Plant Physiol, December 2001, Vol. 127, pp. 1524-1532

UPDATE ON C4 PHOTOSYNTHESIS
Environmental Regulation of C₃ and C₄ Differentiation in the Amphibious Sedge Eleocharis vivipara¹

Department of Plant Physiology, National Institute of Agrobiological Sciences, Tsukuba, Ibaraki 305-8602, Japan

	INTRODUCTION

TOP INTRODUCTION C3 AND C4 PHOTOSYNTHESIS DISCOVERY OF C3 AND... STRUCTURAL AND BIOCHEMICAL... PLASTIC EXPRESSION OF C3... HORMONAL REGULATION OF C3... EXPRESSION OF C3 AND... VARIATION IN EXPRESSION OF... CONCLUSIONS AND FUTURE... LITERATURE CITED

The biochemical and physiological characteristics of C₄ photosynthesis have been researched and clarified in detail. We now know that the differentiation of two cell types, mesophyll cells (MC) and bundle sheath cells (BSC), is required for efficient C₄ photosynthesis. Thus, the leaves of C₄ plants have more complicated structural and functional features than those of C₃ plants (Hatch, 1999; Kanai and Edwards, 1999). Current studies are focused on elucidating the regulatory mechanisms of genetic and developmental events in C₄ photosynthesis, but most of them are as yet poorly understood (Dengler and Nelson, 1999; Sheen, 1999). There is much indirect evidence that C₄ plants have evolved in parallel from C₃ plants among diverse taxonomic groups (Kellogg, 1999). Some C₃-C₄ intermediate plants, such as Flaveria spp., provide a suitable system for studying the possible process of evolution of C₄ plants (Ku et al., 1991; Westhoff et al., 1997). It is becoming clear that there is diversity not only in the structural and biochemical features, but also in the genetic and developmental aspects of C₄ photosynthesis (Dengler and Nelson, 1999; Sheen, 1999; Edwards et al., 2001).

This article reviews the available data on the differentiation of photosynthetic characteristics in some amphibious species of Eleocharis in the Cyperaceae, with particular reference to the sedge Eleocharis vivipara. E. vivipara has a unique nature that expresses C₄ characteristics under terrestrial conditions and C₃ characteristics under submerged aquatic conditions (Ueno et al., 1988). This characteristic is unknown in other C₄ plants, and this plant provides an excellent opportunity to investigate the development of C₄ photosynthesis in response to environmental factors. The amphibious species of Eleocharis are also useful for increasing our understanding of the ecological and adaptive aspects of C₄ plants.

	C3 AND C4 PHOTOSYNTHESIS

TOP INTRODUCTION C3 AND C4 PHOTOSYNTHESIS DISCOVERY OF C3 AND... STRUCTURAL AND BIOCHEMICAL... PLASTIC EXPRESSION OF C3... HORMONAL REGULATION OF C3... EXPRESSION OF C3 AND... VARIATION IN EXPRESSION OF... CONCLUSIONS AND FUTURE... LITERATURE CITED

In the C₃ pathway, CO₂ is fixed by Rubisco and is synthesized into carbohydrate. This metabolic pathway operates only in the MC. Leaves of C₄ plants display Kranz anatomy, in which vascular bundles are surrounded by an outer layer of MC and an inner layer of BSC (Dengler and Nelson, 1999). In the C₄ pathway, CO₂ is fixed initially by phosphoenolpyruvate carboxylase (PEPC), which is localized in the MC, forming C₄ acids (malate and Asp). The C₄ acids are transported to the BSC, where they are decarboxylated by C₄-acid-decarboxylating enzymes. The released CO₂ is incorporated in the C₃ cycle by the operation of Rubisco. In the process of decarboxylation of the C₄ acids, pyruvate is also formed. This C₃ compound is returned to the MC and used for regeneration of phosphoenolpyruvate by pyruvate, Pi dikinase (PPDK). The operation of the C₄ cycle results in the increased concentration of CO₂ at the active site of Rubisco in the BSC and suppression of the oxygenase reaction of Rubisco. As a consequence, C₄ photosynthesis is more efficient than C₃ photosynthesis under some environmental conditions (Hatch, 1999; Kanai and Edwards, 1999).

In general, most plants use one of these photosynthetic modes: Leaves of rice and maize fix carbon through the C₃ pathway and the C₄ pathway, respectively, although the reproductive organs may use different photosynthetic pathways (Imaizumi et al., 1990; Langdale and Nelson, 1991). Environmental factors influence the expression of photosynthetic machinery in some plants. Well-known examples occur in plants with Crassulacean acid metabolism (CAM), such as common ice plant (Mesembryanthemum crystallinum), and in some submerged aquatic plants, such as Hydrilla verticillata. In the former, a switch from C₃ to CAM mode is induced by NaCl stress (Winter and Smith, 1996), whereas in the latter a change from C₃ to C₄ metabolism occurs if CO₂ is limited in the water (Bowes and Salvucci, 1989; Reiskind et al., 1997). In these facultative CAM plants and aquatic plants, the photosynthetic carbon metabolism operates in a single cell, and no differentiation of two cell types is required. In contrast, C₄ photosynthesis requires structural differentiation and biochemical specialization of photosynthetic cells. Therefore, conversion between C₃ and C₄ modes may not be as easy for plants as those between other modes.

