in more eutrophic coastal and estuarine systems, seagrasses may be functionally more important through the provision of substrate for epiphytic micro-and macro-algae, which then serve as the


3 primary food sources for herbivores (Kitting et al., 1984; Fry et al., 1987) Seagrasses also bind sediments, thereby reducing erosion and improving water quality, and they are pivotal in many coastal nutrient and geochemical cycles (Pulich, 1987; Zieman, 1987; Moriarity and Boon, 1989). Seagrass beds occur in a relatively narrow range of depths, generally extending from the low intertidal zone to less than ten meters. Their distribution may be controlled by numerous interrelated factors, such as nutrient and light availability, salinity, depth, temperature, substrate, and currents (Zieman and Zieman, 1989). However, their shallow distribution places many seagrass beds in proximity to major coastal population centers, and subjects them to a variety of man-induced stresses. Man's increasing use of coastal and estuarine habitats for residential, industrial and recreational purposes has resulted in dramatic alteration and loss of many seagrass beds (see review by Shepard et al. 1989). Concurrent recognition of their trophodynamic importance to coastal marine systems has led to an increased awareness of the need to understand the nutritional requirements and metabolic characteristics of these important plants. Currently, views on the nutritional ecology of seagrasses are in a state of flux. Past in situ nutritional studies have provided equivocal information regarding nutrient requirements. For example, nitrogen has traditionally been


4 considered the most limiting nutrient for many marine and estuarine seagrass ecosystems (Patriquin, 1972; Nixon et al., 1976; McRoy and McMillan, 1977; Short, 1983; Boon; 1986). However, recent investigations suggest that nitrogen limitation in seagrass systems is probably very rare because rates of microbially-mediated sediment NH4+ regeneration and nitrogen fixation usually exceed plant requirements (Dennison et al., 1987; Zimmerman et al., 1987). Other studies suggest that under certain circumstances (e.g. when growing in carbonate sediments) seagrasses may be phosphorus limited (Short et al., 1985; Short, 1987), but this view does not consider the ability of seagrass rhizosphere bacteria to solubilize inorganic phosphate in sediments via organic acid release (Craven and Hayasaka, 1982) A third concept, which is gaining increasing acceptance, is that carbon supply is most limiting to submerged marine macrophyte photosynthesis and growth, especially for evolutionarily recent re-invaders of the aquatic habitat like seagrasses (Abel, 1984; Millhouse and Strother, 1986; Beer and Shragge, 1987; Holbrook et al., 1988) Unlike terrestrial plants (from which they presumably evolved), seagrasses are surrounded by a medium in which inorganic carbon is present in three available forms: free C021 HC03-, and co3 2 In seawater {pH 8. 2) 89% of the inorganic carbon is present as HCo3 (approx. 2.0-2.5 mM, compared to 10 C02 in air) and this is the ionic form


5 which has been suggested as the most likely source of photosynthetic carbon (Benedict and Scott, 1976; Beer et al., 1977) In this regard, the relatively high o13C values1 of seagrasses were initially attributed to HCo3 utilization (Park and Epstein, 1961) and possession of the C4 carboxylation pathway (Benedict and Scott, 1976). However, later studies confirmed that most seagrasses were C3 plants and that their relatively heavy isotopic signatures reflected high diffusional barriers and a relatively "closed" carbon fixation system (Andrews and Abel, 1979; Benedict et al., 1980) In c3 plants, inorganic carbon must be in the form of free C02 at the site of carboxylation. Thus, seagrasses should be unable to directly access seawater HCo3 Abel (1984) demonstrated, using buffered seawater media of various pH and total inorganic carbon values, that Thalassia hemprichii (Ehrenb.) Aschers. utilized only free C02 as its photosynthetic substrate. She suggested that this species was carbon-limited based on kinetic data in which the estimated K for free C02 ranged from 0.176 to 0.331 rnM compared to a concentration in the water column of about 0. 015 rnM. As stated above, photosynthetic and growth limitation of submerged macrophytes by inorganic carbon availability is a concept gaining increasing acceptance (Beer and Shragge, 10'13C = [ (RsamptefRstandard) -1] X 1000 / ooi R=13C/12C; s t andard i s a cretaceous belemnite from the Peedee Formation (PDB), South C arolina, which i s given an arbitrary o13C value of 0.0 /00


6 1987). The reliance of some seagrasses on C02 means that they may still resemble their presumed ancestors in this aspect of their physiology. Raven (1970) suggested that submerged plants which utilize c3 type carbon fixation may also be carbon limited due to diffusional limitations, and this factor may be responsible, seagrasses. in large part, for the anomalous 6C values of Keeley et al. (1986) reported that 613C was unrelated to photosynthetic pathway in 22 species of aquatic plants, but rather, seemed to be related to diffusional resistances. Current hypotheses propose that because of the slow diffusion of C02 across the boundary layer surrounding leaves, carbon fixation may draw from an intermediate pool of inorganic carbon (e.g., a lacunae) that is not in reservoir of respired C02 in the equilibrium with the surrounding medium and as a result of this "closed" system there is less isotopic fractionation by RuBP carboxylase. Osmond et al. (1981) argued that, in aquatic plants which use C02 and rely exclusively on RuBP carboxylase, as the diffusional resistances to C02 increase, the 61 3C value of the biomass should approach that of the source carbon. Therefore, the &13c value of the plant may vary according to C02 concentration, expressed as pC02 (andjor C02

7 values under various conditions (Osmond et al., 1981). This has not been done experimentally with seagrasses. 13Cf12C ratios in seagrasses can also be affected by the influence of carbon source, oxygen concentration, light intensity and temperature (Smith et al., 1976; McMillan, 1980, 1982; Zieman, et al., 1984), resulting in between-leaf, betweensite, and seasonal variability in isotopic composition in the same species (Fry et al., 1987). These variations need to be understood in order to utilize seagrass 6'13C information for tracing carbon flow in trophodynamic analyses (i.e., to obtain source information as opposed to process information, see Peterson and Fry, 1987). There is now general agreement that levels of atmospheric C02 will rise significantly over the next 50 to 100 years. Carbon dioxide makes up a small, but vitally important part of the global carbon cycle which circulates among three active resevoirs: the atmosphere, the oceans, and the terrestrial system. global Post et al. (1990) stated that the largest pool of carbon is the ocean with about 37,000 gigatons (billions of metric tons) of dissolved inorganic carbon (DIC}, over 2,000 gigatons of dissolved organic carbon (DOC), and about 30 gigatons of particulate organic carbon (POC) compared to an atmospheric resevoir of 671 gigatons. However, these authors did not consider the relatively vast amount of carbon in sedimentary limestone 1. 8 X 107 gigatons I Stumm and Morgan, 1981). It is estimated that the overall fluxes of


8 carbon into and out of the atmosphere comprise more the 25 percent of the total atmospheric resevoir. Thus, it is to be expected that much of the excess atmospheric C02 will enter the oceans because of the rapid exchange of C02 across the air-sea interface. This rapid exchange, which is facilitated by the action of winds over the oceans' vast surface area (Kanwisher, 1963a,b), results in an approximate equilibrium between the partial pressures of C02 in the atmosphere and in the surface waters of the ocean. All previous evidence, which is based on laboratory photosynthetic experiments, indicates that seawater dissolved inorganic carbon (DIC) concentrations under natural conditions are presently limiting to seagrass photosynthesis (see review by Beer, 1989). Wetzel and Grace {1983) used this indirect evidence to predict that increasing pC02 in seawater, brought about by elevated atmospheric C02 levels, will increase seagrass productivity in the near future. understanding the carbon cycle and For the purposes of predicting future atmospheric C02 understanding of levels, it the carbon is essential to gain an metabolism of marine and terrestrial vegetation and to determine responses to changes in C02 over short time scales {Post et al., 1990). In view of this data need, the goal of this study was to elucidate various aspects of the carbon dynamics of Thalassia testudinum Banks ex KSnig (turtle grass) Particular objectives included determinations of: ( 1) the effects of


9 dissolved inorganic and organic carbon sources on stable carbon isotope composition and growth, (2} the effects of pC02 on stable carbon isotope composition and growth, ( 3) the inorganic carbon source(s) for photosynthesis, and (4) the presence and activity of carbonic anhydrase. Thalassia testudinum is the dominant seagrass species in the Gulf of Mexico and the subtropical-tropical Atlantic (Thayer and Ustach, 1981; Zieman, 1982; Iverson and Bittaker, 1986). It is the largest and most robust seagrass in this region and is generally considered to be the climax successional species (Zieman, 1982) It is estimated that there are hundreds-to-thousands of species of associated flora and fauna that inhabit Thalassia-dominated seagrass meadows and utilize the food, substrate, and shelter provided by these meadows (Zieman and Zieman, 1989}. These characteristics are indicative of its importance to coastal marine systems and are, in part, responsible for the relatively large amount of research that has been done concerning the synecology and physiological ecology of this species. Yet, there is very little information on basic plant metabolic characteristics (Durako and Moffler, 1987) Many biological and distributional patterns of seagrasses, such as T testudinum, are currently enigmatic, due to a general lack of physiological and autecological information. Therefore, a refinement of our understanding of the effects of environmental influences at the plant level is a prerequisite


10 for effective understanding of community and system level processes.


CHAPTER 2 INFLUENCE OF CARBON SOURCE ON GROWTH AND STABLE CARBON ISOTOPIC COMPOSITION OF THE SEAGRASS Thalassia testudinum. Introduction Seagrass c513C values can be affected by 11 oxygen concentration, light intensity, and temperature (Benedict and Scott, 1976; Smith et al., 1976; McMillan, 1980; McMillan and Smith, 1982; Cooper and DeNiro, 1989). However, the relatively high ratios of 13C to 12c characteristic of seagrasses are thought to result primarily from physical constraints on the supply of carbon used in photosynthesis (Abel, 1984) External (boundary layer) and internal (cellular) resistances to C02 diffusion, the concentration of free C02 and the degree to which photorespired C02 is recycled all limit the amount of isotopic fractionation which occurs during photosynthetic carbon fixation in submerged plants (Raven, 1970; Benedict et al., 1980; Osmond et al., 1981; Keeley et al., 1986). Isotopic variations in dissolved inorganic and organic carbon sources may also affect &13C signatures of seagrasses. Parker and Calder (1970) observed diel changes in the &13c of dissolved inorganic carbon (DIC) in natural and enclosed seagrass beds. These changes were attributed to the effects of isotopic fractionation, which were associated with diel


12 variations in photosynthesis and respiration, on the DIC pool. Zieman et al. {1984) reported that seagrass and macroalgae c513c values were more negative at a bay site containing large amounts of fine particulate organic matter derived from the flushing of mangrove stands than they were at a well-flushed site characterized by clear water and low nutrient concentrations. These researchers suggested that the more negative bay-site values were probably due to remineralization of isotopically light mangrove carbon, which may have lowered the of the DIC available for photosynthesis. The present study examined the effects of carbon source on the growth and stable carbon isotope composition of the seagrass Thalassia testudinum Banks ex Konig (turtle grass) Thalassia is the largest and most robust seagrass occurring in the subtropical-tropical Atlantic and Gulf of Mexico regions. Experimental treatments consisting of additions to sediments of isotopically heavy organic matter, isotopically light organic matter and C02 enrichment were investigated because of their potential importance in contributing to between-leaf, between-site {sensu Zieman et al., 1984), and seasonal variability in c513C values within a species {see review by Fry et al., 1987). Elucidating the sources of isotope variability in seagrasses is critical both to an understanding of their carbon metabolism and to the interpretation of c513C data for tracing carbon flow in food web/trophic analyses.


13 Methods Plant material Thalassia testudinum seedlings were collected from shoreline drift material at Matheson Hammock (25'N, 80'W) in Biscayne Bay, Florida on 10 August 1988 and 26 July 1989. Seedlings were placed in plastic bags containing seawatersaturated paper towels (to maintain a water-saturated atmosphere) transported to the Florida Marine Research Laboratory in St. Petersburg, FL at ambient temperature (27300C), and placed in 71 1 aquaria filled with synthetic seawater (Instant Ocean) at ambient salinity (30 /00) within 24 h of collection. The holding and treatment aquaria were located in a temperature-controlled culture room maintained at 27C; illumination was provided for each aquarium by a bank of 4, 70 em cool-white fluorescent lamps on a 14/10 light/dark photoperiod with a PAR photon flux density of approximately 125 J.i.E sec1 m2 Seedlings were cultured without substrate until the initiation of the experimental treatments (one to two weeks). Experimental For the 1988 seedling crop, the experimental setup consisted of six substrate treatments: 1} natural seagrass sediments, 2) combusted (ashed) seagrass sediments (500C for 4 h), 3) combusted sediments amended with isotopically heavy organic matter (3% wt:wt dried Thalassia leaves, o13C=-10.0 0 /00), 4) combusted sediments amended with isotopically light


14 organic matter (3% wt:wt dried Avicennia qerminans (L.) Stearn, o13C=-26.6 /00), 5) combusted sediments enriched with C02 and 6) horticultural peat pellets (peat moss o13C=-26.8 o 1 00) Peat moss is very refractory when compared to the readily degradable Thalassia and Avicennia leaf material (Zieman et al., 1984). carbon dioxide enrichment was accomplished using subsurface perfusers (airstones) connected to a C02 cylinder. The o13C of the C02 supply used at the beginning of the experiment was -11.3 /00 After three months, the cylinders were changed and the C02 supplied for the remainder of the experiment had a o13C of -49.6 /00 Gas delivery was regulated via a two-stage pressure regulator and flowmeter [ o. 5 cc C02 min1 pot1 @ 0. 28 bars (g) ) The perfusers were located below the open bottoms of 2-x 2-x 12-cm plastic culture pots. Seedling growth was determined from monthly measurements of green leaf area (Durako and Moffler, 1981). At the initiation of the experiment, and after three, six,and nine months in culture, four randomly selected seedlings were harvested for biomass and leaf o13C determinations. Individual seedlings were removed from the substrate, rinsed in deionized water, and separated into shoot, root and seed fractions. Each fraction was dried at 60C for 48 h and weighed to the nearest 0.1 mg. Water samples obtained just prior to the seedling harvests were analyzed to determine pH, and the concentration


15 and o13c of the total dissolved inorganic carbon. The samples were immediately filtered through 0.45 Nucleopore filters and the pH measured. Dissolved inorganic carbon concentration was determined for triplicate 500 water-sample aliquots from each treatment aquarium using an Oceanography International Model 524 Total Carbon Analyzer. Samples for o13C01c determinations were stored at 4C in acid-washed BOD bottles with no head space. Because of the isotope exchange problems in the 1988/89 experiments, the experimental design was modified and the study was repeated using the 1989 seedling crop. In this study, the experimental design consisted of three substrate treatments: 1) natural seagrass sediments, 2) natural sediments amended with 3% (wt:wt) dried Thalassia leaves, and 3) natural sediments amended with 3% (wt:wt) dried Avicennia leaves. Leaf growth, biomass and leaf 6'13C were sampled quarterly using the same methods outlined above. For both years, sixteen replicate seedlings were cultured individually in culture pots containing one of the treatment substrates. 6'13C analysis Samples for stable carbon isotope analyses were prepared as described by Macko (1981). Dried leaf samples of 6-15 mg were ground with a mortar and pestle in approximately 2 g of copper oxide and 150 mg Cuprox (Perkin Elmer) which had been precombusted at 800 C for 1 h. This mixture was placed in precombusted ( 550 C for 4 h) pyrex tubes. The tubes were


16 evacuated to high vacuum (<30 hermetically sealed, and mixed on a vortex mixer. Samples were then combusted at 550C for 24 h. The resulting C02 was collected and purified using an evacuated glass purification/collection system employing liquid nitrogen and alcohol/liquid nitrogen traps. The DIC in water samples was converted in vacuo to C02 by adding 8 5% phosphoric acid, which was purified and collected as above. Carbon dioxide was analyzed for stable carbon isotope o13C values using a Finnigan Mat 250 isotope ratio mass spectrometer. All results are expressed as 0 /00 relative to the international PDB standard. Typical overall errors in sample preparation and measurement are 0 .1-0. 2 /00 for o13C (Fry et al., 1987). Results 1988 seedling crop Mean leaf areas of seedlings in all six treatments increased during the first three to four months of culture (Fig. 1}. This increase reflected growth of the initial two to three blades which are present when the viviparous seedlings of this species are released by fruit dehiscence. After the initial peak, leaf area then declined and fluctuated at a reduced level reflecting senescence and abscision of the original blades and the reduced size and growth rate of subsequent leaves. Leaf areas of seedlings in the co2 -enriched treatment continued to decline and by eight months


17 6 0 N AT U RAL ASH ED 5 o + THALASS I A ,.-.... v N +AV I CE NNI A E () 4 ...__., :, +C02 0 Q) ... PEA T PELLET 1..... 0 '+-3 0 Q) _j 2 1 0 50 100 150 200 250 300 Days i n culture Figure 1 Mean leaf areas o f 1988 T testudinum seedling c u ltures in six sediment treatments (n=4, s.e. n o t shown for clarity). were approaching zero as their condition deteriorated. Seedlings in the Avicennia-amended sediments maintained greater leaf areas than all other treatments after three months in culture Total biomass of seedlings declined temporally for all treatments except the T halassia-amended sediment which had peak total biomass after six months in culture (Table 1). T otal biomass and seedlin g (root+shoot) biomass were generally greater for seedlings in the natural sediments and Avicennia-amended sediments. Seedling b iomass decline d with time i n


18 natural sediment and C0 2-enriched treatments, but increased in Thalassia-amended sediments. Seedling biomass was relatively unchanged after 3 months in peat pellets and ashed sediments. Between three and six months, seedling biomass increased in the Avicennia-amended sediments; between six and nine months it decreased. Root-to-shoot ratios were generally lowest in C02-enriched, Avicennia-and Thalassia-amended treatments, and generally highest in natural-and ashed-sediment treatments. Seedling leaves became increasingly depleted in 13C in all six treatments over the nine month experimental period (Table 2). In all treatments except the C0 2-enriched treatment, o13C values declined from -9.1 /00 at t=O to between -18.3 to -22.2 0 /00 after nine months in culture. This decline was linear and could be described by a single first order regression line with a coefficient of determination of 0.95, suggesting no treatment effect (Fig. 2) o13C values of seedling leaf tissue in the CQ2-enriched treatment alSO declined linearly (r2=0 97) I but from -9.1 to -57.1 /00a rate almost five times that of the other treatments ( -0.19 versus -o. 04 I oo day-1 ) Isotopic fractionation (L113C) in seedling leaf tissue generally increased temporally in the non-C02 enriched treatments (Table 2). Fractionation in the C02-bubbled treatment was highest after three months then decreased, although both the media DIC and seedling leaf material continued to become increasingly depleted in 13C. Initial media DIC o13C values ranged from -7.0 to -10.1


19 Table 1. Biomass (mg) and resource allocation characteristics in Thalassia testudimum aquarium seedling cultures [means ( s. e. ) n=4]. Treatment Age Root Shoot Seed Total Seedling Root: (mos) (rt+sht) Shoot Natural 3 83.1 69. 8 156.1 309.0 152.9 1.19 sediments (13.4) (5.3) (31.2) 6 60.4 58.3 99.3 218.0 118.7 1. 04 (7.4) ( 11. 4) (21.2) 9 43.4 44.4 47.0 148.6 102.0 0.98 (8.9 ( 4. 5) (7. 7) Ashed 3 57.6 42.6 65.0 165.2 100.2 1. 35 sediments (10.2) (7 .1) (10.6) 6 50.1 50.4 58.3 158.8 100.5 0.99 ( 3. 3) ( 5. 0) (8.9) 9 48.7 53.3 46.6 148.6 102.0 0.91 (7. 2) (9.7) (3.9) Ashed 3 22.0 48.0 45.6 115.6 70.0 0.46 sediments + (5.9) (9.6) ( 7. 6) Thalassia 6 35.3 66.4 64.8 166.5 101.7 0.53 ( 8. 1) (9. 0) (13.2) 9 47.7 64.5 43.0 155.2 112.2 0.74 ( 12. 0) (4.3) (6.2) Ashed 3 28.8 70.5 169.9 269.2 99.3 0.41 sediments + (6.2) {20.6) {101.2) Avicennia 6 46.6 77.8 55.2 179.6 124.4 0.60 ( 8. 5) (10.6) ( 2. 5) 9 35.2 69.9 49.8 154.9 105.1 0.50 (8.8) ( 8. 6) (2.7) Ashed 3 31.8 61.7 86.4 179.9 93.5 0.51 sediments (12.0) ( 3 3) ( 3. 0) + C02 6 31.6 46.0 65.8 143.4 77.6 0.69 (3.9) ( 4. 1) (9.0) 9 15.6 33.2 42.4 91.1 48.7 0 .47 (15.5) (14.8) ( 3 2) Peat 3 46.9 45.9 74.3 167.1 92.8 1. 02 pellets (9. 1) (2.9) (7.8) 6 27.2 44.2 49.8 121.2 71.4 0.64 ( 3. 8) (4.4) (8.7) 9 31.1 51.4 36.7 119.2 82.5 0.64 ( 3. 5) ( 8. 8) ( 2. 8)


