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 difficult 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] fluorometry 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 Forster 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 first stable product of carbon dioxide fixation 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 fixation, 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
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.W. Touchette, J.M. Burkholder J. Exp. Mar. Biol. Ecol. 250 2000 169 –205
21 21
Zostera marina 2.62 and 1.97 mmol CO min mg
chl, respectively are comparable
2
to those reported for submersed freshwater angiosperms 21 species; mean |2.3 mmol
21 21
CO min mg
chl, and for marine green and brown macroalgae six chlorophyte and
2 21
21
pheophyte species; mean |2.5 and 2.4 mmol CO min mg chl
, respectively.
2
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 CO min
mg chl ; Beer et al.,
2
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, O
is consumed and inorganic
2
phosphate P and CO are released, as well as byproducts such as peroxide and
i 2
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 first appeared in the fossil record Phillips and Menez, 1988, C
losses from photorespiration were likely to be minimal because atmospheric CO O
2 2
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
benefits remain unclear. However, the process may benefit 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 CO availability
2
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 difficult 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 C source, and some also have developed
i
various mechanisms to concentrate relatively high levels of CO around Rubisco active
2
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 HCO , and those that do are typically less efficient than macroalgae
3
and cyanobacteria blue-green algae; Beer et al., 1991. There is experimental evidence for photorespiration, as data for increasing CO loss
2
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 modified 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,
14
1976; Donton et al., 1976. Pulse-chase experiments with 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 fluorimetry to evaluate photosynthetic
rates also have shown curvilinear relationships between estimated fluorometric photo- synthesis and O -evolving photosynthesis. For example, in Halophila stipulacea and
2
Zostera marina during high irradiance, the rate of O release decreased relative to the
2
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 — 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.
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.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 CO O ratio near
2 2
active C-fixation sites of the Rubisco enzyme. Such mechanisms would enhance carbon fixation in environments where CO diffusion is slow e.g., submersed habitats, while
2
also depressing wasteful photorespiration. Known carbon concentrating mechanisms include C4 photosynthesis with Crassulacean acid metabolism [CAM] as a modi-
2 2
fication, CO HCO pumps, and HCO
dehydration catalyzed by enzymes such as
2 3
3
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 fixation to a 4-C product is spatially separated from C3 metabolism.
For example, in terrestrial plants possessing Kranz anatomy e.g., corn, carbon fixation 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 CO which depress
2
the oxygenase photorespiratory function of Rubisco. The resulting CO is then
2
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 efficiency
f , as O evolution per unit of absorbed photons under
a 2
light-limited photosynthesis for submersed angiosperms including the seagrass, Zostera marina. They reported that the
f values more closely resembled those for terrestrial C4
a
than C3 plants. In other investigations, direct measurements of photosynthetic products
13
and or d C 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
13 13
d C-derived classification is based on studies involving terrestrial plants, wherein d C values were typically ca. 228 for C3 plants, and 214 for C4 species. In seagrasses the
13
d C 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
13
Drew, 1989. Interpretations about C4 status from d C data should be made with caution, since the values observed may reflect 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
fixation system Benedict and Scott, 1976; O’Leary, 1988; Abel and Drew, 1989; Durako, 1993.
14
Other investigations using 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 first stable product of carbon fixation. 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 C label was found in malate
i
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
14
initial increase of C-malate and other organic acids was reported following addition of
14
C label; but subsequent decline in labeled malate did not occur with increased chase
i
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
14
and aspartate aminotransferase, as well as C-labeled C4 products aspartate and alanine,
Waghmode and Joshi 1983 concluded that Halophila beccaeii may also fix C via a
i
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 C , H
.
i
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 fixation processes in an analogous
manner. From immunocytochemical gold labeling and fluorescence 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 CO through cytosolic PEP-carboxylase, the CO concentrating site in hydrilla is the
2 2
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 CO concentrating cites in cyanobacteria, or chloroplasts in microalgae Badger and
2
Price, 1992; Reiskind et al., 1997. In H . verticillata, C -limited plants that were induced
i
to conduct C4 metabolism increased 5-fold in internal dissolved C DIC.2000 mmol,
i
relative to C3 plants that were not subjected to C limitation Reiskind et al., 1997.
