Thioredoxins o1 and h2 jointly adjust mitochondrial dihydrolipoamide dehydrogenase-dependent pathways towards changing environments.

Thioredoxins (TRXs) are central to redox regulation, modulating enzyme activities to adapt metabolism to environmental changes. Previous research emphasized mitochondrial and microsomal TRX o1 and h2 influence on mitochondrial metabolism, including photorespiration and the tricarboxylic acid (TCA) cycle. Our study aimed to compare TRX-based regulation circuits towards environmental cues mainly affecting photorespiration. Metabolite snapshots, phenotypes and CO2 assimilation were compared among single and multiple TRX mutants in the wild-type and the glycine decarboxylase T-protein knockdown (gldt1) background. Our analyses provided evidence for additive negative effects of combined TRX o1 and h2 deficiency on growth and photosynthesis. Especially metabolite accumulation patterns suggest a shared regulation mechanism mainly on mitochondrial dihydrolipoamide dehydrogenase (mtLPD1)-dependent pathways. Quantification of pyridine nucleotides, in conjunction with 13C-labelling approaches, and biochemical analysis of recombinant mtLPD1 supported this. It also revealed mtLPD1 inhibition by NADH, pointing at an additional measure to fine-tune it's activity. Collectively, we propose that lack of TRX o1 and h2 perturbs the mitochondrial redox state, which impacts on other pathways through shifts in the NADH/NAD+ ratio via mtLPD1. This regulation module might represent a node for simultaneous adjustments of photorespiration, the TCA cycle and branched chain amino acid degradation under fluctuating environmental conditions.

Plant NADPH-dependent thioredoxin reductases are crucial for the metabolism of sink leaves and plant acclimation to elevated CO2.

Plants contain three NADPH-thioredoxin reductases (NTR) located in the cytosol/mitochondria (NTRA/B) and the plastid (NTRC) with important metabolic functions. However, mutants deficient in all NTRs remained to be investigated. Here, we generated and characterised the triple Arabidopsis ntrabc mutant alongside with ntrc single and ntrab double mutants under different environmental conditions. Both ntrc and ntrabc mutants showed reduced growth and substantial metabolic alterations, especially in sink leaves and under high CO2 (HC), as compared to the wild type. However, ntrabc showed higher effective quantum yield of PSII under both constant and fluctuating light conditions, altered redox states of NADH/NAD+ and glutathione (GSH/GSSG) and lower potential quantum yield of PSII in sink leaves in ambient but not high CO2 concentrations, as compared to ntrc, suggesting a functional interaction between chloroplastic and extra-chloroplastic NTRs in photosynthesis regulation depending on leaf development and environmental conditions. Our results unveil a previously unknown role of the NTR system in regulating sink leaf metabolism and plant acclimation to HC, while it is not affecting full plant development, indicating that the lack of the NTR system can be compensated, at least to some extent, by other redox mechanisms.

Phycobilisome breakdown effector NblD is required to maintain the cellular amino acid composition during nitrogen starvation.

Small proteins are critically involved in the acclimation response of photosynthetic cyanobacteria to nitrogen starvation. NblD is the 66-amino-acid effector of nitrogen-limitation-induced phycobilisome breakdown, which is believed to replenish the cellular amino acid pools. To address the physiological functions of NblD, the concentrations of amino acids, intermediates of the arginine catabolism pathway and several organic acids were measured during the response to nitrogen starvation in the cyanobacterium Synechocystis sp. PCC 6803 wild type and in an nblD deletion strain. A characteristic signature of metabolite pool composition was identified, which shows that NblD-mediated phycobilisome degradation is required to maintain the cellular amino acid and organic acid pools during nitrogen starvation. Specific deviations from the wild type suggest wider-reaching effects that also affect such processes as redox homeostasis via glutathione and tetrapyrrole biosynthesis, both of which are linked to the strongly decreased glutamate pool, and transcriptional reprogramming via an enhanced concentration of 2-oxoglutarate, the metabolite co-regulator of the NtcA transcription factor. The essential role played by NblD in metabolic homeostasis is consistent with the widespread occurrence of NblD throughout the cyanobacterial radiation and the previously observed strong positive selection for the nblD gene under fluctuating nitrogen supply. Importance Cyanobacteria play important roles in the global carbon and nitrogen cycles. In their natural environment, these organisms are exposed to fluctuating nutrient conditions. Nitrogen starvation induces a coordinated nitrogen-saving program that includes the breakdown of nitrogen-rich photosynthetic pigments, particularly phycobiliproteins. The small protein NblD was recently identified as an effector of phycobilisome breakdown in cyanobacteria. In this study, we demonstrate that the NblD-mediated degradation of phycobiliproteins is needed to sustain cellular pools of soluble amino acids and other crucial metabolites. The essential role played by NblD in metabolic homeostasis explains why genes encoding this small protein are conserved in almost all members of cyanobacterial radiation.

