Plants exposed to incidences of excessive temperatures activate heat-stress responses to cope with the physiological challenge and stimulate long-term acclimation(1,2). The mechanism that senses cellular temperature for inducing thermotolerance is still unclear(3). Here we show that TWA1 is a temperature-sensing transcriptional co-regulator that is needed for basal and acquired thermotolerance in Arabidopsis thaliana. At elevated temperatures, TWA1 changes its conformation and allows physical interaction with JASMONATE-ASSOCIATED MYC-LIKE (JAM) transcription factors and TOPLESS (TPL) and TOPLESS-RELATED (TPR) proteins for repressor complex assembly. TWA1 is a predicted intrinsically disordered protein that has a key thermosensory role functioning through an amino-terminal highly variable region. At elevated temperatures, TWA1 accumulates in nuclear subdomains, and physical interactions with JAM2 and TPL appear to be restricted to these nuclear subdomains. The transcriptional upregulation of the heat shock transcription factor A2 (HSFA2) and heat shock proteins depended on TWA1, and TWA1 orthologues provided different temperature thresholds, consistent with the sensor function in early signalling of heat stress. The identification of the plant thermosensors offers a molecular tool for adjusting thermal acclimation responses of crops by breeding and biotechnology, and a sensitive temperature switch for thermogenetics.

SUMMARYPhytohormones are essential signaling molecules regulating various processes in growth, development, and stress responses. Genetic and molecular studies, especially using Arabidopsis thaliana (Arabidopsis), have discovered many important players involved in hormone perception, signal transduction, transport, and metabolism. Phytohormone signaling pathways are extensively interconnected with other endogenous and environmental stimuli. However, our knowledge of the huge and complex molecular network governed by a hormone remains limited. Here we report a global overview of downstream events of an abscisic acid (ABA) receptor, REGULATORY COMPONENTS OF ABA RECEPTOR (RCAR) 6 (also known as PYRABACTIN RESISTANCE 1 [PYR1]‐LIKE [PYL] 12), by integrating phosphoproteomic, proteomic and metabolite profiles. Our data suggest that the RCAR6 overexpression constitutively decreases the protein levels of its coreceptors, namely clade A protein phosphatases of type 2C, and activates sucrose non‐fermenting‐1 (SNF1)‐related protein kinase 1 (SnRK1) and SnRK2, the central regulators of energy and ABA signaling pathways. Furthermore, several enzymes in sugar metabolism were differentially phosphorylated and expressed in the RCAR6 line, and the metabolite profile revealed altered accumulations of several organic acids and amino acids. These results indicate that energy‐ and water‐saving mechanisms mediated by the SnRK1 and SnRK2 kinases, respectively, are under the control of the ABA receptor‐coreceptor complexes.

Soil water uptake by roots is a key component of plant water homeostasis contributing to plant growth and survival under ever-changing environmental conditions. The water transport capacity of roots (root hydraulic conductivity; Lpr ) is mostly contributed by finely regulated Plasma membrane Intrinsic Protein (PIP) aquaporins. In this study, we used natural variation of Arabidopsis for the identification of quantitative trait loci (QTLs) contributing to Lpr . Using recombinant lines from a biparental cross (Cvi-0 x Col-0), we show that the gene encoding class 2 Sucrose-Non-Fermenting Protein kinase 2.4 (SnRK2.4) in Col-0 contributes to >30% of Lpr by enhancing aquaporin-dependent water transport. At variance with the inactive and possibly unstable Cvi-0 SnRK2.4 form, the Col-0 form interacts with and phosphorylates the prototypal PIP2;1 aquaporin at Ser121 and stimulates its water transport activity upon coexpression in Xenopus oocytes and yeast cells. Activation of PIP2;1 by Col-0 SnRK2.4 in yeast also requires its protein kinase activity and can be counteracted by clade A Protein Phosphatases 2C. SnRK2.4 shows all hallmarks to be part of core abscisic acid (ABA) signaling modules. Yet, long-term (>3 h) inhibition of Lpr by ABA possibly involves a SnRK2.4-independent inhibition of PIP2;1. SnRK2.4 also promotes stomatal aperture and ABA-induced inhibition of primary root growth. The study identifies a key component of Lpr and sheds new light on the functional overlap and specificity of SnRK2.4 with respect to other ABA-dependent or independent SnRK2s.

