Chlamydomonas mutants lacking chloroplast TRIOSE PHOSPHATE TRANSPORTER3 are metabolically compromised and light sensitive.

Modulation of photoassimilate export from the chloroplast is essential for controlling the distribution of fixed carbon in the cell and maintaining optimum photosynthetic rates. In this study, we identified chloroplast TRIOSE PHOSPHATE/PHOSPHATE TRANSLOCATOR 2 (CreTPT2) and CreTPT3 in the green alga Chlamydomonas (Chlamydomonas reinhardtii), which exhibit similar substrate specificities but whose encoding genes are differentially expressed over the diurnal cycle. We focused mostly on CreTPT3 because of its high level of expression and the severe phenotype exhibited by tpt3 relative to tpt2 mutants. Null mutants for CreTPT3 had a pleiotropic phenotype that affected growth, photosynthetic activities, metabolite profiles, carbon partitioning, and organelle-specific accumulation of H2O2. These analyses demonstrated that CreTPT3 is a dominant conduit on the chloroplast envelope for the transport of photoassimilates. In addition, CreTPT3 can serve as a safety valve that moves excess reductant out of the chloroplast and appears to be essential for preventing cells from experiencing oxidative stress and accumulating reactive oxygen species, even under low/moderate light intensities. Finally, our studies indicate subfunctionalization of the TRIOSE PHOSPHATE/PHOSPHATE TRANSLOCATOR (CreTPT) transporters and suggest that there are differences in managing the export of photoassimilates from the chloroplasts of Chlamydomonas and vascular plants.

Three consecutive cytosolic glycolysis enzymes modulate autophagic flux.

Autophagy serves as an important recycling route for the growth and survival of eukaryotic organisms in nutrient-deficient conditions. Since starvation induces massive changes in the metabolic flux that are coordinated by key metabolic enzymes, specific processing steps of autophagy may be linked with metabolic flux-monitoring enzymes. We attempted to identify carbon metabolic genes that modulate autophagy using VIGS screening of 45 glycolysis- and Calvin-Benson cycle-related genes in Arabidopsis (Arabidopsis thaliana). Here, we report that three consecutive triose-phosphate-processing enzymes involved in cytosolic glycolysis, triose-phosphate-isomerase (TPI), glyceraldehyde-3-phosphate dehydrogenase (GAPC), and phosphoglycerate kinase (PGK), designated TGP, negatively regulate autophagy. Depletion of TGP enzymes causes spontaneous autophagy induction and increases AUTOPHAGY-RELATED 1 (ATG1) kinase activity. TGP enzymes interact with ATG101, a regulatory component of the ATG1 kinase complex. Spontaneous autophagy induction and abnormal growth under insufficient sugar in TGP mutants are suppressed by crossing with the atg101 mutant. Considering that triose-phosphates are photosynthates transported to the cytosol from active chloroplasts, the TGP enzymes would be strategically positioned to monitor the flow of photosynthetic sugars and modulate autophagy accordingly. Collectively, these results suggest that TGP enzymes negatively control autophagy acting upstream of the ATG1 complex, which is critical for seedling development.

Short-term salt stress reduces photosynthetic oscillations under triose phosphate utilization limitation in tomato.

Triose phosphate utilization (TPU) limitation is one of the three biochemical limitations of photosynthetic CO2 assimilation rate in C3 plants. Under TPU limitation, abrupt and large transitions in light intensity cause damped oscillations in photosynthesis. When plants are salt-stressed, photosynthesis is often down-regulated particularly under dynamic light intensity, but how salt stress affects TPU-related dynamic photosynthesis is still unknown. To elucidate this, tomato (Solanum lycopersicum) was grown with and without sodium chloride (NaCl, 100 mM) stress for 13 d. Under high CO2 partial pressure, rapid increases in light intensity caused profound photosynthetic oscillations. Salt stress reduced photosynthetic oscillations in leaves initially under both low- and high-light conditions and reduced the duration of oscillations by about 2 min. Besides, salt stress increased the threshold for CO2 partial pressure at which oscillations occurred. Salt stress increased TPU capacity without affecting Rubisco carboxylation and electron transport capacity, indicating the up-regulation of end-product synthesis capacity in photosynthesis. Thus salt stress may reduce photosynthetic oscillations by decreasing leaf internal CO2 partial pressure and/or increasing TPU capacity. Our results provide new insights into how salt stress modulates dynamic photosynthesis as controlled by CO2 availability and end-product synthesis.