Thioredoxin-mediated regulation of (photo)respiration and central metabolism.

Thioredoxins (TRXs) are ubiquitous proteins engaged in the redox regulation of plant metabolism. Whilst the light-dependent TRX-mediated activation of Calvin-Benson cycle enzymes is well documented, the role of extraplastidial TRXs in the control of the mitochondrial (photo)respiratory metabolism has been revealed relatively recently. Mitochondrially located TRX o1 has been identified as a regulator of alternative oxidase, enzymes of, or associated with, the tricarboxylic acid (TCA) cycle, and the mitochondrial dihydrolipoamide dehydrogenase (mtLPD) involved in photorespiration, the TCA cycle, and the degradation of branched chain amino acids. TRXs are seemingly a major point of metabolic regulation responsible for activating photosynthesis and adjusting mitochondrial photorespiratory metabolism according to the prevailing cellular redox status. Furthermore, TRX-mediated (de)activation of TCA cycle enzymes contributes to explain the non-cyclic flux mode of operation of this cycle in illuminated leaves. Here we provide an overview on the decisive role of TRXs in the coordination of mitochondrial metabolism in the light and provide in silico evidence for other redox-regulated photorespiratory enzymes. We further discuss the consequences of mtLPD regulation beyond photorespiration and provide outstanding questions that should be addressed in future studies to improve our understanding of the role of TRXs in the regulation of central metabolism.

The impact of photorespiration on plant primary metabolism through metabolic and redox regulation.

Photorespiration is an inevitable trait of all oxygenic phototrophs, being the only known metabolic route that converts the inhibitory side-product of Rubisco's oxygenase activity 2-phosphoglycolate (2PG) back into the Calvin-Benson (CB) cycle's intermediate 3-phosphoglycerate (3PGA). Through this function of metabolite repair, photorespiration is able to protect photosynthetic carbon assimilation from the metabolite intoxication that would occur in the present-day oxygen-rich atmosphere. In recent years, much plant research has provided compelling evidence that photorespiration safeguards photosynthesis and engages in cross-talk with a number of subcellular processes. Moreover, the potential of manipulating photorespiration to increase the photosynthetic yield potential has been demonstrated in several plant species. Considering this multifaceted role, it is tempting to presume photorespiration itself is subject to a suite of regulation mechanisms to eventually exert a regulatory impact on other processes, and vice versa. The identification of potential pathway interactions and underlying regulatory aspects has been facilitated via analysis of the photorespiratory mutant phenotype, accompanied by the emergence of advanced omics' techniques and biochemical approaches. In this mini-review, I focus on the identification of enzymatic steps which control the photorespiratory flux, as well as levels of transcriptional, posttranslational, and metabolic regulation. Most importantly, glycine decarboxylase (GDC) and 2PG are identified as being key photorespiratory determinants capable of controlling photorespiratory flux and communicating with other branches of plant primary metabolism.

Redox-Regulation of Photorespiration through Mitochondrial Thioredoxin o1.

