Journal of Experimental Marine Biology and Ecology 250 (2000) 169±205 www.elsevier.nl/locate/jembe

Overview of the physiological ecology of carbon metabolism in seagrasses

Brant W. Touchette* , JoAnn M. Burkholder Department of Botany Box 7510, North Carolina State University, Raleigh, NC 27695-7510, USA

Abstract

The small but diverse group of angiosperms known as seagrasses form submersed meadow communities that are among the most productive on earth. Seagrasses are frequently light-limited and, despite access to carbon-rich seawaters, they may also sustain periodic internal carbon limitation. They have been regarded as C3 plants, but many species appear to be C3±C4 intermediates and/or have various carbon-concentrating mechanisms to aid the Rubisco enzyme in carbon acquisition. Photorespiration can occur as a C loss process that may protect photosynthetic electron transport during periods of low CO2 availability and high light intensity. Seagrasses can also become photoinhibited in high light (generally . 1000 mE m22 s21 ) as a protective mechanism that allows excessive light energy to be dissipated as heat. Many photosynthesis± irradiance curves have been developed to assess light levels needed for seagrass growth. However, most available data (e.g. compensation irradiance Ic ) do not account for belowground tissue respiration and, thus, are of limited use in assessing the whole-plant carbon balance across light gradients. Caution is recommended in use of Ik (saturating irradiance for photosynthesis), since seagrass photosynthesis commonly increases under higher light intensities than Ik; and in estimating seagrass productivity from Hsat (duration of daily light period when light equals or exceeds Ik ) which varies considerably among species and sites, and which fails to account for light-limited photosynthesis at light levels less than Ik. The dominant storage carbohydrate in seagrasses is sucrose (primarily stored in rhizomes), which generally forms more than 90% of the total soluble carbohydrate pool. Seagrasses with high Ic levels (suggesting lower ef®ciency in C acquisition) have relatively low levels of leaf carbohydrates. Sucrose-P synthase (SPS, involved in sucrose synthesis) activity increases with leaf age, consistent with leaf maturation from carbon sink to source. Unlike terrestrial plants, SPS apparently is not light-activated, and is positively in¯uenced by increasing temperature and salinity. This response may indicate an osmotic adjustment in marine angiosperms, analogous to increased SPS activity as a cryoprotectant response in terrestrial non-halophytic plants. Sucrose synthase (SS, involved in sucrose metabo- lism and degradation in sink tissues) of both above- and belowground tissues decreases with tissue age. In belowground tissues, SS activity increases under low oxygen availability and with increasing temperatures, likely indicating increased metabolic carbohydrate demand. Respiration

*Corresponding author. Tel.: 11-919-515-2726; fax: 11-919-513-3194. E-mail address: joann [email protected] (B.W. Touchette). ] 0022-0981/00/$ ± see front matter  2000 Elsevier Science B.V. All rights reserved. PII: S0022-0981(00)00196-9 170 B.W. Touchette, J.M. Burkholder / J. Exp. Mar. Biol. Ecol. 250 (2000) 169 ±205 in seagrasses is primarily in¯uenced by temperature and, in belowground tissues, by oxygen availability. Aboveground tissues (involved in C assimilation and other energy-costly processes) generally have higher respiration rates than belowground (mostly storage) tissues. Respiration rates increase with increasing temperature (in excess of 408C) and increasing water-column nitrate enrichment (Z. marina), which may help to supply the energy and carbon needed to assimilate and reduce nitrate. Seagrasses translocate oxygen from photosynthesizing leaves to belowground tissues for aerobic respiration. During darkness or extended periods of low light, belowground tissues can sustain extended anerobiosis. Documented alternate fermentation pathways have yielded high alanine, a metabolic `strategy' that would depress production of the more toxic product ethanol, while conserving carbon skeletons and assimilated nitrogen. In comparison to the wealth of information available for terrestrial plants, little is known about the physiological ecology of seagrasses in carbon acquisition and metabolism. Many aspects of their carbon metabolism Ð controls by interactive environmental factors; and the role of carbon metabolism in salt tolerance, growth under resource-limited conditions, and survival through periods of dormancy Ð remain to be resolved as directions in future research. Such research will strengthen the understanding needed to improve management and protection of these environmentally important marine angiosperms.  2000 Elsevier Science B.V. All rights reserved.

Keywords: Carbon; Light; Photosynthesis; Respiration; Seagrass; Temperature

1. Introduction

Seagrasses are a small but diverse group of mostly submersed marine angiosperms which inhabit environments that are characterized by periodic light limitation. These monocots are taxonomically restricted to two families, 12 genera, and 55 species (here, including the species Ruppia maritima), all of which have evolved from land pre- decessors that returned to the sea approximately 100 million years ago during the Cretaceous (McRoy and Helfferich, 1977; Larkum and den Hartog, 1989). Few angiosperms have evolved the osmoregulatory capacity to exist in marine waters. Nevertheless, the high productivity of seagrass meadows, sometimes exceeding 15 g C m22 d21 , places them among the most productive of all marine ecosystems (Phillips and McRoy, 1980; Hillman et al., 1989). Seagrass meadows provide both habitat and a nutritional base for ®n®sh, shell®sh, waterfowl, and herbivorous mammals (Klumpp et al., 1989; Phillips and Menez,Ä 1998). Seagrass meadows also function in stabilizing bottom sediments and clearing the water of suspended sediments and nutrients (Terrados and Duarte, 2000). Most seagrasses grow rooted in nutrient-rich sediments of shallow coastal lagoons and embayments, where the water column sustains periodic increased turbidity from sediment loading/resuspension, phytoplankton, and macroalgal growth (Harlin, 1993; Morris and Tomasko, 1998). Epiphytic algal development on the macrophyte leaves can signi®cantly reduce available light for growth, as well (Sand-Jensen, 1977). Moreover, during low tide some intertidal species undergo hours of atmospheric exposure with associated desiccation and increased UV radiation (Trocine et al., 1981; Leuschner et al., 1998). B.W. Touchette, J.M. Burkholder / J. Exp. Mar. Biol. Ecol. 250 (2000) 169 ±205 171

Since the sediments typically provide high supplies of most nutrients, with the water column as a secondary source (Short and McRoy, 1984; Harlin, 1993), seagrasses generally are not primarily nutrient-limited (Zimmerman et al., 1987). However, seagrasses obtain their carbon supply from the water rather than the sediments (Sand- Jensen, 1977). Since carbon dioxide diffuses through water |10 000-fold more slowly than through air (Stumm and Morgan, 1996), carbon acquisition is more dif®cult for submersed plants. Whereas there is a wealth of information about the ecology of seagrasses under varying light regimes, there has been no effort to present an overview and conceptual framework about carbon uptake and metabolism in seagrasses from physiological and ecological perspectives. Here, we synthesize available information on the interplay between carbon and light in the carbon metabolism of this ecologically important group of aquatic angiosperms.

2. Seagrass photosynthesis

2.1. Basic photosynthetic characteristics

Investigations into the basic photosynthetic processes in seagrasses (for example, photosystem II studies using pulsed amplitude-modulated [PAM] ¯uorometry) suggest that seagrasses have the basic photosynthetic biochemistry reported for other angios- perms (Goodwin and Mercer, 1983; Beer et al., 1998). The pigment composition in seagrasses is similar to that of most angiosperms and includes chlorophylls a and b which function directly in photosynthesis, and carotenoids which assist in ultraviolet light and excess oxygen absorption, and in other protective roles (Beer and Waisel, 1979; Beer, 1998). Within the chloroplast, the chlorophylls absorb light for photo- synthesis, with excitation energy transferred from one pigment molecule to another by a nonradiative process referred to as resonance or ForsterÈ transfer. The antenna system is highly varied among photosynthetic organisms, but the central reaction site (chloro- phyll±protein complex; P680 and P700) is highly conserved (Wales et al., 1989). Four independent protein complexes that reside within the thylakoid membrane carry out the majority of the chemical processes that occur in the light reactions, including photo- system II, cytochrome b6-f complex, photosystem I, and an ATP synthase (Taiz and Zeiger, 1991). The `dark reactions' of photosynthesis (photosynthetic carbon reduction [PCR] or Calvin±Benson cycle) also occur in the chloroplast and are fairly conserved among photosynthetic eukaryotes. Seagrasses have previously been considered to be mostly C3 plants (but see below), meaning that the ®rst stable product of carbon dioxide ®xation is the 3-carbon structure, 3-phosphoglycerate (PGA, formed by carboxylation or attach- ment of carbon to a 5-carbon sugar, ribulose 1,5-bisphosphate, which produces an unstable product that splits into two 3-carbon PGA molecules; Goodwin and Mercer, 1983). The activity of Rubisco (ribulose bisphosphate carboxylase/oxygenase), the primary carboxylase involved in carbon ®xation, is generally lower in submersed aquatic plants than in emergent wetland or terrestrial plants (Farmer et al., 1986; Beer et al., 1991). For example, reported Rubisco activities in the seagrasses Ruppia maritima and 172 B.W. Touchette, J.M. Burkholder / J. Exp. Mar. Biol. Ecol. 250 (2000) 169 ±205

21 21 Zostera marina (2.62 and 1.97 mmol CO2 min mg chl, respectively) are comparable to those reported for submersed freshwater angiosperms (21 species; mean |2.3 mmol 21 21 CO2 min mg chl), and for marine green and brown macroalgae (six chlorophyte and | 21 21 pheophyte species; mean 2.5 and 2.4 mmol CO2 min mg chl , respectively). However, Rubisco activities in seagrasses are lower than those reported for marine red macroalgae and freshwater emergent angiosperms (three rhodophyte species and six 21 21 emergent angiosperm species; 8.6 and 7.4 mmol CO2 min mg chl ; Beer et al., 1991).

