Photosynthesis and photorespiration in freshwater organisms: Amphibious plants

Aquatic Botany, 34 (1989) 267-286

267

Elsevier Science Publishers B.V., Amsterdam - - Printed in The Netherlands

P H O T O S Y N T H E S I S A N D P H O T O R E S P I R A T I O N IN FRESHWATER ORGANISMS: AMPHIBIOUS PLANTS

S.C. MABERLY and the late D.H.N. SPENCE

Department of Biology and Preclinical Medicine, Sir Harold Mitchell Building, The University, St. Andrews, Fife, KY16 9TH (Gt. Britain) {Accepted for publication 29 June 1987)

ABSTRACT Maberly, S.C. and Spence, D.H.N., 1989. Photosynthesis and photorespiration in freshwater organisms: amphibious plants. Aquat. Bot., 34: 267-286. Amphibious macrophytes are remarkable for their ability to photosynthesise in air and water, since these two media are very different. Specifically, water has a higher water potential, greater thermal stability, but reduced availability of light, C02 and 02 than air. In heterophyllous amphibious plants, production of an appropriate leaf form is triggered by water potential, temperature, photoperiod, ratio of red to far-red light, or C02 concentration in some species. These effects may be mediated by endogenous growth regulators. High concentrations of C02 are necessary to saturate photosynthesis of submerged shoots of amphibious species underwater, largely because of boundary-layer resistance. The low rate of C02 supply underwater is reflected in low activities of carboxylating and other enzymes involved in photosynthesis in submerged shoots. Any limitation of photosynthesis by C02 is rarely overcome by HCOa use; rather, the aerial portion may allow access to an environment with greater availability of C02. Alternatively, submerged-CAM found in amphibious species such as Isoetes howeUii Engelm. may reduce C02 limitation of photosynthesis by exploiting the higher C02 concentration often found at night. Photorespiration is present in submerged and aerial shoots, but incubation underwater at high temperature (30 ° C ) and long days ( 14 h) reduces photorespiration in both shoot types in MyriophyUum brasiliense Cambess. and Proserpinaca palustris L. Higher relative activities of phosphoenolpyruvate carboxylase in submerged compared with emerged shoots of some amphibious species may be a response which reduces photorespiration. Submerged shoots require less light to saturate photosynthesis and have generally lower light compensation points at air levels of C02. It is unclear whether or not this is partly caused by the interacting effect of C02. Amphibious macrophytes are poorly understood, and future work should distinguish between the effects of air and water, environmental pretreatment, and linkage to changes in leaf form when photosynthetic and photorespiratory responses of submerged and aerial shoots are compared.

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268 INTRODUCTION Amphibious plants are remarkable for their ability to photosynthesise naturally in air and water, and in terms of their vertical zonation, they usually inhabit the intermediate environments between land and water (Spence, 1982 ). If freshwater vascular plants are descended from terrestrial predecessors, as evidenced by possession of a very thin cuticle, functionless stomata and poorly lignified vascular tissue (Sculthorpe, 1967), then the amphibious habit is the precursor of the fully submerged habit. The range of adaptation of plants to life in water or air reflects the gradual transition between aquatic and terrestrial environments, accentuated by seasonally fluctuating water tables. Amphibious plants of low stature tend to bear leaves of one type (homophyllous) which can photosynthesize, either in air or in water at any one time. Amphibious plants with longer stems tend to bear leaves of more than one kind (heterophyllous), often with one leaf type adapted for photosynthesis in air and another leaf type adapted for photosynthesis in water. Given that vegetative propagation is the rule rather than the exception in freshwater macrophytes (Sculthorpe, 1967 ) then any phenotypic plasticity, particularly marked in heterophyllous species, will tend to be the response of a given genotype, either through the ontogeny of individual shoots, or through positive responses of given shoots to environmental conditions. The latter type of plasticity, at least, requires the perception of environmental conditions and its translation to an appropriate response, which may involve anatomical, morphological, physiological and biochemical changes. These responses of amphibious plants are reviewed here, but we exclude surface-floating (acropleustophytes sensu Luther, 1949) or emergent species, which in terms of their photosynthesis are largely land plants. A COMPARISONOF AIR AND WATERAS ENVIRONMENTS FOR PHOTOSYNTHESIS AND PHOTORESPIRATION