Photorespiration sustains photosynthesis in the presence of oxygen due to rapid metabolization of 2-phosphoglycolate, the major side-product of the oxygenase activity of Rubisco that also directly impedes carbon assimilation and allocation. Despite the fact that both the biochemical reactions and the underlying genetics are well characterized, information concerning the regulatory mechanisms that adjust photorespiratory flux is rare. Here, we studied the impact of mitochondrial-localized thioredoxin o1 (TRXo1) on photorespiratory metabolism. The characterization of an Arabidopsis (Arabidopsis thaliana) transfer DNA insertional line (trxo1-1) revealed an increase in the stoichiometry of photorespiratory CO2 release and impaired Gly-to-Ser turnover after a shift from high-to-low CO2 without changes in Gly decarboxylase (GDC) gene or protein expression. These effects were distinctly pronounced in a double mutant, where the TRXo1 mutation was combined with strongly reduced GDC T-protein expression. The double mutant (TxGT) showed reduced growth in air but not in high CO2, decreased photosynthesis, and up to 54-fold more Gly alongside several redox-stress-related metabolites. Given that GDC proteins are potential targets for redox-regulation, we also examined the in vitro properties of recombinant GDC l-proteins (lipoamide dehydrogenase) from plants and the cyanobacterium Synechocystis species strain PCC6803 and observed a redox-dependent inhibition by either artificial reducing agents or TRXo1 itself. Collectively, our results demonstrate that TRXo1 potentially adjusts photorespiration via redox-regulation of GDC in response to environmental changes.

Thioredoxin h2 contributes to the redox regulation of mitochondrial photorespiratory metabolism.

Thioredoxins (TRXs) are important proteins involved in redox regulation of metabolism. In plants, it has been shown that the mitochondrial metabolism is regulated by the mitochondrial TRX system. However, the functional significance of TRX h2, which is found at both cytosol and mitochondria, remains unclear. Arabidopsis plants lacking TRX h2 showed delayed seed germination and reduced respiration alongside impaired stomatal and mesophyll conductance, without impacting photosynthesis under ambient O2 conditions. However, an increase in the stoichiometry of photorespiratory CO2 release was found during O2 -dependent gas exchange measurements in trxh2 mutants. Metabolite profiling of trxh2 leaves revealed alterations in key metabolites of photorespiration and in several metabolites involved in respiration and amino acid metabolism. Decreased abundance of serine hydroxymethyltransferase and glycine decarboxylase (GDC) H and L subunits as well as reduced NADH/NAD+ ratios were also observed in trxh2 mutants. We further demonstrated that the redox status of GDC-L is altered in trxh2 mutants in vivo and that recombinant TRX h2 can deactivate GDC-L in vitro, indicating that this protein is redox regulated by the TRX system. Collectively, our results demonstrate that TRX h2 plays an important role in the redox regulation of mitochondrial photorespiratory metabolism.

Photorespiration-how is it regulated and how does it regulate overall plant metabolism?

Under the current atmospheric conditions, oxygenic photosynthesis requires photorespiration to operate. In the presence of low CO2/O2 ratios, ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) performs an oxygenase side reaction, leading to the formation of high amounts of 2-phosphoglycolate during illumination. Given that 2-phosphoglycolate is a potent inhibitor of photosynthetic carbon fixation, it must be immediately removed through photorespiration. The core photorespiratory cycle is orchestrated across three interacting subcellular compartments, namely chloroplasts, peroxisomes, and mitochondria, and thus cross-talks with a multitude of other cellular processes. Over the past years, the metabolic interaction of photorespiration and photosynthetic CO2 fixation has attracted major interest because research has demonstrated the enhancement of C3 photosynthesis and growth through the genetic manipulation of photorespiration. However, to optimize future engineering approaches, it is also essential to improve our current understanding of the regulatory mechanisms of photorespiration. Here, we summarize recent progress regarding the steps that control carbon flux in photorespiration, eventually involving regulatory proteins and metabolites. In this regard, both genetic engineering and the identification of various layers of regulation point to glycine decarboxylase as the key enzyme to regulate and adjust the photorespiratory carbon flow. Potential implications of the regulation of photorespiration for acclimation to environmental changes along with open questions are also discussed.

The Photorespiratory Metabolite 2-Phosphoglycolate Regulates Photosynthesis and Starch Accumulation in Arabidopsis.

