Amax, maximum CO2 assimilation rate CAB, genes encoding chlorophyll a/b binding proteins Ci, intercellular CO2 concentration PGK, the gene encoding 3-phosphoglycerate kinase PRK, the gene encoding phosphoribulokinase PSAB, the gene encoding the 83 kDa apoprotein of the PSI reaction centre PSBA, the gene encoding the D1 protein of photosystem II RBCS, genes encoding the Rubisco small subunit protein RBCL, the gene encoding the Rubisco large subunit protein Rubisco, ribulose-1,5-bisphosphate carboxylase/ oxygenase SBP, the gene encoding sedoheptulose-1,5-bisphosphatase
There have been many recent exciting advances in our understanding of the cellular processes that underlie photosynthetic acclimation to rising atmospheric CO2 concentration. Of particular interest have been the molecular processes that modulate photosynthetic gene expression in response to elevated CO2 and the biochemical processes that link changes in atmospheric CO2 concentration to the production of a metabolic signal. Central to this acclimation response is a reduction in ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) protein content. Studies indicate that this reduction results from species-dependent variation in the differential use and temporal control of molecular processes. We present a model for the control of Rubisco protein accumulation that emphasizes the role of subunit message translation as well as the abundance of subunit messages as components of the acclimation response. Many studies indicate that photosynthetic acclimation to elevated CO2 results from adjustments in leaf carbohydrate signalling. The repression of photosynthetic gene expression is considered to occur primarily by hexokinase functioning as a hexose flux sensor that ultimately affects transcription. Leaf hexoses may be produced as potential sources of signals primarily by sucrose cycling and secondarily by starch hydrolysis. An increased rate of sucrose cycling is suggested to occur at elevated CO2 by enhanced provision of sucrose to leaf acid invertases. Additionally, sink limitations that accentuate photosynthetic acclimation may result from a relative decrease in the export of leaf sucrose and subsequent increase in cellular sucrose levels and sucrose cycling.
The concentration of CO2 in the earth's atmosphere, currently 360 μL L–1, is approximately 30% higher than the pre-industrial level and is conservatively projected to double by the end of the 21st century. Since the activity of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) in C3 plants is CO2-limited under present atmospheric conditions, the rate of CO2 assimilation by photosynthesis at twice ambient CO2 concentration increases by 50% or more in the short term (minutes to hours; Fig. 1). However, in the longer-term (days to weeks) this initial stimulation of carbon uptake is often followed by biochemical and molecular changes that result in a substantial decrease in photosynthetic capacity (for reviews see van Oosten & Besford 1996; Griffin & Seemann 1996; Drake, Gonzalez-Meier & Long 1997). Increased atmospheric CO2 concentration is known to primarily affect components of photosynthesis in an indirect manner through secondary plant metabolic adjustments, at least in part as a response to increased carbohydrate production and metabolism. During growth at twice-ambient CO2, leaf soluble carbohydrate content increases on average by 52% and starch content by 160% (Long & Drake 1992). An increase in leaf carbohydrates has long been associated with an inhibition of photosynthesis (e.g. Neals & Incoll 1968), and carbohydrates are known to modulate the expression of many photosynthetic and non-photosynthetic genes (for reviews see Sheen 1994; Graham 1996; Koch 1996; Jang & Sheen 1997). Exposure of plants to elevated CO2 can result in reduced levels of mRNAs encoding certain photosynthetic genes, and this repression response is similar to that which occurs by feeding glucose or sucrose to leaf tissue (Krapp et al. 1993; van Oosten, Wilkins & Besford 1994). However, despite correlations between increased carbohydrate metabolism and reduced photosynthetic gene expression at elevated CO2, only recently have we substantially improved our understanding of the cellular processes that underlie CO2-driven changes in photosynthetic capacity. In this review, we consider the influence of elevated CO2 on (1) the molecular mechanisms that modulate photosynthetic gene expression; and (2) the signalling mechanism by which regulatory carbohydrates ultimately affect photosynthetic gene transcription. Since this acclimation response varies considerably between species (Stitt 1991; Sage 1994) and is modulated by organismal factors such as sink and tissue development (Xu, Gifford & Chow 1994), we also consider the molecular basis for differential species adjustments in their photosynthetic properties when grown at elevated CO2.
PHOTOSYNTHETIC GENE RESPONSES TO ELEVATED CO2
Influence of elevated CO2 on transcripts and proteins of photosynthetic genes
Certain characteristics of leaf Rubisco were recognized a number of years ago as possibly being modulated during plant growth at elevated CO2 (Wong 1977; Porter & Grodzinski 1984; Sage, Sharkey & Seemann 1989). In some cases, enzyme activation state decreases, but in other cases it may actually increase. Whether decreased activation is associated with a decrease in Rubisco activase protein level has not been determined, but at least in tomato, activase mRNA levels do decline at elevated CO2 (van Oosten et al. 1994). Since chloroplast CO2 levels may directly affect the activity of Rubisco activase (Lan & Mott 1991), it would be useful to comprehensively evaluate the response to elevated CO2 of the various components that control Rubisco activation state. For example, carbonic anhydrase facilitates diffusion of CO2 from intercellular air spaces (Edwards & Walker 1983), and generally the level of mRNA (when examined) and enzyme activity of carbonic anhydrase are reduced during growth at elevated CO2[e.g. pea (Majeau & Coleman 1996), cucumber (Peet, Huber & Patterson 1986), and bean (Porter & Grodzinski 1984); but see also unchanged or increased carbonic anhydrase expression in tobacco (Sicher, Kremer & Rodermel 1994) and Arabidopsis (Raines et al. 1992)]. However, whether these decreases specifically affect chloroplast CO2 levels or the Rubisco activation state at elevated CO2 is not known.
