Plant Physiology

Differential transpiration is a newly discovered acclimation strategy of annual plants to a combination of water deficit (WD) and heat stress (HS). Under these conditions (i.e., WD+HS), transpiration of vegetative tissues is suppressed in plants such as soybean and tomato, while transpiration of reproductive tissues is not (termed Differential Transpiration; DT). This newly discovered acclimation process enables the cooling of reproductive organs under conditions of WD+HS, limiting HS-induced damage to plant reproduction. However, at what temperature and WD extremes will this process be active and functional at reducing the internal temperature of reproductive tissues, and at what developmental stages of the plant is it activated, remain unknown. Here, we report that DT occurs at most nodes (leaf developmental stages) of soybean plants subjected to WD+HS, and that it can function under extreme conditions of WD+HS (i.e., 18% of field water capacity and 42{degrees}C combined). Our findings reveal that DT is an effective acclimation strategy that protects reproductive processes from extreme conditions of WD+HS, at almost all developmental stages. In addition, our findings suggest that under field conditions DT could also be active in plants subjected to low or mild levels of WD during a heat wave.

The hydrogen peroxide (H2O2) scavenging enzyme, catalase, plays a critical role in the photorespiratory pathway by maintaining the balance of H2O2, a reactive oxygen species (ROS), in the peroxisome. H2O2 acts as both a signaling molecule and a potential source of ROS depending on its accumulation in the peroxisome. Additionally, H2O2 can also drive non-enzymatic decarboxylation (NED) reactions as well as other decarboxylation reactions, leading to increased CO2 release that is linked to a severe growth phenotype. However, the exact cause of this stunted growth phenotype is not fully understood, and it remains unclear whether the capacity of catalase is critical for minimizing these decarboxylating reactions. Here we elucidate the mechanism behind the decrease in plant growth due to the accumulation of H2O2 from photorespiration using cat2 knock-out lines of Arabidopsis thaliana rescued with transgenic expression lines of Heliobacter pylori catalase. These experiments demonstrated that while one of the three heterologous lines expressing H. pylori catalase isoform (Hp615) had greater catalase activity than cat2-KO and rescued the severe growth and photosynthetic phenotype, its catalase activity was still far below wild type levels. These findings suggest that catalase plays a crucial role in maintaining H2O2 homeostasis within the peroxisome and minimizing decarboxylation reactions, both of which are linked to plant growth. Moreover, once a threshold capacity is reached, increasing catalase capacity further may offer limited benefits in enhancing net carbon fixation. HighlightWe show that peroxisomal catalase is important for maintaining high rates of net carbon fixation associated with plant growth. Native catalase levels in Arabidopsis thaliana are in excess of that which is required to minimize alternative decarboxylation reactions. Therefore, efforts to optimize catalase-mediated degradation of H2O2 may be of limited benefit.

Methionine (Met) is a central metabolite in plants, as it serves as a precursor for S-adenosylmethionine (SAM), a key methyl donor for epigenetic and metabolic processes. Met is also an essential amino acid that limits the nutritional value of plant-based diets. Understanding how altered Met levels affect the metabolome, transcriptome, and epigenetic regulation of plant leaves remains an open challenge. This study investigates the impact of ectopic Met accumulation in SSE Arabidopsis leaves of transgenic lines expressing a deregulated form of AtCGS (AtD-CGS) under the seed-specific phaseolin promoter. Unexpected activation of the phaseolin promoter in leaves led to AtD-CGS expression and variable Met accumulation among progeny, despite genetic homozygosity. High-Met (HM) plants showed elevated amino acid and sugar levels, enrichment of stress-related transcripts, and suppression of Met biosynthetic genes, while Low-Met (LM) plants showed reduced Met levels and increased non-CG DNA methylation, especially in centromeric and promoter regions. Integrated transcriptome and methylome analyses revealed that high Met levels were associated with the upregulation of stress hormone pathways (abscisic acid, jasmonate, salicylic acid, and ethylene), downregulation of key epigenetic regulators (e.g., MET1, CMTs), and broader transcriptional reprogramming. By contrast, low Met (LM) lines displayed similar expression levels of genes as control plants. Our findings reveal a complex regulatory network whereby Met accumulation reprograms metabolism, gene expression, and DNA methylation patterns. These results suggest feedback between sulfur-carbon metabolism, stress adaptation, and epigenetic control, positioning Met as both a nutrient and a signaling hub in plant physiology.