20 "'loo {Table 2), indicating a relatively high degree of 13c depletion compared to the total inorganic carbon {TIC) in the synthetic seawater salts { o13C=-4. 0 o I 00) Media DIC o13C values in the non-C02-enriched treatments decreased an additional two to four per mil during the experimental period, except in the peat pellet treatment where they increased one per mil. DIC o13C decreased 44.6 I 00 in the co2-enriched treatment. Concentrations of DIC in the non-co2-enriched treatment media fluctuated between 1.51 to 3.35 mM, and were generally higher at the end of the experiment; DIC concentrations in the C0 2-enriched medium increased from 2.21 to 13.4 mM over this same time period. Media pH exhibited fluctuations of about one pH unit {7.5 to 8.5) with the C0 2 -enriched medium being about one unit lower. 1989 seedling crop After 110 days in culture, leaf areas for seedlings in the natural sediments and Thalassia-amended sediments were two to three times greater than both those i n the Avicenniaamended sediments and those of all six treatments in the 1988 studies {compare F igs. 1 and 3). Leaf areas in the former two treatments then decreased by half after 1 9 5 days, attaining levels comparable to those of the previous year. In contrast, leaf area increased from 2.9 to 7.6 cm2 between 110 and 195 days in the Avicennia-amended treatment. Because of a breakdown in the environmental control system of the culture


21 Table 2 o Mean o13C values of the DIC in the synthetic seawater media and Thalassia testudinum seedling leaf tissue, and A13C ( o13C1cat o13C01c) for the 1988 experiments o Treatment Natural sediments A shed sediments Ashed sediments + Thalassia Ashed sediments + Avicennia A shed sediments + C02 Peat pellets Time (months} 0 3 6 9 0 3 6 9 0 3 6 9 0 3 6 9 0 3 6 9 0 3 6 9 pH 7o53 8o31 8o31 8o03 8o21 8o64 8.24 8o00 8.13 8.40 8.39 8.12 8.00 8.47 8.28 8.37 7.64 7.00 7.10 7.97 8.18 8.33 8.28 8.09 DIC (mM} 2o25 1. 71 1. 65 2o23 1.89 1. 65 1. 23 3o12 2o02 2.11 2o05 2o68 1. 99 2o94 1.51 1. 78 2o21 11o11 16o32 13.39 2.18 2.00 2.04 3.35 r13c 0 DIC -10.1 -11.5 -10o6 -12o5 -9.1 -11.8 9o0 -11.2 -9o4 -8.4 -11.6 -13.2 -8.8 -9.2 -13.0 -15o4 -8.2 -11. 3" -39o9 -52.8 7o0 -9.8 -9.7 6.0 -9.1 -13.3 -1. 8 -16.7 -6.1 -20o1 -7.6 -9o1 -11.9 0.1 -17.2 -8o2 -19.2 8.0 -9.1 -11.5 -3.1 -16.6 5.0 -22.2 9.0 -9.1 -13.5 -4.3 -18o8 -5.8 -21.0 -5o6 -9.1 -24.7 -13.4 -50.8 -10o9 -57.1 -4.3 -9.1 -13.1 -3o3 -17.8 -8.1 -18.3 -12.3 Value estimated assuming equilibrium with C02 gas ( o13C= -11.3 /00) used for bubbling. After 3 months, the C02 gas had a o13C of -49.6 /ooo


-60 -50 ..........., 0 0 -40 "--. 0 -....__.u <'") -30 '0 u.... <( w -20 _) -10 0 Y=-0 .19X-8.87 2 R =0.97 50 100 Y=-0.04X-8.95 2 R =0.95 150 200 DAYS I N CULTURE 22 <> NATURAL ASH ED 0 +THALASSIA +AVICENNIA 6. +C02 ... PEAT PELLE T 250 .300 Figure 2 c513C values of 1988 T testudinum seedling cultures in six sediment treatments. room, this experiment was terminated after 195 days. Shoot and root biomass exhibited the same type of pattern as leaf area; declining in the natural sediment and Thalassia-amended treatments between 110 and 195 days in culture. Shoot biomass increased and root biomass remained constant in the Avicennia-amended treatment over this same time period (Fig. 4}. The Avicennia-amended treatment had the lowest shoot biomass after 110 days, but the highest after 195 days in culture. Root biomass for both time periods was greatest in the natural sediments and lowest in the Avicennia-amended


23 sediments. As in 19 88, root: shoot ratios were highest in natural sediments (0.54 and 1.25 at 110 and 195 days, respectively) and lowest in sediments with organic matter amendments (+Thalassia: 0.39 and 0.66, +Avicennia: 0.23 and 0. 22 at 110 and 195 days, respectively) Seed biomass decreased in all three treatments, declining 23% (from 80.5 to 62.3 mg dwt) in the natural sediment treatment, 37% (from 72.8 to 46.8 mg dwt) in the Thalassia-amended treatment, and 39% (from 82.9 to 51.0 mg dwt) in the Avicennia-amended treatment. After 110 days in culture, seedlings in the Thalassiaamended sediments had the most 13c-depleted leaves, while those in the Avicennia-amended treatment were isotopically the heaviest (Fig. 5). This pattern was reversed after 195 days and the seedlings in the Avicennia-amended treatment exhibited the greatest change in o1 3C I decreasing from -8.4 to -19.3 I oo In contrast, leaf o13C values increased slightly (i.e. became isotopically heavier) in the Thalassia-amended treatment. Seedlings in the natural sediments had leaf o13C values that were intermediate at both time periods, decreasing slightly from -16.1 to -17.0 /00 (Fig. 5). Discussion Seedling growth Leaf growth rates of the 1988 seedlings for the first four months of culture were similar to those previously reported for aquarium-cultured Thalassia testudinum seedlings


24 20 Natura l sediments Thalassia-ame nded Avicennia-amended ,--....... 15 N E 0 .......__, 0 10 Q) I-0 0 Q) _j 5 0 11 0 195 Days 1n cultur e Figure 3 Leaf area (mean s .e.) of 1989 T. testudinum seedlings after 110 and 195 days in culture in three sediment treatments. (Durako and Moffler, 1981). The dramatically higher leaf areas for the 1989 seedlings in the natural sediments and Thalassia-amended sediments after three months in culture, suggests possible annual seed crop variability. This variability may be due to annual differences in maternal investment into seed reserves. There is abundant evidence that plants allocate differing proportions of their net annual assimilated income into reproductive structures (see reviews by Harper et al., 1970; stephenson, 1981). This income can be


25 150 Natural sediments ihalassi a -amended Av icennia-amended 100 -j-J v OJ E 50 '-._../ (f) (f) 0 E Shoot 0 -0 m Root 50 1 1 0 195 Days 1 n culture F igure 4 Root and shoot b iomass of 1989 T testudinum seedlings after 110 and 195 days in culture in three sediment treatments. partitioned in a number of ways, such as in differing numbers and/or sizes of inflorescences and seeds. Since early growth of seedlings is dependent on nutrients, including carbon, derived from storage compounds within the seed (Dalling and Bhalla, 1984), early growth p atterns may vary as a function of


,.-...... 0 0 0 .........., u t') 25 -20 1 5 c.o -10 -5 110 195 Days 1n cul t ure 26 [22!Noturol se diments IS:S)Tholossio-omended 1522lAvicen n ia -amended Figure 5 o13C values (mean s.e.) of 1989 T testudinum seedlings after 110 and 195 days in culture in three sediment treatments. maternal investment and resource allocation. The general decrease in leaf area after three to four months in culture for both years reflects a decline in the physiological state of the seedlings which may have been the result of nutrient limitation as seed reserves were depleted (Moffler and Durako, 1984; Durako and Moffler, 1987) Seagrasses can become nutrient-limited even when growing in nutrient-rich media because diffusion and uptake of nutrients may not equal demand (Thursby, 1984). The possibility that nutrient limitation occurred is supported by the observation that, after nine months in culture, 1988 seedlings in


27 combusted sediments without organic amendments and those with the relatively refractory organic matter of peat pellets had lower total biomass and were reduced in size compared with seedlings in natural sediments or in sediments with labile organic matter enrichments. In addition, 1989 seedlings in natural sediments without organic matter amendments exhibited the greatest decline in biomass between 110 and 195 days in culture. In contrast, shoot and root biomass of 1989 seedlings in Avicennia-amended natural sediments increased during this same time period. The dramatic decline of 1988 seedlings in the C02-enriched treatment indicates that the limiting nutrient was not carbon. Leaf stable carbon isotope composition Leaf SC values of 1988 Thalassia seedlings after nine months in the C02-enriched treatment are the most C-depleted values ever reported for a higher plant. Leaf SC values for terrestrial plants generally range between -8 and -16 /00 for C4 plants and between -20 and -30 /00 for C3 plants; marine plant S13C values typically range from -6 to -20 /00 (Sackett, 1989). Plant SC values reflect the contribution of both source and process effects during carbon assimilation (Peterson and Fry, 1987). The carbon source sets an isotopic baseline. Leaf isotopic composition can subsequently be altered from the carbon source by processes associated with carbon fixation that affect fractionation, such as resistance to C02 diffusion and enzyme isotope descrimination (Wong et


al., 1979; Osmond et al., 1981) Small 28 isotopic fractionations (sometimes approaching 0 /00) have been observed for aquatic plants when C02 is limiting, whereas fractionations of 20 /00 or more occur when C0 2 availability is high (O'Leary, 1988). In this case, media DIC that was enriched with Be-depleted C0 2 gas was more Be-depleted than DIC in other treatments or in DIC that would usually be encountered in situ. After 3 months in culture, the high isotopic fractionation in the co2-enriched treatment was about half of that which would be expected if C0 2 supply to RuBP carboxylase was unlimited (i.e. 29 /00, Wong et al., 1979). It is unclear why fractionation then decreased even though DIC concentration continued to increase. The change in isotopic composition of the C02 gas after 3 months (from -11.3 /00 to -49.6 /00) and the deteriorating condition of the seedlings, as indicated by the decline in leaf area and biomass, may have been contributing factors. Fractionation of the DIC stable carbon isotopes by leaf tissue of the 1988 seedlings increased temporally in the nonco2-enriched treatments, and the observed d epletion in leaf 13c over time was greater than that which could be attributed solely to the concomitant 1 3C depletion in the media DIC (Table 2). The reasons are agai n unclear. In contrast to the C0 2 -enriched treatment, media DIC concentrations in the non-C02 enriched treatments varied only slightly. The apparent increase in fractionation may reflect a decrease in the stored


29 seed reserve (seed tissue o13C=-7 to -10 /00) contribution to leaf carbon as these reserves become depleted with time, coupled with an increasing contribution by fixation and fractionation of exogenous carbon. Alternatively, the increasing descrimination against 13c in the media with time may indicate that the exogenous carbon supply became less limiting over the experimental period (O'Leary, 1988). Depletion of 13C in the non-C02 enriched media during the 1988 experiments, was probably due to the release and exchange of isotopically light C02 from the C02-enriched aquarium within the relatively closed environment of the culture chamber. This isotopic exchange may have overshadowed any effect on the o13C signatures of the seedling leaves that might have been imparted by remineralization of the isotopically distinctive organic carbon sources which were added to the sediments. Recycling of C02 in an enclosed area such as a greenhouse or growth chamber can result in significant 13C depletion of artificial seawater DIC and a 5-9 /00 depletion in seagrass leaf 13c relative to the same species growing in situ (Smith et al., 1976). It was this confounding effect of the C02-enriched treatment on the other treatments' o13C values that made it necessary to essentially re-do the experiment, eliminating this treatment. The reasons for the relatively high o13c values of the seedlings in Avicennia-amended sediment treatment after 110 days in culture are unclear, although the


30 values are indicative of seed reserve mobilization to the leaves (see above). However, the dramatic decrease that occurred between 110 and 195 days suggests that the 13C depleted isotopic composition of the sediment organic matter did have an effect on the seedlings' stable carbon isotope composition after seed reserves were depleted. The increase in leaf o13C values for seedlings in the isotopically heavy Thalassia-amended sediment treatment also supports this conclusion. Thus, the results of this study provide experimental evidence supporting the suggestion by Zieman et al. (1984) that the isotopic composition of allochthonous and autochthonous organic matter in seagrass systems can affect the isotopic composition of the seagrasses. This probably occurs via remineralization of the organic matter and subsequent fixation of the resulting isotopically distinct C02 The large range in o13C values of Thalassia leaf material observed here also illustrates the high degree of variability that is possible. This variability may place limits on the interpretation of o13C data from seagrass ecosystems and may limit the utility of employing the carbon isotope method for ecosystem carbon flow analysis.


CHAPTER 3 EFFECTS OF pC02 ON THE GROWTH AND CARBON ISOTOPIC COMPOSITION 31 OF THE SEAGRASS Thalassia testudinum (HYDROCHARITACEAE) Introduction Photosynthetic carbon fixation is accompanied by fractionation of the stable isotopes of carbon. Isotopic fractionation is a mass-dependent phenomenon that is useful for investigating the efficiency of C02 uptake associated with photosynthesis (Peterson and Fry, 1987) During photosynthesis, plants discriminate against 13C relative to 12C because of small differences in chemical and physical properties imparted by the mass difference. This discrimination can be used to differentiate various photosynthetic groups in terrestrial plants (C3 versus C4 Whelan et al., 1970). However, in submerged plants the degree of isotopic fractionation that occurs during photosynthesis is more difficult to understand because of the importance of C02 diffusion in aquatic systems (O,Leary, 1988). In addition, there are differences in the o13C value, availability, and use of dissolved C02 and dissolved HC03 -(O'Leary, 1988). Seagrass o13C values reflect relatively high proportions of the heavy stable carbon isotope 13C and they are much less negative than o13c values of terrestrial C3 plants (-6 to -12 0 /00 versus -23 to -35 /00, respectively, Fry et al., 1987).


32 These "heavy" o13C values were initially attributed to HCo3 utilization (Park and Epstein, 1960; Steeman Nielsen, 1975) and, later, to possession of the C4 carboxylation pathway (Benedict and Scott, 1976). Terrestrial C4 plants also have high o13C values (Smith and Epstein, 1971) and Hco3 is the substrate for the primary carboxylating enzyme of the C4 pathway. However, the C4 carboxylation pathway is generally associated with species adapted to high temperatures and intermittent water stress (Doliner and Jolliffe, 1979) so its occurrence in a submerged plant would not be expected. Accordingly, more recent studies have confirmed that most seagrasses are indeed C3 plants and use ribulose bisphosphate (RuBP) carboxylase as the initial carboxylating enzyme for photosynthesis (Beer et al., 1977, 1980; Andrews and Abel, 1979; Benedict et al., 1980). In C3 plants, inorganic carbon must be in the form of C02 at the site of carboxylation. Thus, seagrasses are unable to directly access the relatively abundant HCo3 present in seawater. All evidence from controlled experiments suggests that seagrasses are carbon limited under natural conditions (see review by Beer, 1989). Availability of carbon for photosynthesis is limited because of the low concentration of free C02 at seawater pH (10 to 15 compared to 2 mM for HC03.) and the presence of extracellular and intracellular barriers to the supply of free C02 and HCo3 (Smith and Walker, 1980; Larkum et al., 1989). The primary external barrier is


33 the presence of an unstirred water layer surrounding seagrass leaves (Larkum et al., 1989). Internally, there are potential constraints imposed by differing cuticle and membrane permeabilities and cellular diffusion resistances on the pathway to C02 fixation in the chloroplasts (Larkum et al., 1989) In this investigation the effects of elevated seawater free-C02 concentration (pC02 ) on the growth and stable carbon isotope composition of the seagrass Thalassia testudinum Banks ex Konig (turtle grass) were examined. This species is the dominant and most robust seagrass of the subtropical-tropical Atlantic and Gulf of Mexico regions, and it is generally considered the climax successional species. (Zieman, 1982; Iverson and Bittaker, 1986; Zieman and Zieman, 1989). There is a relatively large amount of information on the synecology and physiological ecology of 1_. testudinum, however, very little information exists on basic metabolic characteristics (Durako and Moffler, 1987) There are no reports on the effects of elevated medium pC02 on seagrasses, although it has been predicted that increasing pC02 in seawater, brought about by elevated atmospheric C02 levels, will increase seagrass productivity in the near future (Wetzel and Grace, 1983). Elucidation of the effects of increased pC02 on the fractionation of carbon isotopes photosynthesis may provide valuable information regarding photosynthetic during seagrass source and process carbon uptake and


34 fixation in this unique group of plants (Peterson and Fry, 1987; O'Leary, 1988). Methods Plant material Thalassia testudinum seedlings were collected and maintained as described in Chapter 2. Seedlings were again cultured without substrate until the initiation of the experimental treatments. Four 1988 and twelve 1989 testudinum seedlings were randomly harvested from the culture room aquaria for determination of pretreatment (t=O) composition ( o13C) of leaf material. stable carbon isotope Epiphytes were removed from leaf blade surfaces by gentle scraping with a metal spatula while individual seedlings were rinsed in distilled water. Rinsed individuals were weighed (fresh weight) separated into shoot, root, and seed fractions, dried for 48 h at 60C and re-weighed (dry weight) Dried fractions were stored in a vacuum desiccator until processed for o13C determinations. An additional 16 -1988 and 32 -1989 seedlings were randomly selected for placement in the four treatment microcosms. Leaf lengths and widths, root lengths, and fresh weights were measured for each seedling (t=O). Individual seedlings were then placed in labeled 60 ml glass (25 mm x 150 mm) culture tubes containing enough media to completely


35 immerse the seedling and capped with a vented closure. The processed seedlings were transported to the Marine Ecosystems Research Laboratory (MERL) of the University of Rhode Island and placed in the treatment microcosms within 48 h of removal from the aquaria. Experimental setup The experiment was performed using four adjacent MERL microcosms 1977). The (for description of microcosms, see Pilson et al., microcosms were run in batch mode during the experimental period rather than using the normal flow-through regime. Experimental treatments consisted of an unmanipulated (= control) seawater medium, with near-ambient pC02 (which was expected to be 330 J.Latm), and three treatments with elevated pC02 levels of approximately 660, 1320, and 3300 J.LAtm pC02 (designated 2X, 4X, and lOX, respectively). A detailed description of the methodology employed in obtaining the elevated pC02 levels will be the subject of a subsequent report (K. Hinga, M. Pilson, and M. Arthur, in preparation). Briefly, elevated pC02 levels were achieved by "spiking" the microcosms with C02 derived from the acid hydrolysis of shell material of the Northern Quahog, Mercinaria mercinaria L. This source material was used in order to achieve a o13C value of approximately zero for the dissolved inorganic carbon (DIC) in the seawater media. Alkalinity was adjusted with NaOH to maintain near-ambient pH levels. Water quality, including measurement of total dissolved C02 and alkalinity, was


36 Table 3. Water quality characteristics of pC02 treatment microcosms. T s pH T(C02] I TA2 pC02 Treatment Date (oC) ( 0 / oo) (mM) (mMolfkg) (J..LAtm) Control 08/03/89 22.0 29.5 7.61 n.d. n.d. n.d. 08/08/89 22.0 29.5 7.56 1.84 1.81 1663 08/15/89 22.0 29.5 7.68 1.81 1.81 1233 08/22/89 22.5 29.5 7.78 1. 71 1. 74 936 08/29/89 19.5 29.5 7.91 1. 73 1. 79 669 09/05/89 19.5 29.5 7.99 1. 67 1. 76 529 09/12/89 21.0 29.5 7.89 1. 71 1. 77 711 09/26/89 18.1 29.0 7.83 1. 68 1. 71 766 2X 08/03/89 22.0 29.5 8.02 2.29 2.41 704 08/08/89 22.0 29.5 8.01 2.34 2.46 736 08/15/89 22.0 29.5 8.03 2.30 2.43 693 08/22/89 22.5 29.5 7.86 2.31 2.37 1047 08/29/89 19.5 29.5 7.90 2.28 2.34 893 09/05/89 19.5 29.5 7.96 2.24 2.33 766 09/12/89 21.0 29.5 7.91 2.21 2.28 868 09/26/89 18.1 29.0 7.98 2.11 2.19 683 4X 08/03/89 22.0 29.5 7.69 4.48 4.45 3023 08/08/89 22.0 29.5 7.75 4.40 4.40 2565 08/15/89 22.0 29.5 7.85 4 0 4.36 1987 08/22/89 22.5 29.5 7 .91 4.18 4.29 1659 08/29/89 19.5 29.5 7.95 4.19 4.30 1488 09/05/89 19.5 29.5 8.06 4.06 4.24 1105 09/12/89 21.0 29.5 8.10 3.97 4.20 980 09/26/89 18.1 29.0 7.99 3.97 4.08 1260 lOX 08/03/89 22.0 29.5 7.54 n.d. n.d. n.d. 08/08/89 22.0 29.5 7.60 11.09 10.86 9145 08/15/89 22.0 29.5 7.72 10.83 10.76 6738 08/22/89 22.5 29.5 7.82 10.58 10.64 5307 08/29/89 19.5 29.5 7.92 10.49 10.64 3991 09/05/89 19.5 29.5 8.02 10.26 10.56 3053 09/12/89 21.0 29.5 8.00 10.16 10.46 3226 09/26/89 18.1 29.0 7.93 10.05 10.19 3645 I Total dissolved inorganic carbon 2 Total alkalinity


37 monitored by MERL personnel weekly (Table 3). Four 1988 and eight 1989 seedlings were randomly assigned to one of 12 -5 em x 5 em x 10 em compartments in each of four 25 em x 20 em floating racks (one rack/treatment microcosm x four treatments x sixteen compartments= 48 seedlings). The racks were perforated on the top, sides and bottom to allow water and gas exchange with the surrounding medium. They were tethered to rings located on the northeast sides of the microcosms to restrict horizontal movement. Seedlings were cultured in the microcosms for a period of 57 days -approximately four plastochrone intervals (PITMussia 14 to 16 days, Zieman, 1982) -which should have resulted in the replacement of all leaf material during the course of the experiment (seedlings had two to four leaves at t=O}. At the conclusion of the experiment, seedlings were harvested and water samples from each microcosm were obtained. Individual seedlings were rinsed in ambient seawater, epiphytes were removed, leaves and roots were measured, and fresh weights were obtained as described above. The seedlings were then placed in culture tubes and transported back to St. Petersburg to be processed for dry weight and o13C determinations. Each seedling was immersed in 10% HCl (to remove any carbonates present) followed by two distilled water rinses, then separated into shoot, seed, and root fractions, dried for 48 h at 60C, and weighed. Dried fractions were stored in a vacuum desiccator as described above.


