Activity of the Calvin cycle enzyme sedoheptulose-1,7-bisphosphatase (SBPase) was increased by overexpression of an Arabidopsis (Arabidopsis thaliana) cDNA in tobacco (Nicotiana tabacum) plants. In plants with increased SBPase activity, photosynthetic rates were increased, higher levels of Suc and starch accumulated during the photoperiod, and an increase in leaf area and biomass of up to 30% was also evident. Light saturated photosynthesis increased with increasing SBPase activity and analysis of CO 2 response curves revealed that this increase in photosynthesis could be attributed to an increase in ribulose 1,5-bisphosphate regenerative capacity. Seedlings with increased SBPase activity had an increased leaf area at the 4 to 5 leaf stage when compared to wild-type plants, and chlorophyll fluorescence imaging of these young plants revealed a higher photosynthetic capacity at the whole plant level. Measurements of photosynthesis, made under growth conditions integrated over the day, showed that mature plants with increased SBPase activity fixed 6% to 12% more carbon than equivalent wild-type leaves, with the young leaves having the highest rates. In this paper, we have shown that photosynthetic capacity per unit area and plant yield can be increased by overexpressing a single native plant enzyme, SBPase, and that this gives an advantage to the growth of these plants from an early phase of vegetative growth. This work has also shown that it is not necessary to bypass the normal regulatory control of SBPase, exerted by conditions in the stroma, to achieve improvements in carbon fixation.The photosynthetic carbon reduction (Calvin) cycle is the primary pathway for fixation of atmospheric CO 2 . This cycle plays a central role in plant metabolism, providing intermediates not only for starch and Suc biosynthesis, but also for isoprenoid metabolism and shikimic acid biosynthesis (Geiger and Servaites, 1994). Recently, a major focus has been to identify the individual steps that control carbon flux through the Calvin cycle. To address this question, antisense plants have been produced in which the levels of individual enzymes in the cycle have been reduced and the response of photosynthesis in these plants has been measured. This has allowed the contribution that individual enzymes exert over the control of flux through the Calvin cycle to be quantified (for review, see Stitt and Schulze, 1994;Raines, 2003). A number of interesting and surprising results have emerged from these analyses. Firstly, it was shown that for plants grown in moderate light and temperature conditions, more than 50% of wild-type levels of Rubisco could be removed before any significant effect on ambient photosynthesis was observed. However, under conditions of high light, reductions in photosynthesis were proportionate to decreases in Rubisco activity Stitt et al., 1991;Hudson et al., 1992;Krapp et al., 1994;Stitt and Schulze, 1994). Reductions of greater than 50% in the levels of glyceraldehyde-3-P dehydrogenase, Fru-1,6-bisphosphatase (FBPase)...

The Arabidopsis mutant pho3 accumulates sucrose and other carbohydrates to high levels, providing a means of investigating the genomic response to sucrose accumulation using microarray analysis. Wild-type and mutant plants were grown in soil to the mature rosette stage for the analysis of gene expression using the Affymetrix ATH1 chip, containing more than 22,500 probe sets. Small, but significant, decreases were observed in the expression of many genes encoding enzymes and regulatory proteins involved in primary carbon assimilation, suggesting that, in mature leaves of Arabidopsis, there is limited feedback regulation on gene expression by sugars. The study revealed a striking increase in the expression of the plastid glucose 6-phosphate/phosphate translocator, characteristically expressed only in heterotrophic tissues. This indicated a change in the nature of metabolite exchange between the plastid and the cytosol in the pho3 mutant. The expression of enzymes of starch synthesis also increased significantly. Very large increases were observed in the expression of transcription factors and enzymes involved in anthocyanin biosynthesis. This finding reinforces the emerging picture of an important role for primary metabolism in regulating secondary metabolism.

