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The current atmospheric CO2 concentration is suboptimal for C3 photosynthesis, which suggests that any future increase should benefit plant growth. However, it is often observed that plants acclimate to elevated CO2 conditions through a reduction in their photosynthetic activity, which is generally associated with an increase in soluble leaf carbohydrates and starch (Drake et al., 1997). This circumstantial evidence is substantiated by several studies showing that hexose accumulation or cycling within the cells can repress photosynthetic gene expression through a hexokinase-controlled signalling pathway (Krapp et al., 1993; Jang et al., 1997; Moore et al., 1999). More recently, hydrolytic conversion of starch to sucrose has also been thought to contribute to this sugar-sensing pathway (Sharkey et al., 2004). Other studies suggested that accumulation of large starch grains disrupt chloroplast integrity (Pritchard et al., 1997) or hinder CO2 diffusion from the intercellular space to the chloroplast stroma (Sawada et al., 2001).
Both soluble sugar and starch metabolisms can affect photosynthesis. However, the role played by carbohydrates in mechanisms controlling plant acclimation to elevated CO2 remains unclear, as their accumulation in photosynthetic tissues does not always correlate with photosynthetic acclimation to elevated CO2 (Moore et al., 1998; Sims et al., 1998; Geiger et al., 1999). The emerging paradigm is that of whole-plant-level control of photosynthesis through a signalling network that integrates the global source–sink status of the plant (Coruzzi & Zhou, 2001; Paul & Foyer, 2001). In this context, transient starch becomes functionally important as an overflow product for photosynthesis in plants with low sink capacity. Indeed, the sink-regulation hypothesis suggests that starch accumulation in leaves during the light period is enhanced when the photosynthetic production rate exceeds that of growth and transport processes. Grimmer & Komor (1999) report that carbon export from leaves of Ricinus communis L. during the light period was identical for plants exposed to 350 and 700 ppm CO2, while night-time exports of 700-ppm plants doubled those of their 350-ppm counterparts. They hypothesized that Ricinus plants grown under ambient CO2 conditions are sink-limited during daytime and source-limited at night. Starch accumulation would be just a symptom that sugar is available in excess of plant growth demand and phloem transport capacities.
Observations conducted on Arabidopsis plants affected in starch synthesis and breakdown rates suggest that it is not the starch content itself, but the ability to sustain a steady supply of soluble sugar, that is crucial for normal plant growth. The same phenotypic response of substantially reduced growth compared with the wild type (WT) was reported for both starch-excess (Caspar et al., 1991) and starch-deficient mutants (Caspar et al., 1985; Schulze et al., 1991; Sun et al., 1999). The capacity of these mutants to benefit from elevated CO2 conditions in terms of plant growth is also decreased compared with that of the WT. In Arabidopsis starch-deficient mutants, Sun et al. (1999) have shown that the growth response to atmospheric CO2 enrichment was closely related to starch turnover. Moreover, they observed that, in the case of a starchless mutant – when the capacity of the plant to use starch as transient carbon storage is null – increased atmospheric CO2 concentration did not stimulate growth at all. The photosynthetic response to elevated CO2 of starch-deficient transgenic potatoes was also found to be positively correlated to their capacity to produce leaf starch (Ludewig et al., 1998).
Arabidopsis mutants impaired in their starch metabolism display a sharply reduced growth rate and response to elevated CO2 compared with their nonmutated counterpart. Starch contents of A. thaliana leaves display a pronounced diurnal pattern, where starch is converted back to sugar during the night to sustain maintenance and growth (Cheng et al., 1998; Zeeman & ap Rees, 1999; Gibon et al., 2004). If the main difference between ambient and elevated CO2-grown plants happens during the night, as suggested by Grimmer & Komor (1999), then the ability of the plant to regulate a steady flux of available sugar during the night appears pivotal to normal growth and sustained response to elevated CO2.
In the present study, we hypothesize that reduced sugar availability during the night in both starch-deficient and starch-excess mutants of A. thaliana is responsible for their lower growth rates and reduced responses to elevated CO2 compared with the WT. To test this hypothesis, we developed an experimentally based model of the starch metabolism of A. thaliana as it relates to its growth pattern. The objective of the present research was to demonstrate that such a model can predict differential growth patterns between the WT and mutants based on single rate modifications of starch synthesis and breakdown in leaves.
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Plant carbohydrate allocation is known to exert a major regulatory role on growth response to elevated CO2 (Stitt, 1991; Makino & Mae, 1999). In the present study, we showed that single-parameter modifications of the starch synthesis and breakdown equations had profound effects on both the simulated growth of A. thaliana and its response to elevated CO2 (Figs 4–6). A reduction in STc was sufficient to model the reduced growth of sex1 compared with WT, which is in agreement with both our measured values (Fig. 4) and the literature (Caspar et al., 1991; Zeeman & ap Rees, 1999). Similarly, forcing the sugar-to-starch conversion to zero in the model produced realistic pgm : WT growth ratios compared with measured values (Fig. 4). This finding concurs with several studies reporting reduced growth of starchless vs WT (Schulze et al., 1991). Our results were obtained with a noncalibrated model containing only parameters that were either directly measured or computed from indirect measurements and literature data (Fig. 1; Table 2).