Climate change and overexploitation of groundwater resources cause constraints on water demand for agriculture, thus threatening crop productivity. For future food security, there is an urgent need for crops of high water use efficiency combined with high crop productivity, i.e. having high water productivity. High water productivity means efficient biomass accumulation at reduced transpiration. Recent studies show that plants are able to optimize carbon uptake per water transpired with little or no trade-off in yield. The phytohormone abscisic acid (ABA) plays a pivotal role in minimizing leaf transpiration and mediating enhanced water productivity. Hence, ABA and more chemically stable ABA agonists have the potential to improve crop water productivity. Synthesis, screening, and identification of suitable ABA agonists are major efforts currently undertaken. In this study, we used yeast expressing the plant ABA signal pathway to prescreen ABA-related cyano cyclopropyl compounds (CCPs). The yeast analysis allowed testing the ABA agonists for general toxicity, efficient uptake, and specificity in regulating different ABA receptor complexes. Subsequently, promising ABA-mimics were analyzed in vitro for ligand-receptor interaction complemented by physiological analyses. Several CCPs activated ABA signaling in yeast and plant cells. CCP1, CCP2, and CCP5 were by an order of magnitude more efficient than ABA in minimizing transpiration of Arabidopsis plants. In a progressive drought experiment, CCP2 mediated an increase in water use efficiency superior to ABA without trade-offs in biomass accumulation.

We present unresolved questions in plant abiotic stress biology as posed by 15 research groups with expertise spanning eco-physiology to cell and molecular biology. Common themes of these questions include the need to better understand how plants detect water availability, temperature, salinity, and rising carbon dioxide (CO2) levels; how environmental signals interface with endogenous signaling and development (e.g. circadian clock and flowering time); and how this integrated signaling controls downstream responses (e.g. stomatal regulation, proline metabolism, and growth versus defense balance). The plasma membrane comes up frequently as a site of key signaling and transport events (e.g. mechanosensing and lipid-derived signaling, aquaporins). Adaptation to water extremes and rising CO2 affects hydraulic architecture and transpiration, as well as root and shoot growth and morphology, in ways not fully understood. Environmental adaptation involves tradeoffs that limit ecological distribution and crop resilience in the face of changing and increasingly unpredictable environments. Exploration of plant diversity within and among species can help us know which of these tradeoffs represent fundamental limits and which ones can be circumvented by bringing new trait combinations together. Better defining what constitutes beneficial stress resistance in different contexts and making connections between genes and phenotypes, and between laboratory and field observations, are overarching challenges.

Abstract Altering plant water use efficiency (WUE) is a promising approach for achieving sustainable crop production in changing climate scenarios. Here, we show that WUE can be tuned by alleles of a single gene discovered in elite maize (Zea mays) breeding material. Genetic dissection of a genomic region affecting WUE led to the identification of the gene ZmAbh4 as causative for the effect. CRISPR/Cas9-mediated ZmAbh4 inactivation increased WUE without growth reductions in well-watered conditions. ZmAbh4 encodes an enzyme that hydroxylates the phytohormone abscisic acid (ABA) and initiates its catabolism. Stomatal conductance is regulated by ABA and emerged as a major link between variation in WUE and discrimination against the heavy carbon isotope (Δ13C) during photosynthesis in the C4 crop maize. Changes in Δ13C persisted in kernel material, which offers an easy-to-screen proxy for WUE. Our results establish a direct physiological and genetic link between WUE and Δ13C through a single gene with potential applications in maize breeding.