Species that have a decreased photosynthetic capacity at elevated CO2 generally have reduced levels of Rubisco protein by up to 40% and often also have reductions in other components of the photosynthetic apparatus (Sage, Sharkey & Seemann 1989; Bowes 1993; Webber, Nie & Long 1994; Moore et al. 1998). Although Rubisco has been studied more extensively than other components, leaf chlorophyll content frequently declines at elevated CO2 (e.g. Moore et al. 1998) as do transcript levels of thylakoid-related, nuclear-encoded genes such as CAB (van Oosten et al. 1994). However, a common acclimation response is that adjustments in the expression of Calvin cycle proteins often occur differentially relative to adjustments in components of leaf photochemistry (Sage 1994; Nie et al. 1995b). This response may reflect different levels and efficiencies of N deployment within the photosynthetic apparatus according to prevailing growth limitations (Xu et al. 1994). Furthermore, the expression of specific Calvin cycle genes also may be differentially affected at elevated CO2. For example, in wheat flag leaves, transcripts for Rubisco subunits and phosphoglycerate kinase are particularly sensitive to a moderate increase in atmospheric CO2, but sedoheptulose-1,7-bisphosphatase (SBP) and phosphoribulokinase (PRK) mRNAs are not (Fig. 2, Nie et al. 1995a). The significance and species variation in such responses have not yet been determined.
In a broader context, a number of other proteins may be considered as photosynthetic genes, but the available information on their responses to elevated CO2 is even more limited. Despite the large accumulation of leaf starch that often occurs at elevated CO2, transcripts for ADP glucose pyrophosphorylase (β-subunit) show little change (van Oosten et al. 1994). Although photorespiration decreases at elevated CO2 (Stitt 1991), the associated responses of proteins and mRNAs involved in photorespiration have not been well investigated. Elevated CO2 had little effect on the activities of several photorespiratory enzymes in tobacco [although different responses of specific catalase forms were noted (Havir & McHale 1989)], little effect on glycolate oxidase mRNA level in tomato (van Oosten et al. 1994), but did modestly repress mRNA accumulation of hydroxypyruvate reductase during illumination of dark-adapted cucumber seedlings (Bertoni & Becker 1996).
Influence of leaf development on photosynthetic responses to elevated CO2
Photosynthetic acclimation to elevated CO2 generally varies with leaf age, with young leaves commonly showing no acclimation to elevated CO2 (e.g. wheat, Nie et al. 1995a; but for acclimation occurring in young pea leaves see Xu et al. 1994). In a particularly thorough set of experiments, van Oosten & Besford (1995) examined the influence of varying CO2 levels on leaf photosynthesis and gene expression during development of the fifth leaf of tomato. Decreases in leaf photosynthetic rates and Rubisco content (as inferred from activity measurements) were not observed until leaves were more than 60% expanded. These adjustments occurred along with decreases in Rubisco small subunit (rbcS) mRNA (Fig. 3) and large subunit (rbcL) mRNA. Jang & Sheen (1994) suggested that the lack of acclimation in young leaves may be associated with their particularly active sucrose metabolism, resulting in an already repressed state of photosynthetic gene expression. However, this response does not result from control at the level of transcript abundance since Rubisco subunit mRNAs are higher in young leaves (tomato, Fig. 3; tobacco, Cheng, Moore & Seemann unpublished results). In part, leaf age-dependent responses to elevated CO2 may be associated with altered hormonal conditions. Plant sugar and hormone signalling pathways do show cross-talk (deWald, Sadka & Mullet 1994; Jang & Sheen 1997; Zhou et al. 1998).
Photosynthetic acclimation of mature leaves to elevated CO2 may allow the optimization of whole plant N use by redistributing N from photosynthetic proteins of source leaves to sink tissues (Bowes 1991; Stitt 1991). The lack of acclimation commonly observed in young leaves then may be due, in part, to plant developmental programmes not generally being sufficiently fine-tuned to permit a redistribution of N away from Rubisco in a leaf that is not yet fully competent as a source leaf to one that is even much less developed. Miller et al. (1997) have suggested that growth at elevated CO2 may result in a temporal shift in the normal leaf maturation processes, but evaluation of their data is complicated by their having done an analysis of leaf growth and photosynthesis following whole plant transfer to elevated CO2 (a shock-type experiment) rather than examining such during continuous growth at high CO2. Furthermore, the possibility that leaf development merely progresses more rapidly to a senescent condition at elevated CO2 (e.g. Miller et al. 1997) is inconsistent with the observation that loss of photosynthetic capacity in mature leaves at elevated CO2 is rapidly reversible upon plant exposure to lower levels of atmospheric CO2 (Sasek, DeLucia & Strain 1985; Majeau & Coleman 1996).