The Calvin-Benson cycle and its photorespiratory repair shunt are in charge of nearly all biological CO2 fixation on Earth. They interact functionally and via shared carbon flow on several levels including common metabolites, transcriptional regulation, and response to environmental changes. 2-Phosphoglycolate (2PG) is one of the shared metabolites and produced in large amounts by oxidative damage of the CO2 acceptor molecule ribulose 1,5-bisphosphate. It was anticipated early on, although never proven, that 2PG could also be a regulatory metabolite that modulates central carbon metabolism by inhibition of triose-phosphate isomerase. Here, we examined this hypothesis using transgenic Arabidopsis thaliana lines with varying activities of the 2PG-degrading enzyme, 2PG phosphatase, and analyzing the impact of this intervention on operation of the Calvin-Benson cycle and other central pathways, leaf carbohydrate metabolism, photosynthetic gas exchange, and growth. Our results demonstrate that 2PG feeds back on the Calvin-Benson cycle. It also alters the allocation of photosynthates between ribulose 1,5-bisphosphate regeneration and starch synthesis. 2PG mechanistically achieves this by inhibiting the Calvin-Benson cycle enzymes triose-phosphate isomerase and sedoheptulose 1,7-bisphosphate phosphatase. We suggest this may represent one of the control loops that sense the ratio of photorespiratory to photosynthetic carbon flux and in turn adjusts stomatal conductance, photosynthetic CO2 and photorespiratory O2 fixation, and starch synthesis in response to changes in the environment.

Effects of prolonged food limitation on energy metabolism and burrowing activity of an infaunal marine bivalve, Mya arenaria.

Benthic organisms are subject to prolonged seasonal food limitation in the temperate shallow coastal waters that can cause energetic stress and affect their performance. Sediment-dwelling marine bivalves cope with prolonged food limitation by adjusting different physiological processes that might cause trade-offs between maintenance and other fitness-related functions. We investigated the effects of prolonged (42 days) food deprivation on bioenergetics, burrowing performance and amino acid profiles in a common marine bivalve, Mya arenaria collected in winter and spring. Food limitation of >15 days decreased respiration of the clams by 80%. Total tissue energy content was higher in spring-collected clams (reflecting higher lipid content) than in their winter counterparts. Prolonged food deprivation decreased the tissue energy content of clams, especially in winter. The levels of free amino acids transiently increased during the early phase of food deprivation possibly reflecting suppression of the protein synthesis or enhanced protein degradation. The levels of amino acids considered essential for bivalves were more tightly conserved than those of non-essential amino acids during starvation. The burrowing capacity of clams was negatively affected by food deprivation so that the time required for a burial cycle increased by 35-50% after 22-42 days of starvation. During the early phase of starvation, clams preferentially used lipids as fuel for burrowing, whereas carbohydrates were used at the later phase. These findings suggest that although M. arenaria can withstand prolonged food deprivation by lowering their basal maintenance costs and switching their fuel usage, their ecological functions (e.g. bioturbation and the energy transferable to the next trophic level) could be negatively impacted by starvation.

Effects of hypoxia and reoxygenation on intermediary metabolite homeostasis of marine bivalves Mytilus edulis and Crassostrea gigas.

Marine benthic invertebrates are frequently exposed to fluctuating oxygen levels resulting in hypoxia-reoxygenation (H/R) stress in the intertidal, estuarine and shallow coastal habitats. H/R stress can strongly affect the organisms' physiological performance due to the negative shifts in bioenergetics and redox balance. H/R stress commonly leads to the depletion of energy substrates and accumulation of anaerobic end products, but the effects of H/R stress on the homeostasis of the intermediate nitrogenous compounds are not well understood. We studied the effects of the short-term and long-term hypoxia (1 and 6 days, respectively) and subsequent reoxygenation on the metabolite profiles of free amino acids (FAAs), as well as the intermediates of the urea cycle and purine metabolism in two species of hypoxia-tolerant intertidal bivalves, the blue mussels Mytilus edulis and the Pacific oysters Crassostrea gigas. Accumulation of succinate was assessed to determine the role of anaerobiosis in the metabolic responses to H/R stress. Our study showed that the more hypoxia-tolerant of the two studied species (C. gigas) had lower rate of succinate accumulation during hypoxia (indicating stronger metabolic rate suppression) and was better able to maintain the homeostasis of nitrogenous intermediates during H/R stress compared with the less hypoxia-tolerant M. edulis. Furthermore, analysis of the metabolite profiles indicate that the oysters maintain high levels of cytoprotective compounds (such as taurine and GABA), accumulate lower levels of potential prooxidants (such as succinate and hypoxanthine) and experience less damage to oxidation-prone thiol-containing amino acids such as cysteine, homocysteine and methionine during hypoxia and reoxygenation compared with the blue mussels. This study indicates a potentially important role of intermediate metabolite homeostasis in the tolerance to prolonged hypoxia and H/R stress in marine organisms and opens avenue for further testing of this hypothesis in a broader comparative framework.