2.2. Photorespiration

Rubisco can function as an oxygenase rather than a carboxylase, to oxygenate ribulose 1,5-bisphosphate in a carbon dioxide-releasing or carbon loss process that directly opposes photosynthesis (Lorimer, 1981). Photorespiration, also called the C2 cycle (wherein the products of oxygenation include one PGA molecule and one 2-carbon molecule, 2-phosphoglycolate), involves three types of organelles (chloroplasts, mito- chondria, and peroxisomes; Fig. 1). In this process, O2 is consumed and inorganic phosphate (Pi2 ) and CO are released, as well as byproducts such as peroxide and ammonia. The oxygenase function of Rubisco is favored under increasing oxygen levels, increasing temperatures, and high light (Taiz and Zeiger, 1991). In the Cretaceous period when seagrasses ®rst appeared in the fossil record (Phillips and Menez,Ä 1988), C losses from photorespiration were likely to be minimal because atmospheric CO22 /O ratios were much higher (Ivany et al., 1991; Kuypers et al., 1999). For plants under present conditions, photorespiration is considered a wasteful process, and its functional bene®ts remain unclear. However, the process may bene®t contemporary seagrasses as a mechanism to remove excess products of the light reactions (i.e., ATP and NADPH), and/or to protect photosynthetic electron transport from photoinactivation, thereby limiting damage to the photosynthetic apparatus during periods of low CO2 availability and high light intensity (Heber et al., 1996). Rates of photorespiration activity are considerably lower in most submersed aquatic plants than in terrestrial plants (Abel and Drew, 1989; Beer et al., 1991; Frost- Christensen and Sand-Jensen, 1992), although the process is more dif®cult to measure accurately in aquatic plants because of confounding effects of gas accumulation in the lacunae, and other factors (Abel and Drew, 1989). Environmental conditions such as current velocity and reduced light intensity would tend to favor Rubisco as a carboxylase rather than as oxygenase because these conditions decrease the potential for oxygen accumulation during photosynthesis (Frost-Christensen and Sand-Jensen, 1992). More- over, in marine waters many photosynthetic organisms (including some seagrasses) use bicarbonate as an additional inorganic carbon (Ci ) source, and some also have developed various mechanisms to concentrate relatively high levels of CO2 around Rubisco active sites (somewhat analogous to C4 photosynthesis; Bidwell and McLachlan, 1985; Raven, 1985; Beer et al., 1990; Beer et al., 1991; Madsen et al., 1993). However, not all 2 seagrasses utilize HCO3 , and those that do are typically less ef®cient than macroalgae and cyanobacteria (blue-green algae; Beer et al., 1991).

There is experimental evidence for photorespiration, as data for increasing CO2 loss B.W. Touchette, J.M. Burkholder / J. Exp. Mar. Biol. Ecol. 250 (2000) 169 ±205 173

Fig. 1. Three organelles involved in photorespiration. This C2 process is initiated when the oxygenase component of Rubisco oxidizes ribulose-1,5-bisphosphate to produce 3-phosphoglycerate and a 2-carbon compound, 2-phosphoglycolate. The bulk of this pathway is dedicated to the energy-costly conservation of the 2C product, and requires both ATP and NADH (modi®ed from Taiz and Zeiger, 1991). and/or photosynthesis inhibition, under increasing oxygen regimes during light periods in seagrasses Cymodocearotundata, Halophila ovata, and Posidonia australis (Hough, 1976; Donton et al., 1976). Pulse-chase experiments with14 C showed increased labeling of photorespiratory intermediates glycolate, glycine, and serine during elevated oxygen conditions in seagrasses H. ovata and Thalassia hemprichii (Burris et al., 1976; Andrews and Abel, 1979). Studies using PAM ¯uorimetry to evaluate photosynthetic rates also have shown curvilinear relationships between estimated ¯uorometric photo- synthesis and O2 -evolving photosynthesis. For example, in Halophila stipulacea and Zostera marina during high irradiance, the rate of O2 release decreased relative to the rate of electron transport in PSII (Beer et al., 1998). This deviation from linearity was believed to indicate photorespiration. A curivelinear response was not observed in Cymodocea nodosa, however, suggesting that photorespiration did not occur under similar conditions Ð possibly because this seagrass species has developed a carbon- concentrating mechanism that enables it to maintain high photosynthetic rates under elevated oxygen levels (Beer and Waisel, 1979; Beer, 1989; Beer et al., 1998). 174 B.W. Touchette, J.M. Burkholder / J. Exp. Mar. Biol. Ecol. 250 (2000) 169 ±205

2.3. Carbon concentrating mechanisms

Many plants apparently have evolved mechanisms to increase the CO22 /O ratio near active C-®xation sites of the Rubisco enzyme. Such mechanisms would enhance carbon

®xation in environments where CO2 diffusion is slow (e.g., submersed habitats), while also depressing wasteful photorespiration. Known carbon concentrating mechanisms include C4 photosynthesis (with Crassulacean acid metabolism [CAM] as a modi- 22 ®cation), CO23 /HCO pumps, and HCO 3 dehydration (catalyzed by enzymes such as carbonic anhydrase; Eighmy et al., 1991; Funke et al., 1997). C4 plants actually have C4 followed immediately by C3 metabolism; however, the initial product of carbon assimilation is a 4-C acid, oxaloacetate (OAA). Typically, the process of carbon ®xation to a 4-C product is spatially separated from C3 metabolism. For example, in terrestrial plants possessing Kranz anatomy (e.g., corn), carbon ®xation occurs in leaf mesophyll cells wherein phosphoenol pyruvate (PEP) is carboxylated by the enzyme PEP carboxylase (Goodwin and Mercer, 1983). The 4-C product, OAA, is converted to malate or aspartate, and then transported to bundle sheath cells where decarboxylation occurs, generating a 3-carbon product (alanine, pyruvate, or PEP, transported back to the mesophyll cells) and high concentrations of CO2 which depress the oxygenase (photorespiratory) function of Rubisco. The resulting CO2 is then reassimilated to form two 3-PGA molecules via typical Rubisco/C3 reactions. In seagrasses as in other aquatic angiosperms, the distinction between C3 and C4 metabolism is not always clear, and some species behave as C3±C4 intermediates (Bowes et al., 1978; Beer et al., 1980; Beer and Wetzel, 1981; Waghmode and Joshi, 1983). For example, Frost-Christensen and Sand-Jensen (1992) determined the photo- synthetic quantum ef®ciency (fa2, as O evolution per unit of absorbed photons under light-limited photosynthesis) for submersed angiosperms including the seagrass, Zostera marina. They reported that the fa values more closely resembled those for terrestrial C4 than C3 plants. In other investigations, direct measurements of photosynthetic products and/or d13C values indicated that, Cymodocea nodosa and Thalassia testudinum were C4 plants, whereas Thalassia hemprichii, Thalassodendron ciliatum, Halophila spinul- osa, Halodule uninervis and Syringodium isolifolium were C3 plants (Benedict and Scott, 1976; Andrews and Abel, 1979; Beer and Waisel, 1979; Beer et al., 1980). This d13C-derived classi®cation is based on studies involving terrestrial plants, wherein d13C values were typically ca. 228 for C3 plants, and 214 for C4 species. In seagrasses the d13C values are at ca. 211, suggesting that these plants more closely resemble C4 terrestrial species (Smith and Epstein, 1971; McMillan et al., 1980; but see Abel and Drew, 1989). Interpretations about C4 status from d13C data should be made with caution, since the values observed may re¯ect other features of the aquatic plants and their habitat (e.g., C limitation with slower diffusion from higher viscosity of water than air, thicker boundary layers, and lacunar oxygen storage) rather than an actual C4 carbon ®xation system (Benedict and Scott, 1976; O'Leary, 1988; Abel and Drew, 1989; Durako, 1993). Other investigations using14 C and/or less direct inferences have indicated contrasting C3/C4 metabolism in seagrasses. For example, Benedict et al. (1980) concluded that T. testudinum is a C3 plant, with 3-PGA as the ®rst stable product of carbon ®xation. By B.W. Touchette, J.M. Burkholder / J. Exp. Mar. Biol. Ecol. 250 (2000) 169 ±205 175

14 contrast, in Cymodocea nodosa nearly 50% of added Ci label was found in malate after a 5-s pulse, indicating C4 metabolism (Beer et al., 1980). Zostera noltii may also have C4-like metabolism, based on lack of observable photorespiration as well as high light saturation values (Raven, 1984; Jimenez et al., 1987). In Halophila stipulacea,an initial increase of14 C-malate and other organic acids was reported following addition of 14 Ci label; but subsequent decline in labeled malate did not occur with increased chase time as would be expected in C4 plants, suggesting that this seagrass may be a C3±C4 intermediate (Beer et al., 1980). Based on the activities of C4 enzymes PEP-carboxylase and aspartate aminotransferase, as well as14 C-labeled C4 products aspartate and alanine,

Waghmode and Joshi (1983) concluded that Halophila beccaeii may also ®x Ci via a C4-like pathway under certain conditions. Although various investigations indicate that C4 metabolism may occur in some seagrasses, and that this process may be induced under certain environmental conditions, it has not been widely accepted. Those scientists who do not accept the premise of C4 metabolism in seagrasses cite the general uncertainty in interpreting unexpected results (as in C3±C4 intermediates), uncertainties as to whether assumptions about metabolic similarities hold between terrestrial plants and seagrasses, and/or uncertainties about the validity of interpretations from indirect approaches wherein assessment of C4 metabo- lism was not the original focus of the research (Abel and Drew, 1989; Frost-Christensen and Sand-Jensen, 1992). The most detailed research on C4 metabolism in submersed aquatic angiosperms has emphasized the freshwater submersed angiosperm and invasive aquatic weed, Hydrilla verticillata. Insights from carbon acquisition in this plant may provide insights about the potential for C4-like metabolism in seagrasses. During periods of low dissolved Ci , H. verticillata switches from C3- to C4-like metabolism, depressing photorespiration (Reiskind et al., 1997). The leaves of this plant are only two cells thick; thus, it lacks classic terrestrial plant C4 anatomical features of mesophyll and bundle sheath cells (Kranz anatomy). Nonetheless, it can separate carbon ®xation processes in an analogous manner. From immunocytochemical gold labeling and ¯uorescence studies, Reiskind et al. (1989) demonstrated that PEP-carboxylase was primarily localized in the cytosol, physically separated from Rubisco. It was hypothesized that to reduce `futile' cycling of