The molecular control of Rubisco protein and subunit message levels at elevated CO2
Moore et al. (1998) found that long-term growth at elevated CO2 (1000 μL L–1) affected the relative expression of Rubisco protein and mRNA levels in a number of ways (Table 1). For convenience, the examined species were divided into four groups based on the relative reduction in Rubisco protein that occurred at elevated CO2. In group (A) species, Rubisco content was unchanged and transcript levels of both RBCS and RBCL (Rubisco small and large subunit) genes modestly increased at elevated CO2. In group (B) species, there occurred a small reduction in Rubisco content (≈ 15%), but a large decrease (> 30%) in rbcS mRNA and no decline in rbcL mRNA. In species of groups (C) and (D), Rubisco content was reduced at elevated CO2 by 25 to 40%, but there occurred no corresponding decreases in the levels of either transcript with the exception of Arabidopsis. The response pattern of Arabidopsis was that the levels of rbcS and rbcL mRNAs and Rubisco content all substantially decreased at elevated CO2. Thus, Rubisco protein content in Arabidopsis may be largely controlled by message availability (Cheng et al. 1998). The control of Rubisco content in the other species of groups C and D is likely to be rather complex, involving perhaps multiple affects on subunit mRNA translation and/or protein turnover. Interestingly, the decrease in small but not large subunit mRNA levels in species of group B may indicate that Rubisco protein content is controlled in certain species at elevated CO2 in large part by the abundance of small subunit protein.
Table 1. . Influence of long-term growth at 1000 μL L–1CO2 on Rubisco protein and subunit message levels of 16 species (Moore et al. 1998). Mature leaves were collected at mid-day into liquid N2. Rubisco protein content is expressed as nmol of CABP-binding sites. Values are means of species’ averages determined on a fresh weight basis (n = 3). Plants were grouped according to the range of Rubisco protein reduction at elevated CO2 (A, 0–10%; B, 10–20%; C, 20–30%; D, 30–44%)
We have further probed the basis for the decrease in Rubisco protein in tobacco (group D) that occurred in the absence of corresponding changes in rbcS and rbcL mRNAs. Decreased tobacco Rubisco protein was not due to increased turnover of holoenzyme, but instead was associated with a decreased synthesis of both subunits (Cheng, Moore & Seemann, unpublished results). When grown at ambient CO2, about 60% of both subunit message pools were associated with polysomes (Fig. 4). However, at elevated CO2 the level of rbcL mRNA associated with polysomes was less than 40%, in contrast to the lack of change observed in rbcS, cab, and psbA mRNAs. Thus, the relatively specific, decreased initiation of rbcL mRNA translation at elevated CO2 is likely to account for a decreased synthesis of large subunit protein in tobacco. The decreased level of small subunit protein synthesis may be due to an inhibition of message translation after the initiation step (elongation or termination). Rodermel et al. (1996) noted that a decreased association of rbcL mRNA with polysomes also occurred in transgenic tobacco containing rbcS antisense DNA. They suggested that decreased small subunit protein abundance may regulate the initiation of rbcL mRNA translation, possibly by several different mechanisms. One conclusion from these studies is that decreased Rubisco content at elevated CO2 (groups B, C, D; Table 1) may result largely from the regulation of small subunit protein levels.
RbcS gene family transcript accumulation and diurnal expression are differentially affected by elevated CO2
In higher plants, rbcS mRNAs are encoded by a multigene family and specific transcripts are expressed differentially even at ambient CO2 (Dean et al. 1989). In Arabidopsis, the RBCS gene family consists of four members designated 1A, 1B, 2B, and 3B (Krebbers et al. 1988). During long-term growth of Arabidopsis at high CO2, expression of the 1A gene was repressed by about 45% relative to plants grown at ambient CO2, whereas the other three genes were repressed by 60 to 70% (Cheng et al. 1998). Thus, there was a differential expression of individual RBCS genes in Arabidopsis during long-term growth at high CO2, and this may occur in other species as well. Such a response could either enhance or diminish the actual rate of synthesis of small subunit protein depending on the relative translatability of each gene-specific rbcS message (e.g. Johnson et al. 1998).
Short-term exposure to elevated CO2 has been found to affect the diurnal pattern of rbcS transcript accumulation in a species-dependent fashion. In Arabidopsis, the level of rbcS mRNA normally declined throughout the daytime and then increased throughout the night (maximum values at sunrise; Fig. 5a, Cheng et al. 1998). Transfer to high CO2 did not affect the diurnal timing of rbcS mRNA expression, but did differentially affect the normal, night-time increase in mRNA levels of specific RBCS genes (the rbcS 3B gene mRNA was most reduced). In tobacco, the diurnal expression of rbcS mRNA at ambient CO2 did not vary by a simple day/night pattern (Fig. 5b; see also Paulsen & Bogorad 1988). Short-term exposure of tobacco to elevated CO2 altered the overall daytime increase in rbcS mRNA such that the maximum accumulation level occurred at mid-morning rather than at sunset. Notably, the diurnal pattern of tobacco cab mRNA expression was not affected by elevated CO2 in these experiments although its levels were reduced (data not shown). In a similar manner to the response by Arabidopsis, transfer of tobacco to high CO2 may have differentially repressed expression of a specific RBCS gene(s) whose transcript normally accumulates in the late afternoon. Such species-dependent temporal variation in CO2 effects on rbcS mRNA levels are important not only for their likely modulation of Rubisco protein level, but also suggests that there can be substantial temporal variation in the production and/or transduction of the primary signal that initiates such responses (see below).