New oxotetrahydroquinolinyl- and oxindolinyl sulfonamides interacting with RCAR/(PYR/PYL) receptor proteins were identified as lead structures against drought stress in crops starting from protein docking studies of a sulfonamide lead structure, followed by in-depth SAR studies. Optimized five to six step synthetic approaches via substituted amino oxo-tetrahydro-quinolines and amino oxo-indolines as essential intermediates gave access to the envisaged oxo-tetrahydroquinolinyl and oxindolinyl sulfonamides. Whilst oxo-tetrahydroquinolinyl sulfonamides with additional carbon substituents or spiro-cycloalkyl groups exhibited only low to moderate target affinities, the corresponding spiro-oxindolinyl and oxo-tetrahydroquinolinyl sulfonamides carrying optimized N-substituents revealed strong interactions with RCAR/(PYR/PYL) receptor proteins in Arabidopsis thaliana. Remarkably, the in vitro activity observed for these new compounds was on the same level as observed for the naturally occurring plant hormone in line with strong efficacy against drought stress in-vivo (canola and wheat as broad-acre crops).

The phytohormones salicylic acid (SA) and jasmonic acid (JA) promote two, mutually antagonistic immune pathways respectively protecting plants from biotrophic pathogens and necrotrophic pathogens or insects. This trade-off largely precludes the exploitation of SA and JA immune components for crop protection, raising the interest in immune signalling components that disrupt SA-JA antagonism. A local pathogen infection primes SA-dependent immunity in systemic tissues. This so-called systemic acquired resistance (SAR) ensures a long-lasting, broad-spectrum disease resistance that is not subject to SA-JA antagonism. Here, we show that two sequence-related LEGUME LECTIN-LIKE PROTEINs (LLPs) promote SAR through spatially separated functions with JA promoting local SAR signal generation through LLP3. In concert with LLP1, which is important for systemic recognition and propagation of SAR signals, LLP3 promotes both SA-dependent SAR and JA-mediated immunity. Thus, exploitation of LLP-associated signalling cues might allow application of plant innate immune signals to promote (crop) plant health.

Plant hormones coordinate responses to environmental cues with developmental programs1, and are fundamental for stress resilience and agronomic yield2. The core signalling pathways underlying the effects of phytohormones have been elucidated by genetic screens and hypothesis-driven approaches, and extended by interactome studies of select pathways3. However, fundamental questions remain about how information from different pathways is integrated. Genetically, most phenotypes seem to be regulated by several hormones, but transcriptional profiling suggests that hormones trigger largely exclusive transcriptional programs4. We hypothesized that protein-protein interactions have an important role in phytohormone signal integration. Here, we experimentally generated a systems-level map of the Arabidopsis phytohormone signalling network, consisting of more than 2,000 binary protein-protein interactions. In the highly interconnected network, we identify pathway communities and hundreds of previously unknown pathway contacts that represent potential points of crosstalk. Functional validation of candidates in seven hormone pathways reveals new functions for 74% of tested proteins in 84% of candidate interactions, and indicates that a large majority of signalling proteins function pleiotropically in several pathways. Moreover, we identify several hundred largely small-molecule-dependent interactions of hormone receptors. Comparison with previous reports suggests that noncanonical and nontranscription-mediated receptor signalling is more common than hitherto appreciated.