A Calvin cycle multiprotein complex including phosphoribulokinase (PRK), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and a small protein, CP12, has previously been identified. In this article, we have studied this complex in leaves and have shown that dissociation and reassociation of the PRK/GAPDH/CP12 complex occurs in a time frame of minutes, allowing for rapid regulation of enzyme activity. Furthermore, we have shown that the extent of formation and dissociation of the PRK/GAPDH/CP12 complex correlates with the quantity of light. These data provide evidence linking the status of this complex with the rapid and subtle regulation of GAPDH and PRK activities in response to fluctuations in light availability. We have also demonstrated that dissociation of this complex depends on electron transport chain activity and that the major factor involved in the dissociation of the pea complex was thioredoxin f. We show here that both PRK and GAPDH are present in the reduced form in leaves in the dark, but are inactive, demonstrating the role of the PRK/GAPDH/CP12 complex in deactivating these enzymes in response to reductions in light intensity. Based on our data, we propose a model for thioredoxin f-mediated activation of PRK and GAPDH by two mechanisms: directly through reduction of disulfide bonds within these enzymes and indirectly by mediating the breakdown of the complex in response to changes in light intensity.Calvin cycle ͉ photosynthesis ͉ protein-protein interactions ͉ redox

Transgenic tobacco (Nicotiana tabacum L. cv. Samsun) plants with reduced levels of the Calvin cycle enzyme sedoheptulose-1,7-bisphosphatase (SBPase; EC 3.1.3.37) were produced using an antisense construct in which the expression of a tobacco SBPase cDNA clone was driven by the cauli¯ower mosaic virus (CaMV) promoter. The reduction in SBPase protein levels observed in the primary transformants correlated with the presence of the antisense construct and lower levels of the endogenous SBPase mRNA. No changes in the amounts of other Calvin cycle enzymes were detected using Western blot analysis. The SBPase antisense plants with less than 20% of wild-type SBPase activity were observed to display a range of phenotypes, including chlorosis and reduced growth rates. Measurements of photosynthesis, using both light-dosage response and CO 2 response curves, of T1 plants revealed a reduction in carbon assimilation rates, which was apparent in plants retaining 57% of wild-type SBPase activity. Reductions were also observed in the quantum eciency of photosystem II. This decrease in photosynthetic capacity was re¯ected in a reduction in the carbohydrate content of leaves. Analysis of carbohydrate status in fully expanded source leaves showed a shift in carbon allocation away from starch, whilst sucrose levels were maintained in all but the most severely aected plants. Plants with less than 15% of wild-type SBPase activity were found to contain less than 5% of wild-type starch levels. The results of this preliminary analysis indicate that SBPase activity may limit the rate of carbon assimilation.Abbreviations: A = rate of CO 2 assimilation; CaMV = cauliower mosaic virus; C i = intercellular CO 2 concentration; FBPase = fructose-1,6-bisphosphatase; GAPDH = glyceraldehyde-3-phosphate dehydrogenase; LSU = large subunit; PRKase = phosphoribulokinase; RuBP = ribulose-1,5-bisphosphate; SBP = sedoheptulose-1,7-bisphosphate; SBPase = sedoheptulose-1,7-bisphosphatase Correspondence to: C.A. Raines;

We show here that light stimulates the expression of nuclear genes in wheat leaves for chloroplast fructose-1,6-bisphosphatase (FBPase) and describe a sequence of amino acids in this enzyme which may be responsible, via thioredoxin, for the light regulation of its activity. This data results from (a) our isolation and characterization of a cDNA of this enzyme which contains its entire coding sequence, and (b) our use of this cDNA as a probe to detect mRNA levels in wheat plants subjected to different light regimes. The similarity in amino acid sequence of the encoded enzyme from diverse sources suggests that the FBPase genes all had a common origin. However, their control sequences have been adjusted so that they are appropriately expressed and their coding sequences modified so that the enzymic activity of their products are suitably regulated in the particular cellular environment in which they must function. The light-activated regulatory sequences in the gene for the chloroplast protein have probably come together by a shuffling of DNA segments.