Toward the apoplast metabolome: Establishing a leaf apoplast collection approach suitable for metabolomics analysis.

The leaf apoplast contains several compounds that play important roles in the regulation of different physiological processes in plants. However, this compartment has been neglected in several experimental and modelling studies, which is mostly associated to the difficulty to collect apoplast washing fluid (AWF) in sufficient amount for metabolomics analysis and as free as possible from symplastic contamination. Here, we established an approach based in an infiltration-centrifugation technique that use little leaf material but allows sufficient AWF collection for gas chromatography mass spectrometry (GC-MS)-based metabolomics analysis in both tobacco and Arabidopsis. Up to 54 metabolites were annotated in leaf and apoplast samples from both species using either 20% (v/v) methanol (20% MeOH) or distilled deionized water (ddH2O) as infiltration fluids. The use of 20% MeOH increased the yield of the AWF collected but also the level of symplastic contamination, especially in Arabidopsis. We propose a correction factor and recommend the use of multiple markers such as MDH activity, protein content and conductivity measurements to verify the level of symplastic contamination in MeOH-based protocols. Neither the concentration of sugars nor the level of primary metabolites differed between apoplast samples extracted with ddH2O or 20% MeOH. This indicates that ddH2O can be preferentially used, given that it is a non-toxic and highly accessible infiltration fluid. The infiltration-centrifugation-based approach established here uses little leaf material and ddH2O as infiltration fluid, being suitable for GC-MS-based metabolomics analysis in tobacco and Arabidopsis, with great possibility to be extended for other plant species and tissues.

Abscisic acid-mediated guard cell metabolism regulation.

Abscisic acid (ABA) is crucial for plant water deficit (WD) acclimation, but how the interplay between ABA and guard cell (GC) metabolism aids plant WD acclimation remains unclear. Here, we investigated how ABA regulates GC metabolism and how this contributes to plant WD acclimation using tomato wild type (WT) and the ABA-deficient sitiens mutant. These genotypes were characterized at physiological, metabolic, and transcriptional levels under recurring WD periods and were used to perform a13C-glucose labelling experiment using isolated guard cells following exogenously applied ABA. ABA deficiency altered the level of sugars and organic acids in GCs in both irrigated and WD plants and the dynamic of accumulation/degradation of these compounds in GCs during the dark-to-light transition. WD-induced metabolic changes were more pronounced in sitiens than WT GCs. Results from the 13C-labelling experiment indicate that ABA is required for the glycolytic fluxes toward malate and acts as a negative regulator of a putative sucrose substrate cycle. The expression of key ABA-biosynthetic genes was higher in WT than in sitiens GCs after two cycles of WD. Additionally, the intrinsic leaf water use efficiency increased only in WT after the second WD cycle, compared to sitiens. Our results highlight that ABA deficiency disrupts the homeostasis of GC primary metabolism and the WD memory, negatively affecting plant WD acclimation. Our study demonstrates which metabolic pathways are activated by WD and/or regulated by ABA in GCs, which improves our understanding of plant WD acclimation, with clear consequences for plant metabolic engineering in the future.

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.

Impairment in photosynthesis induced by CAT inhibition depends on the intensity of photorespiration and peroxisomal APX expression in rice.

We have previously shown that rice plants silenced for peroxisomal ascorbate peroxidase (OsAPX4-RNAi) display higher resilience to photosynthesis under oxidative stress and photorespiratory conditions. However, the redox mechanisms underlying that intriguing response remain unknown. Here, we tested the hypothesis that favorable effects triggered by peroxisomal APX deficiency on photosynthesis resilience under CAT inhibition are dependent on the intensity of photorespiration associated with the abundance of photosynthetic and redox proteins. Non-transformed (NT) and OsAPX4-RNAi silenced rice plants were grown under ambient (AC) or high CO2 (HC) conditions and subjected to 3-amino-1,2,4-triazole (3-AT)-mediated CAT activity inhibition. Photosynthetic measurements evidenced that OsAPX4-RNAi plants simultaneously exposed to CAT inhibition and HC lost the previously acquired advantage in photosynthesis resilience displayed under AC. Silenced plants exposed to environment photorespiration and CAT inhibition presented lower photorespiration as indicated by smaller Gly/Ser and Jo/Jc ratios and glycolate oxidase activity. Interestingly, when these silenced plants were exposed to HC and CAT-inhibition, they exhibited an inverse response compared to AC in terms of photorespiration indicators, associated with higher accumulation of proteins. Multivariate and correlation network analyses suggest that the proteomics changes induced by HC combined with CAT inhibition are substantially different between NT and OsAPX4-RNAi plants. Our results suggest that the intensity of photorespiration and peroxisomal APX-mediated redox signaling are tightly regulated under CAT inhibition induced oxidative stress, which can modulate the photosynthetic efficiency, possibly via a coordinated regulation of protein abundance and rearrangement, ultimately triggered by crosstalk involving H2O2 levels related to CAT and APX activities in peroxisomes.