Non-canonical localization of RubisCO under high-light conditions in the toxic cyanobacterium Microcystis aeruginosa PCC7806.

The frequent production of the hepatotoxin microcystin (MC) and its impact on the lifestyle of bloom-forming cyanobacteria are poorly understood. Here, we report that MC interferes with the assembly and the subcellular localization of RubisCO, in Microcystis aeruginosa PCC7806. Immunofluorescence, electron microscopic and cellular fractionation studies revealed a pronounced heterogeneity in the subcellular localization of RubisCO. At high cell density, RubisCO particles are largely separate from carboxysomes in M. aeruginosa and relocate to the cytoplasmic membrane under high-light conditions. We hypothesize that the binding of MC to RubisCO promotes its membrane association and enables an extreme versatility of the enzyme. Steady-state levels of the RubisCO CO2 fixation product 3-phosphoglycerate are significantly higher in the MC-producing wild type. We also detected noticeable amounts of the RubisCO oxygenase reaction product secreted into the medium that may support the mutual interaction of M. aeruginosa with its heterotrophic microbial community.

Glycolate Induces Redox Tuning Of Photosystem II in Vivo: Study of a Photorespiration Mutant.

Bicarbonate removal from the nonheme iron at the acceptor side of photosystem II (PSII) was shown recently to shift the midpoint potential of the primary quinone acceptor QA to a more positive potential and lowers the yield of singlet oxygen (1O2) production. The presence of QA- results in weaker binding of bicarbonate, suggesting a redox-based regulatory and protective mechanism where loss of bicarbonate or exchange of bicarbonate by other small carboxylic acids may protect PSII against 1O2 in vivo under photorespiratory conditions. Here, we compared the properties of QA in the Arabidopsis (Arabidopsis thaliana) photorespiration mutant deficient in peroxisomal HYDROXYPYRUVATE REDUCTASE1 (hpr1-1), which accumulates glycolate in leaves, with the wild type. Photosynthetic electron transport was affected in the mutant, and chlorophyll fluorescence showed slower electron transport between QA and QB in the mutant. Glycolate induced an increase in the temperature maximum of thermoluminescence emission, indicating a shift of the midpoint potential of QA to a more positive value. The yield of 1O2 production was lowered in thylakoid membranes isolated from hpr1-1 compared with the wild type, consistent with a higher potential of QA/QA- In addition, electron donation to photosystem I was affected in hpr1-1 at higher light intensities, consistent with diminished electron transfer out of PSII. This study indicates that replacement of bicarbonate at the nonheme iron by a small carboxylate anion occurs in plants in vivo. These findings suggested that replacement of the bicarbonate on the nonheme iron by glycolate may represent a regulatory mechanism that protects PSII against photooxidative stress under low-CO2 conditions.

Efficient 2-phosphoglycolate degradation is required to maintain carbon assimilation and allocation in the C4 plant Flaveria bidentis.

Photorespiration is indispensable for oxygenic photosynthesis since it detoxifies and recycles 2-phosphoglycolate (2PG), which is the primary oxygenation product of Rubisco. However, C4 plant species typically display very low rates of photorespiration due to their efficient biochemical carbon-concentrating mechanism. Thus, the broader relevance of photorespiration in these organisms remains unclear. In this study, we assessed the importance of a functional photorespiratory pathway in the C4 plant Flaveria bidentis using knockdown of the first enzymatic step, namely 2PG phosphatase (PGLP). The isolated RNAi lines showed strongly reduced amounts of PGLP protein, but distinct signs of the photorespiratory phenotype only emerged below 5% residual PGLP protein. Lines with this characteristic were stunted in growth, had strongly increased 2PG content, exhibited accelerated leaf senescence, and accumulated high amounts of branched-chain and aromatic amino acids, which are both characteristics of incipient carbon starvation. Oxygen-dependent gas-exchange measurements consistently suggested the cumulative impairment of ribulose-1,5-bisphosphate regeneration with increased photorespiratory pressure. Our results indicate that photorespiration is essential for maintaining high rates of C4 photosynthesis by preventing the 2PG-mediated inhibition of carbon utilization efficiency. However, considerably higher 2PG accumulation can be tolerated compared to equivalent lines of C3 plants due to the differential distribution of specific enzymatic steps between the mesophyll and bundle sheath cells.