In C4 photosynthesis, incoming CO2 is incorporated in mesophyll cells (MC) into 4-carbon acids that diffuse to bundle sheath cells (BSC) and decarboxylated to generate a high CO2 concentration that suppresses the oxygenation reaction of Rubisco. Decarboxylation can occur by NADP-malic enzyme, (NADP-ME), NAD-malic enzyme (NAD-ME) or phosphoenolpyruvate carboxykinase (PEPCK). NADP-ME generates NADPH in the BSC chloroplast and species that use it as the major route for decarboxylation typically have dimorphic BSC chloroplasts with little or no photosystem II. They operate an energy shuttle: much of the 3-phosphoglycerate formed in the Calvin-Benson cycle diffuses to the MC, enters the chloroplasts and is reduced to triose phosphates that return to the BSC. In species where carboxylation occurs mainly via NAD-ME or PEPCK, BSC chloroplasts possess photosystem II. Indirect evidence indicates they nevertheless have the capacity to operate an energy shuttle. We show here that NAD-ME and PEPCK species possess large pools of 3PGA and triose phosphates and, for two examples of each subtype, opposed concentration gradients of 3-phosphoglycerate and triose phosphates to drive rapid exchange between the BSC and MC. Reasons for and consequences of the widespread operation of the intercellular energy shuttle in C4 plants are discussed. Highlight StatementAn intercellular energy shuttle in which 3-phosphoglycerate moves from the bundle sheath to the mesophyll and triose phosphates return to the bundle sheath is a general feature of C4 photosynthesis.

Photorespiration is a mandatory metabolic repair shunt of carbon fixation by the Calvin-Benson (CB) cycle in oxygenic phototrophs. Its extent depends mainly on the CO2/O2 ratio in chloroplasts, which is regulated via stomatal movements. However, despite comprehensive understanding on the role of photorespiration in mesophyll cells (MC), its role in guard cells (GC) is yet unknown. To analyze this issue, the key enzyme of photorespiration, glycine decarboxylase (GDC), was manipulated through overexpression and antisense suppression of the GDC H-protein gene. A positive correlation of GDC-H expression with growth, photosynthesis and carbohydrate biosynthesis was observed in the transgenic lines, demonstrating active photorespiration is involved in stomatal regulation. This view is supported by gas exchange measurements showing that optimized GC photorespiration improves plant acclimation towards conditions requiring a high photorespiratory capacity, including high light. Microscopic analysis revealed that altered photorespiratory flux also affected starch accumulation patterns in GC, eventually serving as the underlying mechanistic for altered stomatal behavior. Collectively, our data suggest that photorespiration is a key component of the regulatory circuit that coordinates stomatal movements with external and internal CO2 availability. Thus, manipulation of photorespiration in GC has the potential to engineer crops maintaining growth and photosynthesis under future climates. One-sentence summaryGuard-cell-specific manipulation of photorespiratory glycine decarboxylase reveals photorespiration is active in guard cells and makes a major contribution to stomatal metabolism and movements.

Interactions between plant energy organelles, the chloroplasts and the mitochondria, are crucial for plant development and acclimation. These interactions occur at different levels including exchange of metabolites and reducing power, organelle signaling pathways and intracellular gas exchange. Mitochondrial retrograde stress signaling activates expression of nuclear genes encoding mitochondrial components, including alternative oxidases. High abundances of these respiratory enzymes coincide not only with the changes in plant respiration but also with alterations in the chloroplast. For example, plants that overexpress alternative oxidases are tolerant to methyl viologen, a redox-active compound that catalyzes transfer of electrons from Photosystem I to molecular oxygen. The mechanism of this inter-organelle interaction is unclear but could be related to diminished availability of tissue oxygen. Here we assessed respiration, photosynthesis and in vivo levels of oxygen in a set of Arabidopsis lines with perturbations in diverse mitochondrial functions, including defects in respiratory complex I, mitochondrial protein processing, transcription, nucleoid organization, altered fission and architecture or suppressed ATP synthase activity. In these lines, the increased abundance and activity of alternative oxidases strongly correlated with higher oxygen consumption in darkness, lower oxygen re-accumulation in light, and diminished effects of methyl viologen in chloroplasts. These results support the hypothesis that increased mitochondrial oxygen sink capacity affects photosynthesis by decreasing oxygen levels in tissues. This phenomenon can be one of the reasons for the impact that stressed mitochondria have on chloroplasts and photosynthesis. It contributes to our understanding of the mechanisms of hypoxia establishment and acclimation in plants.