CO22 through cytosolic PEP-carboxylase, the CO concentrating site in hydrilla is the chloroplast, rather than the entire cell as in terrestrial plants (Bowes and Salvucci, 1989; Reiskind et al., 1997). This mechanism is somewhat analogous to use of carboxysomes as CO2 concentrating cites in cyanobacteria, or chloroplasts in microalgae (Badger and Price, 1992; Reiskind et al., 1997). In H. verticillata, Ci -limited plants that were induced to conduct C4 metabolism increased 5-fold in internal dissolved Ci (DIC.2000 mmol), relative to C3 plants that were not subjected to Ci limitation (Reiskind et al., 1997). If C4 metabolism does occur in seagrasses, it may be an inducible response to low internal DIC levels, and may be metabolically separated intracellularly (analogous to C4 metabolism in H. verticillata), rather than through multiple cells as in (terrestrial plant) Kranz anatomy. C4 inducabilty may also help to explain why researchers have reported C4-like metabolism in some seagrass species, whereas other studies have indicated only C3 metabolism (Abel and Drew, 1989). Note that CAM, in which C4 and C3 ®xation are separated temporally rather than spatially, occurs in desert plants and a few freshwater 176 B.W. Touchette, J.M. Burkholder / J. Exp. Mar. Biol. Ecol. 250 (2000) 169 ±205

angiosperms in extremely Ci -limited softwater habitats (Beer and Wetzel, 1981; Keeley, 1982) but has not been reported in seagrasses. In desert plants it apparently is used as a mechanism to minimize water loss; CO2 is taken in through open stomata at night and temporarily incorporated into C4 products (Goodwin and Mercer, 1983). C3 ®xation subsequently is completed with energy generated from the light reactions of photo- synthesis, while the stomata are closed to prevent water loss during high-temperature light periods. Submersed freshwater angiosperms with CAM lack functional stomata, and ®x carbon at night when CO2 levels are highest from ecosystem respiration (Beer and Wetzel, 1981; Keeley, 1982). 2 Use of CO23 /HCO pumps in concentrating C i is con®ned to aquatic plants, mostly algae (Taiz and Zeiger, 1991). The pumps occur on the plasma membrane and appear to be inducible during periods of low CO2 availability. The energy necessary to drive these pumps is believed to come from the light reactions of photosynthesis. Although many 2 seagrass species have been demonstrated to utilize HCO3 , they are not nearly as ef®cient as marine macroalgae (Beer, 1994; BjorkÈ et al., 1997). This reduced ability to 2 use HCO3 , together with consideration of certain properties of seawater (low free CO2 | | 2 concentrations [ 12 mMat208C, 150-fold lower than HCO32 ], low CO diffusion 2 rates, and slow conversion rates between CO23 and HCO ) Ð and the low or negligible 22 ability of seagrasses to utilize the common CO3i ion in marine waters as a C source (Steeman-Nielsen, 1960; Raven, 1970; Prins and Elzenga, 1989; Durako, 1993; Stumm and Morgan, 1996) Ð has led some researchers to regard submersed seagrasses as potentially Ci -limited for growth (Beer, 1996; BjorkÈ et al., 1997; Zimmerman et al., 1997). 2 Some seagrasses have been shown to directly use HCO3i as a C source in photosynthesis (e.g., Halophila ovalis, Cymodocea rotundata, Syringodium isoetifolium, Thalassia testudinum, Zostera marina Ð Sand-Jensen and Gordon, 1984; Durako, 1993; 2 Beer and Rehnberg, 1997; BjorkÈ et al., 1997). Moreover, in many plants this HCO3 2 utilization is not restricted to dehydration of HCO3 via carbonic anhydrase (CA; BjorkÈ 2 et al., 1997). These species may instead directly transport HCO3 into photosynthesizing cells in an active, energy-costly process Ð indicated, for example, by signi®cantly depressed photosynthetic rates in Zostera marina following application of ATPase inhibitors (N,N9-dicyclohexylcarbodiimide and sodium orthovanadate; Beer and Re- hnberg, 1997). 2 As an alternative or supplement to direct HCO3 uptake, some seagrass species utilize 2 CA as an extracellular/membrane enzyme to dehydrate HCO32 and liberate free CO prior to its uptake (Table 1). However, the determination of CA in these plants was

Table 1 Carbonic anhydrase (CA) activity (mequiv. H12 min1 mg21 chl) reported in seagrass species; values are based on direct enzymatic measurements Species CA activity Source Cymodocea nodosa No detection Beer et al. (1980) Halophila stipulacea 6 Beer et al. (1980) Syringodium isoetifolium 11 Beer et al. (1980) Thalassodendron ciliatum 8 Beer et al. (1980) Zostera muelleri No detection Millhouse and Strother (1987) B.W. Touchette, J.M. Burkholder / J. Exp. Mar. Biol. Ecol. 250 (2000) 169 ±205 177

Table 2 Photosynthetic suppression reported in seagrasses by a speci®c carbonic anhydrase (CA) inhibitor, acetazola- mide (pH also indicated). Greater suppression of photosynthesis indicates higher reliance on CA in acquiring inorganic carbon for photosynthesis. Data are given as means61 S.E. for temperate and tropical/subtropical seagrass species (as designated by Phillips and Menez,Ä 1988) Species Photosynthetic pH (n) Source suppression (%) Temperate Posidonia australis 25 7.6±8.8 James and Larkum (1996) Posidonia oceanica 53 8.2±8.5 Invers et al. (1999) Zostera marina 60 8.2 Beer and Rehnberg (1997) Grand mean61 S.E. 46.0610.7% n53 species Tropical/subtropical Cymodocea nodosa 35 8.2±8.5 Invers et al. (1999) Cymodocea rotundata 55 8.2 BjorkÈ et al. (1997) Cymodocea serrulata 50 8.2 BjorkÈ et al. (1997) Enhalus acoroides 60 8.2 BjorkÈ et al. (1997) Halodule wrightii 50 8.2 BjorkÈ et al. (1997) Halophila ovalis 25 8.2 BjorkÈ et al. (1997) Syringodium ioetifolium 45 8.2 BjorkÈ et al. (1997) Thalassia hemprichii 40 8.2 BjorkÈ et al. (1997) Thalassodendron ciliatum 20 8.2 BjorkÈ et al. (1997) Grand mean61 S.E. 42.264.5% n59 species made either through direct enzymatic measurements, or through indirect decline in photosynthesis following addition of a CA-speci®c inhibitor (acetazolamide). Decreases measured in photosynthesis following application of acetazolamide have ranged from 0 2 to as much as 75%, suggesting that the degree at which seagrasses utilize HCO3 via membrane-bound CA is highly variable (Table 2).

3. Photosynthesis±irradiance relationships

Most of the research that has been conducted on seagrass physiology has focused on photosynthesis±irradiance (P±I) relationships (Fig. 2), in efforts to determine light levels needed to maintain healthy growth. Such curves have provided estimates for photosynthetic capacity (Pmax), photosynthetic quantum ef®ciency (a; moles of carbon ®xed per mole of PAR absorbed), saturating irradiance for photosynthesis (Ikmax5 P /a), compensation irradiance (Ic ), and other variables (Tables 3 and 4). The parameter Ic represents the light intensity at which oxygen production is equivalent to oxygen demand during respiration in photosynthetic tissues. Whole-plant respiratory oxygen demand is higher than the respiratory demand of photosynthetic tissues only; thus, Icp represents the (additional) light required for whole-plant compensation irradiance

(Tomasko, 1993). Most of the available data (for Iccp, rather than I ) do not consider belowground and non-photosynthetic tissues, and are of limited use in predicting whole-plant carbon balance (Dunton and Tomasko, 1991; Tomasko, 1993; Burd and 178 B.W. Touchette, J.M. Burkholder / J. Exp. Mar. Biol. Ecol. 250 (2000) 169 ±205 Masini et al. (1995) Masini et al. (1995) Masini et al. (1995) (light-limited slope or quantum

a CC Masini and Manning (1997) Masini and Manning (1997) C Masini and Manning (1997) C Masini and Manning (1997) 8 8 8 8 P P P 2 Condition(s) Source (saturating irradiance), I 1 S.E. for temperate and tropical/subtropical species, or as 6 1 2 1 1 1 1 2 2 2 2 1 2 2 2 1 1 1 1 2 2 2 2 1 1 1 1 1 2 2 2 values, since they were expressed with common units across studies) were 2 1 g chl hg chl h Variable temp. 13±23 Gross g chl h Variable temp. 13±23 2 2 2 m m m I 23 2 2 2max 1 1 1 1 1 2 M O dm min Apex (young/intermed. leaf) Mazzella and Alberte (1986) 2 2 2 2 2 2 1 m g chl hg chl h Gross Variable temp. 13±23 g chlg h chl h Gross Variable temp. 13±23 gO gO gO and 2 m m m m 2 m m m 2 ck 2max 2 2max 2 I 2 (compensation irradiance), m O g fw min Seasonal variations Zimmerman et al. (1995) gO gO m O m s Leaf segments, artif. seawater Flanigan and Critchley (1996) gO gO ck m I m m m m m

a 1a 2 2 2 E m s ) (Photosyn. units) m 1 2 2 2 Em s ) ( m ck 2517±20372420±2545No data 90 35±507±1310 25728 55±59 38±5585 100±290 182 0.015±0.024; 0.009; 40± 55 0.01; 100 mg 0.016±0.019; O 0.015; g 230 0.0035; dw 450 h 0.018; 0.0020±0.0053; No data No data 0.005±0.008; mg O Yearly g and tissue age means dw h Alcoverro et al. (1998) NO enrichment Leaf segments Whole shoots Touchette (1999) Dennison and Alberte (1982) Drew (1979) II ( 17±232015±17 32±40 70 25±56 0.039±0.054; 0.039; 0.035; ), and growing and/or measurement conditions. Data are given as means I / max k P P P P 5 P P P P a P | b Amphibolis antarctica Amphibolis grif®thii Amphibolis grif®thii Posidonia australis Posidonia australis Posidonia oceanica Posidonia sinuosa Posidonia sinuosa Zostera capricorni Zostera marina Zostera marina Zostera marina Zostera marina Zostera marina Table 3 Photosynthetic±irradiance parameters reported foref®ciency; seagrass species including ranges if means werecalculated not using available. midrange Grand values means for (con®nedSpecies to those consideration cases of Temperate B.W. Touchette, J.M. Burkholder / J. Exp. Mar. Biol. Ecol. 250 (2000) 169 ±205 179 light dry weight. 5 Â Â È Dunton and Tomasko (1994) C Marsh et al. (1986) C Terrados and Ros (1995) 8 8 chlorophyll; and dw 5 33 m depth Parnik et al. (1992) 2 , yearly means Dunton (1996) In situ In situ (Parnik et al., 1992) were not used to calculate the grand mean, ash-free dry weight; chl 1 2 1 5 2 1 1 2 2 1 1 2 2 1 1 1 2 2 2 1 1 2 2 2 2 1 2 2 2 2 2 2 1 1 2 2 M O mg chl min Variable temp. 15±35 2 2 m 2 g O g AFD min Seasonal variations Vermatt and Verhagen (1996) m mO g dwh mO g dwh m m m O mg chl min Variable soil sul®de Goodman et al. (1995) m Thalassodendron ciliatum 8 species) 7 species) 5 5 n n 2 2 2 2 38.8 ( 71.1 ( 6 6 ÂÈ ). I (Jimenez et al., 1987) and , and cp kp I 3.3 146.0 7.6 284.6 Zostera noltii 6 6 0.01±43 26±230 0.005±0.63; mg O g dw h Variable temp. 10±30 10± 1530± 3512± 6098±30030± 35 65 250 198±210 222±39020±40 3508581 0.002±0.004; 10±60 0.003; 0.008; mg C 0.23±0.63; g20±40 dw10±60 h 5010±60 0.008; mg C g 319 dw h 319 430±500 100 430±500 430±500 No data Young leaf segments No 0.5±2.4; data 0.5±2.4; Young No leaf No data segments data No data Jimenez et al. (1987) Jimenez et al. (1987) Variable water depth Seasonal and salinity response Seasonal and Variable salinity water response depth Seasonal and salinity response Chan et al. Beer (1987) and Waisel (1982) Chan et al. (1987) Chan et al. (1987) Beer and Waisel (1982) | No data 1.5±5 W m No data Plants from 0.5 ) are ratios and, consequently, are dependent on units; therefore, units of photosynthesis are also provided (note that light units were standard