An efficient triose phosphate synthesis and distribution in wheat provides tolerance to higher field temperatures.

High temperatures in the field hinder bread wheat high-yield production, mainly because of the adverse effects of heat over photosynthesis. The Yaqui Valley, the main wheat producer region in Mexico, is a zone prone to have temperatures over 30°C. The aim of this work was to test the flag leaf photosynthetic performance in 10 bread wheat genotypes grown under high temperatures in the field. The study took place during two seasons (2019-2020 and 2020-2021). In each season, control seeds were sown in December, while heat-stressed were sown in late January to subject wheat to heat stress (HS) during the grain-filling stage. HS reduced Grain yield from 20 to 58% in the first season. HS did not reduce chlorophyll content and light-dependent reactions were unaffected in any of the tested genotypes. Rubisco, chloroplast fructose 1,6-biphosphatase (FBPase), and sucrose phosphate synthase (SPS) activities were measured spectrophotometrically. Rubisco activity did not decrease under HS in any of the genotypes. FBPase activity was reduced by HS indicating that triose phosphate flux to starch synthesis was reduced, while SPS was not affected, and thus, sucrose synthesis was maintained. HS reduced aerial biomass in the 10 chosen genotypes. Genotypes SOKWB.1, SOKWB.3, and BORLAUG100 maintained their yield under HS, pointing to a potential success in their introduction in this region for breeding heat-tolerant bread wheat.

Remodeling of Carbon Metabolism during Sulfoglycolysis in Escherichia coli.

Sulfoquinovose (SQ) is a major metabolite in the global sulfur cycle produced by nearly all photosynthetic organisms. One of the major pathways involved in the catabolism of SQ in bacteria such as Escherichia coli is a variant of the glycolytic Embden-Meyerhof-Parnas (EMP) pathway termed the sulfoglycolytic EMP (sulfo-EMP) pathway, which leads to the consumption of three of the six carbons of SQ and the excretion of 2,3-dihydroxypropanesulfonate (DHPS). Comparative metabolite profiling of aerobically glucose (Glc)-grown and SQ-grown E. coli cells was undertaken to identify the metabolic consequences of the switch from glycolysis to sulfoglycolysis. Sulfoglycolysis was associated with the diversion of triose phosphates (triose-P) to synthesize sugar phosphates (gluconeogenesis) and an unexpected accumulation of trehalose and glycogen storage carbohydrates. Sulfoglycolysis was also associated with global changes in central carbon metabolism, as indicated by the changes in the levels of intermediates in the tricarboxylic acid (TCA) cycle, the pentose phosphate pathway (PPP), polyamine metabolism, pyrimidine metabolism, and many amino acid metabolic pathways. Upon entry into stationary phase and the depletion of SQ, E. coli cells utilize their glycogen, indicating a reversal of metabolic fluxes to allow glycolytic metabolism. IMPORTANCE The sulfosugar sulfoquinovose is estimated to be produced on a scale of 10 billion metric tons per annum, making it a major organosulfur species in the biosulfur cycle. The microbial degradation of sulfoquinovose through sulfoglycolysis allows the utilization of its carbon content and contributes to the biomineralization of its sulfur. However, the metabolic consequences of microbial growth on sulfoquinovose are unclear. We use metabolomics to identify the metabolic adaptations that Escherichia coli undergoes when grown on sulfoquinovose versus glucose. This revealed the increased flux into storage carbohydrates through gluconeogenesis and the reduced flux of carbon into the TCA cycle and downstream metabolism. These changes are relieved upon entry into stationary phase and reversion to glycolytic metabolism. This work provides new insights into the metabolic consequences of microbial growth on an abundant sulfosugar.

Enrichment of hepatic glycogen and plasma glucose from H218 O informs gluconeogenic and indirect pathway fluxes in naturally feeding mice.