Physical factors Air has a variable but often large negative water potential (Table 1 ) which in leaves of terrestrial plants requires barriers to water loss, such as a cuticle and stomata. These barriers also reduce CO2 uptake so that water supply may be a major factor limiting photosynthesis. Obviously, water supply does not limit photosynthesis of freshwater submerged leaves, and the water potential is at or close to zero. Additional benefits of water include its greater density compared with air (Table 1 ) which obviates the need for substantial non-photosynthetic supporting tissue in water plants. The greater heat capacity of water than air on a volume basis (Table 1 ), in addition to the thermostatic effects of

15.1 0.1 - 10 9.35 ~ constant

147

0.016

16 CO2

Proctor ( 1982 ) Nobel (1983) Hutchinson (1957)

Proctor {1982)

S t u m m a n d M o r g a n {1970)

Nimbostratus ( -~ 13% )2

1.4 Nobel (1983) 93.6 Nobel (1983) 1.22 Weast (1981) -~ 1.0 Weast (1981) 1.2 Calculated Clear sky ( -~ 84% )2

-

1.0 <0.1-0.7 0.31 Variable

-~0.016 5.9 -~ 1000 0.018

34.0 0.6 16 free C02, HCO~-, CO~variable (2-11)

2.92 0.05

0 0 999.1 4.2 4186.2

Value

Value

Reference

Water

Air

Proctor ( 1982 ) Westlake {1975) Hutchinson (1957)

Reynolds (1980) Talling (1985) Proctor (1982)

Smith et al. (1973) Dokulil (1979) S t u m m and Morgan (1970) S t u m m and Morgan (1970)

Smith et al. (1973) Dokulil (1979)

Weast (1981) Weast (1981) Calcuhted

Nobel(1983)

Reference

~Calculated depth at which transmission of PAR (excluding surface losses) is equivalent to transmission through types of air mass using data from two contrasting lakes. 2Calculated transmission from data in Monteith ( 1973 ) for a solar elevation of - 40 °.

Kinematic viscosity (20°C m 2 s -1 × 10 -6) Typical range of fluid velocity (m s - 1) [02] at air-equilibrium (15°C m M ) Temporal and spatial stability of (O2) and (CO2)

10 -7)

Crater Lake Neusiedlersee [CO2 ] at air-equilibrium (15 ° C ~M) Forms of available inorganic carbon pH of medium Total inorganic carbon at air-equilibrium (15°C mM) Zero alkalinity Quoisley Little Mere, Shropshire L. Nakuru, Africa Diffusion coefficient C02 (20°C, m 2 s -1 X

Water potential (MPa) 99% relative humidity 50% relative humidity Density (15°C, kg m -3) Heat capacity (15°C J g-1 ( k - l ) (15°C kJ m -3 k -~) Light attenuation (depth in metres of equivalent light transmission) Crater Lake Neusiedlersee

Character

A comparison of air and water as environments for photosynthesis and photorespiration

TABLE 1

t~ ¢.O

270 phase changes, and the lower density of ice than water at 4 ° C, all tend to reduce the daily and seasonal variation in temperatures found in most freshwaters. The absence of a water deficit in freshwater allows plant surfaces underwater to become colonised by epiphytes, and the density of water allows planktonic organisms to remain in suspension. Epiphytes and phytoplankton may have detrimental effects on macrophyte photosynthesis by competing for light and dissolved inorganic carbon (DIC), and epiphytes may reduce the supply of DIC by increasing boundary-layer thickness (Sand-Jensen, 1977).

Light In terms of quantity, all but the most shallow water is a shade environment (Spence, 1981 ). A variable amount (about 10% ) of light is lost at the air-water interface, and more importantly, dissolved material absorbs and suspended particles absorb and scatter light, causing an approximately logarithmic attenuation of light with depth. Even in lakes approaching the clarity of distilled water, the rate of attenuation of photosynthetically available radiation (PAR, 400-700 nm) is much higher than for the atmosphere, even under heavy cloud cover (Table 1 ). Since given wavelengths of light are attenuated differentially, the quality of light changes with depth. The significance of this for photosynthesis and photorespiration is unknown in freshwaters, but an increase in the ratio of red light (660 nm) to far-red light (730 nm) with depth, caused by differential attenuation, may play a role in plant morphogenesis.