	DISCOVERY OF C3 AND C4 DIFFERENTIATION IN E. VIVIPARA

TOP INTRODUCTION C3 AND C4 PHOTOSYNTHESIS DISCOVERY OF C3 AND... STRUCTURAL AND BIOCHEMICAL... PLASTIC EXPRESSION OF C3... HORMONAL REGULATION OF C3... EXPRESSION OF C3 AND... VARIATION IN EXPRESSION OF... CONCLUSIONS AND FUTURE... LITERATURE CITED

Many C₄ species occur in the monocotyledonous family Cyperaceae, and most of them thrive in relatively wet habitats. Thus, the C₄ group of the Cyperaceae represents ecologically unusual C₄ plants (Ueno and Takeda, 1992). Several genera that include both C₃ and C₄ species, such as Cyperus, Rhynchospora, and Eleocharis, are found in this family (Takeda et al., 1980; Bruhl et al., 1987). At the New York Botanical Garden, I extensively screened the photosynthetic modes of Eleocharis spp. by examining herbarium specimens. This study revealed that most species in the genus are C₃ plants, but some species possess characteristics of C₄ and C₃-C₄ intermediate plants (Ueno et al., 1989). Furthermore, this study led to a more important discovery that the amphibious species E. vivipara exhibits Kranz anatomy in the photosynthetic tissues of the terrestrial form, but exhibits non-Kranz anatomy in those of the submerged form. Subsequent biochemical studies with fresh plants in Dr. Miyachi's laboratory demonstrated that E. vivipara can display C₃ or C₄ characteristics, depending on the environmental conditions (Ueno et al., 1988).

E. vivipara occurs in the margins of ponds, marshes, swamps, and wet ditches in Florida (Wunderlin, 1998). The plants grow continuously in conditions ranging from completely aerial to semi-aerial and semi-submerged, to completely submerged aquatic (Fig. 1A).

View larger version (105K): [in this window] [in a new window]   Figure 1.   A, The submerged form of E. vivipara growing in a creek (about 50 cm deep). B, Gross morphologies of the terrestrial form (left) and the submerged form (right) of E. vivipara.

	STRUCTURAL AND BIOCHEMICAL CHARACTERISTICS OF PHOTOSYNTHESIS IN E. VIVIPARA

TOP INTRODUCTION C3 AND C4 PHOTOSYNTHESIS DISCOVERY OF C3 AND... STRUCTURAL AND BIOCHEMICAL... PLASTIC EXPRESSION OF C3... HORMONAL REGULATION OF C3... EXPRESSION OF C3 AND... VARIATION IN EXPRESSION OF... CONCLUSIONS AND FUTURE... LITERATURE CITED

In E. vivipara, the leaf blades are reduced, and the culms function as photosynthetic tissues, as seen in all other members of Eleocharis spp. Some amphibious plants exhibit heterophylly between aerial and aquatic leaves (Sculthorpe, 1967; Smith and Hake, 1992). This is the case in culms of E. vivipara (Fig. 1B). The terrestrial form has erect, hard culms. In contrast, the submerged form shows a hair-like morphology consisting of slender, soft culms, and reproduces new culms by proliferation from sterile spikelets at the apex of the culms.

The dimorphism is clearer in the anatomical features of the culms (Ueno et al., 1988; Ueno, 1996a). The culms of the terrestrial form show Kranz anatomy, but the structure is complex: There are three bundle sheathsthe outermost parenchymatous sheath, the middle mestome sheath, and the innermost organelle-rich sheathtogether with the MC (Fig. 2A). The innermost BSC (Kranz cells) include many granal chloroplasts and large mitochondria. In contrast, the culms of the submerged form lack Kranz anatomy (Fig. 2B). The MC have a spherical shape, forming one or two layers inside the epidermis. The innermost BSC are small and include only a few small chloroplasts and mitochondria. As a result, the MC to BSC volume ratio is much higher in the submerged form than in the terrestrial form. The culms of the submerged form lack stomata, and the vascular bundles are reduced both in size and number compared with those in the terrestrial form.

View larger version (70K): [in this window] [in a new window]   Figure 2.   Structural and biochemical features of the terrestrial and submerged forms of E. vivipara. A, Culm anatomy of the terrestrial form (Kranz type). B, Culm anatomy of the submerged form (non-Kranz type). M, MC; OBS, outermost BSC; MS, mestome sheath cells; and IBS, innermost BSC (Kranz cells). C, Cellular localization of C3 and C4 enzymes and proposed scheme for photosynthetic carbon metabolism in the terrestrial and submerged forms. Only the MC and the innermost BSC (Kranz cells) are shown. The outermost BSC and the middle mestome sheath cells are omitted, because the former are functionally equivalent to the MC and the latter lack photosynthetic enzymes. Solid and dashed lines represent the proposed major and minor fluxes, respectively, of photosynthetic intermediates. The circles show the C3 pathway. , Rubisco; , PEPC; , PPDK; ×, NAD-ME; C3, C3 compound.

The ¹⁴C pulse-¹²C chase experiment indicates that the terrestrial form shows a pattern of ¹⁴C labeling characteristic of C₄ plants, forming Asp and malate as initial photosynthetic products. However, the submerged form shows a C₃ pattern of labeling, forming C₃ compounds as initial products (Ueno et al., 1988). The terrestrial form has high activities of C₄ enzymes, such as PEPC, PPDK, and NAD-malic enzyme (ME), whereas in the submerged form these activities are low. Rubisco activity in the submerged form is almost the same as, or higher than, that in the terrestrial form (Ueno et al., 1988; Ueno, 1998a). These trends in enzymatic activity have also been confirmed by the amounts of these enzyme proteins present after immunoblotting (Ueno, 1996b).