Our experimentally based model was possible with A. thaliana because of mutant availability, and because this species presents fairly simple starch and sugar cycles, which are mostly controlled by activities occurring within the leaves. Arabidopsis preferentially transforms excess photosynthates into leaf carbohydrates rather than increasing exports to sink organs such as roots (Geiger et al., 2000). In contrast, allocation models that have tried to incorporate multiple storage and structural sink terms require numerous nonmeasurable parameters left to calibration exercises (Escobar-Gutiérrez et al., 1998; Bonato et al., 1999; Yang & Midmore, 2005). In plants with larger nonleaf sink capacity than Arabidopsis, translocation might play a more substantial role. This potentially explains why woody plants with large storage capacity respond more to elevated CO2 than their herbaceous counterparts, as reported by Nowak et al. (2004). This does not mean, however, that the mechanisms described in our Arabidopsis model are not relevant to more complex plant architectures. Not only are the principles inherently similar, but within-leaf allocation processes remain potentially crucial in larger-storage plants because sugar export from leaves can be transport-rate limited, as reported by Grimmer & Komor (1999).
Starch and sugar pools in plant leaves are regulated by complex enzymatic machinery (Zeeman et al., 2004; Lloyd et al., 2005). Sugar and starch accumulation in leaves can have direct effects on photosynthesis through physiological mechanisms (Pritchard et al., 1997), biochemical feedbacks (for review see Paul & Foyer, 2001) and gene-expression control (Krapp et al., 1993; Jang et al., 1997; Moore et al., 1999). The interplay of these putative controllers under elevated CO2 conditions appears highly complex and uncertain. Here we argue that, in addition to potential biochemical feedbacks, the response of starch mutants to elevated CO2 might be controlled essentially by their capacity to supply a constant rate of soluble sugars that matches optimal growth demand. This capacity is affected very differently in sex1 and pgm mutants, and we need to consider the two cases separately.
For the sex1 starch-excess mutant, our model does not invoke any special relationship beyond the obvious fact that a portion of assimilates is sequestered in excessive starch reserves and therefore is not readily available for growth. Implemented in the model, this simple relationship was sufficient to explain our experimental results of a much reduced growth response of sex1 vs that of WT under both ambient and elevated CO2 conditions.
In the pgm starch-deficient mutant, all assimilates remain as growth-available soluble sugars. This calls for a feedback mechanism of soluble sugar content on growth in order to explain the reduced growth response of pgm vs that of the WT under both ambient and elevated CO2 conditions. Literature information suggests that an increase in soluble sugar concentration can directly downregulate photosynthesis in pgm mutants of Arabidopsis (Sun et al., 1999). However, both the magnitude and the mechanism of downregulation remain uncertain. Sun et al. (2002) reported that genotypes of starch mutants and wild types of Arabidopsis were similar in total activity and Rubisco content in response to elevated CO2 conditions. Kofler et al. (2000) reported no significant decrease in photosynthetic electron transport in pgm mutants compared with WT. These studies exemplify that the response mechanism remains elusive, which prevented us from integrating direct downregulation of photosynthesis into our model. Our experimental results indicate that maintenance respiration correlates positively with soluble sugar concentration, which provided us with the needed quantifiable feedback of soluble sugar concentration on pgm plant growth. This does not mean that direct downregulation does not happen, rather it suggests that the simple relationship linking soluble sugar concentration to maintenance respiration is a good mathematical approximation of the global contribution of both mechanisms (increased maintenance respiration and downregulation of photosynthesis) to the apparent reduction of CO2 assimilation and growth rate observed in starchless mutants. The exact importance of each remains to be determined, but our results emphasize the need for further evaluation of the role played by maintenance respiration in response to elevated CO2, at least in sink-limited conditions.
While several studies reported a link between maintenance respiration and tissue N concentration (Ryan, 1991; Ryan et al., 1996), it is clear that N concentration is only a proxy for biochemical processes, which are likely to include soluble sugar production. Overall, maintenance respiration remains a poorly understood process. Hirai et al. (2002) reported large differences in maintenance respiration rates among rice species, independent of N concentrations. The initial response of maintenance respiration to temperature changes is large, but subsequent acclimation occurs (Wythers et al., 2005). Most importantly, our results agree with those of Ogren (2000), who reported that maintenance respiration correlates with soluble sugar but not with nitrogen concentration in dormant plants. This author suggests that increases in maintenance respiration in response to increases in soluble sugar concentrations are directly linked to the costs of maintaining concentration gradients.