a P P 1 S.E. 28.5 1 S.E. 38.5 P 6 6 Superscript letter P indicates measurements using whole plants (thus, considered the in¯uence, or demand, of both above- and belowground tissues on Slope values ( a b Zostera marina Zostera marina Zostera marina Zostera noltii Zostera noltii Grand mean Cymodocea nodosa Halodule uninervis Halodule wrightii Halodule wrightii Halophila engelmannii Halophila stipulacea Syringodium ®liforme Thalassia testudinum Thalassodendron ciliatum Grand mean due to use of extreme light levels and different units (Watts m ), respectively. Note that AFD across studies). Light values from requirements to sustain the plants (e.g., Tropical/subtropical 180 B.W. Touchette, J.M. Burkholder / J. Exp. Mar. Biol. Ecol. 250 (2000) 169 ±205 Masini et al. (1995) Masini et al. (1995) Masini et al. (1995) CC Masini and Manning (1997) Masini and Manning (1997) CC Masini and Manning (1997) Masini and Manning (1997) 8 8 8 8 max max max P P P (minimum light for maximum photosynthesis), and growing 2 3 Condition(s) Source I 1a 2 2 2 Em s ) m ( (maximum photosynthesis), max max P 1 S.E. for temperate and tropical/subtropical species, or as ranges if means were not available 6 1 1 1 2 2 2 1 1 1 1 1 2 2 1 2 2 1 1 1 1 1 2 2 2 2 2 1 1 1 2 2 1 1 2 2 2 1 2 1 1 2 2 2 2 2 2 g chl h No data Gross 2 1 1 1 2 2 2 2 2 m 2 2 2 1 1 g chl h No data Gross 2 2 2 2 g chl h No data Gross 2 m 2 2 2 2 2 2 m 2 gO mol O gfw min 200±900 Seasonal variations Zimmerman et al. (1995) 2 mol O g dw min 100 Leaf segments Dennison and Alberte (1982) 2 m m m gO M O dm min 200 Apex of young/intermed. leaf Mazzella and Alberte (1986) gO g C mg dw hmol O m smol O dm min No data 450 230 Highest seasonal value, mid-leaf Leaf segments, artif. seawater Modigh et al. (1998) Whole shoots Flanigan and Critchley (1996) Drew (1979) m m m m m m max max PI 1±1.5 mg O2.4 g h1±3.5 mg O g h No data No data Variable temp. 13±23 Variable temp. 13±23 0.84 0.8±2.0 mg O7.7 g mg O h g2.2 dw0.8±1.1 h0.6±1.2 mg O4.2 g h0.5±1.7 0.66 1.2±1.5 2.0 400±7005±6.2 mg O 350 g dw h 100±800 Variable temp. 13±23 Yearly and tissue 600 age means Variable temp. 13±23 Alcoverro et al. (1998) NO enrichment Touchette (1999) P P P P P P P P P b Amphibolis antarctica Amphibolis grif®thii Amphibolis grif®thii Posidonia australis Posidonia australis Posidonia oceanica Posidonia oceanica Posidonia sinuosa Posidonia sinuosa Zostera capricorni Zostera marina Zostera marina Zostera marina Zostera marina Zostera marina Table 4 Photosynthetic parameters reported forand/or seagrass species measurement including conditions. Data areSpecies given as means Temperate B.W. Touchette, J.M. Burkholder / J. Exp. Mar. Biol. Ecol. 250 (2000) 169 ±205 181 ynthesis and  dry weight. È Dunton and Tomasko (1994) 5 1 1 2 2 chlorophyll; and dw 5 0.64 cm s Koch (1994) 0.25 cm s Kock (1994) C Marsh etC al. (1986) Terrados and Ros (1995) 8 8 . . , yearly means Dunton (1996) In situ In situ (Parnik et al., 1992) were not used to calculate the grand mean, due ash-free dry weight; chl 5 a a 2 2 5 species) 2 species) 5 5 n n Thalassodendron ciliatum 2 2 1 1 1 1 2 2 1 2 1 2 2 2 1 1 1 1 2 2 1 2 2 1 1 1 values since they were expressed in common units across studies) were calculated using the midrange values for 2 2 2 2 1 1 ÂÈ 1 1 2 1 max 1 1 2 1 2 2 2 2 I 1 2 2 2 1 1 2 2 2 2 (Jimenez et al., 1987) and 2 2 2 2 2 2 2 2 g O g AFD min 150±900 Seasonal variations Vermatt and Verhagen (1996) 2 2 mol CO kg dw s 20±80 W m Plants from 0.5±33 m depth Parnik et al. (1992) m m M O mg chl min 75±150mol O mg chl min Variable temp. 15±35 No data Variable depth Beer and Waisel (1982) mol O gmol O dw h g dw h 520 400±800 31.6 ( 155 ( mol O mg chl min 700±900 Variable soil sul®de Goodman et al. (1995) m m mol O mg chl min No data Variable depth Beer and Waisel (1982) m m m 6 6 m 0.40 0.5 71±236 3±6.5 mg C g dw h3.0 mg O g2.4±8 mg O dw0.12 g h dw374 h422 360040±65 ppm O40 g dw hNo data3.2 mg O gNo data No data dw 100±400 h Young leaf segments No data Saturating Variable light, temp. ¯ow 10±30 No data No data No data Seasonal and salinity response Jimenez et al. (1987) Artif. seawater; ¯ow Seasonal and salinity response Seasonal Chan and et salinity al. response (1987) Chan et al. (1987) Chan et al. (1987) 30±50 Zostera noltii ). max I P P 1 S.E. 452 1 S.E. 405 P±1 6 6 Superscript letter P indicates measurements using whole plants; thus, these data considered the in¯uence or demand of belowground tissues on photos Grand means (con®ned to consideration of a b Zostera marina Zostera marina Zostera noltii Zostera noltii Grand mean Cymodocea nodosa Cymodocea nodosa Halodule uninervis Halodule wrightii Halodule wrightii Halophila engelmannii Halophila stipulacea Syringodium ®liforme Thalassia testudinum Thalassia testudinum Grand mean Thalassodendron ciliatum those cases. Light values from light requirements (e.g. to use of extreme light levels and different units (Watts m ), respectively. Note that AFD Tropical/subtropical 182 B.W. Touchette, J.M. Burkholder / J. Exp. Mar. Biol. Ecol. 250 (2000) 169 ±205

Dunton, 2000). Caution should also be used in interpreting data on saturating irradiance for photosynthesis (Ik ), because seagrass photosynthesis has often been shown to increase under light intensities greater than Ik (Fig. 2; Tomasko, 1993). Geographic comparisons of seagrass photosynthesis are dif®cult because of inconsis- tencies in units used for photosynthetic rates. From the available data, temperate-zone seagrasses have lower Ic values than tropical/subtropical species (means61 standard error [S.E.] as 28.563.3 and 38.567.6 mE m22 s21 , respectively; Table 3), indicating that temperate seagrasses can utilize lower light levels for photosynthesis. Temperate- zone seagrasses also have been reported to have lower Ik values than tropical/ subtropical species (means61 S.E. as 146.0638.8 and 284.6671.1 mEm22 s21 , respectively; Table 3), which would be expected since available ambient light is lower in temperate regions. In addition to the light intensity, the duration of the daily light period at which light equals or exceeds the photosynthetic light saturation point (Hsat) is important in seagrass growth and survival, especially for plants at or near the maximum depth distribution for the species in a given location (Dennison and Alberte, 1982, 1985; Zimmerman et al.,

1995a). A parameter taken from phytoplankton studies, Hsat, has been used to estimate seagrass productivity, mostly in research on Z. marina (Herzka and Dunton, 1998).

Lower Hsat values have been related to signi®cant decreases in productivity and/or increasing mortality in Z. marina (Dennison and Alberte, 1985; Dennison, 1987;

Zimmerman and Alberte, 1991; Zimmerman et al., 1991). However, Hsat is site- as well

Fig. 2. Theoretical photosynthesis±irradiance (P±I) curve, illustrating maximum photosynthesis (Pmax), maximum photosynthetic irradiance (Imax, the minimum irradiance that supports P max), compensation irradiance (Ick), saturating irradiance (I ), and photosynthetic ef®ciency (a). Photosynthetic ef®ciency is expressed as photosynthetic rate per mole of photons (modi®ed from Tomasko, 1993). B.W. Touchette, J.M. Burkholder / J. Exp. Mar. Biol. Ecol. 250 (2000) 169 ±205 183 as species-speci®c (range of 3±12 h reported for Z. marina), with this high variability likely resulting from differences in temperature, metabolic activity, and biomass distribution between C-sink and C-source tissues (Zimmerman and Alberte, 1991). Thus, use of Hsat to predict productivity should not be extrapolated to multiple sites (Dennison and Alberte, 1985; Zimmerman et al., 1989, 1991; Herzka and Dunton, 1998).

The Hsat model assumes that productivity does not occur at light levels below Ik, thus omitting light-limited photosynthesis from consideration (Herzka and Dunton, 1997, 1998). Although the model has been used successfully to estimate productivity of Z. marina, Herzka and Dunton (1998) demonstrated that it is more limited in estimating productivity of the subtropical seagrass, Thalassia testudinum. For example, during a period of low irradiance due to light attenuation, the Hsat model predicted 0±37% of the production that was calculated from numerical integration of empirical data (Herzka and

Dunton, 1998). In this and other seagrass species with higher light requirements, the Hsat model may not be applicable because of the potential for extended periods of light- limited photosynthesis.

