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.
Figure 7. . Leaf photosynthetic acclimation during long-term plant growth at 1000 μL L–1 CO2 as a function of (a) leaf soluble acid invertase activity and (b) leaf hexose-carbon to sucrose-carbon ratio (Moore et al. 1998). Rubisco protein content was measured as an indicator of photosynthetic capacity. Species are those listed in Table 1.
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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).
Figure 8. . A model describing the leaf metabolism associated with the production of leaf carbohydrates as potential signals that affect photosynthetic gene expression at elevated CO2 (developed from Foyer 1987; Huber 1989; Jang & Sheen 1994; Moore et al. 1998). Sucrose is produced by cytosolic sucrose-phosphate synthase and phosphatase. The sucrose may then be maintained in the cytosol, transported to the vacuole, or transported out of the cell. Vacuolar or apoplastic sucrose can be hydrolyzed by acid invertase. Resulting hexoses must be phosphorylated by hexokinase in order to re-enter cellular metabolism, thereby establishing a metabolic cycle. The primary carbohydrate signal would be generated by hexose flux through hexokinase as a result of sucrose cycling. A secondary pathway for hexose production may occur by chloroplastic starch hydrolysis. Sugar sensing of hexose flux by hexokinase may occur by modulation of protein effectors (E, which includes a kinase and phosphatase, Jang & Sheen 1997) that ultimately represses transcription of certain photosynthetic genes. There is also some evidence that carbohydrate signalling may occur by transport of external hexoses or sucrose across the plasma membrane (Smeekens & Rook 1997), but it is not yet clear if the associated transduction processes affect photosynthetic gene expression. Notably, vacuolar invertase is thought not to be metabolically regulated in vivo other than by substrate availability (Kruger 1990), but the activity of apoplastic invertase may be modulated by a protein inhibitor in a process perhaps subject to metabolic control by sucrose (Greiner, Krausgrill & Rausch 1998). See Fig. 6 for the molecular component of this model. TP, triose-phosphates.
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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.
Figure 9. . A conceptual model describing the leaf-level trafficking of sucrose, particularly as related to the generation of potential signals that may affect mesophyll photosynthetic gene expression at elevated CO2 (modified from Chiou & Bush 1996). Transfer of sucrose to the phloem tissue may occur, as shown, by either symplastic or apoplastic routes. Sucrose leakage to and retrieval from the apoplast is shown as possibly occurring along either transfer route, in a process that for simplicity is shown mediated by a single transporter. A relative limitation in the export of sucrose due to insufficient sink utilization of carbohydrate is suggested to result in increased storage in leaf cells and, by mass action effects, to result in increased provision of sucrose to vacuolar and/or apoplastic acid invertases. The integration of sink and source metabolism may occur by feed-forward effects from sucrose translocation to sinks (Farrar & Gunn 1996) and possibly by hormonal or other signals derived from sink tissues (E.G. Morris 1996). CP, chloroplasts; Hex, hexoses; HXK, hexokinase; IVR, invertase; NC, nucleus; Suc, sucrose; VAC, vacuole. Plasmodesmata are shown simply as single, intercellular bridges.
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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.