Inorganic carbon The concentration of CO2 in air is about 350/~l 1-1, equivalent to 16 #M. When water and air are in equilibrium, the (temperature-dependent) concentration of CO2 in water is approximately the same as in air (Table 1 ). In air, C02 is the only form of inorganic carbon available for photosynthesis, whereas in water, three forms are present (Table 1) in a series of equilibria controlled by pH. At air-equilibrium, the concentration of DIC in acid water is equivalent to that in air, but DIC may be many times air-level in water of moderate or high alkalinity (Table 1 ); although not all the forms of DIC are directly available to all submerged plants. Most freshwaters have a small capacity to buffer changes in CO2, because of their relatively small volume (exacerbated by density stratification), and the slow movement of CO2 across the air-water interface (Tailing, 1985). In productive lakes in the summer, the concentration of CO2 may be reduced virtually to zero and pH values may exceed 10 (Tailing, 1976, 1985). In contrast, concentrations of CO2 may exceed air-equilibrium, for example at night (0.26 mM) (Keeley, 1983b), above the sediment surface (0.26 mM) (Maberly, 1985b), or at sites where production of CO2 by decomposition of organic material exceeds photosynthetic demand (3.47 raM) (Prins and de Guia, 1986). The supply of inorganic carbon to photosynthetic organs in air and water is

271

by diffusion through adjacent, still, boundary layers. In most terrestrial plants, the boundary layer is thin, comprising a few/~m (Raven, 1984 ), and boundarylayer resistance is low. Rates of photosynthesis by submerged macrophytes, however, may be limited by the rate of diffusion of CO2 through boundary layers (Browse et al., 1979; Smith and Walker, 1980; Black et al., 1981; Madsen, 1984). This results from a substantial boundary layer in water, because even though kinematic viscosity of water is lower than air, current velocities are generally lower (Table 1 ), and more importantly, the diffusion coefficient of COe in water is about 104 times lower than in air (Table 1 ).

Oxygen When air and water are in equilibrium at 15 ° C, the (temperature-dependent) concentration of oxygen is 0.31 mM, which is about 30 times lower than in air (Table 1). Biological activity causes departure of oxygen concentration from equilibrium and percentage air-saturation of surface waters may lie between 36% and nearly 200% (Hutchinson, 1957). CONTROL OF FORM IN AMPHIBIOUS PLANTS

Fluctuations in the water level of a lake may be the sole determinant of whether amphibious macrophytes of low stature photosynthesize in air or water at any given time. In taller amphibious macrophytes, morphologically distinct leaves are produced in air and water (Arber, 1920; Sculthorpe, 1967) which are presumed to be adapted for photosynthesis in different environments. There is not necessarily a strict relationship between form and habitat: for example Sagittaria sagittifolia L. shows an ontogenetic sequence of development regardless of the environment (Sculthorpe, 1967). In many amphibious species, however, leaf form is determined by features of the terrestrial and aquatic environments outlined in the previous section. Allsopp (1965), Sculthorpe (1967) and Hutchinson (1975) provide reviews of the earlier literature. The presence or absence of water may have a direct effect on leaf form via water deficit and changes in osmotic potential of the leaf. These effects have been found in Proserpinaca palustris L. (McCallum, 1902), CaUitriche intermedia Hoffmann (Jones, 1955), C. heterophylla Pursh (Deschamp and Cooke, 1983) and Hippuris vulgaris L. (McCully and Dale, 1961 ), and may be inferred from the extensive work on Marsilea drummondii A. Braun by Allsopp (1965). Water bodies generally experience lower temperatures than air, particularly at the beginning of the growing season. Johnson (1967) showed that Ranuculus flabellaris Raf. produced very dissected leaves, typical of those underwater at 8 ° C, but apparently aerial leaves at 28 °C underwater, and intermediate forms at intermediate temperatures. Similar temperature effects were found