Signal transduction and sugar response elements
Carbohydrate-dependent regulation of photosynthetic gene expression is thought to occur by hexokinase functioning as a sugar sensor at ambient as well as at elevated CO2 (Jang & Sheen 1994; Jang et al. 1997; see below). Jang & Sheen (1994, 1997) suggested that during hexokinase catalysis, there may be an associated kinase/phosphatase that initiates a transduction response and results in the repression of promoter activities of RBCS and a number of photosynthetic genes. Some components of general plant sugar-related signalling pathways have been identified, including certain protein phosphatases, membrane-bound Ca2+-dependent protein kinases, and transcription factors (Ohto & Nakamura 1995; Mita, Hirano & Nakamura 1997; Smeekens & Rook 1997). To what extent any of these may be involved in the CO2 signal transduction ‘pathway’ and the repression of photosynthetic gene expression is not known.
There has been considerable interest in determining whether sugar-related plant genes may contain a consensus response element within their promoters. However, few groups have yet attempted to identify any cis-acting regulatory sequences that involve sugar repression responses within photosynthetic gene promoters. Sheen (1990) found that sucrose repression of several photosynthetic genes in a maize protoplast transient expression system was mediated by positively acting promoter elements upstream of the TATA box, but there were no apparent specific consensus sequences. In another study, a 123 bp fragment of a malate synthase promoter was shown to mediate sugar repression in a transient cucumber protoplast expression assay (Graham, Baker & Leaver 1994). The cis-element involved in sugar repression was further defined by 5′ deletion analysis and found to be a 16 bp sequence, termed IMH2, in the malate synthase promoter. Maas, Schaal & Werr (1990) reported that within the promoter of the maize Shrunken gene (Sh1, sucrose synthase), there occurs a 26 bp sequence responsive to sucrose repression. Recently, a sucrose repression element was identified in the rbcS2 gene promoter (–203 to –187 bp) of Phaseolus vulgaris (Urwin & Jenkins 1997). This region has little sequence similarity with the IMH2 and Shrunken gene cis-elements, but surprisingly contains sequences resembling elements involved in sucrose induction of genes (SURE, sucrose response element) for certain plant storage proteins (patatin, Grierson et al. 1994; sporamin, Ishiguro & Nakamura 1992) or for certain mammalian genes (ChoRE, carbohydrate response element, Towle 1995). Furthermore, a G-box (CACGTG) located at –200 bp was found to be important for high levels of sucrose-repression of the rbcS2 promoter, indicating a possible involvement of the bZIP class of transcription factors (Urwin & Jenkins 1997). Whether this region of the bean rbcS2 promoter is also involved in the decrease of rbcS mRNA at elevated CO2 (Table 1) remains to be determined.
The regulation of sugar-sensitive promoter elements may also occur by somehow altering the expression of a clock-sensitive element in the same promoter or by altering promoter interactions with clock proteins (Kreps & Kay 1997). In the case of leaf CAB genes, specific promoter regions have been shown to confer clock regulation (Millar et al. 1992) or sugar repression (Sheen 1990) upon a reporter gene. To what extent any of the gene promoters for RBCS and other photosynthetic genes also directly mediate circadian regulation of a reporter gene is apparently not known.
A model for the molecular control of Rubisco expression at elevated CO2
The collective message and protein data from our laboratory and others indicate that in many species grown at elevated CO2, the Rubisco protein content is controlled by subunit message abundance and/or by translation. Based upon this conclusion, we suggest a model for the control of Rubisco protein expression during photosynthetic acclimation to elevated CO2 (Fig. 6). A mesophyll carbohydrate ‘signal’ initiates a transduction response (Smeekens & Rook 1997; see below) that affects transcription of specific nuclear genes including RBCS and/or nuclear-encoded translation factors for rbcS and rbcL mRNAs. The proteins that regulate the initiation of chloroplast translation are not well understood, but involve products of a number of nuclear genes that may activate translation of different organellar classes of mRNA or even gene-specific mRNAs (Gillham, Boynton & Hauser 1994; Danon 1997). Whether achieved by apparent transcriptional or translational control, or by other mechanisms, we suggest that small subunit protein abundance may largely control Rubisco protein level in plants grown at elevated CO2 (see Rodermel et al. 1996 for a more thorough discussion of possible mechanisms).
