Glyceraldehyde-3-phosphate dehydrogenase subunits A and B are essential to maintain photosynthetic efficiency.

In plants, glyceraldehyde-3-phosphate dehydrogenase (GAPDH; EC 1.2.1.12) reversibly converts 1,3-bisphosphoglycerate to glyceraldehyde-3-phosphate coupled with the reduction of NADPH to NADP+. The GAPDH enzyme that functions in the Calvin-Benson cycle is assembled either from 4 glyceraldehyde-3-phosphate dehydrogenase A (GAPA) subunit proteins forming a homotetramer (A4) or from 2 GAPA and 2 glyceraldehyde-3-phosphate dehydrogenase B (GAPB) subunit proteins forming a heterotetramer (A2B2). The relative importance of these 2 forms of GAPDH in determining the rate of photosynthesis is unknown. To address this question, we measured the photosynthetic rates of Arabidopsis (Arabidopsis thaliana) plants containing reduced amounts of the GAPDH A and B subunits individually and jointly, using T-DNA insertion lines of GAPA and GAPB and transgenic GAPA and GAPB plants with reduced levels of these proteins. Here, we show that decreasing the levels of either the A or B subunits decreased the maximum efficiency of CO2 fixation, plant growth, and final biomass. Finally, these data showed that the reduction in GAPA protein to 9% wild-type levels resulted in a 73% decrease in carbon assimilation rates. In contrast, eliminating GAPB protein resulted in a 40% reduction in assimilation rates. This work demonstrates that the GAPA homotetramer can compensate for the loss of GAPB, whereas GAPB alone cannot compensate fully for the loss of the GAPA subunit.

Increased sedoheptulose-1,7-bisphosphatase content in Setaria viridis does not affect C4 photosynthesis.

Sedoheptulose-1,7-bisphosphatase (SBPase) is one of the rate-limiting enzymes of the Calvin cycle, and increasing the abundance of SBPase in C3 plants provides higher photosynthetic rates and stimulates biomass and yield. C4 plants usually have higher photosynthetic rates because they operate a biochemical CO2-concentrating mechanism between mesophyll and bundle sheath cells. In the C4 system, SBPase and other enzymes of the Calvin cycle are localized to the bundle sheath cells. Here we tested what effect increasing abundance of SBPase would have on C4 photosynthesis. Using green foxtail millet (Setaria viridis), a model C4 plant of NADP-ME subtype, we created transgenic plants with 1.5 to 3.2 times higher SBPase content compared to wild-type plants. Transcripts of the transgene were found predominantly in the bundle sheaths suggesting the correct cellular localization of the protein. The abundance of ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit was not affected in transgenic plants overexpressing SBPase, and neither was leaf chlorophyll content or photosynthetic electron transport parameters. We found no association between SBPase content in S. viridis and saturating rates of CO2 assimilation. Moreover, a detailed analysis of CO2 assimilation rates at different CO2 partial pressures, irradiances, and leaf temperatures showed no improvement of photosynthesis in plants overexpressing SBPase. We discuss the potential implications of these results for understanding the role of SBPase in regulation of C4 photosynthesis.

Rieske FeS overexpression in tobacco provides increased abundance and activity of cytochrome b6 f.

Photosynthesis is fundamental for plant growth and yield. The cytochrome b6 f complex catalyses a rate-limiting step in thylakoid electron transport and therefore represents an important point of regulation of photosynthesis. Here we show that overexpression of a single core subunit of cytochrome b6 f, the Rieske FeS protein, led to up to a 40% increase in the abundance of the complex in Nicotiana tabacum (tobacco) and was accompanied by an enhanced in vitro cytochrome f activity, indicating a full functionality of the complex. Analysis of transgenic plants overexpressing Rieske FeS by the light-induced fluorescence transients technique revealed a more oxidised primary quinone acceptor of photosystem II (QA ) and plastoquinone pool and faster electron transport from the plastoquinone pool to photosystem I upon changes in irradiance, compared to control plants. A faster establishment of qE , the energy-dependent component of nonphotochemical quenching, in transgenic plants suggests a more rapid buildup of the transmembrane proton gradient, also supporting the increased in vivo cytochrome b6 f activity. However, there was no consistent increase in steady-state rates of electron transport or CO2 assimilation in plants overexpressing Rieske FeS grown in either laboratory conditions or field trials, suggesting that the in vivo activity of the complex was only transiently increased upon changes in irradiance. Our results show that overexpression of Rieske FeS in tobacco enhances the abundance of functional cytochrome b6 f and may have the potential to increase plant productivity if combined with other traits.

