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• The variation in tolerance to low oxygen is likely explained by divergent sets of molecular and metabolic responses between species. • We analysed the versatility of the response to low oxygen of primary metabolism by comparing nine previously published metabolome profiling studies. Data were juxtaposed with expression profiles of genes encoding enzymes involved in the metabolic pathways of rice, Arabidopsis and poplar. Furthermore, full transcript profiles were compared to determine commonalities in the expression of orthologous genes and genes that serve similar functions. • Activation of fermentation and the accumulation of alanine plus succinate were observed in all species, but transcriptional regulation of these metabolic pathways varied. Global analysis of orthologue expression revealed that most differentially expressed genes either had no orthologues or were not affected in the other species. Expression analysis of nearly all gene clusters with common functions varied significantly between species. • The resemblance of the metabolic response to hypoxia indicates that this occurs independent of the level of tolerance. However, regulation of these processes at transcriptional level varied between species. An important role is suggested for signalling and post-transcriptional regulation to be involved in the mechanisms that lead to tolerance to hypoxia.

Two different thiol-redox-systems exist in plant chloroplasts, the ferredoxin-thioredoxin system, which depends of ferredoxin reduced by the photosynthetic electron-transport chain and, thus, of light, and the NADPH-dependent thioredoxin reductase C (NTRC) system, which relies on NADPH and thus may be linked to sugar metabolism in the dark. Previous studies suggested therefore that the two different systems may have different functions in plants. We now report that there is a previously unrecognized functional redundancy of thioredoxin-f1 and NTRC in regulating photosynthetic metabolism and growth. In Arabidopsis mutants, combined - but not single - deficiencies of thioredoxin-f1 and NTRC led to severe growth inhibition and perturbed light acclimation, accompanied by strong impairments of Calvin-Benson-cycle activity and starch accumulation. Light-activation of key-enzymes of these pathways, fructose-1,6-bisphosphatase and ADP-glucose pyrophosphorylase, was almost completely abolished. The subsequent increase in NADPH/NADP+ and ATP/ADP ratios led to increased nitrogen assimilation, NADP-malate dehydrogenase activation and light-vulnerability of photosystem I core-proteins. In an additional approach, reporter studies show that Trx f1 and NTRC proteins are both co-localized in the same chloroplast substructure. Results provide genetic evidence that light and NADPH dependent thiol-redox systems interact at the level of thioredoxin-f1 and NTRC to coordinately participate in the regulation of Calvin-Benson-cycle, starch metabolism and growth in response to varying light conditions.

Trehalose is the most widespread disaccharide in nature, occurring in bacteria, fungi, insects, and plants. Its precursor, trehalose 6-phosphate (T6P), is also indispensable for the regulation of sugar utilization and growth, but the sites of action are largely unresolved. Here we use genetic and biochemical approaches to investigate whether T6P acts to regulate starch synthesis in plastids of higher plants. Feeding of trehalose to Arabidopsis leaves led to stimulation of starch synthesis within 30 min, accompanied by activation of ADP-glucose pyrophosphorylase (AGPase) via posttranslational redox modification. The response resembled sucrose but not glucose feeding and depended on the expression of SNF1-related kinase. We also analyzed transgenic Arabidopsis plants with T6P levels increased by expression of T6P synthase or decreased by expression of T6P phosphatase (TPP) in the cytosol. Compared with wild type, leaves of T6P synthase-expressing plants had increased redox activation of AGPase and increased starch, whereas TPP-expressing plants showed the opposite. Moreover, TPP expression prevented the increase in AGPase activation in response to sucrose or trehalose feeding. Incubation of intact isolated chloroplasts with 100 μM T6P significantly and specifically increased reductive activation of AGPase within 15 min. Results provide evidence that T6P is synthesized in the cytosol and acts on plastidial metabolism by promoting thioredoxin-mediated redox transfer to AGPase in response to cytosolic sugar levels, thereby allowing starch synthesis to be regulated independently of light. The discovery informs about the evolution of plant metabolism and how chloroplasts of prokaryotic origin use an intermediate of the ancient trehalose pathway to report the metabolic status of the cytosol.

