The triose phosphate use limitation was studied using long-term and short term changes in capacity. The TPU limitation caused increased proton motive force; long-term TPU limitation additionally reduced other photosynthetic components. Photosynthetic responses to CO2 can be interpreted primarily as being limited by the amount or activity of Rubisco or the capacity for ribulose bisphosphate regeneration, but at high rates of photosynthesis a third response is often seen. Photosynthesis becomes insensitive to CO2 or even declines with increasing CO2, and this behavior has been associated with a limitation of export of carbon from the Calvin-Benson cycle. It is often called the triose phosphate use (TPU) limitation. We studied the long-term consequences of this limitation using plants engineered to have reduced capacity for starch or sucrose synthesis. We studied short-term consequences using temperature as a method for changing the balance of carbon fixation capacity and TPU. A long-term and short-term TPU limitation resulted in an increase in proton motive force (PMF) in the thylakoids. Once a TPU limitation was reached, any further increases in CO2 was met with a further increase in the PMF but no increase or little increase in net assimilation of CO2. A long-term TPU limitation resulted in reduced Rubisco and RuBP regeneration capacity. We hypothesize that TPU, Rubisco activity, and RuBP regeneration are regulated so that TPU is normally in slight excess of what is required, and that this results in more effective regulation than if TPU were in large excess.

The exchange of reduced carbon across the inner chloroplast envelope has a large impact on photosynthesis and growth. Under steady-state conditions it is thought that glucose 6-phosphate (G6P) does not cross the chloroplast membrane. However, growth at high CO 2 , or disruption of starch metabolism can result in the GPT2 gene for a G6P/P i translocator to be expressed presumably allowing G6P exchange across the chloroplast envelope. We found that after an increase in light, the transcript for GPT2 transiently increases several 100-fold within 2 h in both the Col-0 and WS ecotypes of Arabidopsis thaliana . The increase in transcript for GPT2 is preceded by an increase in transcript for many transcription factors including Redox Responsive Transcription Factor 1 (RRTF1). The increase in GPT2 transcript after exposure to high light is suppressed in a mutant lacking the RRTF1 transcription factor. The GPT2 response was also suppressed in a mutant with a T-DNA insert in the gene for the triose-phosphate/P i translocator (TPT). However, plants lacking TPT still had a robust rise in RRTF1 transcript in response to high light. From this, we conclude that both RRTF1 (and possibly other transcription factors) and high amounts of cytosolic triose phosphate are required for induction of the expression of GPT2 . We hypothesize that transient GPT2 expression and subsequent translation is adaptive, allowing G6P to move into the chloroplast from the cytosol. The imported G6P can be used for starch synthesis or may flow directly into the Calvin-Benson cycle via an alternative pathway (the G6P shunt), which could be important for regulating and stabilizing photosynthetic electron transport and carbon metabolism.

Glucose-6-phosphate dehydrogenase (G6PDH) can initiate the glucose 6-phosphate (G6P) shunt around the Calvin-Benson cycle. In order to understand the regulation of flux through this pathway, we have characterized the biochemical parameters and redox regulation of the three functional plastidic isoforms of Arabidopsis G6PDH. When purified, recombinant proteins were measured, all three exhibited significant substrate inhibition by G6P but not NADP+, making the determination of enzyme kinetic parameters complex. We found that the half saturation concentration of G6PDH isoform 1 is increased under reducing conditions. The other two isoforms exhibit less redox regulation, however, isoform 2 is strongly inhibited by NADPH. Redox regulation of G6PDH1 can be partially reversed by hydrogen peroxide or protected against by presence of its substrate, G6P. Overall, our results support the conclusion that G6PDH can have significant activity throughout the day and can be dynamically regulated to allow or prevent flux through the glucose 6-phosphate shunt.

Differences between the wild type and treatments were tested by one-way ANOVA followed by Tukey's test in Microcal Origin 8.0. Three levels of significance were tested and indicated as follows: + , a = 0.1; *, a = 0.05; and **, a = 0.01. Box plots are presented with the box encompassing the middle two quartiles, the mean shown as an open square inside the box, the median shown as a line inside the box, and the whiskers showing the SD of the data. Accession NumbersAccession numbers for the mutants used in this study are as follows: hpr1-1, SALK_067724; hpr1-2, SALK_ 143584; and plgg1-1, SALK_053469. ACKNOWLEDGMENTSWe thank Francesco Loreto and Susanna Pollastri for the loan of the 13 CO 2insensitive LI-800 CO 2 analyzer. We also thank Jim Klug and Cody Keilen for their assistance in growing plants, the Arabidopsis Biological Resource Center for seeds, and Andreas Weber for plgg1 seeds. Antje von Schaewen made helpful comments on Figure 9.