LEAF SUGAR SIGNALLING AT ELEVATED CO2
Involvement of hexokinase in carbohydrate signalling at elevated atmospheric CO2
Increased leaf carbohydrate content and decreased Rubisco protein content are common responses of plants exposed to elevated atmospheric CO2. Working with an autotrophic cell suspension culture of Chenopodium, Krapp et al. (1993) examined the influence of added glucose and glucose analogs, in association with light or CO2, on rbcS transcript level. Treatment with glucose reduced rbcS mRNA level by 64% within 3 h. However, treatment with glucose analogs (6-deoxyglucose and 3-O-methylglucose) that are transported into the cell but not phosphorylated by hexokinase did not repress the transcript levels in this study. Furthermore, in the absence of CO2 but in the presence of glucose, the sugar levels were increased and the amount of rbcS mRNA was minimally affected (Table 2). These results suggest that hexose metabolism rather than strictly sugar abundance is involved in the sugar signalling that is related to Rubisco gene expression. The observed 2·5-fold increase in rbcS message level in the absence of glucose and CO2 may indicate that sugar-dependent regulation of photosynthetic gene expression can adjust to the prevailing level of carbohydrate metabolism.
Table 2. . Effect of low CO2 and glucose on rbcS transcript and carbohydrate content in autotrophically growing Chenopodium cells (Krapp et al. 1993). For treatments without CO2, carbonate buffer in the culture medium was replaced by saturated KOH. Sugar treatments were at 50 mM for 10 H. The results are the mean ± SE (n = 5), and the experiment was carried out three times with comparable results. The differential affect of high sugar levels (± CO2) on rbcS mRNA levels (22% versus 82%) indicates that the concentration of sugars per se does not directly regulate rbcS gene expression
Jang & Sheen (1994) proposed that carbohydrate repression of a number of photosynthetic genes, including RBCS, probably involves hexose metabolism via hexokinase. This repression response was sensitive to an inhibitor of hexokinase activity, mannoheptulose. Furthermore, under- or over-expression of either of two Arabidopsis hexokinase genes dramatically altered the seedling sugar sensitivity and resulted in enhanced or reduced expression of rbcS mRNA, respectively (Jang et al. 1997). Following a short-term exposure of Arabidopsis (Cheng et al. 1998) or a long-term exposure of tomato (van Oosten & Besford 1995) to different levels of elevated CO2, the mRNAs for certain photosynthetic genes decreased in dose- and time-dependent patterns with an associated progressive increase in leaf sugars. These findings indicate that changes in gene expression at elevated CO2 can be mediated through a carbohydrate signal that probably affects the hexokinase sensory system.
There also is evidence that carbohydrate signalling in plants can occur by alternate pathway(s) possibly independent of hexokinase (Jang & Sheen 1997; Mita et al. 1997). There is limited evidence that the transport of hexoses or sucrose across the plasma membrane may directly initiate signal transduction processes (Martin et al. 1997; Smeekens & Rook 1997). Unidentifed hexose transporters somehow associated with hexokinase may also be involved in sugar signalling (Herbers et al. 1996; Smeekens & Rook 1997), as occurs for regulation of a few genes in yeast (Özcan et al. 1996). In this sense, hexokinase may function more as a response regulator than as a signal sensor. There is evidence that sucrose also may function directly as a signal metabolite (Urwin & Jenkins 1997; Chiou & Bush 1998). However, as with the previous signal pathways, it is not yet known to what extent the associated transduction processes may affect photosynthetic gene expression at elevated CO2.
Leaf hexokinase occurs in the cytosol, in plastids, and associated with the outer mitochondrial membrane (Miernyk & Dennis 1983; Schnarrenberger 1990). The hexose repression response is presumed to be associated with cytosolic hexokinase (Graham et al. 1994; Sheen 1994; van Oosten & Besford 1996). In plants, the protein may function in part as a sugar sensor (Jang et al. 1997) that can respond to hexose flux (Graham, Denby & Leaver 1994; Koch 1996). Some evidence for this function is the reduced sugar sensitivity of transgenic Arabidopsis seedlings that over-express yeast hexokinase (YHXK2; Jang et al. 1997). This response was attributed to reduced carbohydrate flux through the endogenous, sugar-sensing hexokinase due to substrate competition. Koch (1996) has suggested that flux sensing may occur based on some ratio of the hexokinase reaction components (rather than flux being sensed simply as a concentration-dependent change in an enzyme-product complex), since the formation of hexokinase-reaction intermediates at near equilibrium conditions that occur in the cell may be affected by the concentrations of all substrates and products (and enzyme). As an alternative to flux sensing, hexokinase could function more as a molecular switch according to a given threshold of metabolism (Herbers et al. 1996).
Leaf carbohydrate metabolism at elevated CO2 affects the production of hexoses as potential signals for gene regulation
If hexokinase can function as a flux sensor, then an increase in cytosolic hexose level could result in increased carbohydrate flux to hexose-phosphates and thereby initiate the signal transduction response. Moore, Palmquist & Seemann (1997) tested whether changes in cytosolic glucose content might be involved in carbohydrate signalling and photosynthetic down-regulation by examining mesophyll cytosolic glucose levels during growth at elevated CO2 (1000 μL L–1). Under this condition, leaf Rubisco content declined by at least 30% in tobacco and bean, but was not affected in spinach and parsley (Table 3). In all four species that were grown at high CO2, the total leaf soluble carbohydrates increased substantially, but there was no detectable effect on cytosolic glucose and fructose levels, with both occurring almost entirely in the vacuole (> 99%) irrespective of growth CO2 levels or time of day (sunrise to sunset). These results indicate that it is probably not increased levels of cytosolic hexoses that mediate carbohydrate-dependent gene regulation at elevated CO2.