To understand the photosynthetic pathways operating in the two growth forms, it is important to determine in which cells and compartments the photosynthetic enzymes are localized (Fig. 2C). Immunogold localization studies reveal that in the terrestrial form PEPC is localized in the cytosol of both the MC and the outermost BSC (Fig. 3A), and NAD-ME is found in the mitochondria of the innermost BSC. In the submerged form, these enzymes occur in the same sites as in the terrestrial form, but at low levels (Fig. 3B; Ueno, 1996b, 1998a). PPDK is also distributed in the chloroplasts of the MC and the outermost BSC, and at higher levels in the terrestrial form than in the submerged form. In addition, cytosolic PPDK is found in the photosynthetic cells of both growth forms, which is unusual in C₄ leaves (Ueno, 1996b). In both the terrestrial and submerged forms, Rubisco is distributed in the chloroplasts of the MC, the outermost BSC, and the innermost BSC (Ueno, 1996b). This cellular distribution of Rubisco is unknown in typical C₄ plants. In C₄ species of the Cyperaceae, such as Fimbristylis and Cyperus, which also show unusual Kranz anatomy, Rubisco is restricted to only the innermost BSC (Ueno, 1998b).

View larger version (72K): [in this window] [in a new window]   Figure 3.   Immunogold localization of PEPC in photosynthetic cells of E. vivipara (Ueno, 1998a). A, Outer BSC of the terrestrial form. B, Outer BSC of the submerged form. C, Outer BSC and MC (left margin) of ABA-induced tissue. Scale bar = 0.5 µm. C, Chloroplast; S, starch grain; mt, mitochondrion.

From these studies, it is evident that the NAD-ME-dependent C₄ pathway is operative in the terrestrial form (Fig. 2C). However, the culms of the terrestrial form of E. vivipara show slightly more negative ¹³C values than those of other Eleocharis spp. with Kranz anatomy (Ueno et al., 1988, 1989). An inhibitor of PEPC, 3,3-dichloro-2-(dihydroxyphosphinoylmethyl)- propenoate (Jenkins, 1989), does not completely suppress photosynthesis in the terrestrial form, despite the fact that it completely inhibits photosynthesis of a typical C₄ species of Fimbristylis (O. Ueno and K. Ishimaru, unpublished data). Thus, some CO₂ may be fixed by Rubisco (probably through the C₃ cycle) present in the MC, even though most CO₂ is fixed through the C₄ pathway. Such features are reminiscent of those of C₄-like plants of Flaveria spp. (Cheng et al., 1989), but they may not be identical, because in E. vivipara PEPC and NAD-ME are compartmentalized between the MC and the BSC. In the submerged form the MC are the main photosynthetic tissues, because of the reduction of the BSC. In the MC of the submerged form, both PEPC and Rubisco are present, but the level of PEPC is low relative to that of Rubisco. Thus, it is suggested that CO₂ in water is fixed mainly by Rubisco and the C₃ cycle in the chloroplasts of the MC (Fig. 2C; Ueno, 1996b). Under water conditions of low carbon, the proportion of ¹⁴C incorporated into C₄ compounds is higher than under conditions of high carbon. However, the turnover of ¹⁴C in C₄ compounds is very slow (Ueno, 1998a). Therefore, it is thought that even though the ME-dependent C₄ cycle also operates in the submerged form, the contribution to total carbon flux is not large. It is concluded that cellular regulation of photosynthetic enzyme accumulation, the unusual localization of Rubisco, and the anatomical differentiation of photosynthetic tissues are the main factors responsible for the expression of C₃ and C₄ characteristics in E. vivipara (Ueno, 1996a, 1996b).

	PLASTIC EXPRESSION OF C3 AND C4 CHARACTERISTICS IN E. VIVIPARA IN FLUCTUATING ENVIRONMENTS

TOP INTRODUCTION C3 AND C4 PHOTOSYNTHESIS DISCOVERY OF C3 AND... STRUCTURAL AND BIOCHEMICAL... PLASTIC EXPRESSION OF C3... HORMONAL REGULATION OF C3... EXPRESSION OF C3 AND... VARIATION IN EXPRESSION OF... CONCLUSIONS AND FUTURE... LITERATURE CITED

Transfer experiments demonstrate that the two growth forms of E. vivipara can change reversibly into one another. When submerged plants are exposed to air, the culms wither from the rapid drying. However, plants produce new culms, which possess both Kranz anatomy and the C₄ biochemical traits. When terrestrial plants are immersed in water, the plants develop new culms with intermediate characteristics and finally with C₃ characteristics. It takes several weeks for the switch from the submerged to the terrestrial form and several months for the reverse change (Ueno et al., 1988). When the submerged form is growing underwater, the tips of the culms often reach the water surface. The plants then develop aerial culms at these tips, together with culms floating at the water surface. The aerial culms possess both Kranz anatomy and C₄ biochemical traits, despite the fact that the underwater culms have non-Kranz anatomy and C₃ characteristics. The floating culms show intermediate anatomies between Kranz and non-Kranz types. Therefore, it appears that different photosynthetic modes operate within a single plant and between joined tissues in contrasting environments. Therefore, plants growing in habitats with fluctuating water levels may possess various culms with different anatomical and biochemical features. It seems that the environmental response of E. vivipara represents a very plastic expression of C₃ and C₄ characteristics accompanied by tissue differentiation within a single plant.