Thylakoid membranes in chloroplasts and cyanobacteria harbor the multisubunit protein complexes that catalyze the light reactions of photosynthesis. In plant chloroplasts, the thylakoid membrane system comprises a highly organized network with several subcompartments that differ in composition and morphology: grana stacks, unstacked stromal lamellae, and grana margins at the interface between stacked and unstacked regions. The localization of components of the photosynthetic apparatus among these subcompartments has been well characterized. However, less is known about the localization of proteins involved in the biogenesis and repair of the photosynthetic apparatus, the partitioning of proteins between two recently resolved components of the traditional margin fraction (refined margins and curvature), and the effects of light on these features. In this study, we analyzed the partitioning of numerous thylakoid biogenesis and repair factors among grana, curvature, refined margin, and stromal lamellae fractions of Arabidopsis thylakoid membranes, comparing the results from illuminated and dark-adapted plants. Several proteins previously shown to localize to a margin fraction partitioned in varying ways among the resolved curvature and refined margin fractions. For example, the ALB3 insertase and FtsH protease involved in photosystem II (PSII) repair were concentrated in the refined margin fraction, whereas TAT translocon subunits and proteins involved in early steps in photosystem assembly were concentrated in the curvature fraction. By contrast, two photosystem assembly factors that facilitate late assembly steps were depleted from the curvature fraction. The enrichment of the PSII subunit OE23/PsbP in the curvature fraction set it apart from other PSII subunits, supporting the previous conjecture that OE23/PsbP assists in PSII biogenesis and/or repair. The PSII assembly factor PAM68 partitioned differently among thylakoid fractions from dark-adapted plants and illuminated plants, and was the only analyzed protein to convincingly do so. These results demonstrate an unanticipated spatial heterogeneity of photosystem biogenesis and repair functions in thylakoid membranes, and reveal the curvature fraction to be a focal point of early photosystem biogenesis.

O_LIResearch and rationale: This study investigates whether tissue-specific ethylene biosynthesis regulates stomatal conductance (gs) responses to changing [CO2] in Arabidopsis thaliana. While guard cells sense [CO2], mesophyll-derived signals are also implicated in stomatal control. We aimed to determine if ethylene production in guardcells or mesophyll is the primary driver of CO2-induced gs regulation. C_LIO_LIMethods: An acs octuple mutant with severely reduced ethylene production was complemented with tissue-specific ACS8/ACS11 transgenes driven by guard-cell, spongy-mesophyll, dual palisade/spongy-mesophyll, or whole-leaf promoters. Tissue-specific complementation in the different transgenic lines was confirmed and evaluated by qPCR, tissue-specific NEON expression, microscopic imaging, and ethylene production measurements. Gas-exchange measurements on intact plants recorded gs kinetics, CO2 assimilation, and water-use efficiency, across CO2 shifts. C_LIO_LIKey results: Guard-cell complementation nearly fully restored wild-type gs responses and reversed the mutants aberrant leaf phenotype. Spongy-mesophyll complementation failed to rescue either trait, while dual palisade- and spongy-mesophyll complementation yielded only partial recovery. C_LIO_LIConclusion: Ethylene produced in guard cells is the dominant regulator of CO2-induced stomatal conductance regulation, with mesophyll-derived ethylene contributing secondarily via long-distance signaling or by augmenting the overall ethylene pool. These findings underscore the importance of spatially regulated ethylene biosynthesis in balancing carbon assimilation and transpiration. C_LI

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

Plants exposed to extended periods of darkness experience acute energy stress. This stress is counteracted by mitochondrial metabolism, which requires extensive metabolic reprogramming to sustain survival under carbon-limited conditions. Although lysine metabolism has been associated with energy homeostasis, its specific function under prolonged carbon deprivation remains unclear. Here, we demonstrate that the lysine-biosynthesis mutant dapat exhibits premature senescence and accelerated mortality under an 8h/16h light-dark cycle, whereas it survives under a 12h/12h photoperiod. This suggests a critical dependence on carbon reserves accumulated prior to darkness exposure. Under short-day conditions, dapat plants displayed a pronounced decline in photosystem II efficiency (Fv/Fm), chlorophyll degradation, decreased total protein content and increased levels of free amino acids. Transcriptional analysis of genes encoding amino acid catabolic enzymes, alternative respiratory components, and markers of senescence, starvation, and autophagy revealed a constitutive priming response prior to stress induction. This response was further intensified under carbon-limitation, indicating severe metabolic reprogramming. Notably, growth under a 12h light /12h dark cycle prior to darkness exposure enabled dapat plants to recover following extended darkness. They exhibited a distinctive recovery profile and maintenance of the metabolic reprogramming signature intrinsic to the DAPAT mutation. Collectively, our findings indicate that lysine biosynthesis plays an important role in the coordination of cellular energy status and stress response. Considering the photoperiod-dependent regulation observed, we propose that lysine biosynthesis is essential for plant survival during long-term darkness.