Table 3. . Leaf Rubisco contents and mid-day intracellular amounts of soluble sugars in spinach, parsley, tobacco and bean grown at ambient or 1000 μL L–1 CO2 (Moore et al. 1997; Moore, Cheng & Seemann, unpublished). Rubisco protein content was measured as an indicator of photosynthetic acclimation and intracellular compartmentation of mesophyll sugars was determined using density gradient fractionation with nonaqueous solvents. Chloroplast sugar contents were quite low and for clarity are not shown. Based on subcellular compartment volumes from Heineke et al. (1994), mesophyll cytosolic concentrations of glucose and fructose were ≤100 μM each, regardless of growth CO2 and species
One alternative mechanism for the increased provision of hexoses as a possible source of sugar signals at elevated CO2 could involve their daily mobilization from storage pools. Vacuolar hexoses may be metabolized largely around sunset, while chloroplastic starch may be degraded relatively more during the night. However, after a short-term exposure of tobacco to elevated CO2, rbcS mRNA levels decreased during the late afternoon (Fig. 5b) prior to any decrease in vacuolar hexose or starch levels (Moore, Cheng & Seemann, unpublished results).
A third possible, and most likely source for provision of hexoses as potential signals for the hexokinase-sensing system may involve sucrose cycling. In a mature leaf, sucrose typically is hydrolyzed primarily by vacuolar or apoplastic invertases. The resulting hexoses must be phosphorylated by hexokinase in order to re-enter intermediary metabolism, and then may be utilized for resynthesis of sucrose (Foyer 1987; Huber 1989; Stitt, von Schaewan & Willmitzer 1990). Growth at elevated CO2 certainly generates increased leaf levels of sucrose, and could thus cause an increased rate of sucrose cycling if sufficient hydrolytic activity existed. Increased sucrose cycling through acid invertase and hexokinase might occur without an appreciable increase in cytosolic hexose levels, depending on the rate of hexose transport to the cytosol relative to the catalytic activity of hexokinase. In castor bean cotyledons, sucrose cycling can occur as a result of a small increase in sucrose content that results from an imposed sink limitation to export capacity (Geigenberger & Stitt 1993).
Whether sucrose cycling occurs in leaves at ambient or elevated CO2 has not been directly shown, but it is striking that photosynthetic acclimation to elevated CO2 can be predicted in crop and ornamental species from the measured leaf activity of soluble acid invertase (Fig. 7; Moore et al. 1998). In this study, in 15 of 16 species examined, the occurrence of photosynthetic acclimation to elevated CO2 was related to a threshold level of soluble acid invertase activity; that is, high acid invertase levels (and presumably a high capacity for sucrose cycling) were always associated with a decrease in Rubisco content at elevated CO2. A comparable threshold relationship between photosynthetic capacity and soluble acid invertase activity was previously observed after applying a hot wax girdle to leaf petioles (Goldschmidt & Huber 1992), a treatment that similarly can result in increased leaf carbohydrate levels and decreased photosynthetic capacity. In plants grown at elevated CO2, a decrease of at least 20% in photosynthetic capacity also was associated with relatively high leaf hexose/sucrose ratios (Fig. 7b), which is consistent with the suggested increase in sucrose cycling.
Further studies are needed to definitively prove that sucrose cycling is an important component of carbohydrate signalling and photosynthetic down-regulation at elevated CO2. Tobacco transformed to over-express yeast acid invertase in the apoplasm or vacuole showed phenotypical changes that also suggest that sucrose cycling may have a major role in leaf carbohydrate-dependent gene regulation (Stitt et al. 1990; Sonnewald et al. 1991). Changes in the transformants included a substantial accumulation of hexoses and decreased photosynthetic enzymes in the affected sectors of source leaves. The possible role of leaf acid invertase in carbohydrate signalling is intriguing since vacuolar invertase activities were minimally affected by growth CO2 level (Moore et al. 1998) or by petiole girdling (Goldschmidt & Huber 1992). This occurs in contrast to strong glucose-repression of yeast acid invertase expression (Gancedo 1992) and carbohydrate-dependent regulation of maize invertase genes in certain sink tissues (Koch 1996). One possibility is that leaf vacuolar invertase may function as a pacemaker that modulates cycling activity. The lack of substantial metabolic or molecular regulation of the enzyme may be attributed to its involvement at the same time in other sugar signalling responses that may be up-regulated at elevated CO2.