Deuterated water (2 H2 O) is a widely used tracer of carbohydrate biosynthesis in both preclinical and clinical settings, but the significant kinetic isotope effects (KIE) of 2 H can distort metabolic information and mediate toxicity. 18 O-water (H2 18 O) has no significant KIE and is incorporated into specific carbohydrate oxygens via well-defined mechanisms, but to date it has not been evaluated in any animal model. Mice were given H2 18 O during overnight feeding and 18 O-enrichments of liver glycogen, triglyceride glycerol (TG), and blood glucose were quantified by 13 C NMR and mass spectrometry (MS). Enrichment of oxygens 5 and 6 relative to body water informed indirect pathway contributions from the Krebs cycle and triose phosphate sources. Compared with mice fed normal chow (NC), mice whose NC was supplemented with a fructose/glucose mix (i.e., a high sugar [HS] diet) had significantly higher indirect pathway contributions from triose phosphate sources, consistent with fructose glycogenesis. Blood glucose and liver TG 18 O-enrichments were quantified by MS. Blood glucose 18 O-enrichment was significantly higher for HS versus NC mice and was consistent with gluconeogenic fructose metabolism. TG 18 O-enrichment was extensive for both NC and HS mice, indicating a high turnover of liver triglyceride, independent of diet. Thus H2 18 O informs hepatic carbohydrate biosynthesis in similar detail to 2 H2 O but without KIE-associated risks.

Rapid CO2 changes cause oscillations in photosynthesis that implicate PSI acceptor-side limitations.

Oscillations in CO2 assimilation rate and associated fluorescence parameters have been observed alongside the triose phosphate utilization (TPU) limitation of photosynthesis for nearly 50 years. However, the mechanics of these oscillations are poorly understood. Here we utilize the recently developed dynamic assimilation techniques (DATs) for measuring the rate of CO2 assimilation to increase our understanding of what physiological condition is required to cause oscillations. We found that TPU-limiting conditions alone were insufficient, and that plants must enter TPU limitation quickly to cause oscillations. We found that ramps of CO2 caused oscillations proportional in strength to the speed of the ramp, and that ramps induce oscillations with worse outcomes than oscillations induced by step change of CO2 concentration. An initial overshoot is caused by a temporary excess of available phosphate. During the overshoot, the plant outperforms steady-state TPU and ribulose 1,5-bisphosphate regeneration limitations of photosynthesis, but cannot exceed the rubisco limitation. We performed additional optical measurements which support the role of PSI reduction and oscillations in availability of NADP+ and ATP in supporting oscillations.

Triose phosphate utilization in leaves is modulated by whole-plant sink-source ratios and nitrogen budgets in rice.

Triose phosphate utilization (TPU) is a biochemical process indicating carbon sink-source (im)balance within leaves. When TPU limits leaf photosynthesis, photorespiration-associated amino acid exports probably provide an additional carbon outlet and increase leaf CO2 uptake. However, whether TPU is modulated by whole-plant sink-source relations and nitrogen (N) budgets remains unclear. We address this question by model analyses of gas-exchange data measured on leaves at three growth stages of rice plants grown at two N levels. Sink-source ratio was manipulated by panicle pruning, by using yellower-leaf variant genotypes, and by measuring photosynthesis on adaxial and abaxial leaf sides. Across all these treatments, higher leaf N content resulted in the occurrence of TPU limitation at lower intercellular CO2 concentrations. Photorespiration-associated amino acid export was greater in high-N leaves, but was smaller in yellower-leaf genotypes, panicle-pruned plants, and for abaxial measurement. The feedback inhibition of panicle pruning on rates of TPU was not always observed, presumably because panicle pruning blocked N remobilization from leaves to grains and the increased leaf N content masked feedback inhibition. The leaf-level TPU limitation was thus modulated by whole-plant sink-source relations and N budgets during rice grain filling, suggesting a close link between within-leaf and whole-plant sink limitations.

Engineering of cellobiose phosphorylase for the defined synthesis of cellotriose.