Suppression of metabolite shuttles for export of chloroplast and mitochondrial ATP and NADPH increases the cytosolic NADH:NAD+ ratio in tobacco leaves in the dark.

The communication between chloroplasts and mitochondria, which depends on the inter-organellar exchange of carbon skeletons, energy, and reducing equivalents, is essential for maintaining efficient respiratory metabolism and photosynthesis. We devised a multi-transgene approach to manipulate the leaf energy and redox balance in tobacco (Nicotiana tabacum) while monitoring the in vivo cytosolic redox status of NAD(H) using the biosensor c-Peredox-mCherry. Our strategy involved altering the shuttling capacity of the chloroplast by (1) increasing the chloroplast malate valve capacity by overexpression of the chloroplast malate valve transporter pOMT from Arabidopsis (AtpOMT1) while (2) reducing the activity of the chloroplast triose-phosphate/3-phosphoglycerate shuttle by knocking down the cytosolic NAD-dependent glyceraldehyde 3-phosphate dehydrogenase (NtGAPC). This was accompanied by (3) alterations to the export of reducing equivalents in the mitochondria by knocking down the mitochondrial malate dehydrogenase (NtmMDH) and (4) an increased expression of the mitochondrial fission regulator FIS1A from Arabidopsis (AtFIS1A). The multi-transgene tobacco plants were analysed in glasshouse conditions and showed significant increases in the cytosolic NADH:NAD+ in the dark when transcript levels for NtGAPC or NtmMDH were knocked down. In addition, principal component analysis and Spearman correlation analyses showed negative correlations between average transcript levels for the gene targets and parameters related to chlorophyll fluorescence and plant growth. Our results highlight the importance of the shuttling of energy and reducing equivalents from chloroplasts and mitochondria to support photosynthesis and growth and suggest an important role for the dual 2-oxoglutarate/malate and oxaloacetate/malate transporter (pOMT).

Stimulating photosynthetic processes increases productivity and water-use efficiency in the field.

Previous studies have demonstrated that the independent stimulation of either electron transport or RuBP regeneration can increase the rate of photosynthetic carbon assimilation and plant biomass. In this paper, we present evidence that a multigene approach to simultaneously manipulate these two processes provides a further stimulation of photosynthesis. We report on the introduction of the cyanobacterial bifunctional enzyme fructose-1,6-bisphosphatase/sedoheptulose-1,7-bisphosphatase or the overexpression of the plant enzyme sedoheptulose-1,7-bisphosphatase, together with the expression of the red algal protein cytochrome c6, and show that a further increase in biomass accumulation under both glasshouse and field conditions can be achieved. Furthermore, we provide evidence that the stimulation of both electron transport and RuBP regeneration can lead to enhanced intrinsic water-use efficiency under field conditions.

Overexpression of the Rieske FeS protein of the Cytochrome b6f complex increases C4 photosynthesis in Setaria viridis.

C4 photosynthesis is characterised by a CO2 concentrating mechanism that operates between mesophyll and bundle sheath cells increasing CO2 partial pressure at the site of Rubisco and photosynthetic efficiency. Electron transport chains in both cell types supply ATP and NADPH for C4 photosynthesis. Cytochrome b6f is a key control point of electron transport in C3 plants. To study whether C4 photosynthesis is limited by electron transport we constitutively overexpressed the Rieske FeS subunit in Setaria viridis. This resulted in a higher Cytochrome b6f content in mesophyll and bundle sheath cells without marked changes in the abundances of other photosynthetic proteins. Rieske overexpression plants showed better light conversion efficiency in both Photosystems and could generate higher proton-motive force across the thylakoid membrane underpinning an increase in CO2 assimilation rate at ambient and saturating CO2 and high light. Our results demonstrate that removing electron transport limitations can increase C4 photosynthesis.