	HORMONAL REGULATION OF C3 AND C4 DIFFERENTIATION IN E. VIVIPARA

TOP INTRODUCTION C3 AND C4 PHOTOSYNTHESIS DISCOVERY OF C3 AND... STRUCTURAL AND BIOCHEMICAL... PLASTIC EXPRESSION OF C3... HORMONAL REGULATION OF C3... EXPRESSION OF C3 AND... VARIATION IN EXPRESSION OF... CONCLUSIONS AND FUTURE... LITERATURE CITED

The heterophylly in aquatic plants is controlled by various environmental and hormonal factors (Smith and Hake, 1992). Although abscisic acid (ABA) is considered to be a stress hormone in plants, there is evidence that it is involved in the determination of leaf identity in some heterophyllic aquatic plants (Goliber and Feldman, 1989). It is thought that when plant shoots emerge from underwater into the air, the concomitant osmotic stress and higher light levels induce ABA production, which leads to the development of aerial leaves (Goliber and Feldman, 1989). In fact, when submerged E. vivipara are grown in an aqueous solution of ABA, they begin to develop new culms with Kranz anatomy (Ueno, 1998a). The ABA-induced tissues have several times more C₄ enzyme activity than have tissues of untreated submerged plants and accumulate large amounts of C₄ enzymes at the appropriate cellular sites (Fig. 3C). They exhibit a C₄-like pattern of ¹⁴C fixation under aqueous conditions of low carbon, indicating enhanced C₄ capacity in the tissues (Ueno, 1998a). These facts imply that in E. vivipara ABA acts as a trigger for the regulatory cascade of complex developmental processes that lead to the formation of Kranz anatomy and C₄ biochemical characteristics (Fig. 4).

View larger version (59K): [in this window] [in a new window]   Figure 4.   Environmental and hormonal regulation of the conversion of structural and biochemical traits involved in photosynthetic mechanisms in E. vivipara.

ABA has been reported to induce CAM in some facultative CAM plants (Chu et al., 1990; McElwain et al., 1992; Edwards et al., 1996; Taybi and Cushman, 1999). However, no stimulatory effects of ABA on C₄ enzymes have been observed in maize (Sugiharto et al., 1992), C₄ species of Flaveria, or the obligate CAM species Kalanchoë daigremontiana (Chu et al., 1990). It would be interesting to examine whether a similar signaling system that leads to changes in photosynthetic metabolism in response to environmental stimuli and that is mediated by ABA might have evolved simultaneously in facultative CAM species and E. vivipara (Ueno, 1998a). The induction of PEPC and NADP-ME by ABA has also been reported in a submerged aquatic plant, Egeria densa, which expresses C₄ metabolism under high temperature and light conditions, which limit CO₂ availability (Casati et al., 2000).

There is no evidence that a single gene is capable of setting in motion the entire C₄ machinery. Thus, C₄ photosynthesis appears to be a combination of independently inherited characteristics (Brown and Bouton, 1993). Our understanding of the molecular basis of the control of C₄ differentiation is still limited. However, transcriptional regulators suggesting a specific role in the differentiation of cell types of maize leaves have recently been reported (Hall et al., 1998; Rossini et al., 2001). When gibberellic acid is exogenously applied to terrestrial E. vivipara plants, the plants develop new tissues, without stomata, that are similar to the non-Kranz type tissues of submerged plants. Nevertheless, the tissues show a high accumulation of C₄ enzymes (Fig. 4; O. Ueno and M. Kai, unpublished data). This fact suggests that in E. vivipara, the structural and biochemical characteristics of C₄ photosynthesis are not always differentiated in a coordinated manner, implying that separate signaling systems are responsible for the individual differentiation of structural and biochemical characteristics. Similar results have also been observed in differentiation of these characteristics during the transition from the terrestrial to the submerged form (Uchino et al., 1998).

	EXPRESSION OF C3 AND C4 PHOTOSYNTHETIC GENES IN E. VIVIPARA

TOP INTRODUCTION C3 AND C4 PHOTOSYNTHESIS DISCOVERY OF C3 AND... STRUCTURAL AND BIOCHEMICAL... PLASTIC EXPRESSION OF C3... HORMONAL REGULATION OF C3... EXPRESSION OF C3 AND... VARIATION IN EXPRESSION OF... CONCLUSIONS AND FUTURE... LITERATURE CITED

It is intriguing to address how C₃ and C₄ differentiation is regulated within E. vivipara at the molecular level. At present, this molecular basis remains to be uncovered, but the isolation and expression analysis of several genes encoding C₃ and C₄ enzymes have been performed (Agarie et al., 1997a, 1997b; Baba et al., 1997; Uchino et al., 1998). It is thought that the difference in levels of C₃ and C₄ enzymes between the two growth forms of E. vivipara is regulated largely at a transcriptional level of the corresponding genes (Agarie et al., 1997a, 1997b; Baba et al., 1997; Uchino et al., 1998). This is clearly observed in plants growing under semi-submerged and semi-aerial conditions; the underwater culms show lower expression of genes for PEPC and PPDK, whereas the aerial culms show enhanced expression of the genes. The culms floating at the water surface reveal intermediate expression of the genes (Agarie et al., 1997b). The intercellular patterns of expression of genes for PEPC, PPDK, and the large and small subunits of Rubisco correspond well with the patterns of accumulation of the enzyme proteins (Baba et al., 1997; Uchino et al., 1998).

The kinetic properties of PEPC in E. vivipara differ between the terrestrial and submerged forms. The K_m for phosphoenolpyruvate of the terrestrial form's PEPC is intermediate between those of typical C₃ and C₄ plants, whereas that of the submerged form's PEPC is C₃-like (O. Ueno, unpublished data). It is possible that several isogenes for PEPC are expressed in E. vivipara, as is the case in other plants (Cushman et al., 1989; Kawamura et al., 1992; Westhoff et al., 1997), and that the expression patterns of respective isogenes differ between the terrestrial and submerged forms. Recently, a gene for PEPC, which probably encodes a PEPC expressed most strongly in the terrestrial form, has been isolated (Agarie et al., 1997b). Homology research shows that this PEPC is located between a cluster of C₄-form PEPCs from C₄ grasses and a cluster consisting of C₃-form PEPCs from C₃ and C₄ species and a CAM-form PEPC from the facultative CAM species M. crystallinum.