38 o13c analysis Dried leaf samples of 6-15 mg were prepared for stable carbon isotope analysis as described in the previous chapter. Carbon dioxide was released from seawater samples by addition of 85% H3P04 after evacuation of atmospheric gases from the reaction vessel. Purified C02 was analyzed for stable carbon isotope o13C values using a Varian Mat 250 isotope ratio mass spectrometer. Values are defined as a parts per thousand or per mil (0 /00) difference from a standard reference material: t3cf'2c t3cf' 2 c sample standard X 1000 t3cf'zc s tandard All results are expressed in o13C notation relative to the international PDB standard. Normality of the data and homogeneity of variances were determined using Shapiro-Wilk W statistics and Bartlett's Chi-Square tests, respectively (p< O. 05). Data was log-transformed, if necessary. Within-treatment comparisons of growth, biomass and leaf stable carbon isotopic composition between t=O and t=57 days were assessed by t-tests. Between-treatment effects on these characteristics were assessed by one-way analysis of variance. Where significant effects were determined, means were compared using Duncan's multiple range tests. All calculations were performed using Statistical


39 Analysis System (SAS} programs (SAS Institute, Inc. 1985} Results Because of the restricted period of time when water temperatures in the MERL microcosms are high enough to support the growth of Thalassia, this experiment was initiated shortly after the 2X, 4X, and lOX microcosm tanks had been "spiked" with C02 gas. Consequently, pC02 levels were substantially higher than the desired treatment levels during the first weeks of culture (Table 3}. It wasn't until the latter half of the experimental period that the pC02 levels approached "target" treatment levels. Because of generally lower pH values, the control microcosm maintained pC02 levels which were frequently higher than those of the 2x microcosm even though the dissolved inorganic carbon (DIC} concentrations in the control were lower. Total alkalinity in all four microcosms exhibited an inexplicable gradual, but steady decline during the experimental period; o13C of the DIC in the treatment microcosms showed an enrichment in 13c with increasing pC02 (Table 4}. Year old (1988} T testudi num seedlings generally exhibited declines in growth characteristics after 57 days in the treatment microcosms (Table 5). Leaf areas of seedlings in the 2X treatment and fresh we ights in the lOX treatment did increase during the period, but not significantly. In contrast, almost all growth characteristics of the 1989


increased, the only exception being 40 mean fresh weight of seedlings in the 4X treatment. Leaf area increased significantly only in the control, but was highest for lOX seedlings. Root lengths, which were 0 at the initiation of the experiment for all 1989 seedlings, increased significantly in all but the 2X treatment. Fresh weight significantly increased only in the lOX treatment. Analysis of variance (ANOVA) among treatments at t=57 days indicated a significant treatment effect only on leaf areas of 1988 seedlings (F= 5. 86, df= 3, 12) Between-treatment comparisons indicated mean leaf areas for the 2X and lOX seedlings (4.91 and 4.26 cm2 respectively) were control seedlings respectively). significantly greater than the 4X and at 57 days (1.22 and 0.88 cm2 Biomass characteristics exhibited similar patterns for the 1988 seedlings, i.e. no significant increases (Table 6). Shoot and seed biomasses were lowest for 4X seedlings; root biomass was lowest for lOX seedlings. Shoot and root biomass for all 1989 seedlings w a s significantly higher after the treatment period. ANOVA detected a significant treatment effect only for shoot biomass of 1988 seedlings (F= 5. 61, df=3,12), with lOX seedlings having the greatest shoot biomass. No other biomass treatment effects were detected. The stable carbon isotope composition of 1988 seedling leaf material at t=O was unusually depleted in 1 3C (Table 4) This reflected 13c depletion in the synthetic s eawater DIC


41 Table 4. 6'13C values for Thalassia testudinum leaves (mean s.e: ) and treatment media TIC. Seed year Nominal Treatment pC02 (JJAtm) 1988 Control 929 Control 929 2X 798 4X 1758 lOX 5015 1989 Control 929 Control 929 2X 798 4X 1758 lOX 5015 Time (days) 0 57 57 57 57 0 57 57 57 57 DIC -12. o1 0.4 0.9 + 1. 3 + 1. 9 0.4 0.9 + 1. 3 + 1. 9 Plant -19.3 ( 1. 9) -15.9 ( 1.1) -13.0 (2.2) -14.6 ( 1. 4) -19.1 (0.9) -8.3 ( 1. 6) 9.0 (0.4) -9.8 (0.6) -11. 33 ( 0. 2) -13.5 ( 0. 6) -7.3 -15.5 -12.1 -15.9 -21.0 -8.3 -8.6 -8.1 -12.6 -15.4 1Approximate value, measured c513C01c values ranged from -7.0 to -14 1 I 00 2c513C value for ocean water DIC (Sackett and Moore, 1966) 3t=57 mean significantly different (p< 0.05) than t=O as determined by t-test.


42 Table 5. Growth characteristics (mean s.e.) of Thalassia testudinum seedlings cultured for two months in various pC02 treatments. Seed Treatment Time Leaf Area Root length Fresh weight year (days) (cm2 ) (mm) (g) 1988 Control 0 2.88 46.2 1. 28 (0.88) (14.5) (0.14) 57 0.88 36.2 1.16 (0.38) (18.8) (0.14) 2X 0 3.78 89.2 1.50 (0.94) (36.6) (0.39) 57 4.91 63.5 1. 32 (1.20) (36.7) (0.21) 4X 0 2.35 118.5 1. 21 (0.47) (38.5) (0.12) 57 1.22 97.0 0.97 (0.38) (28.3) (0.06) lOX 0 5.78 58.2 1. 73 (1. 72) (33.3) (0.25) 57 4.26 49.6 1. 75 ( 1. 08) (24.3) (0.33) 1989 Control 0 1.97 bl 0 b 0.57 (0.16) (0.07) 57 2.79 a 8.2 a 0.74 (0.29) (3.3) (0.08) 2X 0 2.16 0 0.59 (0.12) (0.04) 57 2.74 9.4 0.73 (0.26) (5.5) (0.06) 4X 0 2.06 0 b 1. 01 (0.28) (0.54) 57 2.58 13.8 a 0.66 (0.38) (4.8) (0.07) lOX 0 2.41 0 b 0.53 b (0.26) (0.05) 57 3.18 9.5 a 0.81 a (0.49) ( 3. 1) (0.08) 1Means with different letters are significantly different (p<0.05) as determined by t-tests.


43 ( o13C01c ranged from -7 to -14 I 00) of the holding tanks which was probably due to release and exchange of isotopically-light C02 within the relatively closed environment of the culture room. The isotopically-light C02 originated from an adjacent experimental aquarium which was being bubbled with C02 having a o13C of -4 9. 6 I 00 (see chapter 2) Consequently, no significant difference between t=O and t=57 days was observed in the leaf o13C values for any of the treatments. ANOVA among treatments at t=57 days indicated a significant effect of pC02 on leaf o13C (F=3 .16, df=3, 12), and between-treatment comparisons indicated that the lOx seedlings were the most 13C depleted (see Fig. 6). Because 1989 seedlings were held for only a few days before being exposed to the experimental treatments, their initial o13C values were within the range of more-typical natural values [ -8.3 to -12.5 00 (Zieman and Zieman, 1989)], and were considerably higher than those of the 1988 seed crop. The 4X and lOX treatments exhibited a significant decline in leaf o13C during the period (Table 4) ANOVA among treatments at t=57 days indicated a very significant treatment effect (F=l8.79, df=3,28). Between-treatment comparisons indicated a significant step-wise depletion of leaf 13c with increasing pC02 between the control and 2X treatments (not different) and the 4X treatment, and between the 4X treatment and the lOX treatment (Fig. 6).


44 Table 6. Biomass characteristics (mean s.e.) of Thalassia testudinum seedlings cultured for two months in various pC02 treatments. Seed Treatment Time year (days) 1988 Control 0 Control 57 2X 57 4X 57 lOX 57 1989 Control 0 Control 57 2X 57 4X 57 lOX 57 Shoot dwt(g) 51.38 (14.26) 55.46 (7.31) 59.39 ( 8. 68) 29.91 (7.88) 82. 48. {11.82} 13.28 (0.85) 32.09. (4.07) 34. as (4.39) 33.09. (3.40) (3.91) Root dwt(g) 23.48 {10.10} 11.36 (6.62) 15.80 (9.63} 19.45 {3.94) 9.13 (4.64) 0 0 2. 01. (0.98) 2. 04. (1.34} 3. 99. ( 1. 28) 3. 70" (1.43) Seed dwt(g) 69.18 (8.12) 79.84 (28.84) 91.76 (39.47) 49.69 (6.99) 74.00 (10.98) 153.56 (16.25) 155.01 (22.14) 151.27 (22.51} 119.68 (10.49) 140.02 (15.36) T=57 mean significantly different ( p < O.OS) than T=O mean as determined by t-tests.


,-...... 0 0 0 .....__, u n

46 These seedlings appeared stressed at the initiation of the experiment, which may have been due to their extended time of culture without substrate or nutrients. The younger, 1989 seedlings showed consistent growth, but again, the only significant response was an increase in fresh weight in the lOX treatment. Leaf growth rates for the 1989 seedlings were about half of those previously reported for seedlings of this species (Durako and Moffler, 1981). These reduced leaf growth rates probably reflect the relatively low water temperatures in the microcosms. Maximum leaf growth in :r. testudinum occurs around 30 C (Barber and Behrens, 1985), and this species suffers leaf kills when temperature fall below l5 C (Zimmerman and Livingstone, 1976). Microcosm temperatures during the mid-summer experimental period were more similar to those occurring from late-fall to early spring temperatures in the seedlings' natural environment. In contrast to the relatively small degree of growth responses to elevated pC02 fractionation of stable carbon isotopes generally increased as a function of pC02 for both 1988 and 1989 Thalassia seedlings. Unfortunately, the comparatively high degree of 13C depletion exhibited by the 1988 seedlings at the initiation of this study, which represented a pretreatment artifact due to their extended period (one year) of growth in 13C depleted medium (see chapter 2), reduced the statistical significance of the effects of pC02 on isotopic fractionation. A t the conclusion of the


47 experiment, both groups of seedlings (1988 and 1989 seed crops) had leaf o13C values that indicated greater 13c depletion than generally observed for this species. Thalassia o13C values from the Gulf of Mexico and the Caribbean range from 8 3 to -12.5 /00 (Zieman and Zieman, 1989), although Smith et al. (1976) reported a value of -14.8 /00 for Thalassia cultured in a greenhouse. The latter researchers suggested that recycling of C02 within the enclosed area of the greenhouse resulted in a significant 13C depletion of the artificial seawater DIC and a 5-9 /00 depletion in seagrass leaf o13C relative to the same species growing in situ. All of the 1988 seedlings had o13C signatures that were more negative than the natural range, and 1989 seedlings in the lOX treatment were also more negative than this range. Differences in the degree of carbon isotopic fractionation between the 1988 and 1989 seedlings were also probably due to age related factors. Larger "net" fractionations exhibited by the leaves of the older, 1988 seedlings may have reflected reduced contributions of isotopically heavy carbon from stored seed reserves (o13Csecd=7 7 o / oo) Seed reserves in this species are mobilized for initial seedling growth and may be depleted after the first few months of growth (Durako and Moffler, 1987). Lower seed fraction biomass for the 1988 seedlings reflects this depletion of stored reserves. Utilization of stored carbon reserves by seedlings may explain the lack of significant


48 treatment effects on the 1989 seedlings' growth and biomass characteristics, and their relatively lower isotopic fractionations. In these young seedlings, the stored reserves may act as a buffer against varying environmental conditions during initial seedling growth. Carbon isotope fractionation in terrestrial plants is mainly due to differential discrimination between 12C and 13C during the primary carboxylation reactions involving ribulose bisphosphate (RuBP) carboxylase (C3 plants) and phosphoenol pyruvate (PEP) carboxylase (C4 plants, O'Leary, 1981). However, differences in enzymatic discrimination do not explain carbon isotope variability in submerged plants (Keely et al., 1986). Carbon isotope fractionations in algae and aquatic plants are thought to primarily reflect C02 availability (Smith and Walker, 1980; Keeley et al., 1986; O'Leary, 1988). Overall fractionation factors associated with photosynthetic carbon fixation are generally dependent on C02 concentration and range from 0 /00 when C02 is limiting to as high as -28 /00 at C02 concentrations > 5% (vjv in air, Kerby and Raven, 1985). The latter value approaches the maximum discrimination associated with carboxylation by RuBP carboxylase when C02 supply is unlimited (29 /00, Wong et al., 1979) The heavy o13C values which are characteristic of seagrasses reflect the small degree to which source dissolved inorganic carbon is isotopically fractionated during photosynthetic uptake and assimilation (O'Leary, 1988). These


49 characteristically small isotopic fractionations seem to result primarily from physical constraints on the supply of carbon for photosynthesis (Raven, 1970; Benedict et al., 1980; Abel, 1984) Restriction of the external carbon supply coupled with the presence of, and access to, an internal lacunar carbon pool, which is not in isotopic equilibrium with the external medium, may both have the effect of elevating o13C (Andrews and Abel, 1979; Abel, 1984). This is because inorganic carbon may be compartmentalized or pooled within the leaf or in the boundary layer before it is fixed by ribulose bisphosphate carboxylase. The result is a reduction in enzymatic discrimination against 13C02 within this relatively "closed" carbon assimilation system (Berry and Troughton, 1974; Andrews and Abel, 1979; Smith and Walker, 1980). According to Fick' s 1st Law2 there are three ways to increase dissolved inorganic carbon availability (flux). The first way is to increase the concentration gradient between the external source (medium) and the cellular sink (chloroplast) In previous experimental studies, this gradient is usually increased by raising the external C02 concentration (pC02 ) in the bathing medium via bubbling with a co2-enriched gas mixture. Here, the experimental microcosms 2J = [D(C -C))/1, where J is the flux of species j J J s J 2 (mmol m -2s -1), o is the diffusion coeff1c1ent of spec1es J (m s -1), 1 is the dlstance over which diffusion occurs, and c. and c, are the concentration of species j (mmol m -3 ) at the source and sink, respectively.


50 were periodically "spiked" with relatively pure C02 of a known isotopic composition ( o13Ccarbooate o o 1 00) to maintain the desired pC02 During the period of this study, they were spiked only once, before the initiation of the experiment. Because of inter-microcosm variability, especially in pH levels, seedlings in the control microcosm were exposed to pC02 levels which were about 15% higher than those in the 2X microcosm, coincident with total DIC concentrations that were about 24% lower (i.e. less HCo3 ) The comparatively larger carbon isotopic fractionations exhibited by the control seedlings suggests a greater importance for pC02 (andfor C02< v' Rau et al., 1989) and a correspondingly lower relative importance of HCo3 -a s the initial substrate for carbon fixation (Kerby and Raven, 1985). In this regard, Abel (1984) demonstrated that the closely-related species Thalassia hemprichii utilized only C02 as its photosynthetic substrate and she found that c arbon is limiting up to several-f o l d the concentration of C02 in seawater. Thus, it was proposed that the high o13C values o f T hemprichii are the result of restrictions on the supply of free C02 for photosynthesis. In my study, fractionation in T testudinum seedling leaves increased as a function of increasing medium pC02 levels, demonstrating the importance of concentration gradient in mitigating the physical constraints on carbon supply and in increasing carbon availability. Maximum fractionations were observed in seedlings growing in the highest (i.e. lOX ) pC02


51 treatment, and they ranged from -15.4 00 for 1989 seedlings to -21.0 I 00 for the year-old seedlings from the 1988 seed crop. Similarly, a strong relationship between pC02 and stable carbon isotope descrimination (expressed as 6'13C) has been demonstrated for marine algae (Degens et al., 1968; Calder and Parker, 1973; Pardue et al., 1976). Three species of marine phytoplankton exhibited a relatively constant fractionation maximum of about -19 oo when cultured under high C0 2 conditions (5% in air, Degens et al., 1968). Maximum fractionations observed for co2-enriched green and blue-green algae cultures ranged from -18.0 to -23.9 100 and they were obtained where cell censities were low and C02 concentrations in the feed gas were between 1. 5 and 3. 5 %. (Calder and Parker, 1973; Pardue et al., 1976). In contrast, no fractionation occurred at a C02 concentration of 0.2% vlv in air (Calder and Parker, 1973). The maximum fractionations which occur in this diverse array of aquatic plants at saturating C02 levels are quite similar, and they are 5 to 10 01 oo below maximum fractionations exhibited by terrestrial species. Evidently, even when exposed to artificially high pC0 2 conditions there are still some constraints on carbon supply in aquatic photosynthesis which limit enzymatic discrimination against 13C02 at the initial carboxylation step. One of these constraints may be due to limitations imposed by the second factor controlling diffusive carbon flux in submerged plants the distance ( 1) over which


diffusion between the source and sink occurs. 52 External limitations on carbon supply to aquatic plants are primarily due to the presence of an unstirred boundary layer in the medium surrounding photosynthetic tissues. The thickness of this unstirred boundary layer, which is a function of water movement, affects carbon flux rates. Boundary layers may be more than 500 thick under low flow conditions, such as those experienced by the seedlings in the MERL microcosms (the only water movement occurring in the microcosms is associated with the vertical movement of a mixer which is used to prevent stratification of the water column) Boundary layers can be over 60 thick even under well-stirred conditions (Smith and Walker, 1980). Movement within the boundary layer is solely by molecular diffusion and the rate of C02 diffusion in seawater is approximately four orders of magnitude less than in air (Dair=1.56 x 10-5 m2s-1 Fuller et al., 1966; Dseawater=1.55 X 10-9 m2s -1 Joosten and Dankwerts, 1972, both cited in Larkum et al., 1989). Restriction of photosynthetic tissue to outer, epidermal cell layers has been interpreted as an adaptation by seagrasses to minimize the difficulties imposed by the slow diffusion of gases in water (i.e. to reduce diffusion distances, Larkum et al. 1989) Osmond et al. (1981) found that carbon isotope fractionation generally increased as a function of water movement, reflecting a decrease in the thickness of the boundary layer. Accordingly, isotope fractionations are small for plants growing in slow


53 flowing streams or lakes due to boundary-layer imposed diffusion limitation in C02 uptake (Raven et al., 1982). The net diffusion coefficient (Dj) of C02 (or possibly HC03") is the third, and final factor controlling diffusive carbon flux. This coefficient represents the total resistance of the pathway from source to sink. The form of the equation for Fick' s 1st Law assumes that Dj is unchanged over the diffusion distance 1 This is usually not true if plant tissues are involved because of the diffusion resistances imposed by cellular components such as the cuticle (it should be noted that the cuticle is presently the greatest unknown in the movement of DIC because of a general lack of information on seagrass cuticles), cell wall, plasmalemma, cytoplasm, and chloroplast membrane (Larkum et al. 1989) Sorrel and Dromgoole (1986} employed an electrical analogue model to estimate the relative resistance to exchange of oxygen with the external fluid versus the internal lacunae. However, they did not attempt to apportion individual resistances or address effects of differential permeabilities of the pathway components. Larkum et al. (1989 } made a first attempt to examine the possible resistances of_ the unstirred layer and the individual cellular components. Based on their calculate d resistances, simple passive diffusion of C02 at ambient concentrations would not account for the observed rate of total inorganic carbon uptake i n most seagrasses. They hypothesized that Hco3 is a m ajor carrier o f total inorganic


54 carbon to the cuticle, but that it moves across the cuticle as C0 2 and moves both as C0 2 and HCo3 -from thereon to the sites of carboxylation (conversion of HCo3 -to C0 2 may be facilitated by cell-surface carbonic anhydrase, Tsuzuki and Miyachi, 1989). This model may explain another basis for variability in o13C values of seagrasses. In addition to the effects on isotopic fractionation imparted by low C02 concentration and diffusion limitations, seagrass 6'13C values may also reflect the relative proportions of C0 2 and HCo3 -in the intermediate pools and at the site of carboxylation. There are variations in o13C values for C0 2 and HC03 which are due to temperaturedependent fractionations that occur during C0 2(gu> .. C0 2 .. HC03- by about -9 / oo Our observation of a 13c enrichment in the DIC with increasing pC02 may be due to these chemical-exchange induced fractionations (Peterson and Fry, 1987) additional values. Thus, variability in source 6'13C complicating factor in evaluating may pose an seagrass 6'13C To summarize, the data presented here indicate a dominant effect of pC02 relative to HCOi concentration in determining stable carbon isotopic composition of Thalassia testudinum seedlings. Fractionation correlated with pC02 and this is normally interpreted a s reflecting the degree of carbon


limitation. growth and 55 However, the lack of corresponding increases in biomass with increased pC02 indicates other factors, such as endosperm utilization or temperature, may be more dominant in controlling quantitative aspects of early development of seedlings.