Unveiling the dark side of guard cell metabolism.

Evidence suggests that guard cells have higher rate of phosphoenolpyruvate carboxylase (PEPc)-mediated dark CO2 assimilation than mesophyll cells. However, it is unknown which metabolic pathways are activated following dark CO2 assimilation in guard cells. Furthermore, it remains unclear how the metabolic fluxes throughout the tricarboxylic acid (TCA) cycle and associated pathways are regulated in illuminated guard cells. Here we carried out a13C-HCO3 labelling experiment in tobacco guard cells harvested under continuous dark or during the dark-to-light transition to elucidate principles of metabolic dynamics downstream of CO2 assimilation. Most metabolic changes were similar between dark-exposed and illuminated guard cells. However, illumination altered the metabolic network structure of guard cells and increased the 13C-enrichment in sugars and metabolites associated to the TCA cycle. Sucrose was labelled in the dark, but light exposure increased the 13C-labelling and leads to more drastic reductions in the content of this metabolite. Fumarate was strongly labelled under both dark and light conditions, while illumination increased the 13C-enrichment in pyruvate, succinate and glutamate. Only one 13C was incorporated into malate and citrate in either dark or light conditions. Our results indicate that several metabolic pathways are redirected following PEPc-mediated CO2 assimilation in the dark, including gluconeogenesis and the TCA cycle. We further showed that the PEPc-mediated CO2 assimilation provides carbons for gluconeogenesis, the TCA cycle and glutamate synthesis and that previously stored malate and citrate are used to underpin the specific metabolic requirements of illuminated guard cells.

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.

Thioredoxins regulate the metabolic fluxes throughout the tricarboxylic acid cycle and associated pathways in a light-independent manner.

The metabolic fluxes throughout the tricarboxylic acid cycle (TCAC) are inhibited in the light by the mitochondrial thioredoxin (TRX) system. However, it is unclear how this system orchestrates the fluxes throughout the TCAC and associated pathways in the dark. Here we carried out a13C-HCO3 labelling experiment in Arabidopsis leaves from wild type (WT) and mutants lacking TRX o1 (trxo1), TRX h2 (trxh2), or both NADPH-dependent TRX reductase A and B (ntra ntrb) exposed to 0, 30 and 60 min of dark or light conditions. No 13C-enrichment in TCAC metabolites in illuminated WT leaves was observed. However, increased succinate content was found in parallel to reductions in Ala in the light, suggesting the latter operates as an alternative carbon source for succinate synthesis. By contrast to WT, all mutants showed substantial changes in the content and 13C-enrichment in TCAC metabolites under both dark and light conditions. Increased 13C-enrichment in glutamine in illuminated trxo1 leaves was also observed, strengthening the idea that TRX o1 restricts in vivo carbon fluxes from glycolysis and the TCAC to glutamine. We further demonstrated that both photosynthetic and gluconeogenic fluxes toward glucose are increased in trxo1 and that the phosphoenolpyruvate carboxylase (PEPc)-mediated 13C-incorporation into malate is higher in trxh2 mutants, as compared to WT. Our results collectively provide evidence that TRX h2 and the mitochondrial NTR/TRX system regulate the metabolic fluxes throughout the TCAC and associated pathways, including glycolysis, gluconeogenesis and the synthesis of glutamine in a light-independent manner.

Metabolism-mediated mechanisms underpin the differential stomatal speediness regulation among ferns and angiosperms.

