Stomata on leaves determine water loss from plants and carbon uptake for photosynthesis. These key physiological processes affect biomass production, stress sensitivity and water use efficiency in plants, and are also addressed in breeding programs. Abscisic acid (ABA) is an important plant stress hormone, whose signaling module described for Arabidopsis is conserved in land plants: it controls the activity of protein kinase OST1 (Open Stomata 1, also SnRK2.6/SRK2E), which triggers stomatal closure by activating guard cell anion channels. Here, we generated barley double mutants defective in proteins homologous to Arabidopsis OST1 and measured their stomatal conductance and responses to ABA spraying and rising vapor pressure deficit (VPD). These barley mutants showed wider stomatal apertures, higher stomatal conductances and decreased VPD- and ABA-sensitivities compared with wild-type ('Golden Promise') in gas exchange experiments, whereas their net assimilation rates and stomatal densities were only mildly affected. No statistically significant difference between double mutants and wild type was detected in grain yield per plant under well-watered conditions. These results show that OST1-like kinases HvSnRK2.7 and HvSnRK2.9 control stomatal conductance and sensitivity to ABA and VPD in barley. The effect of modified ABA signaling pathway on stress tolerance and yield is discussed in the context of breeding plants that are productive under various conditions of water availability.

Stomatal pores on leaves determine water loss of plants and carbon uptake for photosynthesis. These key physiological processes affect biomass production, stress sensitivity and water use efficiency in plants and are also addressed in breeding programs. Abscisic acid (ABA) is an important plant stress hormone, whose signaling module described for Arabidopsis is conserved in land plants: it controls the activity of protein kinase OST1 (Open Stomata 1, also SnRK2.6/SRK2E), which triggers stomatal closure by activating guard cell anion channels. Here, we generated barley double mutants defective in proteins homologous to Arabidopsis OST1 and measured their stomatal conductance and responses to rising VPD and ABA spraying. These barley mutants showed wider stomatal aperture, higher stomatal conductance and decreased VPD- and ABA-sensitivities compared with wildtype (cv Golden Promise) in gas exchange experiments, whereas their net assimilation rates and stomatal densities were only mildly affected. No statistically significant difference between the lines was detected in grain yield per plant under well-watered conditions. These results show that OST1-like kinases HvSnRK2.7 and HvSnRK2.9 control stomatal conductance and sensitivity to ABA and abiotic factors in barley. The effect of alleviating ABA signalling pathway on stress tolerance and yield is discussed in the context of breeding plants productive under various water availability conditions.

There is a need for ground-breaking technologies to boost crop yield, both grains and biomass, and their processing into economically competitive materials. Novel cereals with enhanced photosynthesis and assimilation of greenhouse gasses, such as carbon dioxide and ozone, and tailored straw suitable for industrial manufacturing, open a new perspective for the circular economy. Here we describe the vision, strategies, and objectives of BEST-CROP, a Horizon-Europe and United Kingdom Research and Innovation (UKRI) funded project that relies on an alliance of academic plant scientists teaming up with plant breeding companies and straw processing companies to use the major advances in photosynthetic knowledge to improve barley biomass and to exploit the variability of barley straw quality and composition. We adopt the most promising strategies to improve the photosynthetic properties and ozone assimilation capacity of barley: (i) tuning leaf chlorophyll content and modifying canopy architecture; (ii) increasing the kinetics of photosynthetic responses to changes in irradiance; (iii) introducing photorespiration bypasses; (iv) modulating stomatal opening, thus increasing the rate of carbon dioxide fixation and ozone assimilation. We expect that by improving our targeted traits we will achieve increases in aboveground total biomass production without modification of the harvest index, with added benefits in sustainability via better resource-use efficiency of water and nitrogen. In parallel, the resulting barley straw is tailored to: (i) increase straw protein content to make it suitable for the development of alternative biolubricants and feed sources; (ii) control cellulose/lignin contents and lignin properties to develop straw-based construction panels and polymer composites. Overall, by exploiting natural- and induced-genetic variability as well as gene editing and transgenic engineering, BEST-CROP will lead to multi-purpose next generation barley cultivars supporting sustainable agriculture and capable of straw-based applications.