CHAPTER 4 GROWTH RESPONSES OF LIGHT-AND DARK-CULTIVATED AXENIC Thalassia testudinum SEEDLINGS TO ORGANIC CARBON AMENDMENT Introduction 56 Ambiguities in seagrass growth responses in many in situ nutritional studies primarily result from relatively rapid alterations in associated microbial communities which compete for and alter nutrient amendments (Harlin and Thorne-Miller, 1981) Rhizosphere and phyllosphere conditions may be modified by utilization and release of inorganic and organic substances during microbial metabolism. Addition of organic compounds may resul t in enhanced bacterial respiration which can raise the C02 concentration i n the medium (Owens and Esaias, 1976). Consequently, relationships between nutrient amendments and nutritional requirements at the plant level are obscured. These requirements can only be critically assessed through culture techniques which eliminate this 'microbial noise' (Wetzel and M cGregor, 1968; Moffler and Durako, 1984 ; Pulich, 1987). Moffler and Durako (1984) recently developed a procedure which achieves axenicity in field-collected Thalassia testudinum Banks ex Konig (Turtle grass) seedlings. Axenic T testudinum seedlings apparently use and deplete stored reserves, including organic carbon, with little or no


57 assimilation or response to a variety of exogenously supplied nutrients (Durako and Moffler, 1987). consequently, growth stops after one to several months in culture. These observations indicate that either rhizosphere microbial associations may play more than a mutualistic role in testudinum's nutrition or that this species may have an unusual nutritional physiology. In an effort to address this latter possibility, within the context of carbon limitation, the effects of sucrose (organic carbon) amendments on growth responses and stable carbon isotopic composition of axenic testudinum seedlings cultured with illumination and in the dark were investigated. Methods Thalassia testudinum fruits were collected from shoreline drift material at Key West in the Florida Keys (1987 seedling crop) or Matheson Hammock in Biscayne Bay (1988 seedling crop) within 48 hr of collection. Fruit collection and processed and processing and axenic culture establishment were accomplished using a modification of the aseptic techniques outlined by Moffler and Durako ( 1984) and Durako ( 1988) Healthy-appearing, green, undehisced fruits were soaked for approximately 12 hr (overnight) in 0.5% (wjv) Captan [n-(trichloromethylthio)-4-cyclohexene-1,2-dicarboximide] fungicide in sterile synthetic seawater (Instant Ocean @ 32 0 /00, IO). Fruits which remained undehisced after the


58 fungicide treatment were rinsed in sterile IO then surfacesterilized for 10 min in 0.6% vfv sodium hypochlorite (i.e. 10% Chlorox) in sterile IO. Seedlings were aseptically excised from fruits, surface-sterilized for 5 min in o. 6% sodium hypochlorite, rinsed in sterile IO and incubated individually in 24-well multidishes on an orbital shaker for 2 hr in 1 ml sterile IO with 100 J.J.g ml-1 each of polymyxin B sulfate and nitrofurantoin. These antibiotics were chosen based on results of antibiotic sensitivity assays of the most frequent bacterial contaminants. The antibiotic treatment was followed by a final rinse in sterile IO. Seedlings were aseptically transferred to 60 ml glass (25 mm X 150 mm) culture tubes containing 50 ml of culture media (Von Stosch's enrichment media in sterile IO at 32 /00, von Stosch, 1964). All processing and transfers were performed at room temperature under a laminar flow hood. Axenicity was verified as previously described (Hoffler and Durako, 1984), and contaminated cultures were discarded. Cultures w ere maintained at 27 C in a positive pressure "clean room" employing uv-sterilized air. A two-way factorial {3 X 2) experimental design was employed to investigate the effects of organic carbon amendment {0, 1 and 3 % sucrose) and presence or absence of light on the growth characteristics, and for the 1988 seedlings the stable carbon isotope composition, of the axenic seedlings. Reagent grade sucrose was the organic carbon


59 source for the 1987 study. Based on the results of the 1987 study, the organic carbon source for the 1988 studies was changed to sucrose derived from the sugar beet -a c3 plant (Beet Sugar Development Foundation, Ft. Collins, CO) Reagent grade sucrose, which is derived from the c4 plant sugar cane, has a o13C value of -10. 5 I oo This is similar to the o13C of Thalassia. In contrast, beet sucrose has a &13c of -24.1 /00, so it could be used as a tracer to determine if the sucrose was actually assimilated by the seedlings. Osmolarity of the media was determined by freezing point depression using a Precision Osmette A osmometer. Light-grown seedling cultures were placed on racks in front of light banks of 48 in fluorescent lamps with a PAR photon flux density of 100 sec -tm-2 on a 14:10 light:dark photoperiod. Dark-grown seedlings were placed in a microbiological incubator within the culture room. Growth characteristics of the seedlings were monitored via monthly leaf and root growth measurements (see Durako and Moffler, 1987). At the termination of the experiment, seedlings were harvested and separated into leaf, root and seed fractions. Each fraction was dried at 60 C to constant weight (24-48 hr) and weighed. Dried leaf samples for stable carbon isotope determinations were soaked three times for 2 h in distilled water to ensure that any sucrose that may have been absorbed as an osmoticum was removed. The samples were then prepared for o13C analysis as decribed in chapter 2.


60 Treatment effects on growth, biomass, and leaf 6'13C were assessed by analysis of variance (ANOVA) Means were compared using Duncan's multiple range tests. All calculations were performed using Statistical Analysis System programs ( SAS Institute, Inc., 1982). Results 1987 seedling cultures Figure 7 and Table 7 summarize the 1987 seedling growth characteristics over the experimental period. With the exception of root production in the dark cultures, all growth and biomass parameters were greatest in seedlings in media amended with 1% sucrose. Total leaf area, which represents the summation of all leaf production up to the sampling period (since senescent leaves remain attached to axenic seedlings and are not decomposed, see Durako and Moffler, 1987), increased over the entire culture period in all of the treatments. ANOVA indicated that sucrose level had a significant effect on this parameter (Table 8). Seedlings in media with 1% sucrose had slightly higher total leaf areas while those in 3 % sucrose had lower areas compared with seedlings in media with no added sucrose. In contrast, green leaf area, an estimate of the physiological state of the seedling, generally increased for the first 2-4 months of culture, then began to decline (Fig. 7). Green leaf area was significantly higher for seedlings in media with 1% sucrose


61 amendment and in dark-cultivated seedlings (Fig. 7 and Table 8); leaf biomass was also significantly affected by sucrose level in the light (F= 2.58, df=2,35), being greatest in the 1% sucrose treatment (Table 7). Dark-cultivated seedlings produced fewer (but longer) leaves compared to the light-cultivated seedlings ( 4-6 versus 8-11 bladesjseedling, respectively, after 160 days in culture). This was because plastochrone intervals for individual seedlings ranged from 26 to 40 days for the former treatment group compared to 14.5 to 20 days for the latter. Leaf widths were significantly different in media with varying sucrose levels and a significant interaction between sucrose and light was detected (Table 8). Leaf widths were narrowest in the highest sucrose amendment media. This media also had the highest osmolarity because of the added sucrose (approximately 990 mosm, compared to 880 and 920 mosm for the o and 1% sucrose amended media, respectively). The most definitive treatment response was an almost total lack of root production in the dark-cultivated seedlings (Fig. 7 and Table 7). Root length was significantly affected by medium sucrose level in the light, and there was a significant interaction between the two (Table 8). In the light, root length was highest in 1% sucrose and lowest in 3% sucrose (Fig. 7) Root biomass in the light was also significantly affected by sucrose level, again being greatest in 1 % sucrose, followed by 0% and 3% levels, respectively. In


u '--' ,.-... N E 0 Q.J "-<( '+-0 ([) _j 0 ...... 0 f-,.-... E E ...... 0' c ([) _j -+--' 0 0 cr: 0

63 Table 7. Biomass (mg dry wt. mean s. e.) characteristics of light-and dark-cultured axenic testudinum seedlings in Von stosch media with varying percent sucrose. Sucrose Leaf Root Seed Seedling Total Light (lf+rt) 1987 (180 days) 0 L 29.52 6.09 41.14 35.61 76.75 (4.58) (0.40) (6.50) (4.73) (10.48) D 22.57 0.81 20.75 23.39 44.14 (7.63} (0.47) (4.85} (7.92} (12.39} 1 L 50.93 8.46 77.14 59.39 136.53 (8.38) ( 1. 11) (17.81) (8.59) (26.06} D 26.39 0.00 48.75 26.39 75.14 (6.55) (0.00} (14.32) (6.55) (19.81} 3 L 30.06 0.43 72.10 30.49 102.59 (8.61) (0.18} (10.73) (8.67) (17.04) D 22.75 0.00 42.45 22.75 65.20 (6.54) 0.00 (7.68) (6.54} (10.51) 1988 (201 days) 0 L 46.07 5.83 54.32 51.90 106.22 (4.93} (3.07) (8.47) (5.84) (13.11} D 51.52 0.75 60.95 52.57 113.22 (8.10} (0.75) (11.35} (7.37) (17.40} 1 L 18.67 0.00 65.52 18.67 84.20 (1. 06) (0.00) (10.06} ( 1. 06) (10.50) D 15.70 0.00 69.83 15.70 85.53 ( 3 07) (0.00} (21.48) (3.07) (84.20} 3 L 10.80 0.00 44.00 10.80 54.80 (4.10} (0.00) (10.70) (4.10) (14.80} D 16.20 0.00 63.10 16.20 79.30 No s.e. because n = 1 for this treatment due to contamination.


64 Table 8. Analyses of variance for treatment effects on 1987 axenic testudinum seedling growth. Dependent Source of variable variation Degrees Total leaf Sucrose area Light (cm2 ) Sucrose X Light Experimental error Total Green leaf Sucrose area Light (cm2 ) Sucrose X Light Experimental error Total Leaf width Sucrose (mm) Light Sucrose X Light Experimental error Total freedom 2 1 2 241 246 2 1 2 241 246 2 1 2 241 246 of Sum of Mean squares square F-Ratio 118.09 59.04 3.94 0.04 0.04 0.00 74.21 37.10 2.48 3609.31 14.97 3801.65 74.82 37.41 12.05 .. 54.88 54.88 17. 68 9.55 4.78 1. 54 748.30 3.10 887.56 29.01 14.50 7. 11 0.02 0.02 0.03 8.85 4.42 5. 3 3 199.85 0.83 237.74 ------------------------------------------------------------Root length Sucrose 2 13081.99 (mm) Light 1 20080.29 Sucrose X Light 2 13023.16 Experimental error 241 221811.31 Total 246 267996.75 Values significant at alpha=0.05. Values significant at alpha=O. 01. 6540.50 7. 11 20080.29 21.82 6511.58 7. 07 920.38


65 the dark, there was also a significant sucrose effect on root biomass (F=5.35, df=2,16), but here it was due to a complete lack of root production in sucrose-amended cultures in the dark (Table 7). 1988 seedling cultures Figure 8 summarizes 1988 seedling growth characteristics. Unlike the 1987 data, total leaf area increased over the entire culture period only for seedlings in media without sucrose. ANOVA indicated a very significant sucrose effect (Table 9) which in this case seemed to be inhibitory. Although the total leaf area patterns of the light-and darkcultured seedlings appear similar ANOVA detected a significant light effect. Green leaf area also was significantly reduced in the sucrose-amended treatments and this parameter also exhibited a significant light effect as well as a sucrose and light interaction (Table 9). In the light, green leaf area declined to zero in the two sucrose-amended media after about three months in culture. This parameter was generally maintained at greater levels in the dark, but even in the O% sucrose treatment green leaf areas were low compared to the 1987 data. Leaf biomass was also significantly lower in the sucrose-amended treatments (F=22.75, df=2,19), but this characteristic was not significantly different between lightand dark-cultured seedlings (Table 9). Leaf width exhibited little variation with time, except in the 3% sucrose treatment where it declined after three


66 months in the light (Fig. 8). This decline may have resulted in the significant sucrose effect detected in the ANOVA (Table 9) As in 1987, dark-cultivated seedlings produced fewer (but longer) leaves compared to the light-cultivated seedlings. The average plastochrone interval in the dark was 32.5 days versus 16.1 days in the light, after 154 days in culture. The most striking treatment responses were again reflected in root production and growth (Fig. 8 and Table 7). Roots were only produced in the absence of sucrose. In addition, root production in the O% sucrose treatment was significantly reduced in the dark (Table 9). This was reflected by a significant sucrose and light interaction in the ANOVA. Observations on the growth characteristics of the darkcultivated seedlings for both years suggested they were not typical of an etiolation response. Compared to the light-cultivated seedlings, leaves of the dark-cultivated seedlings became a paler green and more elongated over time. However, leaf areas or widths were not significantly reduced in the dark, nor was the amount of leaf biomass produced in the dark generally significantly less. The lower seedling biomass of the dark-cultivated seedlings at the end of the experiment was primarily due to the absence of root production in the dark (Table 7). Although the 1988 growth data, unlike those in 1987, did


_.--.. N E u '---" 0 Q) L

68 Table 9. Analyses of variance for treatment effects on 1988 axenic T. testudinum seedling growth. Dependent Source of variable variation Degrees Total leaf Sucrose area Light (cm2 ) sucrose X Light Experimental error Total freedom 2 1 2 281 286 of Sum of Mean squares square F-Ratio 415.12 207.56 41.09 27.17 27.17 5. 38. 15.20 7.60 1.50 1419.52 5.05 1877.02 ------------------------------------------------------------Green leaf Sucrose 2 107.51 53.78 39.62 area Light 1 29.44 29.44 21. 70 .. (cm2 ) sucrose X Light 2 10.74 5.37 3. 96. Experimental error 281 381.22 1.35 Total 286 528.92 ------------------------------------------------------------Leaf width sucrose 2 6.79 3.40 7. 82 (mm) Light 1 1. 65 1. 65 3.80 Sucrose X Light 2 0.78 0.39 0.90 Experimental error 281 121.91 0.43 Total 286 131.14 ------------------------------------------------------------Root length Sucrose 2 4507.20 (mm) Light 1 1756.94 Sucrose X Light 2 2288.86 Experimental error 281 59652.19 Total 286 68205.20 Values significant at alpha=0.05. Values significant at alpha=O. 01. 2253.60 10. 62 1756.94 8. 28 1144.43 5. 39 212.28


69 not indicate enhanced growth with the addition of sucrose to the media, leaf o13C data does conclusively show that the added sucrose was assimilated (Table 10). The 4 to 11 /00 difference between the leaf tissues of seedlings in the sucrose amended media and the o13C of the beet sucrose, probably reflected a variable carbon contribution by isotopically heavy stored reserved in the seed ( 7 0 /00). In addition to the carbon source effect, there was also very significant difference between leaf o13C values for lightand dark-cultured seedlings after three months in culture (F= 21.52, df=1,27). This difference became less well defined after six months in the 1 % sucrose treatment, but it increased in the 3% sucrose treatment (Table 10) Discussion Using a natural-abundance stable carbon isotopic tracer, the results of this study show that Thalassia testudinum can directly assimilate organic carbon. However, the reasons why the sucrose enhanced growth which was exhibited by the 1987 seedlings was not evident in the 1988 data are unclear. A potential contributing factor may be the differing sucrose sources ( i e. C4 cane sugar versus C3 beet sugar) but the basis for this effect is not readily apparent. Smith and Penhale ( 1980) also observed what they termed "heterotrophic" uptake of organic carbon in Zostera marina L. They stated that the removal of dissolved organic carbon was unlikely to


70 .Table 10. Mean leaf &"c values ( s.e.) for 1988 testudinum seedlings after 3 and 6 months in media varying sucrose. Treatment Von Stosch light dark VS + 1% Sucrose light dark vs + 3% sucrose light dark &"c ( 0 / ooPDB) 3 months -7.2 (0.3) -5.1 (0.8) -17.9 ( 1. 0) -15.2 (0.6) -19.7 (0.7) -15.0 ( 1. 3) 6 months -5.4 (0.5) -4.5 (0.8) -17.9 (0.8) -17.0 ( 2. 6) -21.6 -12.5 result in significant growth responses in the plant. However, because their incubations were conducted using non-axenic plants, they indicated that the accumulation of 14C-labeled organic compounds in Zostera leaves may have been due to incorporation of 1 4C02 produced through the respiration of associated epiphytes. Indeed, this has been shown to be one of the main pathways for added organic carbon in non-axenic culture systems (Owens and Esaias, 1976). The growth response of the 1987 axenic seedling cultures to added sucrose much more strongly suggests the existence of heterotrophic uptake in testudinum, and the 1988 &"c data supports this view. Growth and biomass accumulation patterns of dark-cultivated axenic Thalassia testudinum seedlings


71 reinforce previous suggestions regarding the importance of mobilization and reallocation of seed-derived storage reserves for early development of axenic short-shoots and roots (Ourako and Moffler, 1987). Quantitative similarities between leaf characteristics of light-and dark-cultivated seedlings indicated a lack of significant photosynthetically derived production. Rather, light seemed to exert predominantly a photomorphogenic influence as illustrated by lack of root production and doubling of leaf plastochrone intervals in the dark-cultivated seedlings. The presence of roots on several contaminated dark-cultivated seedlings which were not immediately discarded complicates the above interpretation and indicates the possible involvement of a microbially produced growth regulating substance. The difference in plastochrone intervals resulted in distinctive short-shoot morphologies between light-and dark-cultivated seedlings (many short leaves versus few large leaves, respectively). Dennison and Alberte (1982, 1986} also observed an approximate doubling of plastochrone intervals (from 12 to 25 days and 18 to 35 days in the two respective studies) and increased short-shoot leaf size with reduced light in Zostera marina L. in situ. They suggested this change in leaf production rate was a photoadaptive response to balance reduced photosynthesis and carbon fixation. However, total leaf area production (and linear leaf growth rate) for light-and dark-cultivated axenic testudinum seedlings was


essentially identical, so in development without microbes) 72 this situation {i.e., early light may act primarily as a developmental trigger, affecting root and leaf initiation, rather than as an energy source {see Johnson, 1982a) Lack of typical etiolated growth characteristics in dark-grown testudinum seedlings could also reflect this specie's unique reproductive ecology. Embryo development is apparently continuous with seed germination occurring prior to fruit dehiscence {Orpurt and Boral, 1964; Sculthorpe, 1967; Moffler and Durako, 1984). This may be termed 'pseudo-vivipary' since the fruit may, or may not, still be attached to the parent plant. Germination without dormancy and dispersal of seedlings by release from the buoyant fruits precludes seed burial and possibly the selective advantages imparted by etiolation responses typical of terrestrial monocots {whose seeds usually germinate after being buried). In contrast, sucrose amendments to light-and dark-cultivated axenic testudinum seedling cultures elicited quantitative and qualitative responses indicative of enhanced growth and nutritional status in 1987, and an inhibitory influence in 1988. This is unlike the previous almost total lack of growth responses of axenic seedlings to nitrogen enrichment {Durako and Moffler, 1987). Sucrose was used as the organic carbon source because it is the most important transport form of photoassimilate within vascular plants (Richter, 1978). A trend toward increased seed biomass


73 (Table 7) in most of the sucrose-amended cultures suggested quantitatively less mobilization and utilization of endogenous stored reserves for growth when this exogenous organic carbon source was supplied. Rivkin and Putt (1987) recently suggested that the ability to utilize organic compounds to maintain cellular integrity or to grow heterotrophically would be of obvious selective advantage for benthic plants in periodically low irradiance or organically enriched environments such as estuaries and many coastal waters. Although dark growth in angiosperms and marine macroalgae depends on stored reserve materials (Luning, 1970), numerous microalgae in axenic culture exhibit sustained dark-growth if organic carbon substrates are supplied (Hellebust, 1970). Qualitatively, decreasing root:shoot biomass production ratios w ith increasing sucrose level also reflected reduced allocation of seed reserves to nutrient absorptive root tissues, a common response to nutrient sufficiency (Grime, 1979). Both Thalassia and Zostera exhibit decreasing root:shoot ratios in sediments higher in nutrients and organic matter (Burkholder et al., 1959; Kenworthy, 1981). The relatively high osmolarity of the 3% sucrose amendment media may have obscured any beneficial nutritional effects because of secondary osmotic stress responses as suggested by the production significantly narrower leaves. Narrow leaf widths are indicativ e of several type s of environmental stress in testudinum (e.g. high and low salinities; Phillips, 1960;


74 ourako, unpublished data, and low light conditions; McMillan and Phillips, 1979; Phillips and Lewis, 1983; Carlson and Acker, 1985). Responses exhibited by testudinum seedlings to the media and environmental manipulations in this study suggest phenotypic plasticity and photomorphogenetic control of this species' growth dynamics. Phenotypic adjustments in root and leaf size, morphology and ratio in response to nutritional or environmental stresses are characteristic of plants which have evolved a competitive growth strategy (Grime, 1979). This type of strategy is consistent with the view that testudinum is the climax species in low stress and low disturbance Caribbean and sub-tropical Atlantic seagrass ecosystems (den Hartog, 1971; Zieman, 1982).