272 by Deschamp and Cooke (1983) for C. heterophylla, and linked to other environmental factors in P. palustris and P. intermedia Mack. (Wallenstein and Albert, 1963; Kane and Albert, 1982), and H. vulgaris (Bodkin et al., 1980). Photoperiod is unlikely to be linked directly to features of the aquatic environment, unless any effect is via daily light dose rather than photoperiod per se. Cook (1968) argues that photoperiod is a reliable indirect means of controlling seasonal changes in appropriate leaf form. Long days promote the formation of aerial-type leaves, whereas submerged-type leaves are produced under short days in P. palustris (Wallenstein and Albert, 1963 ) and P. intermedia (Kane and Albert, 1982). Similar effects have been found in R. flabellaris (Johnson, 1967) and R. aquatilis L. (Cook, 1968). In P. intermedia flowers are produced under long days on aerial-type leaves. These responses to photoperiod may be mediated by phytochrome (Smith, 1982 ). Alternatively, high temperature and long days may act indirectly allowing greater production and accumulation of more carbohydrates. Janauer and Englmaier (1986) showed that submerged portions of H. vulgaris have higher carbohydrate levels when attached to aerial shoots, and Grainger (1947) suggested that the inability of completely submerged plants of Nuphar lutea (L.) Sm. to flower underwater is caused by lack of carbohydrate. Bodkin et al. (1980), however, found that injection of sucrose into the lacunae of H. vulgaris had no effect on leaf form. Because of differential attenuation, the ratio of red to far-red light increases with depth (see previous section), and a low ratio, believed to have an effect via phytochrome, promotes aerial leaf formation underwater in H. vulgaris (Bodkin et al., 1980). Etiolated plants of Marsilea vestita Hook et Grev. develop into the land form only after a pretreatment with far-red light (Gaudet, 1963). The submerged form of M. vestita (Bristow and Looi, 1968), Myriophyllum brasiliense Cambess. and R. flabellaris (Bristow, 1969) can be induced in air under 5% CO2. The stream from which the latter two species were collected, had high concentrations of CO2 (up to 0.28 mM) during part of the growing season (Bristow, 1969), which suggests that CO2 may act as a natural environmental trigger in some circumstances, but clearly not all (see previous section). This effect could presumably operate via pH. It is possible that changes in levels of endogenous growth regulators may mediate some or all of the responses described above. Anderson (1978, 1982) found that abscisic acid applied to tubers of Potamogeton nodosus Poir. caused floating rather than submerged leaves to be produced. Deschamp and Cooke ( 1983 ) showed that CaUitriche heterophyUa produced aerial leaves underwater when abscisic acid was applied. Osmotic potential effects noted above may operate by desiccation leading to the production of abscisic acid, as shown for C. stagnalis Scop. (Milborrow and Robinson, 1973), and hence production of aerial leaves (Anderson, 1978). Wallenstein and Albert (1963), Davis (1967) and Kane and Albert ( 1982 ) showed that exogenous gibberellic acid promoted the production of aerial leaves in Proserpinaca palustris and P. intermedia. In

273 contrast, gibberellic acid sprayed on aerial shoots of C. heterophylla caused water leaves to be formed (Deschamp and Cooke, 1983). The gaseous plant hormone ethylene appears to be involved in 'depth accomodation' i.e. the increase in petiole length to produce a leaf at the water surface, shown by a number of floating-leaved species (reviewed by Jackson, 1985). Ethylene may interact with auxin (Horton and Samarakoon, 1982; Malone and Ridge, 1983 ) and auxin is implicated in the production of floating leaves by C. intermedia (Jones, 1955). PHOTOSYNTHETIC AND PHOTORESPIRATORY RESPONSE TO ENVIRONMENTAL

FACTORS The different leaf forms triggered by differences between air and water suggest, a priori, that leaves adapted to these two environments will differ in their photosynthetic and photorespiratory responses. These expectations are confronted with the experimental data currently available.

C02 uptake kinetics Aquatic macrophytes require higher concentrations of C02 to saturate photosynthesis underwater than terrestrial plants in air. Amphibious macrophytes provide a natural system to study kinetics of CO2 uptake in air and water. Salvucci and Bowes (1982) found that the aerial leaves of M. brasiliense measured in air were saturated with C02 at 900 pl l-1 ( _ 40 HM), whereas submerged leaves measured in water were not saturated at 2500 HI l-1 ( ~ 112 HM). The concentration of CO2 giving half-maximal rates of photosynthesis (Ko.5(C02)) for aerial shoots in air was 18 HM, compared with 706 HM for submerged shoots measured in water. The cause of the higher Ko.5(CO2) of submerged leaves underwater may be attributed largely to boundary layer resistance, since Ko.5(CO2) of submerged leaves of M. brasiliense fell in watersaturated air to 122 HM (Salvucci and Bowes, 1982), although presumably a water film still surrounded the leaf. Under similar conditions, Lloyd et al. (1977) found apparent resistances to CO2-uptake to be higher in submerged compared with aerial or floating parts of MyriophyUum spicatum L. and Potamogeton amplifolius Tuckerm. The remaining resistance has been attributed to low levels of carboxylating enzymes in submerged leaves (Salvucci and Bowes, 1982 ). Ryan (1985), however, isolated cells or short strands of cells from floating and submerged leaves of P. nodosus and found Ko.5(C02) values of about 33 HM for both cell types, which indicates that any "carboxylation resistance" is similar. Dissected leaves, typical of many submerged shoots, may be a way of reducing boundary-layer thickness, and these and thin, entire leaves may be a response to the low flux of CO2 into the leaf which requires relatively small

274

amounts of photosynthetic machinery for processing (Black et al., 1981 ). These leaves contrast with aerial leaves which are often thick and entire, presumably because considerations such as water deficit are important in air and diffusive resistance is low.