In our study, the simulated increase in maintenance respiration associated with increased sugar concentration substantially affected the pgm mutant only, and not the WT and sex1 mutant. A sensitivity analysis indicates that the simulated plant sizes for WT and sex1 remained nearly unaffected (between 2 and 6%) using this relationship (equation 17) vs considering a fixed basal maintenance respiration value (data not shown). The model therefore suggests that no negative feedback (whether through downregulation or increased respiration) of elevated CO2 on WT growth needs to be invoked to explain the difference in WT growth curves of Fig. 4a vs b. This absence of significant photosynthetic downregulation of Arabidopsis WT growth in response to elevated CO2 conditions under our experimental conditions was demonstrated in a parallel study (Tocquin et al., 2006).
In its present version, the model demonstrates that simple modifications of leaf starch production and degradation rates (themselves the result of complex enzymatic interplay) are bound to carry enormous effects on growth of leaf sink-limited plants and of their response to elevated CO2 conditions. It is certain that the present model is a simplification of the complexity of biochemical regulation. In addition to direct effects on photosynthesis, it is probable that soluble sugar availability modifies allocation to roots, although this effect is likely to be minor in Arabidopsis, as suggested by Geiger et al. (2000). It is also probable that the baseline-and-overflow starch production mechanism is smoothed and fine-tuned through biochemical regulation. As research progresses, we hope the present model will serve as a basis to probe the potential effects of these biochemical fine-tuning mechanisms on Arabidopsis growth and response to elevated CO2.
The maximum rate of leaf growth (GRmax) appeared as the most crucial allocation parameter affecting Arabidopsis response to elevated CO2, while the model suggests a substantial safety margin in the proportion of starch breakdown (STc) before effects on the response to elevated CO2 occur (Fig. 6 vs Fig. 5). In the present study, conducted in constant environmental conditions, starch synthesis was entirely controlled by GRmax because the baseline starch production rate (STbase) was always surpassed and therefore did not modify the simulation results (Fig. 1). Our simulations suggest that small changes in GRmax have a dramatic impact on the response to elevated CO2 (Fig. 6). The existence of a maximum relative growth rate is an important assumption of sugar–starch allocation models (Escobar-Gutiérrez et al., 1998). Without this sink-limited approach, the plant could use its photosynthetic sugar for immediate growth, and would have little advantage to invest in starch storage except for covering maintenance respiration costs during the night.
Plants of the C3 photosynthetic pathway respond positively to short-term increases in atmospheric CO2 concentration because of increased carboxylation efficiency. Nevertheless, in the long term this response varies considerably in magnitude among species (Curtis & Wang, 1998; Nowak et al., 2004). Neither resource-based nor plant functional type models provide a comprehensive explanation for this variability (Nowak et al., 2004). While a common photosynthetic machinery is shared among C3 species, the pattern of starch accumulation varies substantially among species (Zeeman et al., 2004 and references therein). Notwithstanding environmental stress factors and downregulation of photosynthesis as major controls over this variability (Drake & Rasse, 2003; Rasse et al., 2005), our study confirms that regulation of diurnal starch and sugar cycles in leaves can substantially control herbaceous plant response to elevated CO2.
Downregulation of photosynthesis in response to elevated CO2 conditions has been reported in numerous studies (Cheng et al., 1998; Makino & Mae, 1999; Bae & Sicher, 2004 and references therein), although exceptions exist (Sun et al., 2002; Zhao et al., 2004). A decrease in Rubisco enzyme concentration is often associated with a decrease in whole leaf N content (dePury & Farquhar, 1997). A similar enzymatic downregulation associated with N availability is likely to exist for GRmax. At this point we can only speculate as to which of these two enzymatic systems is most affected by downregulation processes. Interestingly, increased enzymatic sucrose synthesis, potentially at the expense of starch, has been shown to stimulate plant yields independently of modifications in photosynthetic activity (Sharkey et al., 2004). In a recent study, Bae & Sicher (2004) reported that the expression of six soluble proteins was modified in Arabidopsis plants subjected to elevated CO2 conditions. Of these six proteins, three were growth-related while only one was photosynthesis-related. These results support our model analysis (Fig. 6) that the photosynthetically induced stimulation of plant growth by elevated CO2 can be restricted by a downregulation of GRmax independently from downregulation of Vcmax.
In conclusion, the model demonstrates that simple modifications of starch synthesis and breakdown rates in Arabidopsis are bound to generate large differences in the photosynthetically induced growth stimulation by elevated CO2. Our results suggest that the night-time starch-to-sugar conversion rate, and even more so the maximum rate of leaf growth, which is a major component of the sink capacity of herbaceous plants, are key parameters needed in comprehensive mechanistic models that aim to predict plant responses to elevated CO2 conditions.