75 CHAPTER 5 PHOTOSYNTHETIC UTILIZATION OF C02 AND HC03 IN THE SEAGRASS Thalassia testudinum (HYDROCHARITACEAE} Introduction Unlike the terrestrial plants from which they presumably evolved, submersed aquatic macrophytes are surrounded by a medium in which dissolved inorganic carbon (DIC} is present in three available forms: free C021 HCo3-, and co3 2 All aquatic plants use C02 for photosynthesis. Carbon dioxide solubility in freshwater is high with a partition coefficient, a (sometimes written as K0), of approximately one between 10 and 20C (Stumm and Morgan, 1981}. In the marine environment, C02 solubility is reduced by 10 to 15% compared with air-equilibrated freshwater due to the effect of salinity on the partition coefficient (Weiss, 1974; Unesco, 1987; Helder, 1988); seawater at 25 C typically contains only about 11 free C02 (Holbrook et al., 1988; Beer, 1989}. In contrast, HCo3 -concentrations are relatively high ( 2 o to 2. 5 mM) in the marine environment because of the high stable pH. Approximately 90% of the total DIC in seawater is present as HCo3 therefore, this ionic carbon species has been suggested as the most likely source of photosynthetic carbon for marine species (Steeman-Nielsen, 1975; Benedict and Scott, 1976; Beer and Waisel, 1979; Sand-Jensen and Gordon, 1984; Millhouse and


76 Strother, 1986; Beer, 1989). A number of seagrasses have been investigated for HCo3 use: Halophila stipulaceae (Forsk.) Aschers., Thalassodendron ciliatum (Forsk.) den Hartog 1 Halodule uninervis (Forsk.) Aschers.1 Syrinqodium isoetifolium (Aschers.) Dandy (Beer et al.1 1977)1 Cyrnodocea nodosa (Ucria) Aschers.1 Halophila oval is (R. Br.) Hook. f. (Beer and Waisel, 1979) Zostera marina L. (Sand-Jensen and Gordon, 1984), and Zostera muelleri Irmisch ex Aschers. (Millhouse and Strother, 1986). All these species were reported to be able to use both HC03 and C02 as carbon sources for photosynthesis. These conclusions were largely based on photosynthetic experiments in which pH, and thus the proportions of HCo3 and C021 were altered in closed systems while monitoring either 02 evolution or 14C uptake rates. The results indicated that these seagrasses possess a high photosynthetic potential which is limited under natural conditions by their relatively inefficient Hco; utilization systems (Beer 1 1989). Although the efficiency of HC03 use at high pH is low, there is no evidence for the use of co; in aquatic photosynthesis (Steeman-Nielsen, 1960; Raven, 1970; Prins and Elzenga, 1989). There are only two reports suggesting that seagrasses utilize only dissolved C02 in photosynthesis, and these are based on experiments with the two species of the genus Thalassia. Benedict et al. ( 1980) performed 14C pulse-chase studies and photosynthesis versus pH measurements on turtle


77 grass, Thalassia testudinum Banks ex Konig. Their finding of phosphoglyceric acid as the first stable product of photosynthetic carbon fixation and a decrease in carbon fixation with increasing pH (and associated decreasing free C02 concentration) led these authors to conclude that this species was unable to use exogenous HCo3-. Abel ( 1984) came to a similar conclusion for Thalassia hemprichii (Ehrenb.) Aschers based on more extensive evidence. In addition to the simple pH response technique, she utilized photosynthetic measurements in buffered media containing various HCo3 -and C02 concentrations within a narrow pH range, and measured 14C uptake by hemprichii leaf segments during the relatively slow equilibration period after spiking media with either 14C02 or H14C03 Photosynthetic carbon fixation in hemprichii was found to be limited by the concentration of free C02 in seawater, in much the same way as for HCo3--using seagrasses (see above). Abel ( 1984) suggested that the question of whether HCo3 -as well as free C02 is taken up during photosynthesis at high pH cannot be resolved by the simple pH response curve technique utilized in most previous studies. This inability is evidenced by the conflicting conclusions regarding C02 versus HCo3 -uptake reached by Beer et al. ( 1977, 1980) and Benedict et al. ( 1980) on the basis of similar results. Another confusing element came from earlier studies of testudinum, using o13C and 14C data, which suggested that this


78 species was a C4 plant and utilized HCo3 -as its photosynthetic substrate {Benedict and Scott, 1976). Later studies (Andrews and Abel, 1979; Beer et al., 1977, 1980; Benedict et al., 1980) confirmed, however, that most seagrasses produce phosophoglyceric acid as the first product of carbon fixation and are, therefore, C3 plants. In C3 plants carbon must be in the form of C02 at the site of carboxylation by ribulose bisphosphate carboxylasejoxygenase. Consequently, seagrasses should be unable to directly assimilate seawater Hco3 Photosynthetic rates of terrestrial C3 plants {Strain, and Bazzaz, 1983) and freshwater submersed macrophytes (Bowes, 1985) are limited by ambient levels of inorganic carbon. Carbon limitations are especially severe for submersed plants because of the very slow rate of diffusion of C02 in water compared to that in air (see Chapter 3) If seagrass photosynthetic rates are DIC limited under natural conditions, then the basis for the reportedly high productivity of seagrasses is not readily understood (Beer, 1989). In order to more reliably determine whether HC03 is a carbon source for photosynthesis in Thalassia testudinum and to assess the degree of carbon limitation in this species, this study employed an approach which allowed separate observations on the effects of total DIC, free C02 and HC03 on the photosynthetic kinetics of exogenous inorganic carbon use. Calculated K values for total DIC, HC03 and free C02 were used to assess the relative affinity and potential for


79 limitation for each carbon species. The results are then compared with previously reported values and discussed with respect to the conflicting and sometimes equivocal interpretations of previous photosynthetic data obtained using a variety of methodologies. Methods Plant material Two types of plant material were used in this investigation: laboratory-cultured Thalassia testudinum seedlings and field-collected testudinum short-shoots. In order to ensure that experimental units represented separate plants (genets) rather than subsamples of the same plant (ramets), .seedlings were used as the basic experimental units for experimental measurements. Field-collected short-shoots were utilized to verify if laboratory-cultured seedling responses were ecologically relevant (i.e. as a check against lab-culture induced artifacts). Seedlings were collected from shoreline drift material at Matheson Hammock Parle (25'N, 80'W) in Biscayne Bay, Florida on 10 August 1988. Seedlings were placed in plastic bags containing seawatersaturated paper towels (to maintain a water-saturated atmosphere) transported to the Florida Marine Research Laboratory in st. Petersburg, Florida at ambient temperature (27-30C), and placed in floating racks in a vault culture system filled with seawater adjusted to ambient salinity (30


80 0 /00) within 24 h. After the seedlings began to form roots (7-14 days) they were planted in peat pellets (Jiffy-7) in liner trays (Jiffy-7 pellet paks) which were placed on the surface of the gravel-bed filtration system of the culture vaults (water depth approximately 50 em). The vaults were located in a greenhouse covered with neutral density nursery cloth which reduced ambient sunlight by approximately 50 %. Experiments were conducted after the seedlings had been in culture for 9 to 16 months. Short-shoots of Thalassia testudinum were collected from a seagrass bed adjacent to Lassing Park (27 45'N, 82'W) in Tampa Bay, Florida. The short-shoots were obtained using a small spade to loosen the substrate from around the base of the short-shoot and to ensure that the shoots remained intact and undamaged. This material was transported back to the laboratory within 2 h of collection and stored in aerated seawater until required. All experiments on this material were conducted within 4 d of collection. Incubation Media Concentrations of individual inorganic carbon species in treatment media were manipulated by altering pH andfor dissolved inorganic carbon (DIC) concentrations according to the principles described by Abel (1983, 1984) and Sand-Jensen ( 1983) Experiments were conducted using 30 o I oo synthetic seawater (Instant Ocean) of known pH and DIC concentrations buffered with 10 mM Bicine (N,N-bis(2-hydroxyethyl) glycine,


81 pK. = 8. 35 at 20C). Preliminary tests confirmed that the addition of 10 mM Bicine did not significantly affect photosynthetic rates (Appendix 1). Buffered synthetic seawater was acidified to below pH 4 with concentrated HCl and purged overnight with N2 The pH of the co2-free medium was adjusted to the required level with carbonate-free NaOH (prepared according to Vogel, 1961) The pH of the medium was determined to the second decimal place using a glass pH electrode connected to an Orion model 901 microprocessor ionanalyzer. The electrode was calibrated using buffers referenced to the N.B.S. (National Bureau of Standards) pH scale. This level of accuracy was necessary to calculate free C02 and Hco3 -concentrations. Inorganic carbon in the form of NaHC03 was then added to the medium to a known final DIC concentration. Free C02 and HCOi concentrations were calculated from the pH and DIC concentration using the first and second dissociation constants for carbonic acid in seawater (Park, 1969; Mehrbach, et al., 1973; UNESCO, 1987). Since Bicine is a chelator of Ca2+ and hence prevents precipitation of caco3 (Good and Izawa, 1972) no CaC03 solubility term was required in the calculations even for media with higher-than-seawater pH and DIC. In addition, total dissolved inorganic carbon concentrations were directly measured by infra-red gas analysis using an Oceanography International Model 524 total carbon analyzer. Two types of media treatments were tested. The first


82 treatment series consisted of media with variable pH but with a constant, near-seawater DIC concentration (Table 11). The second treatment series utilized media at various DIC concentrations with pH values maintained close to that of natural seawater (Table 12) In this series, incubation media of DIC concentrations of 0.75, 2.35, 6.64, and 13.17 mM were made up with pH values of 7. 80, 8. 21, and 8. 61. These combinations were chosen such that an increment in both pH and total carbon maintained an approximately constant free C02 concentration while greatly changing the HCo3 -concentrations (Abel, 1984). Change in total carbon at constant pH has the same proportional effect on both carbon species. Change in pH at constant total carbon concentration has a proportionally greater effect on the concentration of free C02 and a relatively small effect on HCo3 Photosynthesis measurements Photosynthetic rates of Thalassia testudinum leaf tissue were measured by continuous monitoring of dissolved oxygen concentration of the Bicine-buffered assay medium using a Clark-type polarographic oxygen micro-electrode (Microelectrodes, Inc., Londonderry, NH). The electrode was calibrated using N2-sparged and air-saturated media. Most photosynthetic determinations were conducted under well-stirred conditions in a closed temperature-controlled glass reaction cell (cell volume = 34 ml). Irradiance was provided by two 500 watt quartz-halogen bulbs at a photon flux density


83 Table 11. Calculated Co2 and HCO(" concentrations (mM) in media with constant total carbon dioxide concentrations (2.2 mM} but varying pH (Park, 1969; Mehrbach et al., 1973; UNESCO, 1987). pH C02 (mM) 7.25 0.124 7.50 0.071 7.75 0.040 8.00 0.022 8.25 0.012 8.50 0.006 8.75 0.003 [ C02 + H2C03 ] = (h2c) I (h2+hk1+k1k2 ) [HCo3-J = (hk1c) 1 (h2+hk1+k1k2 ) HC03 (mM) 2.052 2.086 2.083 2.043 1.958 1.815 1.602 h = hydrogen ion activity as measured by a pH meter, loP"; c =total carbon dioxide concentration (mM}; k1 = first apparent dissociation constant of carbonic acid at 30 and 25 C 10-603 00 ' k2 = second apparent dissociation constant of carbonic acid at 3000 and 25C, l0-9 18


84 Table 12. Calculated C02 and HCo( concentrations (mM) in media with varying total carbon dioxide concentrations but relatively constant pH (Park, 1969; Mehrbach et al., 1973; UNESCO, 1987). Total C02 0.75 2.35 6.64 13.17 7.80 0 .012 0.708 0.038 2.220 0.106 6.272 0.211 12.440 pH 8.21 0.004 0.673 0.014 2.110 0.039 5.962 0.078 11.825 [ C02 + H2C03 ] = (h2c) I (h2+hk1+k1k2 ) (HCo3 ] = (hk1c) 1 (h2+hk1+k1k2 ) 8.61 0.002 0.590 0.005 1. 848 0.014 5.221 0.027 10.356 h = hydrogen ion activity as measured by a pH meter, 10 P"; c =total carbon dioxide concentration (mM); k1 = first apparent dissociation constant of carbonic acid at 3 0 1 and 2 5 C 1 o -6.03 00 I I k2 = second apparent dissociation constant of carbonic acid at 30 100 and 25C, 109 1 8


85 of approximately 500 J,.E m-2 s -1 PAR, which is above saturation for this species 300 J.E m -2 s -1 Dawes and Tomasko, 1988). Medium temperature was maintained at 25 C during the photosynthetic measurements using a refrigerated recirculating water bath. Leaf tissue used in the photosynthetic determinations consisted of 2 mm long sections of blade material which were cut under water from the mid-section of the youngest mature leaf of a short-shoot using a sterile scapel blade. The leaf segments were placed in N2-sparged buffered synthetic seawater (pH 8.2), aspirated at 20 em Hg vacuum for 30 min, and then allowed to equilibrate at atmospheric pressure overnight to avoid wound respiration (Dawes and Tomasko, 1988) This aspiration procedure floods the lacunae, minimizing problems associated with photosynthetic transients of oxygen exchange and lacunar gas buildup (Sorrell and Dromgoole, 1986). For a typical e xperimental series, treatment media were made up the day before the first photosynthetic determinations and stored with no head space in 300 ml acid-washed glass BOD bottles in a temperature-controlled water bath at 25C. The nex t morning, aspirated leaf segments were placed in the reaction chamber which was filled with the first treatment medium. The chamber was sealed w ith a rubber stopper so that no gas bubbles were present, and the tissue was allowed to equilibrate in the dark for 10 minutes. After dark equilibration, the lights were turned on and the change in

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86 dissolved oxygen was continuously recorded for a 20 minute interval using an X-Y mV recorder connected to a digital pH/mV meter. The lights were then extinguished, the chamber was opened, and the medium was removed using a 50 ml syringe. The next treatment medium was slowly added to the chamber to minimize turbulence and gas exchange. The chamber was resealed and the cycle was repeated. Each set of leaf segments was subjected to the seven (first treatment series) or twelve (second treatment series) treatment media in a predetermined, randomized order. After the completion of a treatment series, the leaf segments were rinsed in deionized water, dried at 60C and weighed for biomass determinations. Four replicate leaf segment groups, each representing leaf material from either an individual plant (seedlings) or a ramet (field collected short-shoots) were run for each media series. In December, photosynthetic determinations were conducted using single 4 em leaf segments under stagnant, unstirred conditions. Leaf segments were collected the day before the experiment from Lassing Park in st. Petersburg, Florida and equilibrated overnight in N2-sparged buffered synthetic seawater (pH 8.2). Treatment media were made up as described above and then distributed into 52 50 ml Erlenmeyer flasks ( 12 treatment media x 4 replicates + 4 blanks) which were stoppered with no head space. in a constant temperature The flasks were placed randomly water bath ( 25C) The next morning, initial dissolved oxygen concentrations in each flask

PAGE 101

87 were determined immediately after an individual leaf segment was placed in the flask. The flask was then resealed and returned to its original position in the now-illuminated water bath (photo flux density about 600 -700 J.LE m2s-1). This procedure was repeated for each flask at 3 minute intervals. After 3.5 h in the light, dissolved oxygen concentration was again determined for each flask, again at 3 minute intervals. Leaf tissue was removed from each flask, rinsed in DI-water, and dried at 60 C for biomass determinations. Photosynthetic rates were calculated using changes in dissolved oxygen concentration in the well-stirred reaction cells during steady-state photosynthesis (i.e. the linear portions of the oxygen concentration versus time curves) or from the difference in dissolved oxygen concentrations between t=O and t=3. 5 h in the flasks (December series) In the reaction cells, steady state photosynthetic rates were usually attained within 2 to 5 minutes after tissue was illuminated. Photosynthetic rates are expressed as mg 02 g dwt-1h "1 The kinetic parameters, Ks (half saturation constant) and Vmax (maximum photosynthetic rate at "infinite" substrate concentration) were calculated using double reciprocal plots and the Lineweaver-Burk equation, which is a linear rearrangement of the Michaelis-Menten equation (see Appendix 2)

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88 Results Photosynthesis at variable pH and constant total carbon concentration The effect of pH on the photosynthetic rate of :r. testudinum at normal seawater dissolved inorganic carbon (DIC) concentration was assessed in June, 1989 (Fig. 9). From pH 7. 25 to 8. 75, free C02 concentration drops by almost two orders of magnitude while HCo3 concentration declines by only about 30% (Table 11) Over the same pH range, photosynthetic rates declined 7-fold suggesting some contribution by HCo3 Bicarbonate use is also suggested by the plateau in the pH response between pH 7.75 to 8.50. Even with this plateau, the overall decline in photosynthetic rate with pH was relatively linear as indicated by a correlation coefficient of 0.859 for the first-order regression. The calculated apparent K ,(C02 ) for photosynthesis (Fig. 10) indicated a relatively high affinity for this substrate and intimated that ambient seawater levels of this substrate would be non-limiting (i.e. the concentration of free-C02 in seawater, is more than twice the K ,). In contrast, apparent K, (HCo3 ) suggested low affinity for this substrate since the K > seawater concentration mM) The apparent negative value for HCo3 reflected a wide variation in photosynthetic rates at the three lowest pH levels. Over this pH range (7.25-7.75) HCo3 concentration is constant while C02 concentration decreases threefold (see Table 11).