Phytochelatin synthase (PCS) is a key component of heavy metal detoxification in plants. PCS catalyzes both the synthesis of the peptide phytochelatin from glutathione and the degradation of glutathione conjugates via peptidase activity. Here, we describe a role for PCS in disease resistance against plant pathogenic fungi. The pen4 mutant, which is allelic to cadmium insensitive1 (cad1/pcs1) mutants, was recovered from a screen for Arabidopsis mutants with reduced resistance to the nonadapted barley fungal pathogen Blumeria graminis f. sp. hordei PCS1, which is found in the cytoplasm of cells of healthy plants, translocates upon pathogen attack and colocalizes with the PEN2 myrosinase on the surface of immobilized mitochondria. pcs1 and pen2 mutant plants exhibit similar metabolic defects in the accumulation of pathogen-inducible indole glucosinolate-derived compounds, suggesting that PEN2 and PCS1 act in the same metabolic pathway. The function of PCS1 in this pathway is independent of phytochelatin synthesis and deglycination of glutathione conjugates, as catalytic-site mutants of PCS1 are still functional in indole glucosinolate metabolism. In uncovering a peptidase-independent function for PCS1, we reveal this enzyme to be a moonlighting protein important for plant responses to both biotic and abiotic stresses.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Plants are essential for life and are extremely diverse organisms with unique molecular capabilities 1 . Here we present a quantitative atlas of the transcriptomes, proteomes and phosphoproteomes of 30 tissues of the model plant Arabidopsis thaliana . Our analysis provides initial answers to how many genes exist as proteins (more than 18,000), where they are expressed, in which approximate quantities (a dynamic range of more than six orders of magnitude) and to what extent they are phosphorylated (over 43,000 sites). We present examples of how the data may be used, such as to discover proteins that are translated from short open-reading frames, to uncover sequence motifs that are involved in the regulation of protein production, and to identify tissue-specific protein complexes or phosphorylation-mediated signalling events. Interactive access to this resource for the plant community is provided by the ProteomicsDB and ATHENA databases, which include powerful bioinformatics tools to explore and characterize Arabidopsis proteins, their modifications and interactions.

The physiological roles of abscisic acid (ABA) as a stress hormone in plant responses to water shortage, including stomatal regulation and gene expression, have been well documented. However, less attention has been paid to the function of basal ABA synthesized under well-watered conditions in recent studies. In this review, we summarize progress in the understanding of how low concentrations of ABA are perceived at the molecular level and how its signaling affects plant metabolism and growth under nonstressed conditions. We also discuss the dual nature of ABA in acting as an inhibitor and activator of plant growth and development.

Improving the water use efficiency (WUE) of crop plants without trade-offs in growth and yield is considered a utopic goal. However, recent studies on model plants show that partial restriction of transpiration can occur without a reduction in CO2 uptake and photosynthesis. In this study, we analyzed the potentials and constraints of improving WUE in Arabidopsis (Arabidopsis thaliana) and in wheat (Triticum aestivum). We show that the analyzed Arabidopsis wild-type plants consume more water than is required for unrestricted growth. WUE was enhanced without a growth penalty by modulating abscisic acid (ABA) responses either by using overexpression of specific ABA receptors or deficiency of ABA coreceptors. Hence, the plants showed higher water productivity compared with the wild-type plants; that is, equal growth with less water. The high WUE trait was resilient to changes in light intensity and water availability, but it was sensitive to the ambient temperature. ABA application to plants generated a partial phenocopy of the water-productivity trait. ABA application, however, was never as effective as genetic modification in enhancing water productivity, probably because ABA indiscriminately targets all ABA receptors. ABA agonists selective for individual ABA receptors might offer an approach to phenocopy the water-productivity trait of the high WUE lines. ABA application to wheat grown under near-field conditions improved WUE without detectable growth trade-offs. Wheat yields are heavily impacted by water deficit, and our identification of this crop as a promising target for WUE improvement may help contribute to greater food security.

The phytohormone abscisic acid (ABA) regulates plant responses to abiotic stress, such as drought and high osmotic conditions. The multitude of functionally redundant components involved in ABA signaling poses a major challenge for elucidating individual contributions to the response selectivity and sensitivity of the pathway. Here, we reconstructed single ABA signaling pathways in yeast for combinatorial analysis of ABA receptors and coreceptors, downstream-acting SnRK2 protein kinases, and transcription factors. The analysis shows that some ABA receptors stimulate the pathway even in the absence of ABA and that SnRK2s are major determinants of ABA responsiveness by differing in the ligand-dependent control. Five SnRK2s, including SnRK2.4 known to be active under osmotic stress in plants, activated ABA-responsive transcription factors and were regulated by ABA receptor complexes in yeast. In the plant tissue, SnRK2.4 and ABA receptors competed for coreceptor interaction in an ABA-dependent manner consistent with a tight integration of SnRK2.4 into the ABA signaling pathway. The study establishes the suitability of the yeast system for the dissection of core signaling cascades and opens up future avenues of research on ligand-receptor regulation.