Redox regulation of plastid gene expression and different metabolic pathways promotes many activities of redox-sensitive proteins. We address the question of how the plastid redox state and the contributing reducing enzymes control the enzymes of tetrapyrrole biosynthesis (TBS). In higher plants, this metabolic pathway serves to produce chlorophyll and heme, among other essential end products. Because of the strictly light-dependent synthesis of chlorophyll, tight control of TBS requires a diurnal balanced supply of the precursor 5-aminolevulinic acid (ALA) to prevent the accumulation of photoreactive metabolic intermediates in darkness. We report on some TBS enzymes that accumulate in a light intensity-dependent manner, and their contents decrease under oxidizing conditions of darkness, low light conditions, or in the absence of NADPH-dependent thioredoxin reductase (NTRC) and thioredoxin f1 (TRX-f1). Analysis of single and double trxf1 and ntrc mutants revealed a decreased content of the early TBS enzymes glutamyl-tRNA reductase (GluTR) and 5-aminolevulinic acid dehydratase (ALAD) instead of an exclusive decrease in enzyme activity. This effect was dependent on light conditions and strongly attenuated after transfer to high light intensities. Thus, it is suggested that a deficiency of plastid-localized thiol-redox transmitters leads to enhanced degradation of TBS enzymes rather than being directly caused by lower catalytic activity. The effects of the proteolytic activity of the Clp protease on TBS enzymes were studied by using Clp subunit-deficient mutants. The simultaneous lack of TRX and Clp activities in double mutants confirms the Clp-induced degradation of some TBS proteins in the absence of reductive activity of TRXs. In addition, we verified previous observations that decreased chlorophyll and heme levels in ntrc could be reverted to WT levels in the ntrc/Δ2cp triple mutant. The decreased synthesis of 5-aminolevulinic acid and porphobilinogen in ntrc was completely restored in ntrc/Δ2cp and correlated with WT-like levels of GluTR, ALAD, and other TBS proteins.

Redox signals generated by the photosynthetic electron transport chain are known to be involved in regulating the Calvin cycle, ATP synthesis, and NADPH export from chloroplasts in response to light. The signal cascade involves transfer of electrons from photosystem I via the ferredoxin–thioredoxin system to target enzymes that are activated by reduction of regulatory disulphide bonds. The purpose of this review is to discuss recent findings showing that this concept can be extended to the regulation of carbon storage and partitioning in plants. Starch is the major carbon store in plants, and ADP-glucose pyrophosphorylase (AGPase) is the key regulatory enzyme of starch synthesis in the plastid. It has been shown that AGPase from potato tubers is subject to post-translational redox modification, and here experimental data will be provided showing that the isozyme from pea leaf chloroplasts is activated by reduced thioredoxin f or m in a similar way. Recent reports will be summarized providing in planta evidence that this mechanism regulates storage starch synthesis in response to light and sugars. Post-translational redox activation of AGPase in response to sugars is part of a signalling mechanism linking the rate of starch synthesis to the availability of carbon in diverse plant tissues. Some of the components of the signalling pathway reporting changes in the cytosolic sugar status to the plastid have been postulated, but detailed work is in progress to confirm the exact mode of action. Recent evidence will be discussed showing that key enzymes of de novo fatty acid synthesis (acetyl-CoA carboxylase) and ammonium assimilation (glutamine synthetase and glutamine:oxoglutarate amino transferase) are regulated by reversible disulphide-bond formation similar to AGPase. Redox regulation is proposed to be the preferred strategy of plastidial enzymes to regulate various metabolic processes such as carbon fixation, starch metabolism, lipid synthesis, and amino acid synthesis in response to physiological and environmental inputs.

Nonsymbiotic hemoglobins are ubiquitously expressed in plants and divided into two different classes based on gene expression pattern and oxygen-binding properties. Most of the published research has been on the function of class 1 hemoglobins. To investigate the role of class 2 hemoglobins, transgenic Arabidopsis (Arabidopsis thaliana) plants were generated overexpressing Arabidopsis hemoglobin-2 (AHb2) under the control of a seed-specific promoter. Overexpression of AHb2 led to a 40% increase in the total fatty acid content of developing and mature seeds in three subsequent generations. This was mainly due to an increase in the polyunsaturated C18:2 (ω-6) linoleic and C18:3 (ω-3) α-linolenic acids. Moreover, AHb2 overexpression led to an increase in the C18:2/C18:1 and C18:3/C18:2 ratios as well as in the C18:3 content in mol % of total fatty acids and in the unsaturation/saturation index of total seed lipids. The increase in fatty acid content was mainly due to a stimulation of the rate of triacylglycerol synthesis, which was attributable to a 3-fold higher energy state and a 2-fold higher sucrose content of the seeds. Under low external oxygen, AHb2 overexpression maintained an up to 5-fold higher energy state and prevented fermentation. This is consistent with AHb2 overexpression results in improved oxygen availability within developing seeds. In contrast to this, overexpression of class 1 hemoglobin did not lead to any significant increase in the metabolic performance of the seeds. These results provide evidence for a specific function of class 2 hemoglobin in seed oil production and in promoting the accumulation of polyunsaturated fatty acids by facilitating oxygen supply in developing seeds.