Abscisic acid (ABA)-induced stomatal closure limits water loss from plants under drought stress. To investigate the signaling pathways involved in ABA-induced stomatal closure, we performed a phosphoproteomic analysis of ABA-treated Arabidopsis guard cell protoplasts (GCPs). We found that ABA-responsive phosphorylation of MITOGEN-ACTIVATED PROTEIN 4 KINASE 1 (MAP4K1) was significantly down-regulated in SnRK2-disrupted mutants. Subsequent biochemical assays showed that Ser479 of MAP4K1 is directly phosphorylated by SRK2E/OST1, a central ABA kinase. Mutational analyses of MAP4K1 and MAP4K2 revealed that both kinases positively regulate ABA-induced stomatal closure and that Ser479 of MAP4K1 is required for this phenotype. In map4k1map4k2 double mutants, stomatal closure was induced by applying exogenous Ca2+ but not H2O2. Electrophysiological experiments showed that MAP4K1/2 is required for ABA-dependent activation of Ca2+-permeable channels in GCPs. Together, our results indicate that SnRK2 and MAP4K function as a signaling module to regulate the Ca2+-mediated pathway in ABA-induced stomatal closure.

Spring wheat (Triticum aestivum L.) remains an important alternative to winter wheat cultivation at Northern latitudes due to high risk of overwintering or delayed sowing of winter wheat. We studied nine major agronomic traits in a set of 299 spring wheat genotypes in trials across 12-year-site combinations in Lithuania, Latvia, Estonia, and Norway for three consecutive years. The dataset analyzed here consisted of previously published phenotypic data collected in 2021 and 2022, supplemented with additional phenotypic data from the 2023 field season collected in this study. We combined these phenotypic datasets with previously published genotypic data generated using a 25K single nucleotide polymorphism (SNP) array that yielded 18,467 markers with a minor allele frequency above 0.05. Analysis of these datasets via genome-wide association study revealed 18 consistent quantitative trait loci (QTL) replicated in two or more trials that explained more than 5% of phenotypic variance for plant height, grain protein content, thousand kernel weight, or heading date. The most consistent markers across the tested environments were detected for plant height, thousand kernel weight, and days to heading in eight, five, and six trials, respectively. No beneficial effect of the semi-dwarfing alleles Rht-B1b and Rht-D1b on grain yield performance was observed across the 12 tested trials. Moreover, the cultivars carrying these alleles were low yielding in general. Based on principal component analysis, wheat genotypes developed in the Northern European region clustered separately from those developed at the southern latitudes, and markers associated with the clustering were identified. Important phenotypic traits, such as grain yield, days to heading, grain protein content, and thousand kernel weight were associated with this clustering of the genotype sets. Interestingly, despite being adapted to the Nordic environment, genotypes in the Northern set demonstrated lower grain yield performance across all tested environments. The results indicate that spring wheat germplasm harbors valuable QTL/alleles, and the identified trait-marker associations might be useful in improving Nordic–Baltic spring wheat germplasm under global warming conditions.

Pectins are enriched in primary cell walls and the middle lamella, determining cell-to-cell adhesion. O-acetylation of pectin backbones influences their physicochemical properties and plays a role in plant development and interactions with the environment. Here we report the isolation and mapping of two trichome birefringence (tbr) mutants from an ozone-sensitivity screen and show that TBR is required for leaf epidermal cell adhesion. We further demonstrate that TBR is an RG-I rhamnose O-acetyltransferase with an acidic pH optimum, and, through structure-function modelling coupled with biochemical analysis determine the significance of mutated amino acid residues. Notably, TBR utilizes multiple O-acetyl donors, and is able to form acyl-enzyme intermediates in the presence of acetylated pectins, suggesting transacetylase activity. We propose that, in tbr, loss of cell adhesion stems from cell wall modifications triggered by decreased RG-I acetylation during leaf cell proliferation. Together, our data suggest that RG-I O-acetylation affects cell adhesion.

Key message QPm.NOBAL-3A is an important QTL providing robust adult plant powdery mildew resistance in Nordic and Baltic spring wheat, aiding sustainable crop protection and breeding. Abstract Powdery mildew, caused by the biotrophic fungal pathogen Blumeria graminis f. sp. tritici , poses a significant threat to bread wheat ( Triticum aestivum L.), one of the world’s most crucial cereal crops. Enhancing cultivar resistance against this devastating disease requires a comprehensive understanding of the genetic basis of powdery mildew resistance. In this study, we performed a genome-wide association study (GWAS) using extensive field trial data from multiple environments across Estonia, Latvia, Lithuania, and Norway. The study involved a diverse panel of recent wheat cultivars and breeding lines sourced from the Baltic region and Norway. We identified a major quantitative trait locus (QTL) on chromosome 3A, designated as QPm.NOBAL-3A , which consistently conferred high resistance to powdery mildew across various environments and countries. Furthermore, the consistency of the QTL haplotype effect was validated using an independent Norwegian spring wheat panel. Subsequent greenhouse seedling inoculations with 15 representative powdery mildew isolates on a subset of the GWAS panel indicated that this QTL provides adult plant resistance and is likely of race non-specific nature. Moreover, we developed and validated KASP markers for QPm.NOBAL-3A tailored for use in breeding. These findings provide a critical foundation for marker-assisted selection in breeding programs aimed at pyramiding resistance QTL/genes to achieve durable and broad-spectrum resistance against powdery mildew.