Plants are frequently subjected to different combinations of abiotic stresses, such as high light intensity and elevated temperatures. These environmental conditions pose an important threat to agriculture production, affecting photosynthesis and decreasing yield. Metabolic responses of plants, such as alterations in carbohydrates and amino acid fluxes, play a key role in the successful acclimation of plants to different abiotic stresses, directing resources towards stress responses and suppressing growth. Here we show that the primary metabolic response of Arabidopsis thaliana plants to high light or heat stress is different than that of plants subjected to a combination of high light and heat stress. We further demonstrate that a combination of high light and heat stress results in a unique metabolic response that includes increased accumulation of sugars and amino acids, coupled with decreased levels of metabolites participating in the tricarboxylic acid (TCA) cycle. Among the amino acids exclusively accumulated during a combination of high light and heat stress, we identified the non-proteinogenic amino acid {gamma}-aminobutyric acid (GABA). Analysis of different mutants deficient in GABA biosynthesis, in particular two independent alleles of glutamate decarboxylase 3 (gad3), reveal that GABA plays a key role in the acclimation of plants to a combination of high light and heat stress. Taken together, our findings identify a new role for GABA in regulating plant responses to stress combination. One sentence summaryThe non-proteinogenic amino acid {gamma}-aminobutyric acid (GABA) is required for plant acclimation to a combination of high light and heat stress in Arabidopsis.

In plants, Fe homeostasis and O2 metabolism are strictly related, indeed several Fe-requiring enzymes catalyze reactions that also involve oxygen, as a reagent, product, entry or end point of the metabolic pathway in which the enzyme takes part. Oxygen sensing itself relies on Fe-dependent enzymes, the Plant cysteine oxidase (PCO) family of 2-OG independent thiol dioxygenases. PCOs are responsible for the degradation of ERFVII ethylene-responsive factors through a proteasomal N-degron pathway that connects hypoxia-inducible responses to the stabilization of the ERFVII transcription factors. Here, we investigated the interplay between low oxygen and Fe-deficiency stresses in A. thaliana. We used plants expressing a genetically encoded reporter of ERFVII protein stability and measured the expression of anaerobic genes to infer PCO activity in vivo. Our results highlight that Fe deprivation can elicit hypoxia-like responses depending on its severity. To test the involvement the ERFVII factors further, we examined the response of a pentuple erfVII mutant to Fe-deficiency stress, individually or combined with low oxygen. Our data indicate that the ERFVIIs might take part to the acclimation to chronic Fe deficiency by acting as positive regulators of starvation-responsive genes. Moreover, our results suggest that the ERFVIIs fine-tune nutrient mobilization to the shoots of submerged plants growing on moderately Fe-deficient substrates. This work expands the known functions of the ERFVII factors and provides new information to understand plant responses to combined environmental stresses.