Although a gene for chloroplastic PPDK is strongly expressed in the leaves of C₄ plants (Sheen, 1999), a gene for cytosolic PPDK and a gene for chloroplastic PPDK are simultaneously expressed in the culms of E. vivipara (Agarie et al., 1997a). Both genes are more strongly expressed in the terrestrial form than in the submerged form. In the terrestrial form, the gene for chloroplastic PPDK is more highly expressed than the other gene, whereas in the submerged form, the reverse trend is found. In general, cytosolic PPDK seems to be involved in functions other than photosynthesis (Moons et al., 1998). However, it has recently been reported that in some CAM plants, both chloroplastic and cytosolic PPDKs are accumulated in the leaves (Kondo et al., 2000). In the facultative CAM plant Kalanchoë blossfeldiana, coordinated accumulation of both PPDKs is observed during enhanced CAM expression, suggesting that they are involved in CAM function (Kondo et al., 2001). At present, it is not known whether the unusual pattern of PPDK expression in E. vivipara is related to its unique C₃/C₄ property.

The ABA-induced culms of the submerged plants exhibit high expression of the genes for PEPC and PPDK. When culms formed before ABA treatment of submerged plants (which lack Kranz anatomy) are exposed to ABA solution, they also exhibit high expression of the genes for PEPC and PPDK (Agarie et al., 1997b). Thus, it seems again that expression of these genes can occur without coordinated differentiation of Kranz anatomy.

	VARIATION IN EXPRESSION OF PHOTOSYNTHETIC MODES IN AMPHIBIOUS SPECIES OF ELEOCHARIS

TOP INTRODUCTION C3 AND C4 PHOTOSYNTHESIS DISCOVERY OF C3 AND... STRUCTURAL AND BIOCHEMICAL... PLASTIC EXPRESSION OF C3... HORMONAL REGULATION OF C3... EXPRESSION OF C3 AND... VARIATION IN EXPRESSION OF... CONCLUSIONS AND FUTURE... LITERATURE CITED

In the genus Eleocharis, two other amphibious species, Eleocharis baldwinii and Eleocharis retroflexa subsp. chaetaria, show C₄ characteristics in their terrestrial forms (Ueno et al., 1989). The responses of these species to aquatic environments differ from that of E. vivipara (Table I; Uchino et al., 1995; Ueno et al., 1998).

The terrestrial forms of E. baldwinii and E. retroflexa possess structural and biochemical characteristics of the NAD-ME-type C₄ plant (Table I). The culms also exhibit unusual Kranz anatomy in which the mestome sheath is interposed between the MC and the BSC. The chloroplasts of the MC, as well as those of the BSC, accumulate Rubisco, but less than in E. vivipara (Ueno, 2000). Consistent with the enzyme distribution, the ¹³C values of the terrestrial forms of E. baldwinii and E. retroflexa are less negative than those of E. vivipara, although the values of the three species are within C₄ range (Ueno et al., 1989). The activities of the C₄ enzymes in the terrestrial forms of all three amphibious species are high, ranging in the following order: E. vivipara less than E. baldwinii less than E. retroflexa. The inhibitory effects of 3,3-dichloro-2-(dihydroxyphosphinoylmethyl)-propenoate on photosynthesis also show the same trend. Thus, the terrestrial forms of E. baldwinii and E. retroflexa also differ from typical C₄ plants, and there is a gradient in the degree of expression of C₄ characteristics among the terrestrial forms of the three species.

When E. baldwinii is growing under submerged conditions, it develops culms with traits intermediate between C₃ and C₄ biochemistry (Uchino et al., 1995). The BSC become small, and the MC develop well. In the MC, the amount of Rubisco becomes higher than that in the terrestrial form, and the amount of PEPC becomes lower (Ueno, 2000). Despite a decrease in NAD-ME activity, NADP-ME activity is maintained or slightly increased in the submerged form. There is evidence that a Kranz-less C₄ metabolism is operative in some submerged aquatic plants such as Hydrilla and Egeria spp. (Bowes and Salvucci, 1989; Reiskind et al., 1997; Casati et al., 2000). A possible unicellular C₄ metabolism has recently been reported in a marine diatom, Thalassiosira weissflogii (Reinfelder et al., 2000). In Hydrilla spp. and Egeria spp., enhanced expression of NADP-ME is observed during induction of C₄ metabolism (Magnin et al., 1997; Casati et al., 2000). It would be interesting to examine whether the submerged form of E. baldwinii fixes some CO₂ through a similar C₄ metabolism. In contrast, E. retroflexa essentially maintains C₄ characteristics even underwater; the culms of the submerged form have both Kranz-like anatomy and C₄ biochemical characteristics (Ueno et al., 1998). Therefore, its features resemble those of some aquatic C₄ grasses in the Orcuttieae (Keeley, 1998). Also, in the submerged form of E. retroflexa, the level of Rubisco in the MC is slightly higher than in the terrestrial form (O. Ueno, unpublished data).

We may well ask why there is variation in the growth-form-specific expression of photosynthetic modes among the amphibious species of Eleocharis. This might be partly explained by the difference in the degree of C₄ expression in the terrestrial forms. From taxonomic study of the photosynthetic modes of Eleocharis spp. it seems that C₄ photosynthesis evolved relatively recently in the genus, generating various intermediate stages (Ueno et al., 1989). According to this scenario, it appears that the terrestrial form of E. vivipara represents an evolutionary stage that is somewhat less advanced toward a full C₄ syndrome than the terrestrial forms of the other two species. This may facilitate the intriguingly plastic expression of photosynthetic modes seen in E. vivipara in contrasting environments.