Stomatal pores that control plant CO2 uptake and water loss affect global carbon and water cycles. In the era of increasing atmospheric CO2 levels and vapor pressure deficit (VPD), it is essential to understand how these stimuli affect stomatal behavior. Whether stomatal responses to sub-ambient and above-ambient CO2 levels are governed by the same regulators and depend on VPD remains unknown. We studied stomatal conductance responses in Arabidopsis (Arabidopsis thaliana) stomatal signaling mutants under conditions where CO2 levels were either increased from sub-ambient to ambient (400 ppm) or from ambient to above-ambient levels under normal or elevated VPD. We found that guard cell signaling components involved in CO2-induced stomatal closure have different roles in the sub-ambient and above-ambient CO2 levels. The CO2-specific regulators prominently affected sub-ambient CO2 responses, whereas the lack of guard cell slow-type anion channel SLOW ANION CHANNEL-ASSOCIATED 1 (SLAC1) more strongly affected the speed of above-ambient CO2-induced stomatal closure. Elevated VPD caused lower stomatal conductance in all studied genotypes and CO2 transitions, as well as faster CO2-responsiveness in some studied genotypes and CO2 transitions. Our results highlight the importance of experimental setups in interpreting stomatal CO2-responsiveness, as stomatal movements under different CO2 concentration ranges are controlled by distinct mechanisms. Elevated CO2 and VPD responses may also interact. Hence, multi-factor treatments are needed to understand how plants integrate different environmental signals and translate them into stomatal responses.

Wheat ( Triticum aestivum L.) has become the most widely grown cereal crop in the Nordic-Baltic region, however, due to climate change its yields are under increasing risk. Here we present results of international effort harnessing available wheat germplasm in the region for identification of tools and genotypes for breeding wheat varieties with improved yield stability. We formed a panel comprising 300 spring wheat genotypes from Nordic-Baltic region and studied their phenotypic diversity across different environments to identify genotypes with high potential for adaptation to changing climate. Field experiments were carried out in 2021 and 2022 in Estonia, Latvia, Lithuania and Norway. The performance and stability of yield (GY), protein content (PC), thousand kernel weight (TKW), test weight (TW), length of growing period (GP), and nine other traits – were studied. Drought and excessive rainfall occurred in the Baltic countries in 2021–2022, whereas weather was more stable in Norway. High variability for most, and significant GxE effect for all the studied traits was identified. We found out genotypes combining yield and quality with stability using the AMMI model-based Weighted Average of Absolute Scores index (WAASB). Finally, we selected nineteen superior genotypes that combined high yield with high values of important quality traits. Thus, combining broad Nordic-Baltic wheat germplasm with extensive field phenotyping, we identified promising breeding material to develop climate ready spring wheat varieties for the region.

Accurate and robust methods are needed to monitor crop growth and predict grain yield and quality in breeding programs, particularly under variable agrometeorological conditions. Field experiments were conducted during two successive cropping seasons (2021, 2022) at four trial locations (Estonia, Latvia, Lithuania, Norway). The focus was on assessment of the grain yield (GY), grain protein content (GPC), and UAV-derived NDVI measured at different plant growth stages. The performance and stability of 16 selected spring wheat genotypes were assessed under two N application rates (75, 150 kg N ha−1) and across different agrometeorological conditions. Quantitative relationships between agronomic traits and UAV-derived variables were determined. None of the traits exhibited a significant (p < 0.05) genotype-by-nitrogen interaction. High-yielding and high-protein genotypes were detected with a high WAASB stability, specifically under high and low N rates. This study highlights the significant effect of an NDVI analysis at GS55 and GS75 as key linear predictors, especially concerning spring wheat GYs. However, the effectiveness of these indices depends on the specific growing conditions in different, geospatially distant locations, limiting their universal utility.