Hydrolytic starch degradation also generates glucose as a possible carbohydrate signal. Starch-accumulating species are often noted for their ability to down-regulate photosynthetic capacity under sink-limited conditions, including elevated CO2 (e.g. Goldschmidt & Huber 1992; Farrar & Williams 1991). Since starch degradation occurs primarily at night and since sucrose cycling could occur during much of the daytime, these dual pathways would potentially allow leaf carbohydrate signalling through hexokinase to affect photosynthetic gene expression over much of the entire 24-h period (i.e. diurnally). While both sucrose cycling and starch hydrolysis may generate hexoses that affect gene expression, the lack of a specific association between photosynthetic acclimation and starch accumulation (Tissue, Thomas & Strain 1993; Moore et al. 1998) indicates that sucrose cycling may be the primary regulatory component involved in leaf sugar sensing at elevated CO2. One extension of the idea that starch metabolism is in some aspects a buffer to sucrose metabolism (Stitt 1984) is the possibility that starch metabolism in part may function to minimize leaf sucrose cycling. From this viewpoint, species that may have enhanced levels of leaf sucrose cycling, experience sink-limited growth conditions and therefore are more active in starch metabolism, as often occurs at high CO2.
A biochemical model for carbohydrate signalling at elevated CO2
We have developed a model (Fig. 8) describing the effect of elevated atmospheric CO2 on gene expression that is based upon the inter-relationship between sucrose cycling and carbohydrate signalling, and that is linked to the previous molecular model (Fig. 6). This model is drawn from results of Foyer (1987), Huber (1989), Jang & Sheen (1994), and Moore et al. (1998). Sucrose cycling may produce leaf hexoses by either an intracellular or extracellular pathway. Vacuolar or apoplastic hexoses produced by sucrose hydrolysis are transported to the cytosol and ultimately are phosphorylated by hexokinase. In this model, the increased provision of hexoses from sucrose cycling at elevated CO2 can bring about the generation of a primary daytime carbohydrate signal by increased hexose flux through hexokinase. The normal daytime compartmentation of leaf hexoses in the mesophyll vacuole may be considered as actually being essential for operation of a cellular hexose-sensing mechanism by regulating the ‘noise’ level for sugar sensing. In Arabidopsis and wheat, diurnal fluctuations in rbcS transcripts and leaf carbohydrates occur in reciprocal patterns, with a night-time increase in rbcS mRNA associated with a decrease in leaf sugars (Nie et al. 1995a; Cheng et al. 1998). In Arabidopsis, both responses are partially repressed after transfer to high CO2 (Cheng et al. 1998). Thus, leaf carbohydrate metabolism potentially can affect photosynthetic gene expression over a large diurnal period. The possible generation of sugar signals over a broad time period is consistent with the gating hypothesis for circadian control of CAB transcription (Kay & Millar 1992). This hypothesis proposes that the circadian clock functions as a negative regulator of gene expression by which windows of possible transcriptional activity are superimposed on a background of ongoing signal production. We anticipate that the CO2 signal-response ‘pathway’ must intersect at a fundamental level with other processes that control plant growth and development, but very little is currently known about such interactions (see also Jang & Sheen 1997).
Effect of leaf and sink physiology on cellular sugar signalling at elevated CO2
Leaf cellular sugar levels are a function not only of their metabolism within the cell, but also are affected by storage processes, metabolic activities of surrounding cells, and activities of distant cells in sink tissues. Farrar (1996) suggested that the control of carbon flux within a plant is distributed to different regulatory components such that the sinks, sources and long-distance transport system all affect plant growth and responses to the environment. The capacity of carbohydrate utilization in distant cells can affect the level of carbohydrate export from source leaves (e.g. Thorne & Koller 1974) and the rate of leaf carbohydrate export can increase in plants grown at elevated CO2 (Zhang & Nobel 1996). However, how these processes interact to affect mesophyll sugar signalling and photosynthetic acclimation at elevated CO2 have not been determined.
In Fig. 9, we present a conceptual model that links the cell-level model for carbohydrate signalling (Fig. 8) to related components that affect leaf-level metabolism. We suggest that a relative limitation in leaf sucrose export and utilization may affect sucrose cycling within mesophyll cells. Increased leaf sucrose levels may produce by mass action effects an increased concentration of sucrose as substrate for vacuolar and/or apoplastic invertases, resulting in increased sucrose cycling and consequent photosynthetic down-regulation. This effect might occur in different ways depending upon the given leaf anatomy and the associated resistances along the leaf transport pathway. Plants are recognized to vary in the transfer of carbohydrates from mesophyll cells to sieve elements of the minor veins, using primarily either an apoplastic pathway or a symplastic pathway (van Bel 1993; Turgeon 1995). Species that use symplastic transport may be less efficient in leaf carbohydrate export particularly at low temperatures (van Bel 1993) and are reported to accumulate, on average, increased levels of starch at both ambient and elevated CO2 in comparison with species that use apoplastic phloem loading (Körner, Pelaez-Riedl & van Bel 1995). However, when grown at least at moderate temperatures at elevated CO2, such species may not acclimate to any different extent (Kingston-Smith, Galtier & Foyer 1998). Interestingly, some species, such as willow, may not concentrate sucrose in the phloem sieve cells (Turgeon & Medville 1998). Instead, sucrose may accumulate to unusually high levels in the mesophyll cells and directly diffuse to the phloem tissue.