Key message A genomic segment on maize chromosome 7 influences carbon isotope composition, water use efficiency, and leaf growth sensitivity to drought, possibly by affecting stomatal properties. Abstract Climate change is expected to decrease water availability in many agricultural production areas around the globe. Therefore, plants with improved ability to grow under water deficit are urgently needed. We combined genetic, phenomic, and physiological approaches to understand the relationship between growth, stomatal conductance, water use efficiency, and carbon isotope composition in maize ( Zea mays L. ). Using near-isogenic lines derived from a maize introgression library, we analysed the effects of a genomic region previously identified as affecting carbon isotope composition. We show stability of trait expression over several years of field trials and demonstrate in the phenotyping platform Phenodyn that the same genomic region also influences the sensitivity of leaf growth to evaporative demand and soil water potential. Our results suggest that the studied genomic region affecting carbon isotope discrimination also harbours quantitative trait loci playing a role in maize drought sensitivity possibly via stomatal behaviour and development. We propose that the observed phenotypes collectively originate from altered stomatal conductance, presumably via abscisic acid.

Phytochelatin synthase (PCS) is a key component of heavy metal detoxification in plants. PCS catalyzes both the synthesis of the peptide phytochelatin from glutathione as well as the degradation of glutathione conjugates via peptidase activity. Here, we describe a hitherto uncharacterized role for PCS in disease resistance against plant pathogenic fungi. The pen4 mutant, which is allelic to cadmium insensitive 1 ( cad1/pcs1 ) mutants, was recovered from a screen for Arabidopsis mutants with reduced resistance to the non-adapted barley fungal pathogen, Blumeria graminis f. sp. hordei . PCS1, which is found in the cytoplasm of cells of healthy plants, translocates upon pathogen attack and colocalizes with the PEN2 myrosinase on the surface of immobilized mitochondria. pcs1 and pen2 mutant plants exhibit a similar metabolic defect in the accumulation of pathogen-inducible indole glucosinolate-derived compounds, suggesting that PEN2 and PCS1 act in the same metabolic pathway. The function of PCS1 in this pathway is independent of phytochelatin synthesis and deglycination of glutathione conjugates, as catalytic-site mutants of PCS1 are still functional in indole glucosinolate metabolism. In uncovering a previously unknown function for PCS1, we reveal this enzyme to be a moonlighting protein important for plant responses to both biotic and abiotic stresses.

Arabidopsis (Arabidopsis thaliana) efficiently synthesizes the antifungal phytoalexin camalexin without the apparent release of bioactive intermediates, such as indole-3-acetaldoxime, suggesting that the biosynthetic pathway of this compound is channeled by the formation of an enzyme complex. To identify such protein interactions, we used two independent untargeted coimmunoprecipitation (co-IP) approaches with the biosynthetic enzymes CYP71B15 and CYP71A13 as baits and determined that the camalexin biosynthetic P450 enzymes copurified with these enzymes. These interactions were confirmed by targeted co-IP and Förster resonance energy transfer measurements based on fluorescence lifetime microscopy (FRET-FLIM). Furthermore, the interaction of CYP71A13 and Arabidopsis P450 Reductase1 was observed. We detected increased substrate affinity of CYP79B2 in the presence of CYP71A13, indicating an allosteric interaction. Camalexin biosynthesis involves glutathionylation of the intermediary indole-3-cyanohydrin, which is synthesized by CYP71A12 and especially CYP71A13. FRET-FLIM and co-IP demonstrated that the glutathione transferase GSTU4, which is coexpressed with Trp- and camalexin-specific enzymes, is physically recruited to the complex. Surprisingly, camalexin concentrations were elevated in knockout and reduced in GSTU4-overexpressing plants. This shows that GSTU4 is not directly involved in camalexin biosynthesis but rather plays a role in a competing mechanism.