Sources of inorganic carbon The high Ko.~(CO2) of submerged shoots underwater suggests that CO2 may be potentially limiting to photosynthesis in the field. In most freshwaters, the submerged leaves have access to C02 and HCO~-, whereas aerial leaves are restricted to CO2 as a source of inorganic carbon for photosynthesis. Ability to use HCO~- is present in about half the species of submerged freshwater macrophytes tested so far (Spence and Maberly, 1985), and may provide a way of overcoming shortages of CO2. Results in Table 2 show carbon-uptake characteristics determined from pHdrift experiments (Maberly and Spence, 1983 ). In these experiments, photosynthetic uptake of CO2 or HCO~- causes the pH of a solution of constant alkalinity to rise, and pH is measured over time until a final, constant pH is achieved. The results can be analysed to allow rates of photosynthesis to be expressed as a function of the DIC species present. Aerial and floating leaves were assayed underwater for HCO~- use, as submergence occurs in nature when TABLE 2 Carbon uptake ability of terrestrial and aquatic forms measured in water using the pH-drift technique 1 Species

Leaf form and position

Alkalinity (equiv. m -3)

Final pH

C02 compensation point (#M)

Potamogeton natans

Broad floating Broad submerged Linear submerged Floating

0.5 0.5 0.5 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0

8.61 8.58 8.72 7.44 7.22 8.66 8.78 7.89 8.00 8.80 8.79 8.81

2.7 2.9 2.1 83.1 43.4 4.3 3.6 29.2 22.6 3.4 3.5 3.3

Nuphar lutea

Submerged

Hippuris vulgaris

Aerial emerged Submerged submerged Aerial submerged

'None of the leaves showed evidence of HCO~--use. Potamogeton natans, broad floating leaf (Maberly and Spence, 1983 ). Others S.C. Maberly, unpublished data, 1980.

275 water level changes, or when aerial-type leaves are produced underwater. The high apparent C02 compensation points for the emerged parts of N. lutea and H. vulgaris underwater may result from the flooding of air spaces. None of the submerged or emerged parts of the three amphibious species tested (Table 2 ) showed evidence for HCO~ use at the alkalinities used. The result for the morphologically aerial shoot of H. vulgaris produced underwater tentatively suggests that, in terms of carbon uptake, the physiology was similar to an underwater shoot, despite the morphology. The apparent lack of ability to utilize HCO~ by the submerged parts of the 3 species in Table 2 is also shown by the amphibious, heterophyllous species P. polygonifolius Pourr. (Allen and Spence, 1981 ), R. flabeUaris and M. brasiliense (Bristow, 1969 ), although there is evidence for HCO~ use in submerged but not floating leaves of Potamogeton distinctus A. Benn. (Kadono, 1980), and in submergedparts ofHygrophilapolysperma (Roxb.) T. Anders. andLimnophila sessiliflora Blume (Bowes, 1985). Inability to use HCO~ is indicated in homophyllous amphibious species with occasional access to CO2 in the atmosphere. Thus, Cinclidotus fontinaloides (Hedw) P. Beauv., Fontinalis antipyretica L., Littorella uniflora (L.) Aschers. Lobelia dortmanna L., Pilularia globulifera L. and Subularia aquatica L. all lacked the ability to use HCO:~ (Spence and Maberly, 1985 ), but Stratiotes aloides L. with seasonal access to atmospheric CO2 did show HCO~ use in submerged but not emerged leaves (Prins and de Guia, 1986). Littorella uniflora and Lobelia dortmanna may overcome restrictions in C02 supply from the water because they obtain most of their CO2 from the sediment (Wium-Andersen, 1971; Sondergaard and SandJensen, 1979).

Carbon fixation and photorespiration The total activity of carboxylating enzymes in emerged parts of amphibious plants is higher than in submerged parts on a chlorophyll (Table 3) and on a fresh-weight basis (Farmer et al., 1986). The lower carboxylation activities in submerged parts may indicate a response to a reduced supply of inorganic carbon underwater. Submerged aquatic macrophytes cannot be classified simply as C3 or C4plants in terms of their carbon-fixation pathway or consequent physiology, since their CO2 compensation point varies, caused by variable rates of photorespiration (Bowes et al., 1978; Holaday et al., 1983; Bowes, 1985). The CO2 compensation point decreases in response to long photoperiod (14 h), high temperature (30 ° C) and possibly also low concentrations of CO2 (Bowes, 1985); factors which also affect leaf form. In the submerged species HydriUa verticillata (L.f.) Royle, photorespiratory activity is correlated with the relative levels of ribulose bisphosphate carboxylase-oxygenase (RuBPCO) and phosphoenolpyruvate carboxylase (PEPC) (Bowes et al., 1978). In the low photorespiration state the CO2 compensation