The ubiquitin-dependent Arg/N-degron pathway relates the stability of a substrate protein to the nature of its N-terminal amino acid residue or its biochemical modifications, with some N-terminal residues being recognized by specific E3 ubiquitin ligases, resulting in the ubiquitylation and degradation of the substrate protein. Work in the model plant Arabidopsis thaliana has shown that the Arg/N-degron pathway is a key regulator of plant responses to hypoxia, which can be either physiological or a stress in the context of waterlogging or submergence. The role of the Arg/N-degron pathway in hypoxia response is mediated via the oxygen-dependent degradation of group VII ETHYLENE RESPONSE FACTOR (ERFVII) transcription factors, which act as the master regulators of the hypoxia response program in plants. Analysis of Arabidopsis mutants for different enzymatic components of the Arg/N-degron pathway has also revealed its roles in the regulation of responses to other abiotic stresses (e.g. salt stress), as well as to pathogens. Although much has been learned from studies in Arabidopsis about the functions of the Arg/N-degron pathway, very little is known about this pathway in crops, including in Brassica crops such as oilseed rape, cabbage or turnip. To determine functional similarities and divergence of the Arg/N-degron pathway between Arabidopsis and Brassica crops, we isolated and characterized the first Arg/N-degron pathway mutants in Brassica rapa (turnip, pak choi), a diploid Brassica crop closely related to oilseed rape. We focused on two enzymatic components, namely the arginine-transferases (ATEs) and the E3 ubiquitin ligase PROTEOLYSIS6 (PRT6). Our results show both similarities and divergence of function for these Arg/N-degron pathway components in B. rapa compared to Arabidopsis. Specifically, ATE mutants in B. rapa arrest their development at the seedling stage, which contrasts with the mild phenotypic defects of the equivalent Arabidopsis mutants. Double mutant lines for two of the three PRT6 genes in B. rapa indicated a constitutive activation of hypoxia response genes at the transcriptional level, as shown in the single prt6 mutant in Arabidopsis. However, contrary to Arabidopsis, the B. rapa double mutants were more sensitive to waterlogging and hypoxia, and did not show differential response to salt stress or to biotic stress compared to the wild type. The functional divergence identified likely reflects variability in each species in the substrate repertoire and/or in the regulation of pathways or targets downstream of Arg/N-degron pathway substrates. Such differences could be driven by direct selective pressures at N-termini (e.g. gain or loss of a destabilizing N-terminal residue), or by species-specific proteases that may generate destabilizing neo-N-termini after cleavage. These similarities and differences highlight the difficulties in translating research findings from Arabidopsis to crops, even within the same plant family (Brassicaceae) and highlight the need to study pathways in crops.

Specialized metabolites mediate diverse plant-environment interactions. Although, recent work has begun to enzymatically characterize entire plant specialized metabolic pathways, little is known about how different pathway components organize and interact within the cell. Here we use acylsugars - a class of specialized metabolites found across the Solanaceae family - as a model to explore cellular localization and metabolic complex formation of pathway enzymes. These compounds consist of a sugar core decorated with acyl groups, which are connected through ester linkages. In Solanum lycopersicum (tomato) four acylsugar acyltransferases (SlASAT1-4) sequentially add acyl chains to specific hydroxyl positions on a sucrose core leading to accumulation of tri and tetraacylated sucroses in the trichomes. To elucidate the spatial organization and interactions of tomato ASATs, we expressed SlASAT1-4 proteins fused with YFP in N. benthamiana and Arabidopsis protoplasts. Our findings revealed a distributed ASAT pathway with SlASAT1 and SlASAT3 localized to the mitochondria, SlASAT2 localized to the cytoplasm and nucleus, and SlASAT4 localized to the endoplasmic reticulum. To explore potential pairwise protein-protein interactions in acylsugar biosynthesis, we used various techniques, including co-immunoprecipitation, split luciferase assays, and bimolecular fluorescence complementation. These complementary approaches based on different interaction principles all demonstrated interactions among the different SlASAT pairs. Following transient expression of SlASAT1-4 in N. benthamiana, we were able to pull down a complex consisting of SlASAT1-4, which was confirmed through proteomics. Size exclusion chromatography of the SlASAT pulldown suggests a heteromultimeric complex consisting of SlASATs and perhaps other proteins involved in this interaction network. This study sheds light on the metabolic coordination for acylsugar biosynthesis through formation of an interaction network of four sequential steps coordinating efficient production of plant chemical defenses.

REPRESSOR OF UV-B PHOTOMORPHOGENESIS (RUP) negatively regulates UV- B signalling, yet its broader physiological roles in crop plants remain largely unexplored. We compared two tomato accessions bearing truncated RUP proteins--rup-1 and rup-2 (rup- variants)--with the cultivar Arka Vikas (AV), which harbours the native RUP protein. Seedlings of rup-variants exhibited enhanced tolerance to supplemental UV-B light. Red ripe (RR) fruits of rup-variants showed significantly elevated carotenoid and folate levels compared to AV. Introgression of rup-variants into AV confirmed that the increased carotenoid accumulation is genetically linked to RUP truncation. Metabolomic profiling of rup-variants revealed a substantial shift in primary metabolic homeostasis, particularly at the breaker stage, marked by a pronounced reduction in sugars and amino acids. Proteomic analyses of rup- variants across ripening stages identified that a significant proportion of differentially expressed proteins belonged to chaperones and ubiquitin-proteasome system (UPS). Upregulation of four key enzymes in the carotenoid biosynthesis pathway likely contributed to increased lycopene content in rup-variants. Elevated folate levels in rup-variants were associated with the upregulation of folate biosynthesis and C1 metabolism enzymes. Despite widespread metabolic reprogramming in rup-variants, hormonal regulatory pathways remained largely unaltered. Our results suggest that RUP modulates metabolic pathways during fruit ripening, and its loss triggers metabolic reprogramming associated with elevated folate/carotenoid levels.