Feeding the world: improving photosynthetic efficiency for sustainable crop production.

A number of recent studies have provided strong support demonstrating that improving the photosynthetic processes through genetic engineering can provide an avenue to improve yield potential. The major focus of this review is on improvement of the Calvin-Benson cycle and electron transport. Consideration is also given to how altering regulatory process may provide an additional route to increase photosynthetic efficiency. Here we summarize some of the recent successes that have been observed through genetic manipulation of photosynthesis, showing that, in both the glasshouse and the field, yield can be increased by >40%. These results provide a clear demonstration of the potential for increasing yield through improvements in photosynthesis. In the final section, we consider the need to stack improvement in photosynthetic traits with traits that target the yield gap in order to provide robust germplasm for different crops across the globe.

Overexpressing the H-protein of the glycine cleavage system increases biomass yield in glasshouse and field-grown transgenic tobacco plants.

Photorespiration is essential for C3 plants, enabling oxygenic photosynthesis through the scavenging of 2-phosphoglycolate. Previous studies have demonstrated that overexpression of the L- and H-proteins of the photorespiratory glycine cleavage system results in an increase in photosynthesis and growth in Arabidopsis thaliana. Here, we present evidence that under controlled environment conditions an increase in biomass is evident in tobacco plants overexpressing the H-protein. Importantly, the work in this paper provides a clear demonstration of the potential of this manipulation in tobacco grown in field conditions, in two separate seasons. We also demonstrate the importance of targeted overexpression of the H-protein using the leaf-specific promoter ST-LS1. Although increases in the H-protein driven by this promoter have a positive impact on biomass, higher levels of overexpression of this protein driven by the constitutive CaMV 35S promoter result in a reduction in the growth of the plants. Furthermore in these constitutive overexpressor plants, carbon allocation between soluble carbohydrates and starch is altered, as is the protein lipoylation of the enzymes pyruvate dehydrogenase and alpha-ketoglutarate complexes. Our data provide a clear demonstration of the positive effects of overexpression of the H-protein to improve yield under field conditions.

Optimizing photorespiration for improved crop productivity.

In C3 plants, photorespiration is an energy-expensive process, including the oxygenation of ribulose-1,5-bisphosphate (RuBP) by ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) and the ensuing multi-organellar photorespiratory pathway required to recycle the toxic byproducts and recapture a portion of the fixed carbon. Photorespiration significantly impacts crop productivity through reducing yields in C3 crops by as much as 50% under severe conditions. Thus, reducing the flux through, or improving the efficiency of photorespiration has the potential of large improvements in C3 crop productivity. Here, we review an array of approaches intended to engineer photorespiration in a range of plant systems with the goal of increasing crop productivity. Approaches include optimizing flux through the native photorespiratory pathway, installing non-native alternative photorespiratory pathways, and lowering or even eliminating Rubisco-catalyzed oxygenation of RuBP to reduce substrate entrance into the photorespiratory cycle. Some proposed designs have been successful at the proof of concept level. A plant systems-engineering approach, based on new opportunities available from synthetic biology to implement in silico designs, holds promise for further progress toward delivering more productive crops to farmer's fields.

Development of pGEMINI, a Plant Gateway Destination Vector Allowing the Simultaneous Integration of Two cDNA via a Single LR-Clonase Reaction.

Gateway technology has been used to facilitate the generation of a large number of constructs for the modification of plants for research purposes. However, many of the currently available vectors only allow the integration of a single cDNA of interest into an expression clone. The ability to over-express multiple genes in combination is essential for the study of plant development where several transcripts have a role to play in one or more metabolic processes. The tools to carry out such studies are limited, and in many cases rely on the incorporation of cDNA into expression systems via conventional cloning, which can be both time consuming and laborious. To our knowledge, this study reports on the first development of a vector allowing the simultaneous integration of two independent cDNAs via a single LR-clonase reaction. This vector "pGEMINI" represents a powerful molecular tool offering the ability to study the role of multi-cDNA constructs on plant development, and opens up the process of gene stacking and the study of gene combinations through transient or stable transformation procedures.