Emerged Submerged Emerged Submerged Emerged Submerged Emerged Submerged Emerged Submerged (high F) a Submerged (low F) a Floating Submerged

Hippuris vulgaris Hygrophila polysperma Limnophila sessiliflora Mentha aquatica Myriophyllum brasiliense 1030 177 154 54 88 78 431 161 154 72 78 417 332

RUBP 36 7 28 13 21 10 13 17 3 6 4 12 29

PEP

Carboxylase activity (/~mol mg - 1chl. h - 1)

1066 184 182 67 109 88 444 178 157 78 82 429 351

TOTAL 28.6 25.3 5.6 4.0 4.2 8.2 33.2 9.5 57.2 12.8 19.5 34.8 11.5

RuBPCO/PEPC

a f = C02 compensation point. High Findicates high, and low findicates low photorespiratory activity.

Potamogeton polygonifolius

Leaf form

Species

Farmer et al. (1986) Farmer et al. (1986) Spencer and Bowes (1985) Spencer and Bowes (1985) Spencer and Bowes (1985) Spencer and Bowes (1985) Farmer et al. ( 1986 ) Farmer et al. (1986) Salvucci and Bowes (1982) Salvucci and Bowes (1982) Salvucci and Bowes (1982) Farmer et al. (1986) Farmer et al. (1986)

Reference

A comparison of activities of RuBP and PEP carboxylase of terrestrial and aquatic forms of amphibious macrophytes

TABLE 3 t~

277

point is low, 02 inhibition of photosynthesis is reduced, and the activity of PEPC and other enzymes involvedin C4metabolism is increased (Bowes, 1985). In contrast, activities of enzymes associated with photorespiration and the kinetic properties of RuBPCO are unchanged (Salvucci and Bowes, 1981; Bowes and Salvucci, 1984). Labelling studies have shown that in the low photorespiration state, over 50% of 14C is incorporated into C4 acids (Salvucci and Bowes, 1983a). There is, however, no evidence of Kranz anatomy (Bowes and Salvucci, 1984). This degree of detail is not available for amphibious plants. Submerged and aerial shoots of amphibious macrophytes show photorespiration (Table 4), as rates of net photosynthesis are reduced under high concentrations of oxygen (Lloyd et al., 1977; Salvucci and Bowes, 1982 ). Rates ofphotorespiration may also be reduced in the amphibious species Hygrophila polysperma and Limnophila sessiliflora (Spencer and Bowes, 1985 ) and MyriophyUum brasiliense and Proserpinaca palustris (Salvucci and Bowes, 1982), but only when the shoots are incubated in water. The aerial form of P. palustris also shows reduced rates of photorespiration underwater, which may enable it to survive short periods of submergence (Salvucci and Bowes, 1981 ). Bowes (1987) notes that the photosynthetic response of aerial leaves of amphibious plants is not comparable with that of truly terrestrial plants. The mechanism which causes the photorespiratory state to change in amphibious macrophytes is not known. It may operate by concentrating CO2 internally, driven by HCO~- uptake with internal carbonic anhydrase (CA) speeding up the conversion of HCO~- to CO2 which is fixed by RuBPCO, as is hypothesised for M. spicatum (Bowes, 1985). The activity of CA doubles in M. spicatum and Hydrilla verticiUata in the low photorespiratory state (Salvucci and Bowes, 1983b). The activity of CA is low, however, in the submerged shoots of these and other submerged and amphibious species, and surprisingly floating-leaved macrophytes had higher CA activity (Weaver and Wetzel, 1980). The low activity of CA in submerged shoots may be related to the generally lower activity of most enzymes in comparison with terrestrial leaves (Van et al., 1976). In the submerged species M. spicatum and amphibious M. brasiliense, PEPC activity does not change with photorespiratory state (Salvucci and Bowes, 1981, 1983a) and the amphibious species Hygrophila polysperma and Limnophila sessiliflora only show small changes in activity (Spencer and Bowes, 1985). The activity of PEPC represents a greater proportion of the total (RuBPCO + PEPC) activity in the submerged compared with the emerged parts of 5 out of the 6 species in Table 3. This may reflect the low C02 and high 02 concentrations which may be found in productive beds of macrophytes (Van et al., 1976), conditions which could otherwise favour photorespiration. Recently, however, a different pattern has been found for amphibious Eleocharis vivipara link with terrestrial leaves showing evidence of C4 carbon