Stomata are pores in the leaf epidermis that regulate the trade-off between CO2 uptake for photosynthesis and water vapor loss to the atmosphere. Stomatal patterning therefore influences water use efficiency and is a target for engineering to avoid drought stress. However, there is limited understanding of how internal leaf anatomy is coordinated with stomatal development, in part due to the technical challenges of assessing three-dimensional anatomy with sufficient resolution. C4 grasses are understudied, and this is a significant knowledge gap given their file-like stomatal distribution and unique mesophyll organization. In this study, wild-type sorghum and a low-stomatal density transgenic line expressing a synthetic Epidermal Patterning Factor (EPFsyn) were studied. High-resolution microCT was paired with machine learning to characterize three-dimensional traits of mesophyll, epidermis, and airspace, which together determine gias. Sorghum internal leaf airspace is an arrangement of large sub-stomatal airspaces with thin air passageways. Adaxial and abaxial surfaces differed in stomatal patterning relative to mesophyll structures, sub-stomatal crypts and airspace CO2 conductance (gias). Surprisingly, adaxial stomata were consistently located above rather than between vascular bundles. Unexpectedly, gias was not significantly different in wild-type versus EPFsyn. EPFsyn plants had larger crypts and shifts in internal leaf anatomy, indicating a potential compensation mechanism for predicted impacts of reduced stomatal density on gias. These findings provide a new understanding of the interplay between leaf surface specific anatomy and internal structural patterning of the mesophyll in a C4 species, and provides knowledge relevant to engineering water use efficiency in crop species.

During oxygenic photosynthesis, oxygen (O2) is generated from water photolysis, which provides reducing power to sustain CO2 assimilation. To date, traditional leaf gas-exchange experiments have been focused on net CO2 exchange (Anet), with limited observations of net oxygen production (NOP). Here, we present the first gas-exchange/fluorescence system, coupling CO2/H2O analysis (photosynthesis and transpiration) with NOP and isoprene emission measurements. This configuration allowed us to calculate the assimilatory quotient (AQ = Anet/NOP) and thus obtain a more complete picture of the photosynthetic redox budget via photosynthetic production of O2, electron transport rate (ETR), and isoprene biosynthesis. We used cottonwood leaves (Populus trichocarpa) and carried out response curves to light, CO2 and temperature along with 18O-labelling with 18O-enriched water. We found that Anet and NOP were linearly correlated across environmental variables with AQ of 1.27 +/- 0.12 regardless of light, CO2, and temperature. Anet and NOP had optimal temperatures (Topt) of 31{degrees}C, while ETR (35{degrees}C) and isoprene emissions (39{degrees}C) had distinctly higher Topt. Leaves labelled with H218O produced labeled (18O16O) oxygen with the same Topt as ETR (35{degrees}C). The results confirm a tight connection between water oxidation and ETR and are consistent with a suppression of Anet and NOP at high temperature driven by an acceleration of (photo)respiration. The findings support the view of isoprene biosynthesis primarily driven by excess photosynthetic ATP/NADPH not consumed by the Calvin cycle during photorespiratory conditions as an important thermotolerance mechanism linked with high rates of CO2 and O2 recycling. KeywordsPhotosynthesis, net oxygen production, gross oxygen production, H218O labeling One sentence summaryA leaf gas-exchange system is presented enabling a more complete picture of the photosynthetic redox budget and calculation of the assimilatory quotient.