Recent results suggest that metabolism-mediated stomatal closure mechanisms are important to regulate differentially the stomatal speediness between ferns and angiosperms. However, evidence directly linking mesophyll metabolism and the slower stomatal conductance (gs ) in ferns is missing. Here, we investigated the effect of exogenous application of abscisic acid (ABA), sucrose and mannitol on stomatal kinetics and carried out a metabolic fingerprinting analysis of ferns and angiosperms leaves harvested throughout a diel course. Fern stomata did not respond to ABA in the time period analysed. No differences in the relative decrease in gs was observed between ferns and the angiosperm following provision of sucrose or mannitol. However, ferns have slower gs responses to these compounds than angiosperms. Metabolomics analysis highlights that ferns have a higher accumulation of secondary rather than primary metabolites throughout the diel course, with the opposite being observed in angiosperms. Our results indicate that metabolism-mediated stomatal closure mechanisms underpin the differential stomatal speediness regulation among ferns and angiosperms, in which the slower stomatal closure in ferns is associated with the lack of ABA-responsiveness, to a reduced capacity to respond to mesophyll-derived sucrose and to a higher carbon allocation toward secondary metabolism, which likely modulates both photosynthesis-gs and growth-stress tolerance trade-offs.

Establishment of a GC-MS-based 13 C-positional isotopomer approach suitable for investigating metabolic fluxes in plant primary metabolism.

13 C-Metabolic flux analysis (13 C-MFA) has greatly contributed to our understanding of plant metabolic regulation. However, the generation of detailed in vivo flux maps remains a major challenge. Flux investigations based on nuclear magnetic resonance have resolved small networks with high accuracy. Mass spectrometry (MS) approaches have broader potential, but have hitherto been limited in their power to deduce flux information due to lack of atomic level position information. Herein we established a gas chromatography (GC) coupled to MS-based approach that provides 13 C-positional labelling information in glucose, malate and glutamate (Glu). A map of electron impact (EI)-mediated MS fragmentation was created and validated by 13 C-positionally labelled references via GC-EI-MS and GC-atmospheric pressure chemical ionization-MS technologies. The power of the approach was revealed by analysing previous 13 C-MFA data from leaves and guard cells, and 13 C-HCO3 labelling of guard cells harvested in the dark and after the dark-to-light transition. We demonstrated that the approach is applicable to established GC-EI-MS-based 13 C-MFA without the need for experimental adjustment, but will benefit in the future from paired analyses by the two GC-MS platforms. We identified specific glucose carbon atoms that are preferentially labelled by photosynthesis and gluconeogenesis, and provide an approach to investigate the phosphoenolpyruvate carboxylase (PEPc)-derived 13 C-incorporation into malate and Glu. Our results suggest that gluconeogenesis and the PEPc-mediated CO2 assimilation into malate are activated in a light-independent manner in guard cells. We further highlight that the fluxes from glycolysis and PEPc toward Glu are restricted by the mitochondrial thioredoxin system in illuminated leaves.

Two mitochondrial phosphatases, PP2c63 and Sal2, are required for posttranslational regulation of the TCA cycle in Arabidopsis.

Protein phosphorylation is a well-established post-translational mechanism that regulates protein functions and metabolic pathways. It is known that several plant mitochondrial proteins are phosphorylated in a reversible manner. However, the identities of the protein kinases/phosphatases involved in this mechanism and their roles in the regulation of the tricarboxylic acid (TCA) cycle remain unclear. In this study, we isolated and characterized plants lacking two mitochondrially targeted phosphatases (Sal2 and PP2c63) along with pyruvate dehydrogenase kinase (PDK). Protein-protein interaction analysis, quantitative phosphoproteomics, and enzymatic analyses revealed that PDK specifically regulates pyruvate dehydrogenase complex (PDC), while PP2c63 nonspecifically regulates PDC. When recombinant PP2c63 and Sal2 proteins were added to mitochondria isolated from mutant plants, protein-protein interaction and enzymatic analyses showed that PP2c63 directly phosphorylates and modulates the activity of PDC, while Sal2 only indirectly affects TCA cycle enzymes. Characterization of steady-state metabolite levels and fluxes in the mutant lines further revealed that these phosphatases regulate flux through the TCA cycle, and that altered metabolism in the sal2 pp2c63 double mutant compromises plant growth. These results are discussed in the context of current models of the control of respiration in plants.

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.

Mesophyll conductance: the leaf corridors for photosynthesis.