	CONCLUSIONS AND FUTURE PERSPECTIVES

TOP INTRODUCTION C3 AND C4 PHOTOSYNTHESIS DISCOVERY OF C3 AND... STRUCTURAL AND BIOCHEMICAL... PLASTIC EXPRESSION OF C3... HORMONAL REGULATION OF C3... EXPRESSION OF C3 AND... VARIATION IN EXPRESSION OF... CONCLUSIONS AND FUTURE... LITERATURE CITED

There is ample evidence to suggest that C₄ photosynthesis evolved from C₃ photosynthesis in parallel among diverse taxonomic groups, thereby, generating extensive diversity in the structural, biochemical, and developmental aspects of C₄ photosynthesis among present-day species (Sheen, 1999; Freitag and Stichler, 2000; Edwards et al., 2001). These include some C₃-C₄ intermediate species of Flaveria that have been useful in elucidating the sequence of events during the evolution in the mode of photosynthesis (Ku et al., 1991). However, the E. vivipara, which has retained its ability to switch between C₃ and C₄ mode of photosynthesis depending upon environmental conditions, provides an attractive opportunity to investigate the developmental process from C₃ to C₄ photosynthesis in its totality.

We still do not understand the molecular mechanism(s) that brings about the switching between photosynthetic modes in E. vivipara. A comparative study of the signaling mechanisms involved in the expression of photosynthetic traits in E. vivipara, facultative CAM plants (Edwards et al., 1996; Taybi and Cushman, 1999), and some submerged aquatic plants (Magnin et al., 1997; Casati et al., 2000) might be of help in determining the universality and/or diversity of the underlying molecular mechanisms. One of the salient features of C₄ photosynthesis is the differentiation of MC and BSC. Although characterization of mutants from maize (Hall et al., 1998; Rossini et al., 2001) and Arabidopsis (Kinsman and Pyke, 1998) can provide some insight into the mechanism, analyses of plants such as E. vivipara and some C₄ dicots that develop cotyledons with a C₃ mode (Voznesenskaya et al., 1999) may provide a better system to understand this process of cell differentiation.

Undoubtedly, the amphibious species of Eleocharis are worth studying if we are to expand our knowledge of the ecological and adaptive aspects of C₄ plants. We still do not know the adaptive significance of the alteration of the photosynthetic characteristics of E. vivipara from C₄ to C₃ and of E. baldwinii from C₄ to C₃-C₄ intermediate when these plants are grown underwater. Further research of the amphibious species of Eleocharis, Hydrilla (Reiskind et al., 1997), and aquatic C₄ grasses (Keeley, 1998) may contribute toward a deeper understanding not only of the diversity of photosynthetic metabolism in aquatic environments but also of evolutionary significance of C₄ photosynthesis.

	ACKNOWLEDGMENTS

I am grateful to Dr. Sanjay Kapoor for a critical reading of the manuscript and to an anonymous reviewer for valuable comments.

	FOOTNOTES

Received August 28, 2001; returned for revision September 9, 2001; accepted September 14, 2001.

¹ This study was supported by the Ministry of Agriculture, Forestry and Fisheries of Japan (grant-in aid no. BDP-01-I-1-6).

^* E-mail uenoos{at}nias.affrc.go.jp; fax 81-298-38-7408.

www.plantphysiol.org/cgi/doi/10.1104/pp.010704.

	LITERATURE CITED

TOP INTRODUCTION C3 AND C4 PHOTOSYNTHESIS DISCOVERY OF C3 AND... STRUCTURAL AND BIOCHEMICAL... PLASTIC EXPRESSION OF C3... HORMONAL REGULATION OF C3... EXPRESSION OF C3 AND... VARIATION IN EXPRESSION OF... CONCLUSIONS AND FUTURE... LITERATURE CITED