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89 ,............_, 18 I _c 16 I -4---' Y=-5.46X+51.98 3 u 14 R=0.859 OJ N 0 1 2 OJ E .........__, 1 0 Q) -4---' 0 L 8 u -4---' Q) _c 6 -+-' c >---(f) 0 4 -+-' 0 ...c Q_ 2 7.0 7 .5 8.0 8.5 9.0 pH Figure 9 Photosynthetic rates of T testudinum leaf segments as a function of pH in June, 1989. These results suggest that free-C02 concentration has a greater effect on photosynthetic rates, but they do not allow an accurate assessment of the contribution of HCOi in photosynthesis, especially at the higher pH values.

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90 Photosynthesis at various DIC concentrations with pH maintained close to that of seawater Three complete media series were run in July, September, and December, 1989. In July, photosynthetic rates of 2.0 mm aspirated leaf segments increased with increasing DIC up to the highest levels tested (Fig. 11). The rate of increase was greatest between the lowest two DIC levels. For a particular DIC concentration, photosynthetic rates were higher as pH decreased (and free-C02 and concentrations increased) .Kinetic data (Fig. 12) indicated that photosynthetic rates at ambient seawater DIC levels were about half of what they would be at saturating DIC concentrations. The apparent Vmu. for DIC in July was greater than the Vmu values for either C02 or HCo3 -in the June determinations. Plotting photosynthetic rates against calculated C02 and HC03 concentrations (Fig. 13) reveals that for a given concentration, rates based on C02 were highest at the highest pH {= highest HCo3 -concentrations). In contrast, rates based on HCo3 -were highest at the lowest pH {= highest C02 concentrations). The kinetic data {Fig. 14) indicate a very high affinity for C02 (although the correlation coefficient for this data is relatively low) and a much lower affinity for similar to what was calculated for the June measurements. However, the Ks values suggest that at seawater concentrations, C02 would be available at saturation levels and HCo3 -would be only slightly limiting (ambient

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> .....,__ Y=0.0006X+0.084 R=0.95 K =0.007 mM s _, -1 vmox=1 1.72 mg 02 g dwt h -500 -250 0 250 500 0 8 0.6 0.4 0 2 0 0 0 2 -0.4 0 6 -0.8 1 / [S] Y-1.376X-0.567 R=0.93 1 / [S] K =2.43 mM s 0 8 1 1 V = -1.76 mg 02 g dwt h max 91 Figure 10 Lineweaver-Burk plots of T testudinum photosynthesis versus C02 (top) and HCo3 -(bottom) in June, 1989.

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I ..c .-I N 0 CJ"l E 0.) ......-0 L () ......-0.) ..c ......c >en 0 ......-0 ..c Cl. 92 30 25 pH 7.80 20 15 10 5 0 0 2 4 6 8 10 1 2 1 4 D issolved inorganic carbon ( mM ) Figure 11 Photosynthetic rates of .T_. testudinum leaf segments at pH 7.80, 8.21, and 8.61 as a function of DIC concentration in July, 1989. concentration twice the K9 ) Photosynthetic rates measured during the September media series (Fig. 15) were much greater than those in June. Photosynthesis increased with increasing DIC at a rate comparable to the July measurements, but the pH dependency of photosynthetic rates versus D.IC was only observed at the

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Y=0.138X+0. 071 R =0.98 -1.5 -1.0 > -0.5 0.75 0.50 0.25 0.0 1 /[S] 93 K=1.94mM s -1 -1 V = 14.04 mg 02 g dwt h max 0 5 1 .0 1 .5 Figure 12 Lineweaver-Burk plot of testudinum photosynthesis as a function of DIC concentration in July, 1989.

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I L N 0 01 E Q) -+-' 0 \....._ u -+-' Q) L -+-' c >.... (f) 0 -+-' 0 ...c Q_ 94 30 2 5 p H 7 .80 pH 8 .2 1 20 pH 8 .61 1 5 I I I 1 0 5 0 0 4 8 12 16 20 24 F ree C02 concentration (mM) X 100 30 25 pH 7.80 20 15 1 0 5 0 L_ __ _L ____ L_ __ 0 2 4 6 8 10 1 2 14 HC03 concentration (mM) Figure 13 Photosynthetic rates of :r. testudinum leaf segments at pH 7 .80, 8.21, and 8.61 as a function of C02 (top) and Hco3 -(bottom) in July, 1989

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higher DIC concentrations. 95 The kinetic data (Fig. 16) indicated a muah greater potential for carbon limitation in September compared to July (the K, was over four times the ambient Die concentration). However, the calculated vmu was almost twice as great as that in July, reflecting potentially high photosynthetic capacity during this period. Plotting photosynthesis against calculated C02 and HCo3 concentrations shows similar trends to those observed in July, although there was less of a pH effect at lower concentrations (compare Figs. 13 and 17). The kinetic data (Fig. 18) indicated that HCo3 is again potentially more limiting than C021 however, the Vmu for HCo3 was one and one half times greater than that for C02 To assess the possibility that the previous photosynthetic and kinetic data reflected artifacts related to the use of aspirated leaf segments in a well-stirred medium, the pH/DIC media series was repeated in December using individual non-aspirated 4 em leaf segments in unstirred media. Photosynthetic characteristics observed during the December runs showed several differences from the previous photosynthetic series. First, the photosynthetic rates in the December series were much lower, being about half those of the previous series (Fig. 19). Second, photosynthetic rates versus DIC consistently leveled off at the higher DIC concentrations, indicating possible substrate saturation or d iffusional limitation. Finally, for a particular DIC

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Y =0.0002X+0.07 R=0.70 -1000 -500 Y=0.072X+0.06 R =0.83 0 1 /[S] 0 .25 0 .15 K =0.00.3 mM 5 -1 1 vmox=13.78 mg 02 g dwt h 500 1000 K = 1.22 mM s 1 -1 V = 17.08 mg 02 g dwt h max -2.0 -1.5 1 0 -0.5 0 0 0.5 1.0 1.5 2 0 1 / [S] 96 Figure 14 Lineweaver-Burk plots of testudinum photosynthesis versus C02 (top) and HCo3 (bottom) in July, 1989.

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97 30 I _c .-1 25 e pH 7.80 N 0 20 (J) E Q) ....... 0 L. u ....... Q) _c -+-' c > (/) 0 ....... 0 _c ()_ 15 1 0 5 0 0 2 4 6 8 10 1 2 14 D issolv ed inorganic carbon ( mM ) Figure 15 Photosynthetic rates of testudinum leaf segments at pH 7.80, 8.21, and 8.61 as a function of DIC concentration in September, 1989. concentration the photosynthetic rate was always higher as pH decreased. The double-reciprocal plots for the kinetic data (Fig. 20) and the calculated kinetic constants revealed a value that was intermediate to those obtained using the aspirated leaf segments from laboratory-cultured seedlings

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Y=0.389X+0.042 R=0.99 > 0.75 0.50 0.25 1 .5 -1.0 -0.5 0 0 1 /[S] 98 K = 9.26 mM s -1 1 V =23.81 mg 02 g dwt h max 0.5 1.0 1 .5 Figure 16 Lineweaver-Burk plot of T testudinum photosynthesis as a function of DIC concentration in September, 1989. (K5=2. 76 mM, compared with 1. 94 and 9. 26 mM for July and September, respectively). Lower photosynthetic rates during the December runs also yielded a relatively low apparent of 7.18 mg 02 g dwt-1h -1 (compared to values of 14.04 and 23.81 for the July and September runs, respectively). When photosynthetic rates were plotted as a function

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I ....c N 0 Q) +-' 0 L u 30 25 20 15 1 0 30 (f) 0 +-' 0 ....c Q_ 20 15 1 0 5 pH 7.80 pH 8.21 4 8 12 16 20 24 Free C02 concentration (mM) X 100 p H 7.80 e 0 0 2 4 6 8 10 1 2 14 HC03 concentration (mM) 99 Figure 17 Photosynthetic rates ofT testudinum leaf segments at pH 7.80, 8.21, and 8.61 as a function of C02 (top) and HCo3 -(bottom) in September, 1989.

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Y=0 .0009X+0.05 R=0.97 -1000 -500 Y =0.216X+0.030 R=0.90 0 6 0 4 0 1/(S) 0 6 0 4 K8-0.018 mt.A -1 -1 vmox-19 .88 mg 02 9 dwt h 500 1000 -2.0 1.5 1.0 0.5 0 0 0 5 1.0 1 5 2.0 1 / (S) 100 Figure 18 Lineweaver-Burk plots of testudinum photosynthesis versus C02 (top) and HCo3 (bottom) in September, 1989.

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101 of calculated C02 and HCo3 concentrations (Fig. 21), the patterns were similar to those for the July and September runs -generally higher rates at higher pH for co2 and highest rates at the lowest pH for HCo3 for C02 and HCo3 (Fig. 22) The calculated constants were, like those for DIC, intermediate to the July and September data; Vmu values were likewise lower. Table 13 summarizes the kinetic parameters calculated for C02 and HC03 using two linear transformations of the Michaelis-Men ten equation, the Lineweaver-Burk equation: 1/v=(Ks/Vmu) (1/[S[)+1/Vmu, and the Eadie-Hofstee equation: v= -Ks(v/[S]} + Vmu. The Lineweaver-Burk equation provided the best fit for the data (r > 0.90 except for July runs). The Ks values calculated for C02 were very similar using both equations, but the calculated Vmu values were slightly higher using the Eadie-Hofstee equation. In contrast, both the calculated Ks and Vmu values for HC03 were lower when calculated using the Eadie-Hofstee equation. Discussion The results of this study indicate that Thalassia testudinum apparently utilizes HC03 as well as C02 as a photosynthetic substrate. The term, apparent, is employed since active H+-Hco3 cotransport (andjor direct uptake of the ionic HCo3) has not been demonstrated in this species. Although Hco3 utilization by submersed aquatic macrophytes is

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I .r:. I N 0 O'l E () ......, Q) .r:. ......, c >... (/) 0 ......, 0 .r:. 0.. 8 6 4 2 0 0 102 pH 7.80 p H 8.21 pH 8 6 1 2 4 6 8 10 1 2 14 Dissolved inorgani c carbon ( mM ) Figure 19 Photosynthetic rates of testudinum segments as a function of DIC concentration in December, 1989. generally considered to be an active, possibly electrogenic, process (Prins et al. 1982; Lucas, 1983), it is also quite possible that there is extracellular conversion of medium HCo3 -into C02 which subsequently enters the leaf by diffusion. This conversion may occur at, or near, the leaf surface possibly by acidification w ithin the unstirred micro layer surrounding the leaf (Beer, 1989; Prins and Elzenga, 1989}, or

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Y=0.384X+0.139 R=0.99 -1.5 1 0 > ""-.. -0.5 0.75 0.50 0.0 1 / [S] 103 K =2.76 mM s 1 -1 V =7.18mg02gdwt h max 0.5 1 0 1 .5 Figure 20 Lineweaver-Burk plot of testudinum photosynthesis as a function of DIC concentration in December, 1989. through the activity of extracellular carbonic anhydrase (Raven, 1970; Tsuzuki and Miyachi, 1989; and following chapter). Benedict and Scott (1976) also suggested that HCo3 -was the photosynthetic substrate for testudinum and that this species utilized a C4 type of carbon metabolism. These conclusions were largely based on indirect evidence of high o13c values. However, as stated in previous chapters, the

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I ....c 0
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Y-0.0013X+0.172 R=0 .95 -1000 -500 Y=0 .304X+0.136 R=0.93 <:. 1 0 0 8 0 6 0 1 / (S) 1 0 0.8 0.6 0 4 K.=0.007 m'-4 -1 1 Vmax=5.83 mg 02 g dwt h 500 1000 K.=2 .22 mM 1 1 vmo.=7 .34 mg 02 9 dwt h -2. 0 -1. 5 -1.0 -0.5 0 0 0 5 1 0 1.5 2 0 1/(S) 105 Figure 22 Lineweaver-Burk plots of T testudinum photosynthesis versus C02 (top} and HCo3 -(bottom} in December, 1989.

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106 relatively "heavy" 6'13C values of seagrasses are primarily a reflection of diffusional limitations on carbon supply and the resulting relatively "closed" carbon fixation system rather than being reflective of their mode of carbon fixation (i.e. C3 versus C4 O'Leary, 1988). Upon their re-examination of this species, Benedict et al. (1980) concluded that testudinum was indeed a C3 plant since phosphoglyceric acid was the first stable product of photosynthetic carbon fixation. They also suggested that C02 was the only species of inorganic carbon utlilized for photosynthesis; a conclusion based on photosynthetic responses of leaf sections at pH levels from 4 to 9. Maximum photosynthesis was observed at pH 5 where 95% of the total DIC was in the form of free C02 and minimum photosynthesis at pH 8 where 97.9% of the DIC is in the form of HC03-. These conclusions are in contrast to those of Beer et al. (1977, 1979, 1980) who interpreted plateaus in the pH response curves from pH 7.5 to 9.2 for the four seagrass species they examined as indicating HC03 uptake. In their studies, pH was changed during the photosynthetic runs by injecting small aliquots of either 0.1 M HCl or NaOH into unbuffered. seawater in a closed system. In the study by Benedict et al. (1980), photosynthetic responses were measured in buffered media using three different buffer systems (pH 4 to 6 -citrate-phosphate, pH 6 to 7 -phosphate, and pH 7 to 9 -tris-HCl) Since buffers can drastically reduce the rate of HC03 utilization

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107 while stimulating the use of C02 (Lucas, 1977, 1983; Walker et al., 1980; Prins et al., 1982; Prins and Zanstra, 1985), the responses observed by Benedict et al. (1980) may have been largely due to a buffer effect. The conflicting conclusions reached by Beer et al. (1977, 1979, 1980) and Benedict et al. (1980) point out the inability to resolve whether HC03 as well as free-C02 are taken up during photosynthesis through the use of the simple technique of examining the pH response curve. In agreement with Abel's (1984) conclusion regarding the limitations of this technique, the photosynthesis versus pH responses for testudinum were not highly correlated with the calculated concentrations for either substrate (rc02 = 0.75, rHco3 = 0.79). The June data (Fig. 9) suggested a strong C02 influence, but it did not preclude HCo3 uptake, especially at the higher pH levels. Indeed, the rate of decline in photosynthetic rate over the pH range tested was much lower than the exponential decline in C02 concentration, suggesting some potential for HC03 uptake. The second approach employed in this study for measuring photosynthetic rates in a series of media of different DIC concentrations at near-ambient pH levels has the potential to show uptake of either free C02 or HC03 or both (Abel, 1983, 1984; Sand-Jensen and Gordon, 1984). A particular advantage to this approach is that different treatment media with similar free co2 concentrations had widely differing HC03 concentrations within a small pH range which would not be

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108 13. of calculated kinetic parameters from l1near transformat1ons of the Michaelis-Menten equation. Lineweaver-Burk Eadie-Hofstee Month parameter June C02 September C02 December C02 r 0.95 0.93 0.70 0.83 0.97 0.90 0.95 0.93 r K, 0.007 11.72 -0.75 0.006 2. 43 -1. 76 -0.99 2.28 0.003 13.78 -0.62 0.004 1.22 17.08 -0.64 0.97 0.018 19.88 -0.70 0.018 7.22 33.36 -0.53 3 .16 0.007 5.83 -0.84 0.009 2.22 7.34 -0.75 1. 68 1K, is the half-saturation constant 2Vmax is the reaction velocity at saturating substrate concentration vmax 11.46 -1.20 15.47 16.95 22.13 23.50 6.54 6.79 expected to affect photosynthesis directly. In assessing the relative contributions of C02 and HC03 to the photosynthetic performance of T testudinum, most of the data obtained in this study suggest characteristics which are quite distinct from those observed by Abel ( 1983, 1984) for the closely related Pacific species, Thalassia hemprichii. Abel (1984) concluded that T hemprichii does not exhibit

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109 appreciable use of Hco; as a carbon source. This conclusion was based on her interpretation of pH independence in photosynthetic responses when plotted against free co2 and on pH-dependent responses when plotted against HCo3 concentrations. In contrast, photosynthetic rates for testudinum exhibited pH dependence when plotted against C02 and HC03-, indicating a contribution by both carbon species. The ability to use HCo3 can vary between species within a genus (e.g. Potamogeton, Kadono, 1980; 1982), and even within the same species (e.g. Elodea, Prins and Elzenga, 1989). However, Hco3 use seems to be related to growing conditions (Spence and Maberly, 1985). Thalassia testudinum and hemprichii both grow in similar environmental conditions (Den Hartog, 1970) so it was somewhat unexpected that they would exhibit differing physiological traits. Upon closer examination of Abel's (1983, 1984) data, the photosynthetic rates of hemprichii based on C02 were higher (if only slightly) at higher pH in over half of the comparisons. The relatively smaller increases, compared to the pH effect when photosynthesis was plotted against HCo3, may reflect the fact that use of HCO; generally results in lower photosynthetic rates than C02 utilization at the same concentrations (Prins and Elzenga, 1989). This seems to be the result of inherently lower transport capacity for ions associated with HCo3 uptake (sand-Jensen, 1983) The distinction of plants as "users" and "nonusers" of

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110 HC03 may in reality be one of degree rather than an absolute {Spence and Maberly, 1985; Bowes and Salvucci, 1989). Both Thalassia species have high rates of primary production {McRoy and McMillan, 1977; Brouns, 1985). Larkum et al. {1989) point out that with this similarity in productivity, coupled with the difficulties in positively delineating C02 versus HCo3 use, it seems likely that HCo3 -is utilized by both species. However, if T hemprichii does truly lack the ability to use HCo3 for photosynthesis, this may be one factor which contributes to its observed inability to outcompete species such as Cymodocea serrulata {R. Br.) Aschers. and Magnus in environments that should be ideal for T hemprichii {den Hartog, 1970). As stated above, the media pH/DIC combinations in this study were chosen such that an increment increase in both pH and DIC resulted in relatively constant C02 concentrations with an increase in HCo3 -concentration. To test if photosynthesis was independent of HC03 only the photosynthetic rates from the three media with a free C02 concentration of 12-14 J..LM were plotted. The effect of HC03 concentration on .'!: testudinum photosynthesis is clearly illustrated in Figure 23. In this figure, The regression lines indicate a positive response to increasing HCO; concentration at constant free-C02 concentration {i.e. the slopes are greater than 0), reflecting Hco3 use. Therefore, the results of this study agree with the conclusions of Benedict and Scott (1976), although for

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111 1 6 JULY SEPTEMBER DECEMBER Q) ........--.. Y=0.84X+ 7 .04 ......., ..12 R=0.96 0 I L. _c u ......., ......., Y=O. 78X+5.40 Q) R=0.75 ...c -o 8 ......., c 01 >(f) N 0 0 ......., 0 01 Y=0.39X+2.58 _c o.__ E 4 R=0.88 ....___,.. 0 ____ _L ____ _J 0 2 3 4 5 6 [HC03 ] (mM) Figure 23 Photosynthetic rates of :r. testudinum leaf segments at relatively constant C02 and increasing HCo3-. different reasons. The results are more similar to those of Beer et al. (19771 19791 1980) 1 Sand-Jensen and Gordon (1984) 1 and Millhouse and Strother ( 1986) in that these authors suggested photosynthetic HCOi use in all the marine macrophytes they examined, based on photosynthetic response data. Beer (1989), in a review of marine angiosperm photosynthesis, stated that all evidence based on controlled

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112 photosynthetic experiments suggests DIC limitation for seagrasses under natural conditions. Likewise, the kinetic data for photosynthesis versus DIC concentration for testudinum indicated that photosynthetic rates would be generally below saturation under natural conditions. A major distinction between the photosynthetic characteristics of testudinum and those of the previously examined submersed aquatic macrophytes is its apparent great affinity for C02 Most submersed aquatic macrophytes exhibit high C02 saturation requirements with K5(C02 ) values ranging from 40 to over 700 J..LM (Steeman Nielsen, 1947; Van et al., 1976; Browse et al., 1979; Allen and Spence; 1981; Salvucci and Bowes, 1982; 1983; Sand Jensen and Gordon, 1984; Millhouse and Strother, 1989). In contrast, terrestrial species exhibit K5(C02 ) values around 10 J..LM (Goldsworthy, 1968). The calculated K5 (C02 ) values for T testudinum ranged from 3 to 18 J..LM, comparable to terrestrial plant values, but almost two orders of magnitude lower than the K5(C02 ) values reported by Abel ( 1984) for Thalassia hemprichii. However, my calculations, using the data from her thesis (Abel, 1983), yielded a K5 (C02 ) of 34 J..LM (Appendix 2) Thus, these two closely related species may both have relatively affinities for C02 high The wide range in substrate affinities reported for submersed aquatic macrophytes may reflect true species andfor environmentally-induced differences, or they could be largely