Besides stomata, the photosynthetic CO2 pathway also involves the transport of CO2 from the sub-stomatal air spaces inside to the carboxylation sites in the chloroplast stroma, where Rubisco is located. This pathway is far to be a simple and direct way, formed by series of consecutive barriers that the CO2 should cross to be finally assimilated in photosynthesis, known as the mesophyll conductance (gm). Therefore, the gm reflects the pathway through different air, water and biophysical barriers within the leaf tissues and cell structures. Currently, it is known that gm can impose the same level of limitation (or even higher depending of the conditions) to photosynthesis than the wider known stomata or biochemistry. In this mini-review, we are focused on each of the gm determinants to summarize the current knowledge on the mechanisms driving gm from anatomical to metabolic and biochemical perspectives. Special attention deserve the latest studies demonstrating the importance of the molecular mechanisms driving anatomical traits as cell wall and the chloroplast surface exposed to the mesophyll airspaces (Sc/S) that significantly constrain gm. However, even considering these recent discoveries, still is poorly understood the mechanisms about signaling pathways linking the environment a/biotic stressors with gm responses. Thus, considering the main role of gm as a major driver of the CO2 availability at the carboxylation sites, future studies into these aspects will help us to understand photosynthesis responses in a global change framework.

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.

The sucrose-to-malate ratio correlates with the faster CO2 and light stomatal responses of angiosperms compared to ferns.

Stomatal responses to environmental signals differ substantially between ferns and angiosperms. However, the mechanisms that lead to such different responses remain unclear. Here we investigated the extent to which leaf metabolism contributes to coordinate the differential stomatal behaviour among ferns and angiosperms. Stomata from all species were responsive to light and CO2 transitions. However, fern stomatal responses were slower and minor in both absolute and relative terms. Angiosperms have higher stomatal density, but this is not correlated with speed of stomatal closure. The metabolic responses throughout the diel course and under different CO2 conditions differ substantially among ferns and angiosperms. Higher sucrose content and an increased sucrose-to-malate ratio during high CO2 -induced stomatal closure was observed in angiosperms compared to ferns. Furthermore, the speed of stomatal closure was positively and negatively correlated with sugars and organic acids, respectively, suggesting that the balance between sugars and organic acids aids in explaining the faster stomatal responses of angiosperms. Our results suggest that mesophyll-derived metabolic signals, especially those associated with sucrose and malate, may also be important to modulate the differential stomatal behaviour between ferns and angiosperms, providing important new information that helps in understanding the metabolism-mediated mechanisms regulating stomatal movements across land plant evolution.

On the role of the plant mitochondrial thioredoxin system during abiotic stress.

Thiol-disulfide redox exchanges are widely distributed modifications of great importance for metabolic regulation in living cells. In general, the formation of disulfide bonds is controlled by thioredoxins (TRXs), ubiquitous proteins with two redox-active cysteine residues separated by a pair of amino acids. While the function of plastidial TRXs has been extensively studied, the role of the mitochondrial TRX system is much less well understood. Recent studies have demonstrated that the mitochondrial TRXs are required for the proper functioning of the major metabolic pathways, including stomatal function and antioxidant metabolism under sub-optimal conditions including drought and salinity. Furthermore, inactivation of mitochondrial TRX system leads to metabolite adjustments of both primary and secondary metabolism following drought episodes in arabidopsis, and makes the plants more resistant to salt stress. Here we discuss the implications of these findings, which clearly open up several research avenues to achieve a full understanding of the redox control of metabolism under environmental constraining conditions.

Insights into ABA-mediated regulation of guard cell primary metabolism revealed by systems biology approaches.

Despite the fact that guard cell abscisic acid (ABA) signalling pathway is well documented, our understanding concerning how and to which extent ABA regulates guard cell metabolism remains fragmentary. Here we have adopted different systems approaches to investigate how ABA modulates guard cell central metabolism by providing genes that are possibly ABA-regulated. By using previous published Arabidopsis guard cell transcript profiling data, we carried out an extensive co-expression network analysis using ABA-related genes and those related to the metabolism and transport of sugars, starch and organic acids. Next, we investigated the presence of ABA responsive elements (ABRE) in the promoter of genes that are highly expressed in guard cells, responsive to ABA and co-expressed with ABA-related genes. Together, these analyses indicated that 44 genes are likely regulated by ABA and 8 of them are highly expressed in guard cells in both the presence and absence of ABA, including genes of the tricarboxylic acid cycle and those related to sucrose and hexose transport and metabolism. It seems likely that ABA may modulate both sucrose transport through guard cell plasma membrane and sucrose metabolism within guard cells. In this context, genes associated with sucrose synthase, sucrose phosphate synthase, trehalose-6-phosphate, invertase, UDP-glucose epimerase/pyrophosphorylase and different sugar transporters contain ABRE in their promoter and are thus possibly ABA regulated. Although validation experiments are required, our study highlights the importance of systems biology approaches to drive new hypothesis and to unravel genes and pathways that are regulated by ABA in guard cells.