Cellodextrins are non-digestible oligosaccharides that have attracted interest from the food industry as potential prebiotics. They are typically produced through the partial hydrolysis of cellulose, resulting in a complex mixture of oligosaccharides with a varying degree of polymerisation (DP). Here, we explore the defined synthesis of cellotriose as product since this oligosaccharide is believed to be the most potent prebiotic in the mixture. To that end, the cellobiose phosphorylase (CBP) from Cellulomonas uda and the cellodextrin phosphorylase (CDP) from Clostridium cellulosi were evaluated as biocatalysts, starting from cellobiose and α-D-glucose 1-phosphate as acceptor and donor substrate, respectively. The CDP enzyme was shown to rapidly elongate the chains towards higher DPs, even after extensive mutagenesis. In contrast, an optimised variant of CBP was found to convert cellobiose to cellotriose with a molar yield of 73%. The share of cellotriose within the final soluble cellodextrin mixture (DP2-5) was 82%, resulting in a cellotriose product with the highest purity reported to date. Interestingly, the reaction could even be initiated from glucose as acceptor substrate, which should further decrease the production costs.Key points• Cellobiose phosphorylase is engineered for the production of cellotriose.• Cellotriose is synthesised with the highest purity and yield to date.• Both cellobiose and glucose can be used as acceptor for cellotriose production.

A Poly(A) Ribonuclease Controls the Cellotriose-Based Interaction between Piriformospora indica and Its Host Arabidopsis.

Piriformospora indica, an endophytic root-colonizing fungus, efficiently promotes plant growth and induces resistance to abiotic stress and biotic diseases. P. indica fungal cell wall extract induces cytoplasmic calcium elevation in host plant roots. Here, we show that cellotriose (CT) is an elicitor-active cell wall moiety released by P. indica into the medium. CT induces a mild defense-like response, including the production of reactive oxygen species, changes in membrane potential, and the expression of genes involved in growth regulation and root development. CT-based cytoplasmic calcium elevation in Arabidopsis (Arabidopsis thaliana) roots does not require the BAK1 coreceptor or the putative Ca2+ channels TPC1, GLR3.3, GLR2.4, and GLR2.5 and operates synergistically with the elicitor chitin. We identified an ethyl methanesulfonate-induced mutant (cytoplasmiccalcium elevation mutant) impaired in the response to CT and various other cellooligomers (n = 2-7), but not to chitooligomers (n = 4-8), in roots. The mutant contains a single nucleotide exchange in the gene encoding a poly(A) ribonuclease (AtPARN; At1g55870) that degrades the poly(A) tails of specific mRNAs. The wild-type PARN cDNA, expressed under the control of a 35S promoter, complements the mutant phenotype. Our identification of cellotriose as a novel chemical mediator casts light on the complex P. indica-plant mutualistic relationship.

No evidence for triose phosphate limitation of light-saturated leaf photosynthesis under current atmospheric CO2 concentration.

The triose phosphate utilization (TPU) rate has been identified as one of the processes that can limit terrestrial plant photosynthesis. However, we lack a robust quantitative assessment of TPU limitation of photosynthesis at the global scale. As a result, TPU, and its potential limitation of photosynthesis, is poorly represented in terrestrial biosphere models (TBMs). In this study, we utilized a global data set of photosynthetic CO2 response curves representing 141 species from tropical rainforests to Arctic tundra. We quantified TPU by fitting the standard biochemical model of C3 photosynthesis to measured photosynthetic CO2 response curves and characterized its instantaneous temperature response. Our results demonstrate that TPU does not limit leaf photosynthesis at the current ambient atmospheric CO2 concentration. Furthermore, our results showed that the light-saturated photosynthetic rates of plants growing in cold environments are not more often limited by TPU than those of plants growing in warmer environments. In addition, our study showed that the instantaneous temperature response of TPU is distinct from temperature response of the maximum rate of Rubisco carboxylation. The new formulations of the temperature response of TPU derived in this study may prove useful in quantifying the biochemical limits to terrestrial plant photosynthesis and improve the representation of plant photosynthesis in TBMs.

Is triose phosphate utilization involved in the feedback inhibition of photosynthesis in rice under conditions of sink limitation?