Interactive effects of osmotic stress and burrowing activity on protein metabolism and muscle capacity in the soft shell clam Mya arenaria.

Bioturbators such as sediment-dwelling marine bivalves are ecosystem engineers that enhance sediment-water exchange and benthic-pelagic coupling. In shallow coastal areas, bivalves are exposed to frequent disturbance and salinity stress that might negatively affect their activity and physiological performance; however, the mechanisms underlying these effects are not fully understood. We investigated the effects of osmotic stress (low and fluctuating salinity) and repeated burrowing on aerobic and contractile capacity of the foot muscle (assessed by the activity of succinate dehydrogenase and myosin ATPase) as well as the levels of organic osmolytes (free amino acids) and biochemical markers of protein synthesis and proteolysis in key osmoregulatory and energy storing tissues (gills and hepatopancreas, respectively) in a common bioturbator, the soft shell clam Mya arenaria. Osmotic stress and exhaustive exercise altered the foot muscle capacity of soft shell clams and had a strong impact on protein and amino acid homeostasis in tissues not directly involved in locomotion. Acclimation to constant low salinity (5 practical salinity units) depleted the whole-body free amino acid pool and affected protein synthesis but not protein breakdown in the gill. In contrast, fluctuating (5-15) salinity increased protein breakdown rate, suppressed protein synthesis, caused oxidative damage to proteins in the gill and selectively depleted whole-body glycine pool. Clams acclimated to normal salinity (15) increased the aerobic capacity of the foot muscle upon repeated burrowing, whereas acclimation to low and fluctuating salinity reduced this adaptive muscle plasticity. Under the normal and low salinity conditions, exhaustive exercise induced protein conservation pathways (indicated by suppression of protein synthesis and catabolism), but this effect was disrupted by fluctuating salinity. These findings indicate that exhaustive exercise and osmotic stress interactively affect whole-body protein homeostasis and functional capacity of the foot muscle in soft shell clams which might contribute to reduced burrowing activity of bivalve bioturbators in osmotically challenging environments such as estuaries and shallow coastal zones.

T-protein is present in large excess over the other proteins of the glycine cleavage system in leaves of Arabidopsis.

MAIN CONCLUSION: T-protein is present in large excess over the other proteins of the glycine cleavage system in leaves of Arabidopsis and therefore, exerts little control over the photorespiratory pathway. T-protein is the aminomethyltransferase of the glycine cleavage multienzyme system (GCS), also known as the glycine decarboxylase complex, and essential for photorespiration and one-carbon metabolism. Here, we studied what effects varying levels of the GCS T-protein would have on GCS activity, the operation of the photorespiratory pathway, photosynthesis, and plant growth. To this end, we examined Arabidopsis thaliana T-protein overexpression lines with up to threefold higher amounts of leaf T-protein as well as one knockdown mutant with about 5% residual leaf T-protein and one knockout mutant. Overexpression did not alter photosynthetic CO2 uptake and plant growth, and the knockout mutation was lethal even in the non-photorespiratory environment of air enriched to 1% CO2. Unexpectedly in light of this very low T-protein content, however, the knockdown mutant was able to grow and propagate in normal air and displayed only some minor changes, such as a moderate glycine accumulation in combination with somewhat delayed growth. Neither overexpression nor the knockdown of T-protein altered the amounts of the other three GCS proteins, suggesting that the biosynthesis of the GCS proteins is not synchronized at this level. We also observed that the knockdown causes less T-protein mostly in leaf mesophyll cells, but not so much in the vasculature, and discuss this phenomenon in light of the dual involvement of the GCS and hence T-protein in plant metabolism. Collectively, this work shows that T-protein is present in large excess over the other proteins of the glycine cleavage system in leaves of Arabidopsis and therefore exerts little control over the photorespiratory pathway.