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due to different analytical methods. 113 The use of buffered media in this study prevented problems associated with pH increases during the photosynthetic assays and the resulting concomitant decreases in C02 concentration. The net result of this type of change would be a reduced apparent affinity for C02 [i.e. an increase in K, (C02)]. Most previous photosynthetic studies of seagrasses have not utilized buffered media. The high apparent K,(C02 ) for submersed aquatic macrophytes has been attributed to the combined effects of slow rates of C02 diffusion in the aqueous environment (Steeman Nielsen, 1947; Abel, 1984; Raven, 1984) and the presence of external and internal resistances to C02 transfer (Sorrell and Dromgoole, 1986; Larkum et al. 1989) As stated earlier, the diffusion coefficients for C02 are approximately 10, 000 times greater in air than in water. Therefore, under quiescent conditions the major resistance to C02 uptake, which accounts for much of the high apparent K ,(C02), is the slow rate of diffusion through the unstirred boundary layer surrounding the leaves (Browse et al., 1979; Smith and Walker, 1980; Wheeler, 1980; Black et al., 1981). This layer can be 50 to over 500 thick under conditions of low water movement (Smith and Walker, 1980). Under wellstirred conditions, the boundary layer thickness decreases, but it can still be from 20 to 60 thick (Smith and Walker, 1980). The apparent K,(C02 ) also decreases under well-stirred

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114 conditions (Browse et al., 1979), and when measured in an aerial humid environment (Salvucci and Bowes, 1982; Bowes, 1987), both of which minimize boundary layer resistance. Even for submersed aquatic plants out of water the apparent K1(C02 ) may remain quite high (70 J.LM for Hydrilla verticillata (L.f.) Royle, Bowes, 1987), suggesting that internal resistances to C02 assimilation can be considerably greater in submersed aquatic plants compared with terrestrial or amphibious plants (Lloyd et al., 1977, Browse et al., 1979; Salvucci and Bowes, 1982) Components of these internal resistances include relatively high Ks(C02 ) for ribulose bisphosphate carboxylase-oxygenase extracted from submersed aquatic plants (Bowes and Salvucci, 1984), photorespiration (Van et al., 1976; Bowes et al., 1979), and dark respiration (Van et al., 1976; Salvucci and Bowes, 1981). The experimental conditions utilized during the December series (unstirred) allowed an indirect assessment of the potential effect of the boundary layer on carbon availability and Ks(C02 ) in T testudinum. The similarity in the kinetic values calculated from this series to those of the previous well-stirred measurements implies little boundary layer effect on Ks (C02 ) using the photosynthetic assay system employed here. However, the leveling off of responses at the higher DIC concentrations implied the existence of some type of limitation which was not observed in the well-stirred assays using aspirated leaf segments. Lower absolute photosynthetic

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115 rates in December may have been due to seasonal influences such as reduced chlorophyll levels (Macaulty et al., 1989) and/or may have reflected lacunar oxygen storage in these larger, non-aspirated tissues. Sorrell and Dromgoole (1986) likewise observed a 17% decrease in apparent photosynthetic rates in non-aspirated compared to aspirated tissues. In contrast to the uniqueness of T testudinum's apparently very high affinity for C02 the apparent K,(HC03") values calculated in this study, which ranged from 1.2 to 7.2 mM, fall within the range of values reported for other submersed aquatic macrophytes (0. 6 to 23 mM, Allen and Spence, 1981; Titus and Stone, 1982; Sand-Jensen and Gordon, 1984; Millhouse and Strother, 1986). Howev er, the apparent Vrrw. values for HCOi for T testudinum were generally higher than those for C02 except in the first pH s eries. As stated above, higher photosynthetic rates at C02 saturation compared with HCOi saturation usually occur, and this is attributed to restrictions associated with HCOi transport across the plasmalemma via an energy requiring electrogenic pump involving counter ions. The comparatively h igher Vmax(C0 2 ) versus vrrw. (HCo3 ) values that have been reported in previous seagrass studies (Beer et al., 1977); Sand-Je n sen and Gordon, 1984; Millhouse and Strother, 1986) may be indicative of active transport of HCo3 in these species Perhaps the pattern in T testudinum reflects rapid e xtracellular conversion of Hco3 to co2 thus raising the C0 2 concentration

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116 around the leaf, rather than an energy-requiring transport system. This would result in an increased concentration gradient across the boundary layer and greater availability at the site of fixation. Using similar types of carbon concentrating systems, cyanobacteria have been shown to exhibit whole-cell K.(C02 ) values of as low as 3 nM, in spite of K.{C02 ) for cynaobacterial ribulose bisphosphate carboxylasejoxygenase of about 200 (Miller et al., 1990). Unfortunately, it is not possible to examine the effects of one inorganic carbon species in the absence of the other. The approach employed here allowed an assessment of the effects of varying the concentration of one carbon species while maintaining a constant concentration of the other. Abel (1984) and Sand-Jensen and Gordon (1984) previously applied this approach to several marine macrophytes and arrived at conflicting conclusions as to whether HCo3 is taken up during photosynthesis. The results of this study also suggested a number of distinctions between the physiological aspects of C02 and HCo3 use by Thalassia testudinum and those reported by previous researchers for other seagrasses. Thalassia hemprichii's apparent reliance on C02 as a carbon source is significant in view of the fact that the ratio of C02 to HCo3 in seawater is about 1:150. Abel (1984) suggested that in its use of the less abundant carbon species, T hemprichii perhaps reflects its presumed evolutionary origin from terrestrial ancestors. Given the dominance of T testudinum along the

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117 coasts of the Caribbean and Gulf of Mexico, it may be that this species' evolution has resulted in the ability to adjust its carbon assimilation capacities so that the K. values are close to the inorganic carbon concentrations of its medium.

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118 CHAPTER 6 CARBONIC ANHYDRASE ACTIVITY IN THE MARINE ANGIOSPERM Thalassia testudinum (HYDROCHARITACEAE). Introduction The kinetics of hydration and dehydration of the various species of inorganic carbon in water have an important influence on aquatic biological processes, especially photosyntheti c carbon fixation. Inorganic carbon occurs in various forms in water in accordance with the following equation: Carbon dioxide can react with water to form either HCo3 -or H2C03 (Johnson, 1982b) However, carbonic acid (H2C03 ) concentration is much less than that of C021d issotvodl because the The rate constants for the interconversion of HC03-+H+ .. H2C03 are seven orders of magnitude greater than the hydration and dehydration rate constants because the former is an ionic reaction (Eigen et al., 1961, cited in Johnson, 1982b). Therefore, it is usually assumed that H2C03 and HC03 are in equilibrium (Johnson, 1982b) In contrast, the rate of conversion betw e e n C02 and HC03 is dependent on factors such as temperature, salinity and pH (Kern, 1960; Johnson, 1982b). At the pH of seawater (7.8 to

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119 8.3), HC03 is the dominant form of inorganic carbon (:::::2 mM, compared to 10-15 for C02). Since most seagrasses are c3 plants and require C02 at the site of carboxylation (Beer and Waisel, 1979; Beer et al., 1980a; Benedict et al., 1980; Beer and Wetzel, 1982; Abel 1983), the relatively slow rates for the uncatalyzed reactions between C02 and H2co3 (hydration kc02:::::0. 03 6 s', dehydration kd:::::3. 5 X 104 mol dm3s1 kHc03:::::1. 4 X 104 ; Johnson, 1982b) may pose a significant limitation on photosynthetic carbon supply in this group of plants (Larkum et al., 1989). Carbonic anhydrase (E.C., carbonate dehydratase, abbreviated CA) catalyzes the reversible hydration of C02 C02 + H20 .. H+ + HC03 at pH > 6. 1, increasing the rate of the forward (and back) reactions. Carbonic anhydrase is widely distributed among bacteria, plants, and animals (Lindskog et al., 1971), and it's occurrence has been documented in freshwater hydrophytes (Weaver and Wetzel, 1980) and several seagrass species (Graham and Smillie, 1976; Beer et al., 1980a). Although the functional role of CA has not been clearly established, it may be important for supplying inorganic carbon in the form of C02 to submersed plants (Graham and Smillie, 1976; Beer, 1989). As stated in earlier chapters, a basic problem in seagrass photosynthesis is the potential limitation imposed by the slow rate of diffusion of C02 across the unstirred layer surrounding the leaves (Smith and Walker, 1980; Larkum et al.,

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120 1989) Given the additional diffusion resistances imposed by cellular structures such as the cuticle, cell wall, and plasmalemma, simple passive diffusion of C02 does not account for the observed rate of total inorganic carbon uptake in most seagrasses (Larkum et al., 1989}. Since HCo3 -is present at such a high concentration compared to co2 in seawater, diffusion of HC03 -across the unstirred layer to the epidermal cells should be more rapid. It is not known which carbon form enters the cells, but the low permeability of the cuticle and plasmalemma to ions seems to rule out passive uptake of HCo3 -(Larkum et al., 1989}. Although there is ample evidence that active transport is involved with HC03 -uptake in some freshwater submersed species (see reviews by Lucas, 1983; Prins and Elzenga, 1989}, this has not been documented for any seagrass species. What is known is that C02 is the substrate for RuBPCO, therefore HCo3 -must be converted to C02 at some point. Thus, it has been speculated that CA may be localized on the cell surface andfor released into the unstirred layer thereby facilitating the rapid conversion of HC03 into C02 at the cell surface, resulting in movement of C02 across the cuticle and from thereon as C02 and HC03 to the sites of carboxylation (Larkum et al. 1989; Tsuzuki and Miyachi, 1989) Because of the demonstrated influence of concentration on the photosynthetic rates of Thalassia testudinum Banks ex Konig (see last chapter), the following

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121 study was performed to determine the presence/absence and relative levels of CA in this species. Methods Approximately 2 em of leaf tissue from the basal, green portion of the youngest mature leaves of four to five Thalassia testudinum short-shoots was cut into 2 mm long segments which were pooled then divided into three subsamples, one each for CA assays, dry weight determinations, and chlorophyll determinations. Carbonic anhydrase was extracted by macerating 0.3 to 0.5 g fresh weight of leaf tissue in an ice-cold mortar with combusted, acid-washed sand and 3 to 4 ml of extraction buffer (0.10 M Tris, 0.010 M 2-mercaptoethanol, and 0.001 M Na2-EDTA adjusted to pH 8.3 with HCl; Weaver and Wetzel, 1980) The subsample for dry weight determination was rinsed in DI-water, dried at 60C and weighed; leaf samples for chlorophyll determinations were immediately frozen in the dark. An electrometric method was used to assay carbonic anhydrase. The principles and procedures for the assay are described in detail by Wilbur and Anderson (1948) and Weaver and Wetzel ( 1980}. This method measures the rate of hydration of C02 ( co2 + H20 .,. HCo3 -+ H+) over time by the reduction in pH. The time course of the reaction was followed on an X-Y recorder. carbon dioxide-saturated water was used as the substrate. This was prepared by bubbling C02 gas through ca.

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122 800 ml of glass-distilled water at 1C for at least one hour before use. For the assay, 1 ml of crude plant extract, prepared as above, and 2 ml of 25 mM Veronal buffer at pH 8.2 were added to a 7 ml temperature-controlled reaction cell at 4 C. The pH was allowed to equilibrate to between 8.30 to 8.45. The reaction was initiated by rapidly injecting 2 ml of co2 -saturated cold water into the plant extract-veronal buffer mixture. The enzymatic time (te) was measured as the time for the pH to drop from 8.0 to 7.0. Non-enzymatic time (tb), was determined using a blank consisting of 2 ml 25 mM Veronal buffer, 1 ml extraction buffer with no plant material, and 2 ml co2-saturated water. Enzyme units were calculated according to the formula given by Wilbur and Anderson (1968): E.U. = 10[(tb/te)-1] g dwt1 (or mg chl B.) where tb = nonenzymatic time in seconds using buffer only and te = enzymatic time in seconds using plant extract. Linearity of the enzymatic assay was determined using varying volumes of purified bovine CA (Sigma Chemical Co.) in extraction buffer. The assay was linear with te values from 11 to over 80 seconds (Figure 24). Initial assays revealed relatively low CA levels in Thalassia leaf tissue. These low levels of activity may have been due to the presence of an inhibitor within the plant cells which was released upon maceration of the leaf tissue.

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123 To test this possibility, plant extracts were combined with an internal CA standard (purified bovine CA) and assayed for CA activity. The combined plant extract and CA internal standard te values were virtually identical to the controls consisting of only the standard CA (11.21 0.40 s.e. versus 11.16 0.22 s.e., respectively), suggesting no inhibition. Chlorophyll was extracted by grinding leaf segments in an ice-cold mortar with combusted, acid-washed sand in 90% spectrophotometric grade acetone. Chlorophyll g content was then measured spectrophotometrically according to Sterman (1988). Results and Discussion The results of this study demonstrate the presence of CA activity in Thalassia testudinum. Table 14 shows the activity of carbonic anhydrase from field-collected T testudinum leaf material. Enzyme activity expressed on a mg chlorophyll g basis was similar to that reported for Halophila ovata Gaud. in Freycin. (540 E.U.) and Cymodocea rotundata Ehrenb. & Hempr. ex Aschers. (500 E.U.), but less than half of that reported for 1'_. hemprichii (Ehrenb.) Aschers. (2160 E.U., Graham and Smillie, 1976). Compared to CA activities measured for most terrestrial (e.g. 500-13,000 E.U. mg chl;1 or g fresh wt-, Atkins et al. 1972) CA acti vi tes iri seagrasses are usually at, or below, the lower end of the range; this is especially true when activities are expressed on a tissue weight basis (Table 14) Graham and Smillie

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124 80 Y=668. 1 X+0.144 ,..--..... R=0.999 ,-, ..--60 Q) I-.D I-L-...J 0 ..--40 (f) +-' c (J) E 20 >.., N c w 0 0.00 0 .02 0 .04 0.06 0 .08 0 10 CA standard volume ( m l ) Figure 24 Linearity of carbonic anhydrase assay over a range of standard volumes. (1976) reported that the presence of CA in marine plant species was rather unexpected because of the h igh concentrations of inorganic carbon in seawater. They suggested it's presence indicated that one role for CA in marine species could be in the conversion of HCo3 to C02 in

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125 photosynthesis. The relatively large differences in CA activities between the two closely related Thalassia species corresponds to an apparent basic difference in their photosynthetic carbon fixation kinetics. Abel (1984) suggested that hemprichii was a strict C02 user and was unable to utilize the abundant HC03 present in seawater. In contrast, photosynthetic rates of T. testudinum are affected by HCo3 concentration (see Fig. 23, Chapter 5). The relatively high CA activity of hemprichii measured by Graham and Smillie (1976) is paradoxical to the results of Abel's study since high CA activity would be expected to enhance conversion of HCo3 to C02 and thus, photosynthetic rates would be affected by the concentration of the former carbon species. et al. (1989) suggested that Abel's (1984) conclusion be treated with caution because of the difficulties associated with differentiating C02 versus HCo3 use. Separations of plants as "users" and "non-users" of HCo3 may be one of degree rather than an absolute (Allen and Spence, 1981; Spence and Maberly, 1985) Alternatively, the decrease in CA activity measured in samples assayed at the end of January compared to the samples col.lected during the middle of the month may be indicative of seasonality in testudinum's CA activity. The degree of decline was dependent on what basis activity was expressed and it ranged from a 12 % decline in activity based on fresh

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126 TaJ;>le 14. Carbonic anhdrase activity expressed as enzyme un1.ts (E. U.) per g fresh weight (mean s. e., n=3), mg chlorophyll or mg protein1 of Thalassia testudinum leaf tissue. E. u. E. u. E. u. Date g FW mg chl mg protein 1/18/90 116.0 9.8 831 22.4 118.8 8.1 971 23.2 1/31/90 107.1 2.9 376 16.9 86.4 5.8 409 13.3 1Assumes leaf protein levels of 10% of dry weight (Dawes, 1986). weight to a 57 % decline based on chlorophyll content. The relatively large chlorophyll-based decline was due to an approximate 45 % increase in chlorophyll content in the leaf material (2.54 versus 3.87 chl dwt for the mid-January collection compared to end of the month, respectively). Seasonal variations in CA activity have previously been observed in the freshwater submersed aquatic macrophyte Myriophyllum spicatum L. (Bowes, 1985). In this species, CA activity was maximal under summer-like conditions (high temperature and long-day length) and marginal under winterlike conditions. In the study by Graham and Smillie (1976), seagrasses were assayed for CA activity during the austral fall. Thus, the differences between the measured CA activities of the two species may not, in reality, be that

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127 pronounced. The presence of CA in T testudinum leaf material may explain the apparently high affinity (i.e. low K,) that this species exhibits for C02 Changes in CA activity and the affinity for C02 in photosynthesis which are observed after transferring high-C02 grown unicellular algae to low C02 conditions, strongly suggest that CA plays an important role in increasing the affinity for C02 in photosynthesis (Ingle and Colman, 1976; Hogetsu and Miyachi, 1979). In addition, the lower apparent K,(C02 ) values for photosynthesis compared to K,(C02 ) values of RuBPCO reported for several unicellular algal species have been shown to be due to the accumulation of DIC within the algal cells (Hogetsu and Miyachi, 1979; Yeoh et al., 1981). This accumulation has been attributed to the presence of an active HCo3 -pump in some species (Beardall, 1981; Kaplan et al., 1982}, and to the formation of C02 from HCo3 -by the action of CA located outside the plasmalemma in other species (Miyachi et al., 1983; Tsuzuki, 1983}. The latter mechanism of CA action seems to accelerate the transport of C02 from outside the cells to the site of the carboxylation reaction (Tsuzuki and Miyachi, 1989), an assumption that has been experimentally proven at the limiting concentration of free C02 (Bird et al., 1980}. In this regard, the necessity of CA for HC03 utilization was implicated in the seagrass Zostera marina through the use of a CA inhibitor (Millhouse and Strother, 1986b}. Enhancement

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128 of the C02 hydration/dehydration reactions by CA contributes to establishing an equilibrium between C02 and HCo3 and thus may minimize the carbon limitational effect of the unstirred boundary layer surrounding seagrass photosynthetic tissue.