i
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 fixation are separated temporally rather than spatially, occurs in desert plants and a few freshwater
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.W. Touchette, J.M. Burkholder J. Exp. Mar. Biol. Ecol. 250 2000 169 –205
angiosperms in extremely C -limited softwater habitats Beer and Wetzel, 1981; Keeley,
i
1982 but has not been reported in seagrasses. In desert plants it apparently is used as a mechanism to minimize water loss; CO is taken in through open stomata at night and
2
temporarily incorporated into C4 products Goodwin and Mercer, 1983. C3 fixation 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 fix carbon at night when CO levels are highest from ecosystem respiration Beer
2
and Wetzel, 1981; Keeley, 1982.
2
Use of CO HCO pumps in concentrating C is confined to aquatic plants, mostly
2 3
i
algae Taiz and Zeiger, 1991. The pumps occur on the plasma membrane and appear to be inducible during periods of low CO availability. The energy necessary to drive these
2
pumps is believed to come from the light reactions of photosynthesis. Although many
2
seagrass species have been demonstrated to utilize HCO , they are not nearly as
3
¨ efficient as marine macroalgae Beer, 1994; Bjork et al., 1997. This reduced ability to
2
use HCO , together with consideration of certain properties of seawater low free CO
3 2
2
concentrations [|12 mM at 208C, |150-fold lower than HCO ], low CO diffusion
3 2
2
rates, and slow conversion rates between CO and HCO — and the low or negligible
2 3
22
ability of seagrasses to utilize the common CO ion in marine waters as a C source
3 i
Steeman-Nielsen, 1960; Raven, 1970; Prins and Elzenga, 1989; Durako, 1993; Stumm and Morgan, 1996 — has led some researchers to regard submersed seagrasses as
¨ potentially C -limited for growth Beer, 1996; Bjork et al., 1997; Zimmerman et al.,
i
1997.
2
Some seagrasses have been shown to directly use HCO as a C
source in
3 i
photosynthesis e.g., Halophila ovalis, Cymodocea rotundata, Syringodium isoetifolium ,
Thalassia testudinum , Zostera marina — Sand-Jensen and Gordon, 1984; Durako, 1993;
2
¨ Beer and Rehnberg, 1997; Bjork et al., 1997. Moreover, in many plants this HCO
3 2
¨ utilization is not restricted to dehydration of HCO
via carbonic anhydrase CA; Bjork
3 2
et al., 1997. These species may instead directly transport HCO into photosynthesizing
3
cells in an active, energy-costly process — indicated, for example, by significantly 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 HCO uptake, some seagrass species utilize
3 2
CA as an extracellular membrane enzyme to dehydrate HCO and liberate free CO
3 2
prior to its uptake Table 1. However, the determination of CA in these plants was
Table 1
1 21
21
Carbonic anhydrase CA activity mequiv. H min
mg 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 specific 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, 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 n 53 species
Tropical subtropical Cymodocea nodosa
35 8.2–8.5
Invers et al. 1999 ¨
Cymodocea rotundata 55
8.2 Bjork et al. 1997
¨ Cymodocea serrulata
50 8.2
Bjork et al. 1997 ¨
Enhalus acoroides 60
8.2 Bjork et al. 1997
¨ Halodule wrightii
50 8.2
Bjork et al. 1997 ¨
Halophila ovalis 25
8.2 Bjork et al. 1997
¨ Syringodium ioetifolium
45 8.2
Bjork et al. 1997 ¨
Thalassia hemprichii 40
8.2 Bjork et al. 1997
¨ Thalassodendron ciliatum
20 8.2
Bjork et al. 1997 Grand mean61 S.E.
42.264.5 n 59 species
made either through direct enzymatic measurements, or through indirect decline in photosynthesis following addition of a CA-specific 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 HCO via
3
membrane-bound CA is highly variable Table 2.
3. Photosynthesis–irradiance relationships