Aerial (high/-)a Submerged (low F) a Submerged (high F)" Aerial Submerged Floating Submerged

Myriophyllum brasiliense Air Water Water Air Air Air Air

Measurement medium

37 17 25 31 7 29 24

Inhibition of net photosynthesis at 21 vs. 2% 02 (%)b

1.4 3.2

2.8 0.5

-

9.0 0.5 3.3 6.8 1.0 19.8 2.9 6.0

Rate (/lmol CO2 mg -1 chl. h -1 ) 1 o r 2 % 02 21% 02

Photorespiration c

11 3 24 39 11 18 34 70

As % of net photosynthesis

a/- = CO2 compensation point. High Findicates high, and low F indicates tow photorespiratory activity. bRates of net photosynthesis were measured at 327/zl 1 - 1 C02 (M. brasiliense) or 350 ~l I 1 CO2 (M. spicatum and P. amplifolius). CPhotorespiration was measured as CO2 evolution into C02-free gas streams in the light.

Myriophyllum spicatum Potamogeton amplifolius

Form

Species

Photorespiratory response of terrestrial and aquatic forms of amphibious macrophytes

TABLE 4

Salvucci and Bowes (1982) Salvucci and Bowes (1982) Salvucci and Bowes (1982) Lloyd et al. (1977) Lloyd et al. (1977) Lloyd et al. (1977) Lloyd et al. (1977) Lloyd et al. (1977)

Reference

OO

b~

279

fixation and a relatively high PEPC activity, and submerged leaves showing C3 carbon fixation and a lower PEPC activity (Veno et al., 1988). Diel changes in titratable acidity are a fairly widespread feature of photosynthesis in submerged macrophytes (Raven et al., 1985), and in a number of cases they approach those found in terrestrial Crassulacean acid metabolism (CAM). The most intensively studied plant showing submerged-CAM is Isoetes howeUii Engelm., an amphibious plant which inhabits pools that dry up in the summer (Keeley, 1981). The amphibious species Littorella uniflora (Keeley and Morton, 1982; Aulio and Salin, 1983; Boston and Adams, 1983) and Stylites andicola (Keeley et al., 1984) also show CAM-like activity. Elevated activities of PEPC may be inferred in species with submerged-CAM, and have been measured in 2 species (Farmer et al., 1986). Terrestrial species of Isoetes lack CAM, even when artificially submerged (Keeley, 1983a), and emergent parts of leaves of I. howeUii lose CAM, whereas it persists in submerged parts of the same leaf (Keeley and Busch, 1984). Terrestrial populations ofL. uniflora have been reported to lack (Aulio, 1985) andpossess (Farmer and Spence, 1985) submerged CAM. This fixation pattern is interpreted as a response to diel changes in degree of carbon limitation (Keeley, 1983b) as, in these pools, the concentration of C02 is high at night, as a result of community metabolism, and low during the day, because of photosynthetic uptake.

Response of photosynthesis to light Most amphibious macrophytes normally grow in relatively shallow water, and they will therefore experience less light reduction than many totally submerged macrophytes. The results in Table 5 suggest that submerged leaves or shoots of amphibious species have slightly lower light requirements than the emergent parts, as in 3 of the 4 species where comparisons are possible, the submerged forms have lower light compensation points than the aerial form. In 5 of the 6 species in Table 5, the submerged leaves require lower levels of photon irradiance than emerged parts to saturate photosynthesis at approximately air-equilibrium levels of CO2. A low light saturation point could be caused by limitation by CO2 rather than a physiological difference in their response to light (Maberly, 1985a). This is indicated by the results of Ryan (1985) (Table 5) where cells from floating and submerged leaves of Potamogeton nodosus at saturating concentrations of C02 showed light saturation at about the same photon irradiance. Further evidence for lack of sun-shade adaptation by amphibious plants is provided by Osmond et al. (1981) who found similar chlorophyllcontent and ratios of chlorophylla to chlorophyll b in aquatic and terrestrial forms.