Stomata play crucial roles in the multilayered defense system against pathogens. Upon pathogen perception, stomata close promptly, establishing the first line of defense known as stomatal immunity. The bacterial pathogen Pseudomonas syringae (Pst) exploits open stomata for entry, however, it can also induce stomatal closure at post-invasive stages to enhance apoplastic hydration. This creates a favorable environment for Pst proliferation, evident as water-soaked lesions on leaves. During the post-invasive stages of Pst infection, plants deploy a second layer of stomatal defense by reopening their stomata, a process termed water immunity. To evaluate the relative importance of stomatal versus water immunity, we utilized a diverse set of Arabidopsis (Arabidopsis thaliana) mutants with impaired stomatal function and monitored bacterial growth, stomatal behavior, and water-soaking capacity after Pst pv. tomato DC3000 infection. Most mutants with constitutively open stomata and disrupted stomatal closure were more resistant against Pst than wild-type plants. Also, while some stomatal mutants displayed similar stomatal behavior at the initial steps of defense, their disease outcomes were the opposite, suggesting that stomatal immunity is not the determining factor in disease resistance. Instead, we discovered that the water-soaking capacity, associated with stomatal status at later stages of infection, i.e., water immunity, dictates the disease outcome. Our results show that water immunity can override the lack of stomatal immunity in plant resistance to Pst. We also address previous discrepancies in literature showing contradicting results for pathogen growth on stomatal mutants, highlighting the challenges in dissecting stomatal effects on plant resistance.

Prolonged drought is a major challenge in plant growth, severely affecting development and yield. Enhancing drought tolerance is thus a highly desired goal for agriculture. Here, we report that the loss-of-function of two drought-induced genes, GASA3 and AFP1, significantly enhances drought tolerance in Arabidopsis thaliana. While constitutive expression of GASA3 and AFP1 increased drought sensitivity compared to wild type (WT) plants, a gasa3afp1 double mutant exhibited superior drought tolerance compared to the single mutants. Enhanced drought tolerance of gasa3, afp1 and gasa3afp1 is likely due to reduced water loss caused by smaller stomatal apertures and thus lower transpiration rates. Moreover, gasa3 and afp1 mutants accumulated higher levels of abscisic acid (ABA) under drought conditions than WT plants, concomitant with a stronger up-regulation of ABA-responsive genes such as RD29A/B, ABF2/3, and ABI5. The stronger ABA increase in the mutants seems to result from hydrolysis of abscisic acid-glucosyl ester (ABA-GE) from vacuolar stores via the {beta}-glucosidase BG2 rather than by de-novo biosynthesis. Promoter analysis revealed the presence of ABA-responsive and drought stress-related cis-acting elements within the GASA3 and AFP1 promoter regions. RT-qPCR confirmed that the expression of both genes increased under drought. However, GASA3 induction was significantly reduced in the absence of AFP1, suggesting that AFP1 is involved in the modulation of GASA3 expression. Our findings identify a novel AFP1/GASA3-driven control circuit that negatively regulates drought tolerance by suppressing stomatal closure and attenuating ABA signalling.

Non-motile plants have evolved regulatory mechanisms to maintain homeostasis for optimal growth. Responses to environmental changes in light are particularly important not only during the diurnal transition from night to day but also to react to light changes caused by passing clouds or by wind. Thioredoxins rapidly orchestrate redox control during environmental change by modifying cysteine residues. Here, we assign a function to regulatory cysteines of PGRL1A, a constituent of the ferredoxin-dependent cyclic electron flow (Fd-CEF) pathway and show their role in the regulation of proton motive force (PMF) and nonphotochemical quenching (NPQ). During step increase of low light intensity (10-60 E*m-2*s-1), the intermolecular disulfide of the PGRL1A 59-kDa complex is reduced transiently within seconds to the 28 kDa form. In contrast, step increases to higher light intensity (60-600 E*m-2*s-1) stimulated a stable partially reduced redox state in PGRL1A. Measurements of NPQ, PMF and resultant photosynthetic controls Y(ND) and Y(NA) were found to correlate with the redox state of PGRL1A during step increases in light intensity but not in PGRL1mutant plants pgrl1ab or PGRL1A cysteine mutant (PGRL1AC1,2A). Continuous light regimes did not affect mutant growth; however, fluctuating regimes of light intensity showed significant growth reduction in the mutants. Inhibitors of photosynthesis placed control of the PGRL1A redox state as dependent on the penultimate ferredoxin redox state that fuels reducing equivalents to the large set of chloroplasts thioredoxins. Our results showed that redox state changes in PGRL1A are crucial to the optimization of photosynthesis and are regulated by the photosynthetic electron flux.