Modulation of auxin signalling through DIAGETROPICA and ENTIRE differentially affects tomato plant growth via changes in photosynthetic and mitochondrial metabolism.

Auxin modulates a range of plant developmental processes including embryogenesis, organogenesis, and shoot and root development. Recent studies have shown that plant hormones also strongly influence metabolic networks, which results in altered growth phenotypes. Modulating auxin signalling pathways may therefore provide an opportunity to alter crop performance. Here, we performed a detailed physiological and metabolic characterization of tomato (Solanum lycopersicum) mutants with either increased (entire) or reduced (diageotropica-dgt) auxin signalling to investigate the consequences of altered auxin signalling on photosynthesis, water use, and primary metabolism. We show that reduced auxin sensitivity in dgt led to anatomical and physiological modifications, including altered stomatal distribution along the leaf blade and reduced stomatal conductance, resulting in clear reductions in both photosynthesis and water loss in detached leaves. By contrast, plants with higher auxin sensitivity (entire) increased the photosynthetic capacity, as deduced by higher Vcmax and Jmax coupled with reduced stomatal limitation. Remarkably, our results demonstrate that auxin-sensitive mutants (dgt) are characterized by impairments in the usage of starch that led to lower growth, most likely associated with decreased respiration. Collectively, our findings suggest that mutations in different components of the auxin signalling pathway specifically modulate photosynthetic and respiratory processes.

The Mitochondrial Thioredoxin System Contributes to the Metabolic Responses Under Drought Episodes in Arabidopsis.

Thioredoxins (Trxs) modulate metabolic responses during stress conditions; however, the mechanisms governing the responses of plants subjected to multiple drought events and the role of Trxs under these conditions are not well understood. Here we explored the significance of the mitochondrial Trx system in Arabidopsis following exposure to single and repeated drought events. We analyzed the previously characterized NADPH-dependent Trx reductase A and B double mutant (ntra ntrb) and two independent mitochondrial thioredoxin o1 (trxo1) mutant lines. Following similar reductions in relative water content (∼50%), Trx mutants subjected to two drought cycles displayed a significantly higher maximum quantum efficiency (Fv/Fm) and were less sensitive to drought than their wild-type counterparts and than all genotypes subjected to a single drought event. Trx mutant plants displayed a faster recovery after two cycles of drought, as observed by the higher accumulation of secondary metabolites and higher stomatal conductance. Our results indicate that plants exposed to multiple drought cycles are able to modulate their subsequent metabolic and physiological response, suggesting the occurrence of an exquisite acclimation in stressed Arabidopsis plants. Moreover, this differential acclimation involves the participation of a set of metabolic changes as well as redox poise alteration following stress recovery.

Impairment of peroxisomal APX and CAT activities increases protection of photosynthesis under oxidative stress.

Retrograde signalling pathways that are triggered by changes in cellular redox homeostasis remain poorly understood. Transformed rice plants that are deficient in peroxisomal ascorbate peroxidase APX4 (OsAPX4-RNAi) are known to exhibit more effective protection of photosynthesis against oxidative stress than controls when catalase (CAT) is inhibited, but the mechanisms involved have not been characterized. An in-depth physiological and proteomics analysis was therefore performed on OsAPX4-RNAi CAT-inhibited rice plants. Loss of APX4 function led to an increased abundance of several proteins that are involved in essential metabolic pathways, possibly as a result of increased tissue H2O2 levels. Higher photosynthetic activities observed in the OsAPX4-RNAi plants under CAT inhibition were accompanied by higher levels of Rubisco, higher maximum rates of Rubisco carboxylation, and increased photochemical efficiencies, together with large increases in photosynthesis-related proteins. Large increases were also observed in the levels of proteins involved in the ascorbate/glutathione cycle and in other antioxidant-related pathways, and these changes may be important in the protection of photosynthesis in the OsAPX4-RNAi plants. Large increases in the abundance of proteins localized in the nuclei and mitochondria were also observed, together with increased levels of proteins involved in important cellular pathways, particularly protein translation. Taken together, the results show that OsAPX4-RNAi plants exhibit significant metabolic reprogramming, which incorporates a more effective antioxidant response to protect photosynthesis under conditions of impaired CAT activity.