The efficiency and characteristics of leaf carbohydrate export processes need to be better defined in order to understand their impact on photosynthetic acclimation to elevated CO2. While the majority of crop plants utilize an obligate apoplastic step in transfer of sucrose to phloem tissue (Giaquinta 1983; Fig. 9), even the mechanism for the release of sucrose from mesophyll cells to the apoplast is not specifically known (van Bel 1993; Sauer et al. 1994). Sucrose release may involve facilitated transport (Laloi, Delrot & M’Batchi 1993) using a carrier analogous to ones thought to occur in certain sink cells (Patrick 1997), but the mesophyll carrier has not yet been identified at the molecular level (Ward et al. 1998). Sucrose is probably released to and then retrieved from the apoplasm along the transfer paths, but this process has not been well characterized (Madore & Lucas 1989). To whatever extent that there may occur natural or imposed variation in these processes, this may be expected to affect leaf-level responses to elevated CO2. The role of the apoplastic compartment in sucrose cycling and the response of apoplastic sugars to plant growth at elevated CO2 should be also determined for species that vary in their degree of photosynthetic acclimation. Notably, after inhibiting phloem export by a cold-induced petiole girdle, leaf apoplastic sucrose levels rapidly and reversibly increased several-fold in Vicia faba (Nitska & Delrot 1986). In addition to possible signal effects (Fig. 8), leaf apoplastic sugars can affect carbon partitioning due to their increased solute potential affecting phloem turgor (Williams, Minchin & Farrar 1991).
It is not known whether or not phloem loading of sucrose is ever limiting for plants at elevated CO2. Phloem loading of sucrose from the apoplast may involve two transport systems, a high-affinity sucrose/H+ symporter (Bush 1993) and a low-affinity facilitator (Overvoorde, Frommer & Grimes 1996). Antisense inhibition of the symporter protein in potato resulted in an altered phenotype with mature leaves having bleached areas, reduced photosynthesis, and an accumulation of carbohydrates that often resulted in plant stunting (Riesmeier, Willmitzer & Frommer 1994; Kühn et al. 1996). Phloem expression of the sucrose symporter protein is enhanced during leaf maturation (Lemoine et al. 1992) and is diurnally regulated at the mRNA and protein levels (Kühn et al. 1997). Furthermore, the mRNA abundance and carrier activity of the sucrose symporter protein may be under metabolic control by sucrose abundance (Chiou & Bush 1998). Under field conditions of suboptimal abiotic conditions, resulting limitations in source leaf metabolism could be manifested in some species at elevated CO2 as a limiting activity of the sucrose symporter protein. However, if plant growth is normally sink-limited at elevated CO2 (Stitt 1991), then the over-expression of this protein in transgenic plants would be predicted to have little affect on photosynthetic acclimation to elevated CO2.
Elucidation of the control of assimilate partitioning by sucrose-mediated regulation of the sucrose symporter activity (Chiou & Bush 1998) will improve our understanding of leaf level responses to elevated CO2, but is unlikely to explain all of the carbohydrate-related responses in part due to long-distance interactions between source and sink tissues (Fig. 9). Sucrose normally constitutes about one-half of the osmotic solutes in sieve elements (e.g. Smith & Milburn 1980). In one study, phloem sugar concentrations are reported from a C11-tracer analysis to change diurnally and in response to different imposed changes in CO2 concentrations (Magnuson, Goeschl & Fares 1986). However, short-term exposure to elevated CO2 has little affect on the rate of leaf sugar export (Grodzinski, Jiao & Leonardos 1998). This may occur because the rate of long-distance carbohydrate transport is a function of a phloem turgor pressure gradient between source and sinks rather than being a function of phloem sugar concentrations (Patrick 1997). Sink growth may facilitate a reduction in phloem turgor pressure during normal, symplastic unloading, thereby influencing translocation rates (Farrar, Minchin & Thorpe 1995) and ultimately photosynthetic acclimation to elevated CO2.
Long-distance signalling between source and sink tissues may involve a number of regulatory components. Sucrose has been suggested to function as a feed-forward signal that originates in source leaves and stimulates sink growth (Farrar & Gunn 1996), but just how this may be linked with increased sink development at elevated CO2 (e.g. Jitla et al. 1997) is not clear. Sink leaves have for long been recognized as preferentially utilizing for protein synthesis carbon derived from photosynthesis within the sink leaf (Joy 1967; Turgeon 1989). Thus, increased protein synthesis in sink leaves due to increased photosynthesis may be an important component of plant responses to elevated CO2. Co-ordination of sink and source activities at elevated CO2 also may involve hormonal signals sent from source to sink tissues (Morris 1996) or from sink to source tissues (Davies & Zhang 1991). Additionally, such co-ordination will be affected by plant nitrogen metabolism (Stitt & Krapp 1999). Nitrate itself may function as a metabolic signal that can affect carbohydrate metabolism (Champigny & Foyer 1992) and acclimation to elevated CO2. Understanding the nature and influence of long-distance signalling events on leaf-level responses to elevated CO2 will be a challenge for future research efforts, as will integrating the affects of other environmental factors that influence photosynthetic acclimation to elevated CO2.
Research by our laboratory described in this review was supported by a grant from the U.S. National Science Foundation (IBN-1940709).