This study aimed to understand the physiological basis of rice photosynthetic response to C source-sink imbalances, focusing on the dynamics of the photosynthetic parameter triose phosphate utilization (TPU). Here, rice (Oriza sativa L.) indica cultivar IR64 were grown in controlled environment chambers under current ambient CO2 concentration until heading, and thereafter two CO2 treatments (400 and 800 µmol mol-1) were compared in the presence and absence of a panicle-pruning treatment modifying the C sink. At 2 weeks after heading, photosynthetic parameters derived from CO2 response curves, and non-structural carbohydrate content of flag leaf and internodes were measured three to four times of day. Spikelet number per panicle and flag leaf area on the main culm were recorded. Net C assimilation and TPU decreased progressively after midday in panicle-pruned plants, especially under 800 µmol mol-1 CO2. This TPU reduction was explained by sucrose accumulation in the flag leaf resulting from the sink limitation. Taking together, our findings suggest that TPU is involved in the regulation of photosynthesis in rice under elevated CO2 conditions, and that sink limitation effects should be considered in crop models.

Triose phosphate utilization and beyond: from photosynthesis to end product synthesis.

During photosynthesis, plants fix CO2 from the atmosphere onto ribulose-bisphosphate, producing 3-phosphoglycerate, which is reduced to triose phosphates (TPs). The TPs are then converted into the end products of photosynthesis. When a plant is photosynthesizing very quickly, it may not be possible to commit photosynthate to end products as fast as it is produced, causing a decrease in available phosphate and limiting the rate of photosynthesis to the rate of triose phosphate utilization (TPU). The occurrence of an observable TPU limitation is highly variable based on species and especially growth conditions, with TPU capacity seemingly regulated to be in slight excess of typical photosynthetic rates the plant might experience. The physiological effects of TPU limitation are discussed with an emphasis on interactions between the Calvin-Benson cycle and the light reactions. Methods for detecting TPU-limited data from gas exchange data are detailed and the impact on modeling of some physiological effects are shown. Special consideration is given to common misconceptions about TPU.

Three glycoside hydrolase family 12 enzymes display diversity in substrate specificities and synergistic action between each other.

PoCel12A, PoCel12B, and PoCel12C are genes that encode glycoside hydrolase family 12 (GH12) enzymes in Penicillium oxalicum. PoCel12A and PoCel12B are typical GH12 enzymes that belong to fungal subfamilies 12-1 and 12-2, respectively. PoCel12C contains a low-complexity region (LCR) domain, which is not found in PoCel12A or PoCel12B and independent of fungal subfamily 12-1 or 12-2. Recombinant enzymes (named rCel12A, rCel12B and rCel12C) demonstrate existing diversity in the substrate specificities. Although most members in GH family 12 are typical endoglucanases and preferentially hydrolyze ß-1,4-glucan (e.g., carboxymethylcellulose), recombinant PoCel12A is a non-typical endo-(1-4)-ß-glucanase; it preferentially hydrolyzes mix-linked ß-glucan (barley ß-glucan, ß-1,3-1,4-glucan) and slightly hydrolyzes ß-1,4-glucan (carboxymethylcellulose). Recombinant PoCel12B possesses a significantly high activity against xyloglucan. A specific activity of rCel12B toward xyloglucan (239 µmol/min/mg) is the second-highest value known. Recombinant PoCel12C shows low activity toward ß-glucan, carboxymethylcellulose, or xyloglucan. All three enzymes can degrade phosphoric acid-swollen cellulose (PASC). However, the hydrolysis products toward PASC by enzymes are different: the main hydrolysis products are cellotriose, cellotetraose, and cellobiose for rCel12A, rCel12B, and rCel12C, correspondingly. A synergistic action toward PASC among rCel12A and rCel12B is observed, thereby suggesting a potential application for preparing enzyme cocktails used in lignocellulose hydrolysis.

Biochemical characterization and low-resolution SAXS structure of two-domain endoglucanase BlCel9 from Bacillus licheniformis.