4. Photoinhibition and photosuppression

Although most seagrasses are regarded as shade- or low light-adapted (Ralph and Burchett, 1995), shallow-water or intertidal species may sustain photoinhibition from high photon ¯ux densities during low tides (Ralph and Burchett, 1995). Photoinhibition is de®ned here as a reduction in photosynthetic rates in response to high light intensities, whereas photosuppression is de®ned as a reduction in photosynthetic rates due to other processes such as toxicological (e.g., herbicides, metals) or physiological effects (e.g., feedback inhibition). Photoinhibition is believed to be a photoprotective mechanism that depresses photosynthetic rates (PSII) and impairs both electron transport and photo- phosphorylation, thus allowing excessive light energy to be dissipated as heat (Krause and Weis, 1991; Hanelt et al., 1994). In seagrasses, this process appears to occur at light intensities between 700 and 1600 mE m22 s21 , most often at.1000 mE m22 s21 , with maximal photoinhibition between 1200 and 1500 h (Table 5; Dawes et al., 1987; Hanelt et al., 1994). The increased energy dissipation in photoinhibition is generally associated with an increase in zeaxanthin levels in plants, and/or with a decrease in the number of active PS II centers (Guenther and Melis, 1990; Adams and Demming-Adams, 1992; Hanelt et al., 1994). Zeaxanthin increases following de-epoxidation of violaxanthin in the xanthophyll cycle, providing the mechanism for the energy dissipation (Demming- Adams and Adams, 1992; Adams et al., 1995; Flanigan and Critchley, 1996). Energy dissipation may also be accomplished through turnover of the D1 protein in the reaction center of PSII. In high light, continuous D1 protein degradation/replacement is believed to occur; but in extremely high light, repair of the reaction center via D1 protein replacement occurs much more slowly than D1 protein degradation, thus producing a photoinhibitory response (Ohad et al., 1984; Guenther and Melis, 1990; Krause and Weis, 1991; Aro et al., 1993). However, in the seagrass Zostera capricorni, maximum synthesis and degradation of D1 occurred at 350 mE m22 s21 , much lower than the light 184 B.W. Touchette, J.M. Burkholder / J. Exp. Mar. Biol. Ecol. 250 (2000) 169 ±205

Table 5 Irradiance-associated photoinhibition reported in seagrass species, including light intensities (mE m22 s21 ) and experimental and/or culture conditions

Species Light Condition(s) Source

Temperate Posidonia sinuosa .1020 Young and basal leaves Masini et al. (1995) Zostera capricorni 1100 Based on ¯uorescence Flanigan and Critchley (1996) 2 Zostera marina .1500 NO3 enrichment Touchette (1999) Zostera marina 1200 Young leaves Jimenez et al. (1987) Zostera marina No inhibition Light levels.1400 Mazzella and Alberte (1987) Zostera noltii No inhibition Light levels.5900 Jimenez et al. (1987)

Tropical/subtropical Halophila engelmannii 700 Culture bottles Dawes et al. (1987) Halophila ovalis 1000 After 120 min Ralph and Burchett (1995) Halophila stipulacea 1000 Based on chl response Drew (1979) Thalassia hemprichii 1600 Low tide Hanelt et al. (1994)

levels considered to photoinhibit this species (1100 mE m22 s21 ; Flanigan and Critchley, 1996). Moreover, D1 protein turnover was not proportional to irradiance, suggesting that the D1 protein levels in this plant may be more in¯uenced by pH and ATP levels in the thylakoid lumen. If so, then Ð at least in this seagrass species Ð D1 protein turnover does not function in photoprotection via photoinhibition (Critchley and Russell, 1994; Flanigan and Critchley, 1996). Chlorophyll ¯uorescence techniques (for example, PAM ¯uorimetry) have enabled non-intrusive study of the behavior of photosystem II and electron transport (Krause and Weis, 1991; Beer et al., 1998; Ralph et al., 1998). Under typical temperature regimes, most chlorophyll a ¯uorescence is attributed to PSII, and can be used to gain information about light conditioning, photosynthetic capacity, photosynthetic ef®ciency, and electron transport of PSII (Krause and Weis, 1991; Ralph et al., 1998). Variable

¯uorescence (Fv ) is related to maximum and initial ¯uorescence (Fmoand F , respective- ly) as: Fv 5 Fmo2 F (Ralph et al., 1998). In `sun' plants that are adapted to grow under high light, Fomremains relatively constant and F ¯uctuates (Demmig and Bjorkman, 1987; Franklin et al., 1992; Ralph and Burchett, 1995). In contrast, `shade' plants that are adapted for growth in low-light conditions tend to ¯uctuate substantially in Fo Ða response that has been linked to photoinhibition and/or other adverse affects on the PSII reaction centers (Demmig and Bjorkman, 1987; Franklin et al., 1992; Ralph and

Burchett, 1995; Dawson and Dennison, 1996). The Fv /Fm ratio (photochemical ef®ciency) is used to evaluate the physiological state (including the extent of photo- inhibition) of the photosynthetic apparatus in various plants, including some seagrasses (Table 6). A decrease in this ratio may be associated with environmental stressors that directly affect PSII ef®ciency (Krause and Weis, 1991).

Seagrasses such as Halophila ovalis and Posidonia australis show variations in Fv that have been interpreted to indicate photosuppression due to UV-B radiation in a photoinhibition-like response (see below; Larkum and Wood, 1993). This UV-B response suggests a lower electron ¯ux through the oxidizing side of reaction center B.W. Touchette, J.M. Burkholder / J. Exp. Mar. Biol. Ecol. 250 (2000) 169 ±205 185 encies under a 3 a 2 1 2 1 1 1 1 2 2 2 2 2 2 E m min ; 2 h Ralph and Burchett (1995) m g Irgarol 1051 dm ; 10 d Scarett et al. (1999) m 1 mg Cd l1 mg Cu ; l 241 h mg Pd ; l 241 h mg Zn ; l 48 h ; 24 h Ralph and Burchett (1998a) Ralph and Burchett (1998a) Ralph and Burchett (1998a) Ralph and Burchett (1998a) . $ $ $ Condition(s) Source m F / v ) reported in seagrass species as an indication of physiological stress m aa F / v F m FF / v F Control0.8100.860 Treatment 0.680 0.470 25% increase UV; 7 25%0.750 d increase UV; 70.730 d0.730 (ls)0.832±0.855 0.600±0.740 Dawson and 0.219 Dennison 0.600 0.300±0.700 (1996) (hs) Dawson and Dennison (1996) Hyposaline (0±50% sw ) Hypersaline (150±250% sw) Variable salinity 25 Ralph (1998) Ralph (1998) Kamermans et al. (1999) 0.7800.780 0.630±0.700 0.650±0.680 Variable oil exposure; 24 Variable h oil dispersant; 24 h Ralph and Burchett Ralph (1998b) and Burchett (1998b) 0.650±0.700 0.2000.7800.7800.7800.780 1000 0.700 0.150±0.650 0.700±0.720 0.300±0.700 0.8400.700±0.8000.8000.840 No change 0.540 0.680 0.710 Light deprivation 25% increase UV; 7 d 25% increase UV; 7 25% d increase UV; 7 d Longstaff et al. (1999) Dawson and Dennison (1996) Dawson and Dennison (1996) Dawson and Dennison (1996) Values are indicated for plants in ambient conditions (controls) and various treatments. The data indicate a general decline in photochemical ef®ci a Cymodocea serrulata Halodule uninervis Halophila ovalis Halophila ovalis Halophila ovalis Halophila ovalis Halophila ovalis Halophila ovalis Halophila ovalis Halophila ovalis Syringodium isoetifolium Zostera capricorni Halophila ovalis treatment conditions. Abbreviations: sw, seawater; ls, low salinity; hs, high salinity. Table 6 Photochemical ef®ciencies (quantum yield, Species Light treatments Halophila ovalis Halophila ovalis Zostera marina Zostera marina Other stressors 186 B.W. Touchette, J.M. Burkholder / J. Exp. Mar. Biol. Ecol. 250 (2000) 169 ±205

P680 in PSII, including the primary donor of P6801 (Z; reaction side of the D1 protein), which would prevent Q from being reduced (Larkum and Wood, 1993). Increases in Fo, apparently result, as well, from UV damage to the PSII reaction centers in the seagrasses Cymodocea serrulata, Halodule uninervis, Halophila ovalis, Syringodium isoetifolium and Zostera capricorni (Dawson and Dennison, 1996). Fv /Fm ratios have been used in seagrasses to demonstrate photosuppression and PSII responses to UV radiation, light deprivation, and other stressors (Table 6).

5. Carbohydrate metabolism

5.1. Major carbohydrate storage compounds

The processes by which starch or sucrose is biosynthesized in angiosperms (from C3 photosynthesis products triose-P, 3-PGA, and dehydroxyacetone) are based on competing reactions that are physically separated within the cell, with starch produced in plastids and sucrose produced in the cytosol (Fig. 3). The relative amount of starch or sucrose produced by plants dependents largely on the available Pi (Goodwin and Mercer, 1983). When high internal Pi levels are available, more triose-P can be exported into the cytosol to form sucrose; when Pi is low, triose-P export decreases with concomitant increase in starch production. Beyond this generalization from general plant biochemistry, however, many plant species have demonstrated preference for storage of one compound (starch, sucrose, or other complex carbohydrates such as ra®nose or stachyose) over another (Brocklebank and Hendry, 1989). Furthermore, the primary storage compound can be in¯uenced by growth status; for example, plants undergoing rapid growth tend to have higher levels of sucrose relative to starch (Taiz and Zeiger, 1991). The dominant storage carbohydrate in most seagrasses is the soluble product, sucrose (Drew, 1983; Pirc, 1989; Vermatt and Verhagen, 1996; Touchette, 1999). In the species that have been examined (Enhalus acoroides, Halodule wrightii, Halophila decipiens, Syringodium ®liforme, Thalassia testudinum, Zostera marina), sucrose forms more than 90% of the total soluble carbohydrate pool. Other soluble carbohydrates have included glucose, fructose, and more complex polysaccharides (Drew, 1983). Although lower in abundance, additional soluble carbohydrates in seagrasses include apiose, arabinose, fucose, galactose, mannose, rhamnose, and xylose (Waldron et al., 1989; Webster and Stone, 1994). On average (basis, 24 studies involving 18 seagrass species), total carbohydrates in stem, leaf, root, and rhizome tissues are ca. 95, 100, 135, and 275 mg g21 dry weight, respectively (Tables 7 and 8). As previously indicated, rhizomatous tissues generally contain most of the stored carbohydrates. Storage of carbohydrate reserves in below- ground structures may minimize carbon loss from herbivory, and also would ensure that high carbohydrate levels were available to sustain the perennating belowground structures during dormant periods (for example, in warm late summer temperatures, for the north temperate seagrass Zostera marina) when aboveground shoot tissues may be inactive and/or senescent (Burke et al., 1996). Leaf and rhizome carbohydrate levels are positively correlated for various seagrass species; that is, species with high leaf sugar B.W. Touchette, J.M. Burkholder / J. Exp. Mar. Biol. Ecol. 250 (2000) 169 ±205 187

Fig. 3. Sucrose and starch biosynthesis and catabolism in plant cells. Reactions of starch biosynthesis occur in plastids, wherein accumulation of triose-phosphate, especially during photosynthesis, initiates the process.