Leaf form

Emerged Submerged Emerged Submerged Emerged Submer~ed Emerged Submerged Floating Submerged Cells from floating leaf Cells from submerged leaf

Species

Hygrophila polysperma Limnophila sessiliflora MyriophyUum brasiliense Myriophyllum spicatum Potamogeton amplifolius Potamogeton nodosus Air Water Air Water Air Water Air Air Air Air Water Water

Measurement medium

327 327 327 327 327 327 350 350 350 350 5440 5440

Measurement [ CO2 ] (pl l - 1)

45 9 16 8 -~ 45 -~ 45 90 45 80 -

S -1 )

S -1 )

600 400 650 250 2000 + 250 1200 + 200 1200 + 200 -~ 350 ~ 350

Light compensation point ( #mol m - 2

Light saturation point (/tmol m - 2

Ryan (1985)

Lloyd et al. ( 1977 )

Lloyd et al. (1977)

Salvucci and Bowes (1982)

Spencer and Bowes ( 1985 )

Spencer and Bowes (1985)

Reference

A comparison of the photosynthetic response to photon irradiance of the terrestrial and aquatic forms of amphibious macrophytes

TABLE 5 b~ O0

281 STRATEGIES OF AMPHIBIOUSPLANTS Although amphibious species are usually restricted to the margins of water bodies, and floating-leaved species may be excluded from exposed sites because of damage, the amphibious habit allows survival in a habitat experiencing seasonal fluctuations in water level; although large fluctuations may prevent or severely restrict the colonization of the margins even by amphibious species (Rorslett, 1984). Various lines of evidence noted in this review suggest that access to air of otherwise submerged species may enhance rates of photosynthesis, by alleviating limitations in supply of DIC underwater. The lower activities of carboxylating and other photosynthetic enzymes in submerged compared with emerged forms of amphibious species (Table 3) is circumstantial evidence for constraints on photosynthesis underwater. The general lack of HCO~ use by submerged parts of amphibious species (Table 2 ) was interpreted by Maberly and Spence (1983) and Spence and Maberly (1985) to indicate that any limitation of photosynthesis underwater by CO2 was overcome by access to air rather than by using HCO~ (see also Bowes, 1985; Sand-Jensen, 1987). A progressive seasonal decline in ~13C (the ratio of 13C to 12C in relation to a standard) in the leaves of Stratiotes aloides, against a more or less constant ~1~C for the source, was interpreted as evidence for carbon limitation in the early season when the leaves were underwater, which was relieved when the leaves emerged into air (Prins and de Guia, 1986). Janauer and Englmaier (1986) have shown that carbohydrate levels in submerged shoots of Hippurus vulgaris are lower than in submerged shoots attached to an aerial shoot. The expectation of greater productivity of amphibious compared with submerged macrophytes has not been widely tested. Bowes (1985) found similar standing crops for Hygrophilapolysperma and Limnophila sessiliflorawhich are amphibious, and HydriUa verticillata which is submerged, although the high standing crops (up to 890 g dry weight m -2) may have been restricted by other factors such as nutrients. Other possible benefits of access to air include enhanced rates of nutrient translocation driven by the transpiration stream (Bowes, 1987), ventilation of underground roots and rhizomes (Dacey, 1981) which may help counter anoxia, and the provision of a platform to facilitate the formation of flowers in the air (Hutchinson, 1975). FUTURE LINES OF RESEARCH Amphibious heterophyllous plants provide a natural system to study, in genetically identical ramets, the stimuli and mechanisms involved in the determination of leaf form, and the photosynthetic response of these leaf forms to the contrasting environments of air and water. These two areas may be linked,

282 since the factors which cause changes in leaf form may also control the photosynthetic response to environmental factors. Despite their advantages as experimental material, there is a dearth of information on the physiology, biochemistry and productivity of these plants. There are specific questions which need to be answered. W h a t is the relative contribution of the various resistances which cause photosynthesis in submerged forms to be saturated at high C02 concentrations? W h a t are the pathways of C02 fixation and photorespiration in amphibious plants? W h a t mechanisms cause rates of photorespiration to vary? Do emerged and submerged shoots differ in their response of photosynthesis to light? Do aerial leaves relieve limitations to photosynthesis underwater, and does this lead to greater productivity in amphibious, compared with submerged macrophytes? H o w do homophyllous amphibious macrophytes respond photosynthetically to air and water? Future work needs to distinguish clearly between those differences in response of aerial and submerged forms caused by the physico-chemical differences between air and water, those caused by environmental pretreatment, and those imposed by linkage to changes in leaf form. ACKNOWLEDGEMENTS Professor D.H.N. Spence died before this review was completed, any misconceptions therefore, are my own. I should like to express my indebtedness to David Spence as supervisor and colleague for his enthusiasm, guidance and friendship (S.C.M).

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