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CHAPTER 7 SUMMARY AND CONCLUSIONS Carbon source and concentration effects 129 Both source-sink (tracer) and process (fractionation) information was obtained on the carbon dynamics of Thalassia testudinum through the application of stable carbon isotope measurements. The carbon source sets an isotopic baseline that can then be shifted by the process of isotopic fractionation (Peterson and Fry, 1987). My study showed experimentally that the source of carbon can have a significant effect on the stable carbon isotope composition of Thalassia testudinum, but it seems to have less of an effect on this specie's growth characteristics, at least at the seedling stage. This last statement requires some elaboration. Because of the clonal nature of testudinum, the use of seedlings provided the advantage of assessment of replicated individual plants (genets) rather than having pseudoreplication of treatments through the use of short-shoots (ramets) which may, or may not have come from the same genetic individual (see Hurlbert, 1984) However, in retrospect, it is evident that seedlings also have some disadvantages as experimental treatment units. A significant problem in the use of seedlings as experimental units is the presence of a

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130 significant seed reserve which allows the seedlings to grow in what may be viewed as a heterotrophic state for the first couple of months (Durako and Hoffler, 1987). Internal nutrient reserves may allow the seedlings to be somewhat independent of the surrounding environmental conditions during their early growth phase. This independence seemed to minimize the effects of some of the treatments which were applied in this study. The observed ability of axenic seedlings to assimilate organic carbon from the medium may also be a reflection of this heterotrophic state. Nevertheless, the carbon isotopic composition of 1:. testudinum leaves was shown to be affected by both the isotopic composition of various inorganic and organic carbon sources (source information) and by pC02 andjor dissolved inorganic carbon concentration (process information, see review by Peterson and Fry, 1987). The comparatively larger isotopic fractionations exhibited by control seedlings in the MERL study and the photosynthetic characterstics of leaf material suggest a greater importance for pC02 (i.e. free-C01 ) and a correspondingly lower relative importance of HCo3 -as the initial substrate for carbon fixation in this species. The source of carbon for photosynthesis In contrast to the results of Abel (1984) for Thalassia hemprichii, evidence presented here suggests that T testudinum photosynthesis is not independent of Hco; levels,

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131 but is affected by both C02 and HCo3 -concentrations. The ability to use HC03 -seems to vary between these two closelyrelated species, as has also been observed in the genus Potamogeton (Kadono, 1980; 1982). At present, there is no evidence for the presence of an active Hco3 -ion pump in any seagrass (Larkum et al., 1989). Therefore, it seems more likely that HC03 -is converted to C02 in the external medium, through the action of leaf-associated carbonic anhydrase (or possibly by extrusion of H+ into the cell wall and the unstirred boundary layer, see Lucas, 1983), moves across the cuticle as C021 then diffuses as C02 and HCo3 -to the site of carboxylation. Abel ( 1984) suggested that hemprichii reflected its presumed evolutionary origin from terrestrial ancestors in not being able to utilize the much more abundant HCo3-. However, as will be discussed below, this physiological trait would be expected to have adapted to ambient conditions under the strong selective pressures that carbon limitation would impose. The high substrate affinities calculated for C02 in this study indicate that testudinum has adapted its carbon assimilation characteristics to ambient seawater conditions. The low K5 ( C02 ) values calculated here compared to previous investigations of submersed aquatic species may also reflect methodological differences and point out the importance of using a buffered media in performing these types of investigations. Use of an appropriate buffer system in the

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132 assay media obviates problems associated with pH increases during photosynthetic measurements and the resulting decreases in C02 concentration. These problems are especially aggravated in the more recently employed microelectrode assay systems which use relatively large tissue:medium volumes. The net results of any pH increases in the medium would be a reduced apparent affinity for C02 [i.e. an increase in K5(C02)] because of the reduction in free C02 in the medium. Is carbon limiting to Thalassia testudinum? It is virtually impossible to definitively single out one factor as limiting photosynthesis and, ultimately the growth of a species in nature because of a dependence on the interaction of both abiotic and biotic factors in a specific habitat (Beer, 1989). The results reported here indicate that the degree of carbon limitation in Thalassia testudinum varies according to the level of organization being considered. At the plant level, leaf stable carbon isotope data, and, to a lesser degree, growth data indicate that dissolved inorganic carbon (DIC) concentration, at natural levels, is limiting. The enhanced growth and leaf o13C responses of axenic seedlings to media sucrose enrichment demonstrated an ability to assimilate dissolved organic carbon. This may be an adaptive response to carbon limitation, although the significance of heterotrophic carbon uptake in situ is probably minimal because of the tremendous competition for organic carbon

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133 substrates from microorganisms. Independent support for the concept of plant-level carbon limitation can be gained by the examination of this question from another standpoint. Leaf o13C values from short-shoots growing in four different light environments are presented in Figure 25. The four environments/treatments are shallow natural light (= shallow control), shallow with light reduced approximately 45% by neutral density nursery cloth (= shallow shaded), deep control, and deep shaded. These experimental treatments were applied in a study of the demographic, morphological, and physiological responses ofT testudinum to in situ light reduction (Hallet al., 1989). Reduction in light by shading resulted in a decrease in leaf o13C at both the shallow and deep sites. This change in leaf o13C is probably due to an increase in isotopic fractionation with a reduction in light. These data indicate that carbon is limiting at the higher light levels based on the relatively heavy isotopic signatures (O,Leary, 1988). However, as light is reduced, which is in the direction of increasing light limitation, carbon appears to become less limiting -i.e. leaf o13c values become more negative. This rationale is basically a variation on Liebig's law of the minimum. In contrast, the photosynthetic measurements, calculated kinetic constants, and carbonic anhydrase activities suggest that _r. testudinum is relatively well adapted to ambient DIC concentrations (cf T hemprichii, Abel,

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,....--.... 0 0 0 ......___., -0 Q) _J u n c.o -14 -12 -10 D Shallow Control Shallow Shade Deep Control E':SSS Deep Shade Period of treatment (months) 134 Figure 25 Leaf & u c values (mean s.e.) forT testudinum short-shoots subjected to in situ light reduction at one and three months post-treatment. 1984) at the tissue level. Although these conclusions seem paradoxical, they can readily coexist when viewed in an evolutionary context. As stated in the introduction, seagrasses are the only group of higher plants to have returned to a completely submerged existence. This probably occurred about one hundred million years ago (den Hartog, 1970). Their apparent success,

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135 as demonstrated by widespread distribution, seems inconsistent with their relative lack of speciation i.e. there are several hundred thousand species of terrestrial and aquatic angiosperms (whose ancestors left the sea some four hundred million years ago, Larkum and den Hartog, 1989), yet only 55 known species of seagrasses. One explanation is that seagrasses exploited their present niche successfully at an early stage with a lack of competition so have changed very little (Larkum and den Hartog, 1989). This explanation implies that seagrasses are well adapted to their environment and thus form stable communities. However, this view has been recently challenged (Larkum and West, 1983) There are a number of reports i n the literature which show instability of seagrass bed communi ties (Rasmussen, 1977; Kirkman, 1978, 1985; Orth, 1976 ; Orth and Moore, 1983; Clarke and Kirkman, 1989). Because of this instability, the fitness of seagrasses for submerged marine existence has recently been questioned to the point that it has been suggested that they can be categorized as stress tolerant plants (sensu Grime, 1979) and, hence, are not as we l l adapted to the marine environment as previously thought (see Table 14). Adaptation, or the hereditary environment, is a universal feature adjustment to the of life. Organisms undergo various types of changes i n response to the selective pressures of their environment. Changes in the hereditary characteristic s of an organism constitute evolution, which

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136 Table 15. Important stresses which limit the distribution and abundance of seagrasses (adapted from Larkum and den Hartog, 1989). Stress High salt environment Temperature change Wave action Pressure Anaerobiosis Nutrient limitation Epiphytes anoxia Light/shade Infection Hebivory Adaptation Sheaths Ion pumps Races Strap leaves Underground rhizomes None? Aerenchyma Fermentative metabolism Root and leaf absorption Continuous leaf growth from base Shade tolerance Phenolics and attack resistence Phenolics Vulnerability Mechanical and biological disturbance. Increased respiratory load and decreased productivity Sudden temperature shifts Blow-outs Sediment movement Impairment of photosynthesis Epiphytes Anoxic water Lack of trace elements Lack of N and/or P (and C02?) Smothering, shading, Decreased productivity Lower productivity Shading by epiphytes Die-back or decreased fitness Removal of critical parts e.g. leaves and meristems

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137 reduced to its barest essentials, is a change in the frequencyof gene alleles. Natural selection can be regarded as the differential and non-random reproduction of different alleles (Grant, 1963). There are different levels and intensities of natural selection. It is generally assumed that natural selection may exert more pressure at the single gene or molecular level since selection for combinations of genes is very costly to a population in terms of genetic deaths (Grant, 1963). As a result, adaptation is generally more rapid for physiological traits than for phenotypic traits because many of the former can be affected by single gene changes, while the latter are generally polygenic and/ or controlled by epistasis (Strickenberger, 1976).

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138 REFERENCES Abel, K., 1983. Photosynthesis in two tropical seagrasses with special reference to carbon metabolism. MSc thesis. James Cook University of North Queensland. 160 pp. Abel, K., 1984. Inorganic carbon source for photosynthesis in the seagrass Thalassia hemprichii (Ehrenb.) Aschers. Plant physiol. 76: 776-781. Allen, E. D. and Spence, D. H. N., 1981. The differential ability of aquatic plants to utilize the inorganic carbon supply in fresh waters. New Phytol. 87: 269-283. Andrews, T. J. and Abel, K. M., 1977. Photosynthetic carbon metabolism in seagrasses. labelling evidence for the C3 pathway. Plant Physiol. 63: 650-656. Atkins, C. A., Patterson, B. D. and Graham, carbonic anhydrases. I. Distribution species. Plant Physiol. 50: 214-217. D., 1972. of types Plant among Barber, B. J. and Behrens, P. J., 1985. Effects of elevated temperature on seasonal in situ leaf productivity of Thalassia testudinum Banks ex Konig and Syringodium filiforme Klitzing. Aquat. Bot. 22: 61-69. Beardall, J., 1981. C02 accumulation by Chlorella saccharophila (Chlorophyceae) at low external pH: evidence for the active transport of inorganic carbon at the chloroplast envelope. J. Phycol. 17: 371-373. Beer, s., 1989. Photosynthesis and photorespiration of marine angiosperms. Aquat. Bot. 34: 153-166. Beer, s. and Shragge, B., 1987. Photosynthetic carbon metabolism in Enteromorpha compressa (Chlorophyta). J. Phycol. 23: 580-584. Beer, s. and Waisel, Y., 1979. Some photosynthetic carbon fixation properties of seagrasses. Aquat. Bot. 7: 129-138. Beer, s. and Wetzel, R. G., 1982. Photosynthetic carbon fixation pathways in Zostera marina and three Florida seagrasses. Aquat. Bot. 13: 141-146.

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139 Bee:, S., Eschel, A. and Waisel, Y., 1977. carbon metabolism 1n I of exogenous inorganic carbon spec1es 1n photosynthes1s. J. Exp. Bot. 28: 1180-1189. Beer, s., Shomer-Ilan, A. and Waisel, Y., 1980. carbon metabolism in seagrasses. II. Patterns of photosynthetic C02 incorporation. J. Exp. Bot. 31: 1019-1026. Benedict, c. R. and Scott, J. R., 1976. Photosynthetic carbon metabolism of a marine grass. Plant Physiol. 57: 876-880. Benedict, c R., Wong, W. W. and Wong, J H., 1980. Fractionation of the stable isotopes of inorganic carbon by seagrasses. Plant Physiol. 65: 512-517. Berry, J. A and Troughton, J. H., 1974. carbon isotope fractionation by C3 and C4 plants in "closed" and "open" atmospheres. Carnegie Inst. Wash. Year Book, pp. 785-790. Bird, I. F., Cornelius, M. J. and Keys, A. J., 1980. Effect of carbonic anhydrase on the activity of ribulose bisphosphate carboxylase. J. Exp. Bot. 31: 365-369. Black, M. A., Maberly, s c. and Spence, D. H. N., 1981. Resistance to carbon dioxide fixation in four submerged freshwater macrophytes. New Phytol. 89: 557-568. Boon, P.I., 1986. Nitrogen pools in seagrass beds of Cymodocea serrulata and Zostera capricorni of Moreton Bay, Australia. Aquat. Bot., 25: 1-19. Bowes, G., 1985. Pathways of C02 fixation by aquatic organisms. In: W. J. Lucas and J. A. Berry (Editors) Inorganic Carbon Uptake by Aquatic Photosynthetic organisms. Am. Soc. Plant Physiol., Rockwell, MD. pp 187210. Bowes, G., 1987. Aquatic plant photosynthesis: strategies that enhance carbon gain. In: R. M. M. Crawford (Editor), Plant Life in Aquatic and Amphibious Habitats. Spec. Publ. No. 5 Br. Ecol. Soc. Blackwell Scientific Publications, Oxford. pp. 79-98. Bowes, G. and Salvucci, M. E., 1984. Hydrilla: inducible C4 -type photosynthesis without Kranz anatomy. In: C. Sybesma (Editor), Progress in Photosynthesis Research. Vol. IV. Martinus Nijhoff, Dordrecht. pp. 345-352. Bowes, G. and Salvucci, M. E., 1989. photosynthetic carbon metabolism of macrophytes. Aquat. Bot. 34: 233-266. Plasticity in the submersed aquatic

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140 Bowes, s. and Haller, w. T., 1979. seasonal var1at1c;m the b1omass, tuber density, and photosynthetic metabol1sm 1n three Florida lakes. J. Aquat. Plant Manage. 17: 61-65. Brix, H. and Lyngby, J. E., 1985. Uptake and translocation of phosphorus in eelgrass (Zostera marina). Mar. Biol. 90: 111-116. Brouns, J. J. P. M., 1985. A comparison of the annual production and biomass in three monospecific stands of the seagrass Thalassia hemprichii (Ehrenb.) Aschers. Aquat. Bot. 23: 149-175. Browse, J. A., Dromgoole, F. I. and Brown, J. M. A., 1979. Photosynthesis in the aquatic macrophyte Egeria densa. III. Gas exchange studies. Aust. J. Plant Physiol. 6: 499-512. Burkholder, P.R., Burkholder, L.M. and Rivero, J.A., 1959. Some chemical constituents of turtle grass, Thalassia testudinum. Bull. Torrey Bot. Club, 86: 88-93. Calder, J. A. and Parker, P. L., 1973. Geochemical implications of induced changes in 13C fractionation by blue-green algae. Geochim. et Cosmochim. Acta 37: 133-140. Carlson, P.R. and Acker, J.G., 1985. Effects of in situ shading on Thalassia testudinum: preliminary experiments. In: F.J. Webb (Editor), Proc. twelfth Ann. Conf. Wetlands Restoration Creation. Hillsborough Comm. Coll., Tampa, Fla. pp. 64-73. Clarke, s. M. and Kirkman, H., 1989. Seagrass Dynamics. In: A. w. Larkum, A. J. McComb and S.A. Shepard (Editors), Biology of Seagrasses, Elsevier, Amsterdam. pp. 304-345. Cooper, L. W. and Deniro, M. J., 1989. Stable carbon isotope variability in the seagrass Posidonia oceanica: evidence for light intensity effects. Mar. Ecol. Prog. Ser. 50: 225-229. Cooper, T. G., Filmer, D., Wishnick, M. and Lane, M.D., 1969. The active species if "C02 utilized by ribulose diphosphate carboxylase. J. Biol. Chern. 244: 1081-1083. craven, P.A. and Hayasaka, S.S., phosphate solubilization by rhizosphere bacter1a 1n a Zostera marina community. Can. J. Microbial., 28: 605-610. Dalling, M. J. and Bhalla, P. L., 1984. nitrogen and phosphorus from endosperm. Mobilization of In: D. R. Murray

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141 (Editor), Seed Physiology, Vol. 2-Germination and Reserve Mobilization. Academic Press, New York, pp. 163-199. Dawes, C. J. 1986. Seasonal proximate constituents and caloric values in seagrasses and algae on the west coast of Florida. J. Coastal Res. 2: 25-32. Dawes, C. J. and Tomasko, D. A., 1988. Depth distribution of Thalassia testudinum in two meadows on the west coast of Florida; a difference in effect of light availability. P.S.Z.N.I. Mar. Ecol. 9: 123-130 Degens, E. T., Guillard, R. L., Sackett, w. M. and Hellebust, J. A., 1968. Metabolic fractionation of carbon isotopes in marine plankton I. Temperature and respiration experiments. Deep-Sea Res. 15: 1-9. Den Hartog, c., 1970. The Seagrasses of the World. NorthHolland, Amsterdam. 275 pp. Den Hartog, C., 1971. The dynamic aspect in the ecology of sea-grass communities. Thalass. Jugoslav., 7: 101-112. Dennison, W. c., 1987. Effects of light in seagrass photosynthesis, growth and depth distribution. Aquat. Bot. 27: 15-26. Dennison, w.c. and Alberte, R.S., 1982. Photosynthetic responses of Zostera marina L. (Eelgrass) to in situ manipulations of light intensity. Oecologia, 55: 137-144. Dennison, W. C. and Alberte, R. S., 1985. Role of daily light period in the depth distribution of Zostera marina (eelgrass). Mar. Ecol. Prog. Ser. 25: 51-61. Dennison, W. c. and Alberte, R. S., 1986. Photoadaptation and growth of Zostera marina L. (eelgrass) transplants along a depth gradient. J. Exp. Mar. Biol. Ecol. 98: 265-282. Dennison, w.c., Aller, R.C. and Alberte, R.S., Sediment ammonium availability and eelgrass (Zostera mar1na) growth. Mar. Biol., 94: 469-477. Deuser, w. G. and Degens, E. T., 1967. Carbon isotope fractionation in the system C02-C02(aqueous>-HC03Nature 215: 1033-1035. Doliner L. H. and Jolliffe, P. A., 1979. Ecological evidence the adaptive significance of the C4 dicarboxylic acid pathway of photosynthesis. Oecologia 38: 23-34.

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C02 by ribulose-1, 5-bisphosphate Physiol. 63: 852-856. carboxylase, 155 Plant Yeoh, H. -H., Badger, M. R. and Watson, L., 1981. Variations in kinetic properties of ribulose-1,5-bisphosphate carboxylases among plants. Plant Physiol. 67: 1151-1155. Zieman, J. C The ecology of the seagrasses of south Florida: A community profile. u.s. Fish and Wildlife Service, Office Biological Services, Washington, D. c. 123 pp. Zieman, J. C., 1987. A review of certain aspects of the life, death, and distribution of the seagrasses of the southeastern United States 1960-1985. In: M. J. Durako, R. C. Phillips and R. R. Lewis (Editors), Proc. Symp. Subtropical-Tropical Seagrasses Southeastern United states, Fla. Mar. Res. Publ. No. 42, St. Petersburg, Florida. pp. 53-76. Zieman, J. C., Thayer, G. w., Robblee, M. B. and Zieman, R. I., 1979. Production and export of seagrasses from a tropical bay. In: R. J. Livingstone (Editor), Ecological Processes in Coastal Marine Systems. Plenum Press, New York, NY. pp. 21-34. Zieman, J. c., Macko, s. A. and Mills, A. L., 1984. Role of seagrasses and mangroves in estuarine food webs: Temporal and spatial changes in stable isotope composition and amino acid content during decomposition. Bull. Mar. Sci. 35: 380-392. Zieman, J. c. and Zieman, R. T., 1989. The ecology of the seagrass meadows of the west coast of Florida: A community profile. u. s. Fish and Wildlife Service, Office Biological Services, Washington, D. C., 155 pp. Zimmerman, M.s. and Livingstone, R. J., 1976. Seasonality and physico-chemical ranges of benthic macrophytes from a north Florida estuary (Apalachee Bay). Contrib. Mar. Sci. 20: 34-45. zimmerman R.C., Smith, R.D. and Alberte, R.S., 1987. Is growth1of eelgrass nitrogen limited? A numerical simulation of the effects of light and nitrogen on the growth dynamics of Zostera marina. Mar. Ecol. Prog. Ser., 41: 167-176.

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157 APPENDIX 1 The Effect of Bicine on the Photosynthetic Rate of Tha1assia testudinum A preliminary series of photosynthetic measurements was conducted to evaluate the effect on photosynthetic rate, if any, of the addition of 10 mM Bicine to the synthetic seawater medium. Five replicate paired photosynthetic assays were done comparing the rate of oxygen exchange of seedling leaf segments (ppm/h) in 30 /00 synthetic seawater at pH 8.2 to that in the same medium with Bicine. A paired observation t-test was run to test the null hypothesis that the mean of the photosynthetic rate differences was zero. Oxygen exchange (ppm/h) Difference IO IO+Bicine (d) (d2 } 1. 07 0.72 0.35 0.12 1. 52 0.99 0.52 0 .28 1. 51 1.19 0.32 0.10 0.65 1. 23 -0.58 0.34 0 .83 0.86 -0.03 0.001 5.58 5.00 0.58 0.84 X 1.12 1. 00

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APPENDIX 1 (Continued) L d2
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159 APPENDIX 2 Calculation of K5(C02 ) for Thalassia hemprichii based on data qiven by Abel (1983). It is difficult to experimentally determine the kinetic constants K and Vmu by directly measuring the rate of the process of interest because of the uncertainty in determining if the maximum rate has been reached. This problem can be solved by rearranging the Michaelis-Menten equation, where v = rate of the process V = the maximum process rate at "infinite" substrate concentration (also written as V ) max [S] = the substrate concentration K = the substrate concentration where the process rate is one-half the maximum rate which numerically expresses the graphic curve of the direct measurements, to yield an equation for a straight line. One type of linear rearrangement is the Lineweaver-Burk equation, which is based on a plot of the inverse of the substrate concentration plotted against the inverse of the process rate

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160 APPENDIX 2 (Continued) 1 Ks+ [S] -= --=-,:--:=v V[S] Ks 1 1 =-.--+-v [S] V (or velocity). It has the advantage that measurements of the process rate can be made at a relatively small number of substrate concentrations and then extrapolated back to infinite concentration at the intercept. The Lineweaver-Burk equation was applied to the photosynthetic data from Abel (1983) to calculate Ks and Vmu as follows: [S] v l. l. (mM C02 ) {J.mol Cjmg chl _g_fh) [S) v 0.001 0.02 1000.0 50.00 0.003 l). 06 333.3 16.67 0.009 0.16 111.1 6.25 0.018 0.25 55.6 4.00 0.020 0.22 50.0 4.54 0.055 0.39 18. 2 2.56 0.078 0.42 12.8 2.38 0.154 0.63 6.5 1. 59 The linear regression (y=bx+a) for double reciprocal data was: y = 0.048x + 1.411 r = 0.999 substituting the appropriate values into the Lineweaver-Burk equation: a = 1/Vmax = 1:411 v = 0.709 Cjmg chl _g_fh mu

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APPENDIX 2 (Continued) b = K./Vmn = 0.048 K = b(Vmn) = 0.048(0.709} K, = 0 0 3 4 mM = 3 4 J,J.M 161


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