Simultaneous stimulation of sedoheptulose 1,7-bisphosphatase, fructose 1,6-bisphophate aldolase and the photorespiratory glycine decarboxylase-H protein increases CO2 assimilation, vegetative biomass and seed yield in Arabidopsis.

In this article, we have altered the levels of three different enzymes involved in the Calvin-Benson cycle and photorespiratory pathway. We have generated transgenic Arabidopsis plants with altered combinations of sedoheptulose 1,7-bisphosphatase (SBPase), fructose 1,6-bisphophate aldolase (FBPA) and the glycine decarboxylase-H protein (GDC-H) gene identified as targets to improve photosynthesis based on previous studies. Here, we show that increasing the levels of the three corresponding proteins, either independently or in combination, significantly increases the quantum efficiency of PSII. Furthermore, photosynthetic measurements demonstrated an increase in the maximum efficiency of CO2 fixation in lines over-expressing SBPase and FBPA. Moreover, the co-expression of GDC-H with SBPase and FBPA resulted in a cumulative positive impact on leaf area and biomass. Finally, further analysis of transgenic lines revealed a cumulative increase of seed yield in SFH lines grown in high light. These results demonstrate the potential of multigene stacking for improving the productivity of food and energy crops.

The CP12 protein family: a thioredoxin-mediated metabolic switch?

CP12 is a small, redox-sensitive protein, representatives of which are found in most photosynthetic organisms, including cyanobacteria, diatoms, red and green algae, and higher plants. The only clearly defined function for CP12 in any organism is in the thioredoxin-mediated regulation of the Calvin-Benson cycle. CP12 mediates the formation of a complex between glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphoribulokinase (PRK) in response to changes in light intensity. Under low light, the formation of the GAPDH/PRK/CP12 complex results in a reduction in the activity of both PRK and GAPDH and, under high light conditions, thioredoxin mediates the disassociation of the complex resulting in an increase in both GAPDH and PRK activity. Although the role of CP12 in the redox-mediated formation of the GAPDH/PRK/CP12 multiprotein complex has been clearly demonstrated, a number of studies now provide evidence that the CP12 proteins may play a wider role. In Arabidopsis thaliana CP12 is expressed in a range of tissue including roots, flowers, and seeds and antisense suppression of tobacco CP12 disrupts metabolism and impacts on growth and development. Furthermore, in addition to the higher plant genomes which encode up to three forms of CP12, analysis of cyanobacterial genomes has revealed that, not only are there multiple forms of the CP12 protein, but that in these organisms CP12 is also found fused to cystathionine-ß-synthase domain containing proteins. In this review we present the latest information on the CP12 protein family and explore the possibility that CP12 proteins form part of a redox-mediated metabolic switch, allowing organisms to respond to rapid changes in the external environment.

Cloning, expression and biochemical characterization of mitochondrial and cytosolic malate dehydrogenase from Phytophthora infestans.

The genes of the mitochondrial and cytosolic malate dehydrogenase (mMDH and cMDH) of Phytophthora infestans were cloned and overexpressed in Escherichia coli as active enzymes. The catalytic properties of these proteins were determined: both enzymes have a similar specific activity. In addition, the natural mitochondrial isoenzyme was semi-purified from mycelia and its catalytic properties determined: the recombinant mitochondrial isoform behaved as the natural enzyme. A phylogenetic analysis indicated that mMDH, present in all stramenopiles studied, can be useful to study the relationships between these organisms. MDH with the conserved domain MDH_cytoplasmic_cytosolic is absent in some stramenopiles as well as in fungi. This enzyme seems to be less related within the stramenopile group. The Phytophthora cMDHs have an insertion of six amino acids that is also present in the stramenopile cMDHs studied, with the exception of Thalassiosira pseudonana cMDH, and is absent in other known eukaryotic cMDHs.