Feeding 14CO2 was crucial to uncovering the path of carbon in photosynthesis. Feeding 13CO2 to photosynthesizing leaves emitting isoprene has been used to develop hypotheses about the sources of carbon for the methylerythritol 4-phosphate pathway, which makes the precursors for terpene synthesis in chloroplasts and bacteria. Both photosynthesis and isoprene studies found that products label very quickly (12C during photosynthesis and isoprene emission. Further, studies with isoprene showed that the proportion of slow label could vary significantly. This was interpreted as a variable contribution of carbon from sources other than the Calvin Benson cycle feeding the methylerythritol 4-phosphate pathway. Here we measured the degree of label in isoprene and photosynthetic metabolites 20 min after beginning to feed 13CO2. Isoprene labeling was the same as labeling of photosynthesis intermediates. High temperature reduced the label in isoprene and photosynthesis intermediates by the same amount indicating no role for alternative carbon sources for isoprene. A model assuming glucose, fructose, and/or sucrose reenters the Calvin Benson cycle as ribulose 5-phosphate through a cytosolic shunt involving glucose 6-phosphate dehydrogenase was consistent with the observations.

Deoxyxylulose 5-phosphate synthase (DXS), a thiamine diphosphate (ThDP) dependent enzyme, plays a regulatory role in the methylerythritol 4-phosphate (MEP) pathway. Isopentenyl diphosphate (IDP) and dimethylallyl diphosphate (DMADP), the end products of this pathway, inhibit DXS by competing with ThDP. Feedback inhibition of DXS by IDP and DMADP constitutes a significant metabolic regulation of this pathway. The aim of this work was to experimentally test the effect of key residues of recombinant poplar DXS (PtDXS) in binding both ThDP and IDP. This work also described the engineering of PtDXS to improve the enzymatic activity by reducing its inhibition by IDP and DMADP. We have designed and tested modifications of PtDXS in an attempt to reduce inhibition by IDP. This could possibly be valuable by removing a feedback that limits the usefulness of the MEP pathway in biotechnological applications. Both ThDP and IDP use similar interactions for binding at the active site of the enzyme, however, ThDP being a larger molecule has more anchoring sites at the active site of the enzyme as compared to the inhibitors. A predicted enzyme structure was examined to find ligand-enzyme interactions, which are relatively more important for inhibitor-enzyme binding than ThDP-enzyme binding, followed by their modifications so that the binding of the inhibitors can be selectively affected compared to ThDP. Two alanine residues important for binding ThDP and the inhibitors were mutated to glycine. In two of the cases, both the IDP inhibition and the overall activity were increased. In another case, both the IDP inhibition and the overall activity were reduced. This provides proof of concept that it is possible to reduce the feedback from IDP on DXS activity.

Phosphoglucoisomerase (PGI) isomerizes fructose 6-phosphate (F6P) and glucose 6-phosphate (G6P) in starch and sucrose biosynthesis. Both plastidic and cytosolic isoforms are found in plant leaves. Using recombinant enzymes and isolated chloroplasts, we have characterized the plastidic and cytosolic isoforms of PGI. We have found that the Arabidopsis plastidic PGI Km for G6P is three-fold greater compared to that for F6P and that erythrose 4-phosphate is a key regulator of PGI activity. Additionally, the Km of spinach plastidic PGI can be dynamically regulated in the dark compared to the light and increases by 200% in the dark. We also found that targeting Arabidopsis cytosolic PGI into plastids of Nicotiana tabacum disrupts starch accumulation and degradation. Our results, in combination with the observation that plastidic PGI is not in equilibrium, indicates that PGI is an important regulatory enzyme that restricts flow and acts as a one-way valve preventing backflow of G6P into the Calvin-Benson cycle. We propose the PGI may be manipulated to improve flow of carbon to desired targets of biotechnology.

Common beans (Phaseolus vulgaris) are highly sensitive to elevated temperatures, and rising global temperatures threaten bean production. Plants at the reproductive stage are especially susceptible to heat stress due to damage to male (anthers) and female (ovary) reproductive tissues, with anthers being more sensitive to heat. Heat damage promotes early tapetal cell degradation, and in beans this was shown to cause male infertility. In this study, we focus on understanding how changes in leaf carbon export in response to elevated temperature stress contribute to heat‐induced infertility. We hypothesize that anther glucose‐6‐phosphate dehydrogenase (G6PDH) activity plays an important role at elevated temperature and promotes thermotolerance. To test this hypothesis, we compared heat‐tolerant and susceptible common bean genotypes using a combination of phenotypic, biochemical, and physiological approaches. Our results identified changes in leaf sucrose export, anther sugar accumulation and G6PDH activity and anther H2O2 levels and antioxidant‐related enzymes between genotypes at elevated temperature. Further, anther respiration rate was found to be lower at high temperature in both bean varieties. Overall, our results support the hypothesis that enhanced male reproductive heat tolerance involves changes in the anther oxidative pentose phosphate pathway, which supplies reductants to critical H2O2 scavenging enzymes.