Lignocellulose feedstock constitutes the most abundant carbon source in the biosphere; however, its recalcitrance remains a challenge for microbial conversion into biofuel and bioproducts. Bacillus licheniformis is a microbial mesophilic bacterium capable of secreting a large number of glycoside hydrolase (GH) enzymes, including a glycoside hydrolase from GH family 9 (BlCel9). Here, we conducted biochemical and biophysical studies of recombinant BlCel9, and its low-resolution molecular shape was retrieved from small angle X-ray scattering (SAXS) data. BlCel9 is an endoglucanase exhibiting maximum catalytic efficiency at pH 7.0 and 60 °C. Furthermore, it retains 80% of catalytic activity within a broad range of pH values (5.5-8.5) and temperatures (up to 50 °C) for extended periods of time (over 48 h). It exhibits the highest hydrolytic activity against phosphoric acid swollen cellulose (PASC), followed by bacterial cellulose (BC), filter paper (FP), and to a lesser extent carboxymethylcellulose (CMC). The HPAEC-PAD analysis of the hydrolytic products demonstrated that the end product of the enzymatic hydrolysis is primarily cellobiose, and also small amounts of glucose, cellotriose, and cellotetraose are produced. SAXS data analysis revealed that the enzyme adopts a monomeric state in solution and has a molecular mass of 65.8 kDa as estimated from SAXS data. The BlCel9 has an elongated shape composed of an N-terminal family 3 carbohydrate-binding module (CBM3c) and a C-terminal GH9 catalytic domain joined together by 20 amino acid residue long linker peptides. The domains are closely juxtaposed in an extended conformation and form a relatively rigid structure in solution, indicating that the interactions between the CBM3c and GH9 catalytic domains might play a key role in cooperative cellulose biomass recognition and hydrolysis.

Toxicity of dihydroxyacetone is exerted through the formation of methylglyoxal in Saccharomyces cerevisiae: effects on actin polarity and nuclear division.

Dihydroxyacetone (DHA) is the smallest ketotriose, and it is utilized by many organisms as an energy source. However, at higher concentrations, DHA becomes toxic towards several organisms including the budding yeast Saccharomyces cerevisiae In the present study, we show that DHA toxicity is due to its spontaneous conversion to methylglyoxal (MG) within yeast cells. A mutant defective in MG-metabolizing enzymes (glo1Δgre2Δgre3Δ) exhibited higher susceptibility to DHA. Intracellular MG levels increased following the treatment of glo1Δgre2Δgre3Δ cells with DHA. We previously reported that MG depolarized the actin cytoskeleton and changed vacuolar morphology. We herein demonstrated the depolarization of actin and morphological changes in vacuoles following a treatment with DHA. Furthermore, we found that both MG and DHA caused the morphological change in nucleus, and inhibited the nuclear division. Our results suggest that the conversion of DHA to MG is a dominant contributor to its cytotoxicity.

Genotype, environment and G × E interaction influence (1,3;1,4)-ß-d-glucan fine structure in barley (Hordeum vulgare L.).

BACKGROUND: The structure of ß-glucan influences its use in cereal-based foods and feed. The objective of this study was to determine the effect of environment (E) and genotype (G) on ß-glucan fine structure and its genetic control in two-row spring barley with normal starch characteristics. RESULTS: A population of 89 recombinant inbred lines, derived from the cross of two-row spring barley genotypes Merit × H93174006 (H92076F1 × TR238), was characterized for concentration and structure of grain ß-glucan in two environments. Results showed that concentrations of ß-glucan, DP3, DP4 and DP3 + DP4 were positively correlated with each other, suggesting no preference for DP3 or DP4 subunit production in high- or low-ß-glucan lines. The concentrations of ß-glucan, DP3, DP4 and DP3:DP4 ratios were significantly influenced by genotype and environment. However, only DP3:DP4 ratio showed a significant effect of G × E interaction. Association mapping of candidate markers in 119 barley genotypes showed that marker CSLF6_4105 was associated with ß-glucan concentration, whereas Bmac504 and Bmac211 were associated with DP3:DP4 ratio. Bmac273e was associated with both ß-glucan concentration and DP3:DP4 ratio. CONCLUSION: The grain ß-glucan concentration and DP3:DP4 ratio are strongly affected by genotype and environment. Single-marker analyses suggested that the genetic control of ß-glucan concentration and DP3:DP4 ratio was linked to separate chromosomal regions on barley genome. © 2016 Society of Chemical Industry.

Triose phosphate use limitation of photosynthesis: short-term and long-term effects.

MAIN CONCLUSION: 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.