Sucrose biosynthesis begins with an exchange of Pi and triose-phosphate at the P-translocator, so that triose-phosphate levels begin to accumulate in the cytosol. Two triose-phosphate (dihydroxyacetone 3- phosphate and glyceraldehyde 3-phosphate) are then combined to form a single 6-C compound, fructose 1,6-bisphosphate (fructose 1,6-bisphosphate), which undergoes dephosphorylation to form fructose 6-phos- phate. This product can then undergo isomerization to form glucose 6-P, which eventually is converted to UDP-glucose. UDP-glucose combines with available fructose 6-P to form sucrose 6-P, via the enzyme sucrose-P synthase (SPS). Sucrose 6-phosphate is dephosphorylated to produce sucrose, which can be used directly in carbon storage, converted to more complex carbohydrates, or translocated to other tissues to aid in other metabolic processes or storage. To initiate sucrolysis, the enzyme sucrose synthase (SS) breaks down sucrose to UDP-glucose and fructose, thus liberating carbon for use in various metabolic pathways (developed from Taiz and Zeiger, 1991). content typically accumulate more rhizome carbohydrates (r 2 5 0.54; Fig. 4A). There is a similar though weaker correlation between root and rhizome tissues (r 2 5 0.35; Fig. 4B).

We note, as well, an interesting trend between light compensation point (Ic ) and leaf soluble carbohydrate levels in seagrasses (Fig. 5). If Ic values represent the light level at which photosynthesis and respiration rates are equivalent, then plants with high Ic values would have either relatively high respiration rates or low photosynthesis rates Ð both would indicate a lower carbon ef®ciency. Accordingly, seagrasses with high Ic levels 188 B.W. Touchette, J.M. Burkholder / J. Exp. Mar. Biol. Ecol. 250 (2000) 169 ±205

Table 7 Nonstructural carbohydrate levels reported in photosynthetic tissues of seagrass species; data include species, tissue (aboveground, leaf, stem), and soluble carbohydratesa

Species Tissue Soluble carbohydrates Source

Temperate Amphibolis antarctica Leaf 11 mg g21 dw Drew (1982) Amphibolis antarctica Stem 7.3 mg g21 dw Drew (1982) Phyllospadix scouleri Leaf 134 mg g21 dw Neighbors and Horn (1991) Phyllospadix torreyi Aboveground 228 mg g21 dw Drew (1982) Ruppia maritima Leaf 119±334 mg g21 dw Lazer and Dawes (1991) Zostera marina Leaf 42 mg g21 fw Zimmerman et al. (1995) Zostera marina Leaf 34 mg g21 fw Zimmerman et al. (1997) Zostera marina Leaf 144 mg g21 dw Drew (1982) Zostera noltii b Aboveground 1±70 mg g21 dw Vermatt and Verhagen (1996)

Grand mean61 S.E. Leaf (n54 species) 128.7644.3 mg g21 dw Stem (n51 species) 7.3 mg g21 dw Aboveground (n52 species) 29.266.3 mg g21 dw Tropical/subtropical Cymodocea nodosa Leaf 55 mg g21 dw Drew (1982) Cymodocea serrulata Leaf 92 mg g21 dw Tomasko (1993) Enhalus acoroides Leaf 25 mg g21 dw Drew (1982) Halodule pinifolia Leaf 163 mg g21 dw Tomasko (1993) Halodule uninervis Leaf 290 mg g21 dw Tomasko (1993) Halodule wrightii Leaf 30 mg g21 dw Drew (1982) Halophila decipiens Leaf 180 mg g21 dw Drew (1982) Halophila engelmanii Leaf 52±124 mg g21 dw Dawes et al. (1987) Halophila engelmanii Stem 72±151 mg g21 dw Dawes et al. (1987) Halophila ovalisb Leaf 15± 42 mg g21 dw Longstaff et al. (1999) Syringodium ®liforme Leaf 185 mg g21 dw Ray and Stevens (1996) Syringodium ®liforme Leaf 22 mg g21 dw Drew (1982) Syringodium isoetifolium Leaf 169 mg g21 dw Tomasko (1993) Thalassia hemprichii Leaf 92 mg g21 dw Tomasko (1993) Thalassia testudinum Leaf 99±161 mg g21 dw Durako and Mof¯er (1985) Thalassia testudinum Leaf 50±70 mg C g21 dw Lee and Dunton (1996) Thalassia testudinum Leaf 50 mg C g21 dw Lee and Dunton (1997) Thalassia testudinum Leaf 60 mg g21 dw Drew (1982) Thalassodendron ciliatum Leaf 24 mg g21 dw Drew (1982) Thalassodendron ciliatum Stem 52 mg g21 dw Drew (1982)

Grand mean61 S.E. Leaf (n514 species) 102.5620.3 mg g21 dw Stem (n52 species) 81.7629.2 mg g21 dw a Note that, where possible, values were converted from percent dry weight to mg g21 dry weight (dw). Other values are reported as fresh weight (fw). The data are given as means 6 1 S.E. for temperate and tropical/subtropical seagrasses, or as ranges if means were not available. Grand means (con®ned to consideration of data reported with the most common unit representation, mg g21 dw), were calculated based on mid-range estimates for those cases. b Starch was also observed in these two species (0±20 mg g21 dw in Zostera noltii; 73±91 mg g21 dw for Halophila ovalis).

tend to have low leaf carbohydrates. Although this approach is simplistic considering the

number of factors that can in¯uence carbohydrate levels, nonetheless, seagrasses with Ic values less than 50 mE m22 s21 have leaf soluble carbohydrates typically in excess of 50 B.W. Touchette, J.M. Burkholder / J. Exp. Mar. Biol. Ecol. 250 (2000) 169 ±205 189

Table 8 Soluble carbohydrate levels reported in non-photosynthetic tissues of seagrass species. Data include species, tissues (belowground, rhizome, root), and soluble carbohydratesa

Species Tissue Soluble carbohydrates Source Temperate Amphibolis antarctica Belowground 33 mg g21 dw Tomasko (1993) Amphibolis antarctica Rhizome 11.4 mg g21 dw Drew (1982) Amphibolis antarctica Root 19 mg g21 dw Drew (1982) Phyllospadix torreyi Belowground 234 mg g21 dw Drew (1982) Posidonia australis Rhizome 390 mg g21 dw Ralph et al. (1992) Ruppia maritima Rhizome 118±520 mg g21 dw Lazar and Dawes (1991) Ruppia maritima Root 95±236 mg g21 dw Lazar and Dawes (1991) Zostera marina Root 5 mg g21 fw Zimmerman et al. (1997) Zostera marina Root 3.5 mg g21 fw Zimmerman et al. (1995) Zostera marina Rhizome 282 mg g21 dw Drew (1982) Zostera marina Root 43 mg g21 dw Drew (1982) Zostera noltii b Belowground 20±150 mg g21 dw Vermatt and Verhagen (1996) Grand mean 6 S.E. Root (n53 species) 75.6645.3 mg g21 dw Rhizome (n54 species) 250.6682.8 mg g21 dw Belowground (n53 species) 120.3662.8 mg g21 dw Tropical/subtropical Cymodocea nodosa Rhizome 213 mg g21 dw Drew (1982) Cymodocea nodosa Root 151 mg g21 dw Drew (1982) Cymodocea serrulata Belowground 367 mg g21 dw Tomasko (1993) Enhalus acoroides Rhizome 84 mg g21 dw Drew (1982) Enhalus acoroides Root 227 mg g21 dw Drew (1982) Halodule pinifolia Belowground 182 mg g21 dw Tomasko (1993) Halodule uninervis Belowground 45 mg g21 dw Tomasko (1993) Halodule wrightii Rhizome 210 mg g21 dw Drew (1982) Halodule wrightii Root 168 mg g21 dw Drew (1982) Halophila decipiens Rhizome 454 mg g21 dw Drew (1982) Halophila decipiens Root 96 mg g21 dw Drew (1982) Halophila engelmanii Rhizome 202±347 mg g21 dw Dawes et al. (1987) Halophila ovalisb Rhizome 40±126 mg g21 dw Longstaff et al. (1999) Halophila ovalisb Root 8±30 mg g21 dw Longstaff et al. (1999) Syringodium ®liforme Rhizome 280 mg g21 dw Rey and Stephens (1996) Syringodium ®liforme Rhizome 804 mg g21 dw Drew (1982) Syringodium ®liforme Root 473 mg g21 dw Drew (1982) Thalassia hemprichii Belowground 45 mg g21 dw Tomasko (1993) Thalassia testudinum Rhizome 194±318 mg g21 dw Durako and Mof¯er (1985) Thalassia testudinum Rhizome 110±200 mg C g21 dw Lee and Dunton (1996) Thalassia testudinum Rhizome 130 mg C g21 dw Lee and Dunton (1997) Thalassia testudinum Root 94±151 mg g21 dw Durako and Mof¯er (1985) Thalassia testudinum Root 65±100 mg C g21 dw Lee and Dunton (1996) Thalassia testudinum Root 71 mg C g21 dw Lee and Dunton (1997) Thalassia testudinum Rhizome 263 mg g21 dw Drew (1982) Thalassia testudinum Root 200 mg g21 dw Drew (1982) Thalassodendron ciliatum Rhizome 139 mg g21 dw Drew (1982) Thalassodendron ciliatum Root 160 mg g21 dw Drew (1982) Grand mean 6 S.E. Root (n58 species) 181.9646.8 mg g21 dw Rhizome (n59 species) 250.8652.5 mg g21 dw Belowground (n54 species) 159.7676.2 mg g21 dw a Some values were converted from percent dry weight to mg g21 dry weight [dw]. Data are given as means 6 1 S.E. for temperate and tropical/subtropical seagrasses, or as ranges if means were not available. Grand means (con®ned to consideration of data reported with the most common unit representation, mg g21 dw) were calculated using mid-range values for those cases. b Starch was also observed in these species (10±40 mg g21 dw for Zostera noltii belowground tissues, 57±85 mg g21 dw for Halophila ovalis rhizome, and 92±118 mg g21 dw for Halophila ovalis roots). 190 B.W. Touchette, J.M. Burkholder / J. Exp. Mar. Biol. Ecol. 250 (2000) 169 ±205

Fig. 4. Relationship between rhizome soluble carbohydrate levels and (A) leaf soluble carbohydrates; or (B) root soluble carbohydrates. Correlations were derived from published literature values, and letters represent different species (see Table 8). In the leaf carbohydrate regression, the far right S.f. value was omitted from the analysis because of its extremely high rhizome carbon content (more than two-fold higher than all other values).

21 mg g dry weight, whereas plants with higher Ic values tend to have much lower leaf carbohydrate content.

5.2. Sucrose metabolism

The key enzyme involved in sucrose biosynthesis is sucrose-P synthase (SPS; Fig. 3). B.W. Touchette, J.M. Burkholder / J. Exp. Mar. Biol. Ecol. 250 (2000) 169 ±205 191

Fig. 5. Relationship between compensation-level irradiance (Ic ) and leaf soluble carbohydrates, including 15 observations on nine seagrass species from the published literature. Letters represent different species (see 22 21 Tables 3 and 8). Note that plants with relatively high Ic values (.50 mE m s tend to have low soluble 21 carbohydrate content (,50 mg g dry weight), whereas species with lower Ic values have the potential to accumulate more soluble carbohydrates. Plants with high Ic values likely have higher respiration-to-photo- synthesis ratios and, thus, may have a higher carbon demand.