Thiol-dependent redox regulation of enzyme activity plays a central role in the rapid acclimation of chloroplast metabolism to ever-fluctuating light availability. This regulatory mechanism relies on ferredoxin reduced by the photosynthetic electron transport chain, which fuels reducing power to thioredoxins (Trxs) via a ferredoxin-dependent Trx reductase. In addition, chloroplasts harbor an NADPH-dependent Trx reductase, which has a joint Trx domain at the carboxyl terminus, termed NTRC. Thus, a relevant issue concerning chloroplast function is to establish the relationship between these two redox systems and its impact on plant development. To address this issue, we generated Arabidopsis (Arabidopsis thaliana) mutants combining the deficiency of NTRC with those of Trxs f, which participate in metabolic redox regulation, and that of Trx x, which has antioxidant function. The ntrc-trxf1f2 and, to a lower extent, ntrc-trxx mutants showed severe growth-retarded phenotypes, decreased photosynthesis performance, and almost abolished light-dependent reduction of fructose-1,6-bisphosphatase. Moreover, the combined deficiency of both redox systems provokes aberrant chloroplast ultrastructure. Remarkably, both the ntrc-trxf1f2 and ntrc-trxx mutants showed high mortality at the seedling stage, which was overcome by the addition of an exogenous carbon source. Based on these results, we propose that NTRC plays a pivotal role in chloroplast redox regulation, being necessary for the activity of diverse Trxs with unrelated functions. The interaction between the two thiol redox systems is indispensable to sustain photosynthesis performed by cotyledons chloroplasts, which is essential for early plant development.

Plants have an unusual plastid-localized NADP-thioredoxin reductase C (NTRC) containing both an NADP-thioredoxin reductase (NTR) and a thioredoxin (Trx) domain in a single polypeptide. Although NTRC is known to supply reductant for detoxifying hydrogen peroxide in the dark, its other functions are unknown. We now report that NTRC plays a previously unrecognized role in the redox regulation of ADP-glucose pyrophosphorylase (AGPase), a central enzyme of starch synthesis. When supplied NADPH, NTRC activated AGPase in vitro in a redox reaction that required the active site cysteines of both domains of the enzyme. In leaves, AGPase was activated in planta either by light or external feeding of sucrose in the dark. Leaves of an Arabidopsis NTRC KO mutant showed a decrease both in the extent of redox activation of AGPase and in the enhancement of starch synthesis either in the light (by 40-60%) or in the dark after treatment with external sucrose (by almost 100%). The light-dependent activation of AGPase in isolated chloroplasts, by contrast, was unaffected. In nonphotosynthetic tissue (roots), KO of NTRC decreased redox activation of AGPase and starch synthesis in response to light or external sucrose by almost 90%. The results provide biochemical and genetic evidence for a role of NTRC in regulating starch synthesis in response to either light or sucrose. The data also suggest that the Trx domain of NTRC and, to a lesser extent, free Trxs linked to ferredoxin enable amyloplasts of distant sink tissues to sense light used in photosynthesis by leaf chloroplasts and adjust heterotrophic starch synthesis accordingly.

Pectin- and hemicellulose-associated structures of plant cell walls participate in defense responses against pathogens of different parasitic lifestyles. The resulting immune responses incorporate phytohormone signaling components associated with salicylic acid (SA) and jasmonic acid (JA). SA plays a pivotal role in systemic acquired resistance (SAR), a form of induced resistance that - after a local immune stimulus - confers long-lasting, systemic protection against a broad range of biotrophic invaders. β-D-XYLOSIDASE 4 (BXL4) protein accumulation is enhanced in the apoplast of plants undergoing SAR. Here, two independent Arabidopsis thaliana mutants of BXL4 displayed compromised systemic defenses, while local resistance responses to Pseudomonas syringae remained largely intact. Because both phloem-mediated and airborne systemic signaling were abrogated in the mutants, the data suggest that BXL4 is a central component in SAR signaling mechanisms. Exogenous xylose, a possible product of BXL4 enzymatic activity in plant cell walls, enhanced systemic defenses. However, GC-MS analysis of SAR-activated plants revealed BXL4-associated changes in the accumulation of certain amino acids and soluble sugars, but not xylose. In contrast, the data suggest a possible role of pectin-associated fucose as well as of the polyamine putrescine as regulatory components of SAR. This is the first evidence of a central role of cell wall metabolic changes in systemic immunity. Additionally, the data reveal a so far unrecognized complexity in the regulation of SAR, which might allow the design of (crop) plant protection measures including SAR-associated cell wall components.