• Agarie S, Kai M, Takatsuji H, Ueno O (1997a) Expression of C₃ and C₄ photosynthetic characteristics in the amphibious plant Eleocharis vivipara: structure and analysis of the expression of isogenes for pyruvate, orthophosphate dikinase. Plant Mol Biol 34: 363-369[Medline]
• Agarie S, Kai M, Takatsuji H, Ueno O (1997b) PEP carboxylase from the amphibious C₃/C₄ sedge Eleocharis vivipara: cDNA cloning and expression analysis (abstract no. 1067). Plant Physiol 114: S-212
• Baba A, Agarie S, Takatsuji H, Ueno O (1997) Expression of C₃ and C₄ characteristics in the amphibious sedge Eleocharis vivipara: cellular pattern of expression of photosynthetic genes (abstract no. 1065). Plant Physiol 114: S-212
• Bowes G, Salvucci ME (1989) Plasticity in the photosynthetic carbon metabolism of submerged aquatic macrophytes. Aquat Bot 34: 233-266[CrossRef]
• Brown RH, Bouton JH (1993) Physiology and genetics of interspecific hybrids between photosynthetic types. Annu Rev Plant Physiol Plant Mol Biol 44: 435-456[Web of Science]
• Bruhl JJ, Stone NE, Hattersley PW (1987) C₄ acid decarboxylation enzymes and anatomy in sedges (Cyperaceae): first record of NAD-malic enzyme species. Aust J Plant Physiol 14: 719-728
• Casati P, Lara MV, Andreo CS (2000) Induction of a C₄-like mechanism of CO₂ fixation in Egeria densa, a submerged aquatic species. Plant Physiol 123: 1611-1621[Abstract/Free Full Text]
• Cheng S-H, Moore B, Wu J, Edwards GE, Ku MSB (1989) Photosynthetic plasticity in Flaveria brownii: growth irradiance and the expression of C₄ photosynthesis. Plant Physiol 89: 1129-1135[Abstract/Free Full Text]
• Chu C, Dai Z, Ku MSB, Edwards GE (1990) Induction of Crassulacean acid metabolism in the facultative halophyte Mesembryanthemum crystallinum by abscisic acid. Plant Physiol 93: 1253-1260[Abstract/Free Full Text]
• Cushman JC, Meyer G, Michalowski B, Schmitt JM, Bohnert HJ (1989) Salt stress leads to differential expression of two isogenes of phosphoenolpyruvate carboxylase during Crassulacean acid metabolism induction in the common ice plant. Plant Cell 1: 715-725[Abstract/Free Full Text]
• Dengler NG, Nelson T (1999) Leaf structure and development in C₄ plants. In RF Sage, RK Monson, eds, C₄ Plant Biology. Academic Press, San Diego, pp 133-172
• Edwards GE, Dai Z, Cheng SH, Ku MSB (1996) Factors affecting the induction of Crassulacean acid metabolism in Mesembryanthemum crystallinum. In K Winter, JAC Smith, eds, Crassulacean Acid Metabolism: Biochemistry, Ecophysiology and Evolution. Springer-Verlag, Berlin, pp 119-134
• Edwards GE, Furbank RT, Hatch MD, Osmond CB (2001) What does it take to be C₄? Lessons from the evolution of C₄ photosynthesis. Plant Physiol 125: 46-49[Free Full Text]
• Freitag H, Stichler W (2000) A remarkable new leaf type with unusual photosynthetic tissue in a central Asiatic genus of Chenopodiaceae. Plant Biol 2: 154-160[CrossRef]
• Goliber TE, Feldman LJ (1989) Osmotic stress, endogenous abscisic acid and the control of leaf morphology in Hippuris vulgaris L. Plant Cell Environ 12: 163-171[CrossRef][Medline]
• Hall LN, Rossini L, Cribb L, Langdale JA (1998) GOLDEN2: a novel transcriptional regulator of cellular differentiation in the maize leaf. Plant Cell 10: 925-936[Abstract/Free Full Text]
• Hatch MD (1999) C₄ photosynthesis: a historical overview. In RF Sage, RK Monson, eds, C₄ Plant Biology. Academic Press, San Diego, pp 17-46
• Imaizumi N, Usuda H, Nakamoto H, Ishihara K (1990) Changes in the rate of photosynthesis during grain filling and the enzymatic activities associated with photosynthetic carbon metabolism in rice panicles. Plant Cell Physiol 31: 835-843[Abstract/Free Full Text]
• Jenkins CLD (1989) Effects of the phosphoenolpyruvate carboxylase inhibitor 3:3-dichloro-2-(dihydroxyphosphinoylmethyl) propenoate on photosynthesis: C₄ selectivity and studies on C₄ photosynthesis. Plant Physiol 89: 1231-1237[Abstract/Free Full Text]
• Kanai R, Edwards GE (1999) The biochemistry of C₄ photosynthesis. In RF Sage, RK Monson, eds, C₄ Plant Biology. Academic Press, San Diego, pp 49-87
• Kawamura T, Shigesada K, Toh H, Okumura S, Yanagisawa S, Izui K (1992) Molecular evolution of phosphoenolpyruvate carboxylase for C₄ photosynthesis in maize: comparison of its cDNA sequence with a newly isolated cDNA encoding an isozyme involved in the anaplerotic function. J Biochem 112: 147-154[Abstract/Free Full Text]
• Keeley JE (1998) C₄ photosynthetic modifications in the evolutionary transition from land to water in aquatic grasses. Oecologia 116: 85-97[CrossRef]
• Kellogg EA (1999) Phylogenetic aspects of the evolution of C₄ photosynthesis. In RF Sage, RK Monson, eds, C₄ Plant Biology. Academic Press, San Diego, pp 411-444
• Kinsman EA, Pyke KA (1998) Bundle sheath cells and cell-specific plastid development in Arabidopsis leaves. Development 125: 1815-1822[Abstract]
• Kondo A, Nose A, Ueno O (2001) Coordinated accumulation of the chloroplastic and cytosolic pyruvate, Pi dikinases with enhanced expression of CAM in Kalanchoe blossfeldiana. Physiol Plant 111: 116-122[CrossRef]
• Kondo A, Nose A, Yuasa H, Ueno O (2000) Species variation in the intracellular localization of pyruvate, Pi dikinase in leaves of Crassulacean-acid-metabolism plants: an immunogold electron-microscope study. Planta 210: 611-621[CrossRef][Web of Science][Medline]