SPS activity has been used as an indicator for sucrose export from source tissues, exempli®ed by an increase in SPS activity in developing leaf tissue during the transition in function from a carbon sink to a carbon source (Giaquinta, 1979; Stitt, 1994; Zimmerman et al., 1995b). In Zostera marina, for example, SPS activity has been shown to increase with leaf age, which is consistent with leaf maturation from carbon sink to source (Zimmerman et al., 1995b). In many terrestrial plants with sucrose as the major storage carbohydrate, SPS activity is strongly in¯uenced by light activation and Pi inhibition; and soluble carbohydrate levels appear to ¯uctuate between light and dark conditions (Huber et al., 1989a). However, the level of SPS activation by light may depend strongly on substrate availability (group I versus group II species; see Huber et al., 1989b). In contrast, SPS activity in starch-accumulating terrestrial plants appears to be unaffected by light or Pi content, and soluble carbohydrate levels remain relatively stable between light and dark transitions (group III species; Huber et al., 1989b). By contrast, Zimmerman et al. (1995b) demonstrated that regulation of SPS activity in the seagrass Zostera marina failed to fall into the typical group classi®cations designated for terrestrial plants. SPS activity in Z. marina was similar to that reported for starch accumulating plants (group III), in demonstrating limited response to light availability. But Z. marina generally does not accumulate high starch content (Smith et al., 1988; Zimmerman et al., 1996; 192 B.W. Touchette, J.M. Burkholder / J. Exp. Mar. Biol. Ecol. 250 (2000) 169 ±205

Touchette, 1999). Moreover, SPS activity in Z. marina leaf tissues appears to be unaffected by extended periods of light deprivation (up to 2 weeks at ,10 mEm22 s21 Touchette et al., 1999), indicating lack of a SPS-light response in this seagrass. Although SPS is highly regulated by physiological processes, certain environmental factors also can in¯uence its activity. In Zostera marina, leaf SPS activities have been 1 correlated with changes in water-column NH42 , CO availability, photosynthesis, salinity, temperature, and grazing (Zimmerman et al., 1996, 1997; Touchette, 1999). In terrestrial plants, SPS activity and/or sucrose levels tend to increase with decreasing temperature (Kaurin et al., 1981; Guy et al., 1992; Hurry et al., 1994). By contrast, in Z. marina a positive relationship has been observed between SPS activity and temperature, and between SPS activity and salinity (Fig. 6). Terrestrial plants that have shown an inverse relationship between SPS activity and temperature appear to increase sucrose accumulation as a cryoprotectant during cooler

Fig. 6. Relationship between (A) temperature and (B) salinity and sucrose-P synthase (SPS) activity in leaf tissue of the seagrass Zostera marina. Data are given as means61 S.E. (from Touchette, 1999). B.W. Touchette, J.M. Burkholder / J. Exp. Mar. Biol. Ecol. 250 (2000) 169 ±205 193 periods (Santarius, 1973; Carpenter et al., 1986), with sucrose functioning as an osmolyte in reducing water stress (Guy et al., 1992). However, halophytes in shallow marine systems sometimes encounter dynamic changes in environmental salinity (for example, during warm periods with increased evaporation), which can signi®cantly alter the osmotic potential of their tissues (Reed and Stewart, 1985; Weimberg, 1987). Increased SPS activity (which has been observed, for example, in Z. marina with increased temperature or salinity) thus may indicate an osmotic adjustment response for marine angiosperms, analogous to increased SPS activity as a cryoprotectant response in terrestrial non-halophytic plants (Touchette, 1999). Sucrose metabolism and degradation in sink tissues involve two key enzymes as invertase (which hydrolyzes sucrose into glucose) and sucrose synthase (SS, which can also carry out the initial step in sucrolysis but forms UDP-glucose and fructose as products; Fig. 3). Activities of these enzymes have been related to sucrose import into sink tissues and sucrose entry into metabolism (Claussen, 1983; Sung et al., 1988; Stitt, 1994; Koch and Nolte, 1995). Based on data from general plant physiology, invertase activity tends to be highest during active growth and declines following maturation (Sung et al., 1994; Koch and Nolte, 1995). In contrast, high SS activity can occur in sink/storage tissues of all ages, and SS appears to be the primary sucrose-metabolizing enzyme in mature storage tissues. SS activity and, thus, sucrose breakdown, may be linked to respiration, given its response to adenylate balance (toward conservation of ATP) and its lack of apparent light activation (Kalt-Torres and Huber, 1987; Koch and Nolte, 1995). Moreover, during periods of low carbon availability, localization of SS activity within speci®c cells and/or tissues may be a mechanism for providing sink priority to the most essential cells. In seagrasses, research on sucrose metabolism has been limited mostly to studies of Zostera marina, a species that stores most of its carbon reserves in belowground (rhizomatous) tissues as mentioned. Root SS activity in Z. marina decreases with tissue age (SS activity in distal root tissuesÐfor example, declined by two-fold in roots from the ®rst to the eighth node; Kraemer et al., 1998). Nonetheless, increased SS activity has been observed in very old root systems (root bundle 10 and beyond; Kraemer et al., 1998). Based on this information, it has been hypothesized that there are two separate SS isozymes in Z. marina, one of which is highly sensitive to low sucrose levels; and that the elevated SS activity in the oldest root tissues may be in response to low sucrose levels (Kraemer et al., 1998). The activity of this SS isozyme may enhance plant survival under periods of carbon limitation by maximizing sucrose utilization during periods when concentrations are low, and during periods of replenishment (Kraemer et al., 1998). In aboveground (leaf) tissue, SS activity also has been reported to decrease with age, a trend that may re¯ect a change in function from carbon sink to source as would be expected (Kraemer et al., 1998). But even the oldest leaves maintain substantial SS activity, suggesting that high levels of carbon metabolism can still occur in these tissues. As in terrestrial angiosperms, SS activity in Z. marina does not appear to be light-activated (Zimmerman et al., 1995b) although, in belowground tissues, SS activity has been reported to vary inversely with Hsat (Alcoverro et al., 1999). Moreover, root-rhizome SS activity in many plants (including this seagrass) tends to increase when 194 B.W. Touchette, J.M. Burkholder / J. Exp. Mar. Biol. Ecol. 250 (2000) 169 ±205 dissolved oxygen concentrations are low, indicating increased carbon metabolism in response to anoxic/hypoxic conditions (Sachs et al., 1996; Germain et al., 1997; Touchette, 1999). In contrast, a positive relationship has been shown between below- ground tissue SS activity and temperature in Z. marina (Touchette, 1999). This temperature-associated increase in SS activity and, thus, higher sucrolysis, may be associated with increased respiratory demand (Touchette, 1999).

5.3. Starch metabolism

Starch synthesis occurs in plastids and, like sucrose production, is activated by an accumulation of triose-P and yields fructose 1,6-bisP as an initial product (Godwin and Mercer, 1983; Fig. 3). It differs from sucrose synthesis in that an ADP-glucose (rather than UDP-glucose) is involved, from which starch is formed by the enzyme starch synthase. The data on starch storage in seagrasses are highly variable. Low starch content has been reported in Thalassia testudinium (Jagels, 1983) and Zostera marina (,5% of the total carbohydrates, US Paci®c Coast, Alcoverro et al., 1999; Touchette, 1999). In contrast, Burke et al. (1996) reported that starch could form more than 65% (up to 140 mg g21 dry weight) of the total nonstructural carbohydrate content in Z. marina from Chesapeake Bay. In Z. noltii, starch levels can approach one-third of the total nonstructural carbohydrates in belowground tissues (Pirc, 1989; Vermatt and Verhagen, 1996). Halophila ovalis has also been found to accumulate up to 90% of its total nonstructural carbohydrates as starch in leaf and root tissue (Longstaff et al., 1999). Moreover, in plants with low shoot and root-rhizome starch, there may be substantial starch accumulation in fruit tissues (e.g., in Halodule spp., Halophila ovalis, Phyllos- padix iwatensis, P. japonicus, Thalassia hemprichii Ð Bragg and McMillan, 1986; Kuo et al., 1990; Kuo et al., 1991; Kuo and Kirkman, 1992).