Mitochondrial Dihydrolipoyl Dehydrogenase Activity Shapes Photosynthesis and Photorespiration of Arabidopsis thaliana.

Mitochondrial dihydrolipoyl dehydrogenase (mtLPD; L-protein) is an integral component of several multienzyme systems involved in the tricarboxylic acid (TCA) cycle, photorespiration, and the degradation of branched-chain α-ketoacids. The majority of the mtLPD present in photosynthesizing tissue is used for glycine decarboxylase (GDC), necessary for the high-flux photorespiratory glycine-into-serine conversion. We previously suggested that GDC activity could be a signal in a regulatory network that adjusts carbon flux through the Calvin-Benson cycle in response to photorespiration. Here, we show that elevated GDC L-protein activity significantly alters several diagnostic parameters of cellular metabolism and leaf gas exchange in Arabidopsis thaliana. Overexpressor lines displayed markedly decreased steady state contents of TCA cycle and photorespiratory intermediates as well as elevated NAD(P)(+)-to-NAD(P)H ratios. Additionally, increased rates of CO2 assimilation, photorespiration, and plant growth were observed. Intriguingly, however, day respiration rates remained unaffected. By contrast, respiration was enhanced in the first half of the dark phase but depressed in the second. We also observed enhanced sucrose biosynthesis in the light in combination with a lower diel magnitude of starch accumulation and breakdown. These data thus substantiate our prior hypothesis that facilitating flux through the photorespiratory pathway stimulates photosynthetic CO2 assimilation in the Calvin-Benson cycle. They furthermore suggest that this regulation is, at least in part, dependent on increased light-capture/use efficiency.

Ozone-Sensitive Arabidopsis Mutants with Deficiencies in Photorespiratory Enzymes.

An ozone-sensitive mutant was isolated from T-DNA-tagged lines of Arabidopsis thaliana. The T-DNA was inserted at a locus on chromosome 3, where two genes encoding glycolate oxidases, GOX1 and GOX2, peroxisomal enzymes involved in photorespiration, reside contiguously. The amounts of the mutant's foliar transcripts for these genes were reduced, and glycolate oxidase activity was approximately 60% of that of the wild-type plants. No difference in growth and appearance was observed between the mutant and the wild-type plants under normal conditions with ambient air under a light intensity of 100 µmol photons m-2 s-1. However, signs of severe damage, such as chlorosis and ion leakage from the tissue, rapidly appeared in mutant leaves in response to ozone treatment at a concentration of 0.2 µl l-1 under a higher light intensity of 350 µmol photons m-2 s-1 that caused no such symptoms in the wild-type plant. The mutant also exhibited sensitivity to sulfur dioxide and long-term high-intensity light. Arabidopsis mutants with deficiencies in other photorespiratory enzymes such as glutamate:glyoxylate aminotransferase and hydroxypyruvate reductase also exhibited ozone sensitivities. Therefore, photorespiration appears to be involved in protection against photooxidative stress caused by ozone and other abiotic factors under high-intensity light.

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.

High serine:glyoxylate aminotransferase activity lowers leaf daytime serine levels, inducing the phosphoserine pathway in Arabidopsis.

Serine:glyoxylate aminotransferase (SGAT) converts glyoxylate and serine to glycine and hydroxypyruvate during photorespiration. Besides this, SGAT operates with several other substrates including asparagine. The impact of this enzymatic promiscuity on plant metabolism, particularly photorespiration and serine biosynthesis, is poorly understood. We found that elevated SGAT activity causes surprisingly clear changes in metabolism and interferes with photosynthetic CO2 uptake and biomass accumulation of Arabidopsis. The faster serine turnover during photorespiration progressively lowers day-time leaf serine contents and in turn induces the phosphoserine pathway. Transcriptional upregulation of this additional route of serine biosynthesis occurs already during the day but particularly at night, efficiently counteracting night-time serine depletion. Additionally, higher SGAT activity results in an increased use of asparagine as the external donor of amino groups to the photorespiratory pathway but does not alter leaf asparagine content at night. These results suggest leaf SGAT activity needs to be dynamically adjusted to ensure (i) variable flux through the photorespiratory pathway at a minimal consumption of asparagine and (ii) adequate serine levels for other cellular metabolism.