• Ku MSB, Wu J, Dai Z, Scott RA, Chu C, Edwards GE (1991) Photosynthetic and photorespiratory characteristics of Flaveria species. Plant Physiol 96: 518-528[Abstract/Free Full Text]
• Langdale JA, Nelson T (1991) Spatial regulation of photosynthetic development in C₄ plants. Trends Genet 7: 191-196[Web of Science][Medline]
• Magnin NC, Cooley BA, Reiskind JB, Bowes G (1997) Regulation and localization of key enzymes during the induction of Kranz-less, C₄-type photosynthesis in Hydrilla verticillata. Plant Physiol 115: 1681-1689[Abstract]
• McElwain EF, Bohnert HJ, Thomas JC (1992) Light moderates the induction of phosphoenolpyruvate carboxylase by NaCl and abscisic acid in Mesembryanthemum crystallinum. Plant Physiol 99: 1261-1264[Abstract/Free Full Text]
• Moons A, Valcke R, Van Montagu M (1998) Low-oxygen stress and water deficit induce cytosolic pyruvate orthophosphate dikinase (PPDK) expression in roots of rice, a C₃ plant. Plant J 15: 89-98[CrossRef][Web of Science][Medline]
• Reinfelder JR, Kraepiel AML, Morel FMM (2000) Unicellular C₄ photosynthesis in a marine diatom. Nature 407: 996-999[CrossRef][Medline]
• Reiskind JB, Madsen TV, van Ginkel LC, Bowes G (1997) Evidence that inducible C₄-type photosynthesis is a chloroplastic CO₂-concentrating mechanism in Hydrilla, a submersed monocot. Plant Cell Environ 20: 211-220[CrossRef]
• Rossini L, Cribb L, Martin DJ, Langdale JA (2001) The maize Golden2 defines a novel class of transcriptional regulators in plants. Plant Cell 13: 1231-1244[Abstract/Free Full Text]
• Sculthorpe CD (1967) The Biology of Aquatic Vascular Plants. Arnold, London
• Sheen J (1999) C₄ gene expression. Annu Rev Plant Physiol Plant Mol Biol 50: 187-217[CrossRef][Web of Science]
• Smith LG, Hake S (1992) Initiation and determination of leaves. Plant Cell 4: 1017-1027[Free Full Text]
• Sugiharto B, Burnell JN, Sugiyama T (1992) Cytokinin is required to induce the nitrogen-dependent accumulation of mRNAs for phosphoenolpyruvate carboxylase and carbonic anhydrase in detached maize leaves. Plant Physiol 100: 153-156[Abstract/Free Full Text]
• Takeda T, Ueno O, Agata W (1980) The occurrence of C₄ species in the genus Rhynchospora and its significance in Kranz anatomy of the Cyperaceae. Bot Mag Tokyo 93: 55-65
• Taybi T, Cushman JC (1999) Signaling events leading to Crassulacean acid metabolism induction in the common ice plant. Plant Physiol 121: 545-555[Abstract/Free Full Text]
• Uchino A, Samejima M, Ishii R, Ueno O (1995) Photosynthetic carbon metabolism in an amphibious sedge, Eleocharis baldwinii (Torr.) Chapman: modified expression of C₄ characteristics under submerged aquatic conditions. Plant Cell Physiol 36: 229-238[Abstract/Free Full Text]
• Uchino A, Sentoku N, Nemoto K, Ishii R, Samejima M, Matsuoka M (1998) C₄-type gene expression is not directly dependent on Kranz anatomy in an amphibious sedge Eleocharis vivipara Link. Plant J 14: 565-572[CrossRef]
• Ueno O (1996a) Structural characterization of photosynthetic cells in an amphibious sedge, Eleocharis vivipara, in relation to C₃ and C₄ metabolism. Planta 199: 382-393
• Ueno O (1996b) Immunocytochemical localization of enzymes involved in the C₃ and C₄ pathways in the photosynthetic cells of an amphibious sedge, Eleocharis vivipara. Planta 199: 394-403
• Ueno O (1998a) Induction of Kranz anatomy and C₄-like biochemical characteristics in a submerged amphibious plant by abscisic acid. Plant Cell 10: 571-583[Abstract/Free Full Text]
• Ueno O (1998b) Immunogold localization of photosynthetic enzymes in leaves of various C₄ plants, with particular reference to pyruvate, orthophosphate dikinase. J Exp Bot 49: 1637-1646[Abstract/Free Full Text]
• Ueno O (2000) Cellular expression patterns of C₃ and C₄ photosynthesis enzymes in the amphibious sedge Eleocharis baldwinii. Plant Cell Physiol Suppl 41: s113
• Ueno O, Samejima M, Koyama T (1989) Distribution and evolution of C₄ syndrome in Eleocharis, a sedge group inhabiting wet and aquatic environments, based on culm anatomy and carbon isotope ratios. Ann Bot 64: 425-438[Abstract/Free Full Text]
• Ueno O, Samejima M, Muto S, Miyachi S (1988) Photosynthetic characteristics of an amphibious plant, Eleocharis vivipara: expression of C₄ and C₃ modes in contrasting environments. Proc Natl Acad Sci USA 85: 6733-6737[Abstract/Free Full Text]
• Ueno O, Takeda T (1992) Photosynthetic pathways, ecological characteristics and the geographical distribution of the Cyperaceae in Japan. Oecologia 89: 195-203
• Ueno O, Takeda T, Samejima M, Kondo A (1998) Photosynthetic characteristics of an amphibious C₄ plant, Eleocharis retroflexa ssp. chaetaria. Plant Prod Sci 1: 165-173
• Voznesenskaya EV, Franceschi VR, Pyankov VI, Edwards GE (1999) Anatomy, chloroplast structure and compartmentation of enzymes relative to photosynthetic mechanisms in leaves and cotyledons of species in the tribe Salsoleae (Chenopodiaceae). J Exp Bot 50: 1779-1795[Abstract/Free Full Text]
• Westhoff P, Svensson P, Ernst K, Blasing O, Burscheidt J, Stockhaus J (1997) Molecular evolution of C₄ phosphoenolpyruvate carboxylase in the genus Flaveria. Aust J Plant Physiol 24: 429-436
• Winter K, Smith JAC (1996) Crassulacean acid metabolism: current status and perspectives. In K Winter, JAC Smith, eds, Crassulacean Acid Metabolism: Biochemistry, Ecophysiology and Evolution. Springer-Verlag, Berlin, pp 389-426
• Wunderlin RP (1998) Guide to the Vascular Plants of Florida. University Press of Florida, Gainesville