6. Respiration

Respiration is considered here as the controlled mobilization and oxidation of stored carbohydrates. During this process free energy is released and incorporated into NADH and ATP, which can then be utilized to support other metabolic processes (Taiz and Zeiger, 1991). Respiration in plants may be considered in three stages as glycolysis and fermentation; the tricarboxylic acid cycle (TCA cycle or Krebs cycle, in mitochondria); and the electron transport chain, yielding ATP in mitochondria. These processes are conserved among many organisms, including seagrasses. In glycolysis, carbohydrates are converted in the cytosol to produce NADH and a 3-carbon product, pyruvate. Under aerobic conditions in glycolysis, two electrons are required to reduce the NAD1 to NADH, which can then be used as a means to store free energy. The NADH eventually drives the synthesis of ATP via the electron transport chain. However, during anaerobic conditions both the TCA cycle and electron transport do not function (Taiz and Zeiger, 1991). In the absence of oxygen, a surplus of NADH may develop, and a concomitant de®cit B.W. Touchette, J.M. Burkholder / J. Exp. Mar. Biol. Ecol. 250 (2000) 169 ±205 195 in NAD11 . Because NAD is a required cofactor for many enzymes, a de®cit in NAD1 can signi®cantly impair important metabolic processes (Taiz and Zeiger, 1991). To alleviate NAD1 depletion, pyruvate may be further metabolized through fermentation. In plants, both lactic acid and alcohol fermentation have been described (Davies, 1980; Roberts et al., 1984; Smith et al., 1988). Alternatively, some plants (including seagrasses) may accumulate other compounds during fermentation such as organic acids (malate, shikimate) or amino acids (alanine, g-amino butyric acid; Smith and Ap Rees, 1979; Davies, 1980; Mendelssohn et al., 1981; Joly and Crawford, 1982; Pregnall et al., 1984). To avoid anaerobic respiration in belowground tissues, seagrasses and many other aquatic angiosperms translocate and release O2 in the rhizosphere during periods of active photosynthesis (Sand-Jensen et al., 1982; Smith et al., 1984; Crawford, 1987; Caffrey and Kemp, 1991). However, during darkness or extended low light (for example, from sustained cloud cover or water turbidity), belowground tissues may undergo periods of anerobiosis (Crawford, 1987). High rates of ethanol synthesis have been shown in excised roots of Zostera marina during anaerobic conditions (from use of 14C-sucrose; Smith et al., 1988). However, typically ethanol does not accumulate. For example, in the above laboratory study, more than 95% of the ethanol produced was released into the rhizosphere, indicating effective removal from belowground tissues. Field populations similarly yielded little or no accumulation of ethanol in root tissues, even during extended periods of sediment anoxia associated with long periods of low light (Penhale and Wetzel, 1983; Pregnall et al., 1984). It is likely that the low ethanol content in belowground tissues re¯ects its release into the rhizosphere. Nonetheless, alternative fermentation pathways have been described with signi®cant increases in alanine and g-amino butyric acid, and decreases in glutamate and glutamine within 2±4 h of anaerobiosis (Pregnall et al., 1984; Smith et al., 1988). It was hypothesized that in Z. marina, pyruvate undergoes transamination via glutamate and/or glutamine to form alanine (Pregnall et al., 1984; Smith et al., 1988). Thus, pyruvate from glycolysis would be converted to alanine, thereby lowering production of the more toxic end product, ethanol. This preference for alanine accumulation would further bene®t the plant by conserving carbon skeletons and assimilated nitrogen (Pregnall et al., 1984). The primary environmental factor believed to in¯uence respiration rates in seagrasses is temperature (Marsh et al., 1986; Zimmerman et al., 1989; Terrados and Ros, 1995; Masini and Manning, 1997). Unlike photosynthesis which increases with temperature up to |5±108C above ambient, respiration rates continue to increase with increasing temperatures in excess of 408C (Drew, 1978; Bulthius, 1983; Marsh et al., 1986). Light and other environmental factors can also signi®cantly in¯uence respiration. For example, seagrasses in deeper water tend to have lower respiration rates (Dennison and Alberte, 1982). Z. marina grown at 5.5 m had respiration rates that were |40% lower than rates observed in plants at 1.3 m (Dennison and Alberte, 1982). Respiration rates in Z. marina have been shown to increase, as well, with increasing water-column nitrate enrichment and tissue NR activity (Touchette, 1999), as has been observed for terrestrial angio- sperms (Bloom et al., 1992). Dark respiration rates in Z. marina leaf tissue increased by as much as 36% following an 8 mM pulse of water-column nitrate. The increased 196 B.W. Touchette, J.M. Burkholder / J. Exp. Mar. Biol. Ecol. 250 (2000) 169 ±205 respiration under nitrate enrichment may help to supply the energy and carbon needed to assimilate and reduce nitrate (Bloom et al., 1992; Touchette, 1999). In seagrasses, aboveground tissues typically have higher respiration rates (Table 9). On a weight basis, respiration rates in root-rhizome tissues have been reported as only | 12±36% of the rates measured for leaves. Respiration rates in root tissues ( 4.5 mgO2 g21 dw min21 ), for example, in Thalassia testudinum, are substantially higher than | 21 21 rhizome respiration ( 1 mg O2 g dw min ; Fourqurean and Zieman, 1991). A cautionary note is warranted, however, because in many studies of seagrass respiration, belowground tissues were maintained under aerobic conditions despite the fact that the natural sediment environment is often anaerobic or microaerobic. Respiration rates in non-photosynthetic tissues can be strongly in¯uenced by sediment anoxia (Smith et al., 1988). For example, sucrose metabolism in belowground tissues of Zostera marina during anoxia was reported as only |65% of rates observed during aerobic periods (Smith et al., 1988). Thus, respiration studies of seagrass belowground tissues under aerobic conditions may overestimate respiration rates in anoxic sediments (Smith et al., 1988; Herzka and Dunton, 1998). Nevertheless, the general differences in respiration that have been reported for above- and belowground tissues likely re¯ect differences in metabolic function. That is, rhizomes tend to be primarily storage tissues, whereas leaf and root tissues are involved in more energy-costly metabolism such as carbon assimilation, nutrient absorption, and reduction.

7. Future research directions

Seagrasses have evolved to tolerate harsh environmental conditions that are charac- teristically unsuitable for most angiosperms. The necessary adaptations for survival and growth in such environments undoubtedly have altered many physiological processes in these plants, including various processes involved in photosynthesis and carbon metabolism. In comparison to the wealth of literature about terrestrial (especially agriculturally important) plants, little is known about these environmentally important marine angiosperms. For example, salt tolerance in agricultural plants recently has received increased research emphasis because of ion accumulation associated with irrigation systems, especially in semiarid regions (Alva and Syvertsen, 1991; Francois et al., 1994). However, many aspects of salt tolerance in seagrasses as well as other halophytes remain to be resolved. Carbohydrates are known to play an important role in osmoregulation of many plant species. Other compounds such as the imino acid, proline, can function similarly. Little is known about the importance of sugars as compatible solutes in seagrasses. Although enzymatic data (e.g., SPS) have documented sucrose production during periods of increased environmental salinity, little increase in sucrose content has been observed. Thus, the fate of this sucrose Ð for example, its potential use for production of more complex carbohydrates in an osmoregulatory response Ð is not known. This as well as other aspects of seagrass osmoregulation, and the role of carbon storage reserves and other compounds in that process, need to be examined. B.W. Touchette, J.M. Burkholder / J. Exp. Mar. Biol. Ecol. 250 (2000) 169 ±205 197 perature, season, and/or C Fourqurean and Zieman (1991) C Fourqurean and Zieman (1991) C; shallow waterC; deep water Dennison and Alberte (1982) Dennison and Alberte (1982) C Fourqurean and Zieman (1991) 8 8 8 8 8 28 C Masini et al. (1995) C Herzka and Dunton (1998) CCCCCC Masini et al. (1995) Masini and Manning (1997) Masini et al. (1995) CCC Marsh et al. (1986) CCC Masini et al.C (1995) Masini et al. (1995) CC Terrados and Ros (1995) C Terrados and Ros (1995) Dunton and Tomasko (1994) Dunton (1996) Dunton (1996) Dawes et al. (1987) Dawes et al. (1987) Herzka and Dunton (1998) Herzka and Dunton (1998) Herzka and Dunton (1998) C Dunton and Tomasko (1994) 2 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 C Marsh et al. (1986) 8 1 1 1 1 1 1 1 2 2 2 2 2 2 2 1 1 1 1 1 1 1 1 1 1 1 2 2 1 2 1 1 2 2 2 1 1 1 13 2 1 1 2 2 2 2 2 2 13 2 2 2 2 2 2 2 1 2 1 1 1 1 1 1 1 1 2 1 2 2 2 2 1 2 2 2 2 1 1 1 2 2 2 1 2 2 2 2 2 2 1 1 1 1 2 2 2 2 2 2 1 1 1 1 2 2 1 1 1 1 1 2 2 2 2 2 2 2 2 2 1 1 1 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 1 mol O g dw h Leaf 30 mol O g dw h Root-rhiz. Aug. Caffrey and Kemp (1991) 2 mol O g dw h Leaf May; variable depth Caffrey and Kemp (1991) 2 2 2 mol O g dw h Leaf Aug.; variable depth Caffrey and Kemp (1991) m m m M O dmM O dm min min Leaf Leaf 5 15 l O g dw h (10 ) Leaf 30 m mol O g dw h Root-rhiz. May Caffrey and Kemp (1991) mol O mgmol O chl mg min chl minmol O mg chl min Leaf Leaf Leaf 20±23 20±23 Mean; variable sul®de Goodman et al. (1995) l O g dw h (10 ) Leaf 10 m m mol O g dw h Leaf 28 m 2 mol O g dw h Root-rhiz. 31 g O g dw ming O g dwmol min O g dw h Leaf Root Root-rhiz. 26±28 26 28 m mol O g dw hmol O g dw hmol O gmol O g dw h dw h Leaf Leaf Leaf Leaf 12 12 28 31 m m m m m m m m m m m m m 0.51 mg O g h Leaf 18 4.3 33±0.88 mg O1±1.4 g mg O0.36±0.57 g h mg O0.39±0.94 g dw mg h O0.08 g h0.13 h0.014 0.073 27± 45 33±51 0.08 0.12±0.21 Leaf mg O0.13 Leaf g mg O Leaf16.2 g h Leaf0.9±7.2 h0.29 mg O0.64 g mg O75 g dw h75±116 18 dw h Yearly37 variations 18 142 Root-rhiz. 18 1±3 2±10 Root-rhiz.7.4 26 35 4.6 Leaf0.9 18 O Leaf Alcoverro g3.7 et al. (1998) dw min 18 10 30 Rhiz. 26±28 Amphibolis grif®thii Posidonia australis Posidonia oceanica Posidonia sinuosa Posidonia sinuosa Zostera marina Zostera marina Zostera marina Zostera marina Zostera marina Zostera marina Zostera marina Posidonia australis Posidonia sinuosa Zostera marina Zostera marina Cymodocea nodosa Cymodocea nodosa Halodule wrightii Halodule wrightii Halodule wrightii Halodule wrightii Halophila engelmannii Halophila engelmannii Thalassia testudinum Thalassia testudinum Thalassia testudinum Thalassia testudinum Thalassia testudinum Thalassia testudinum Table 9 Respiration rates reported intreatments). seagrass species. Note Data that include plants rates, at tissueSpecies higher (leaf, root, temperature rhizome, typically root-rhizome), had and higher experimentalTemperate conditions respiration (tem rates Respiration rateTropical/subtropical Tissue Condition(s)Thalassia testudinum Source 198 B.W. Touchette, J.M. Burkholder / J. Exp. Mar. Biol. Ecol. 250 (2000) 169 ±205

The ecological and physiological signi®cance of starch accumulation in seagrasses has not been assessed. These marine angiosperms generally do not appear to accumulate substantial starch as a storage product, with exception of Halophila ovalis. Based on the general (mostly terrestrial) literature, starch accumulation tends to occur in plants with excessive soluble carbohydrates (Goodwin and Mercer, 1983). By storing excess sugars as an insoluble polysaccharide (starch), plants could alleviate sugar-induced osmotic stress. The available literature indicates that starch can occur in seagrasses, with levels strongly in¯uenced by environmental conditions. Further comparative research on the partitioning of stored carbon between soluble and insoluble carbohydrates in seagrass species would provide valuable insights about their adaptive strategies for carbon utilization during periods of C-limited growth, active growth, and dormancy. Whole- plant carbon budgets, rather than carbon budgets developed on the basis of only aboveground tissues, are also needed as a critical component in efforts to strengthen understanding about carbon metabolism and resource (carbon and light) requirements in seagrasses. Such research, if conducted across gradients of light and temperature, would contribute important information in efforts to assess how seagrass growth and survival will be affected under warming trends in climate change.

Acknowledgements

Funding support for this synthesis of light and carbon metabolism in seagrasses, and for research that strengthened the effort, was provided by the North Carolina General Assembly, the North Carolina Sea Grant College Program, the North Carolina Agricultural Research Service, the North Carolina State University College of Agricul- ture and Life Sciences, and the North Carolina State University Department of Botany. [SS]

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