Protein phosphorylation is a major molecular switch involved in the regulation of stomatal opening and closure. Previous research defined interaction between MAP kinase 12 and Raf-like kinase HT1 as a required step for stomatal movements caused by changes in CO2 concentration. However, whether MPK12 kinase activity is required for regulation of CO2-induced stomatal responses warrants in-depth investigation. We apply genetic, biochemical, and structural modeling approaches to examining the noncatalytic role of MPK12 in guard cell CO2 signaling that relies on allosteric inhibition of HT1. We show that CO2/HCO3- -enhanced MPK12 interaction with HT1 is independent of its kinase activity. By analyzing gas exchange of plant lines expressing various kinase-dead and constitutively active versions of MPK12 in a plant line where MPK12 is deleted, we confirmed that CO2-dependent stomatal responses rely on MPK12's ability to bind to HT1, but not its kinase activity. We also demonstrate that purified MPK12 and HT1 proteins form a heterodimer in the presence of CO2/HCO3- and present structural modeling that explains the MPK12:HT1 interaction interface. These data add to the model that MPK12 kinase-activity-independent interaction with HT1 functions as a molecular switch by which guard cells sense changes in atmospheric CO2 concentration.

Land plants evolved multiple adaptations to restrict transpiration. However, the underlying molecular mechanisms are not sufficiently understood. We used an ozone-sensitivity forward genetics approach to identify Arabidopsis thaliana mutants impaired in gas exchange regulation. High water loss from detached leaves and impaired decrease of leaf conductance in response to multiple stomata-closing stimuli were identified in a mutant of MURUS1 (MUR1), an enzyme required for GDP-l-fucose biosynthesis. High water loss observed in mur1 was independent from stomatal movements and instead could be linked to metabolic defects. Plants defective in import of GDP-l-Fuc into the Golgi apparatus phenocopied the high water loss of mur1 mutants, linking this phenotype to Golgi-localized fucosylation events. However, impaired fucosylation of xyloglucan, N-linked glycans, and arabinogalactan proteins did not explain the aberrant water loss of mur1 mutants. Partial reversion of mur1 water loss phenotype by borate supplementation and high water loss observed in boron uptake mutants link mur1 gas exchange phenotypes to pleiotropic consequences of l-fucose and boron deficiency, which in turn affect mechanical and morphological properties of stomatal complexes and whole-plant physiology. Our work emphasizes the impact of fucose metabolism and boron uptake on plant-water relations.

Little is known about long-distance mesophyll-driven signals that regulate stomatal conductance. Soluble and/or vapor-phase molecules have been proposed. In this study, the involvement of the gaseous signal ethylene in the modulation of stomatal conductance in Arabidopsis thaliana by CO2 /abscisic acid (ABA) was examined. We present a diffusion model which indicates that gaseous signaling molecule/s with a shorter/direct diffusion pathway to guard cells are more probable for rapid mesophyll-dependent stomatal conductance changes. We, therefore, analyzed different Arabidopsis ethylene-signaling and biosynthesis mutants for their ethylene production and kinetics of stomatal responses to ABA/[CO2 ]-shifts. According to our research, higher [CO2 ] causes Arabidopsis rosettes to produce more ethylene. An ACC-synthase octuple mutant with reduced ethylene biosynthesis exhibits dysfunctional CO2 -induced stomatal movements. Ethylene-insensitive receptor (gain-of-function), etr1-1 and etr2-1, and signaling, ein2-5 and ein2-1, mutants showed intact stomatal responses to [CO2 ]-shifts, whereas loss-of-function ethylene receptor mutants, including etr2-3;ein4-4;ers2-3, etr1-6;etr2-3 and etr1-6, showed markedly accelerated stomatal responses to [CO2 ]-shifts. Further investigation revealed a significantly impaired stomatal closure to ABA in the ACC-synthase octuple mutant and accelerated stomatal responses in the etr1-6;etr2-3, and etr1-6, but not in the etr2-3;ein4-4;ers2-3 mutants. These findings suggest essential functions of ethylene biosynthesis and signaling components in tuning/accelerating stomatal conductance responses to CO2 and ABA.

Abscisic acid (ABA) is best known for regulating the responses to abiotic stressors. Thus, applications of ABA signaling pathways are considered promising targets for securing yield under stress. ABA levels rise in response to abiotic stress, mounting physiological and metabolic responses that promote plant survival under unfavorable conditions. ABA elicits its effects by binding to a family of soluble receptors found in monomeric and dimeric states, differing in their affinity to ABA and co-receptors. However, the in vivo significance of the biochemical differences between these receptors remains unclear. We took a gain-of-function approach to study receptor-specific functionality. First, we introduced activating mutations that enforce active ABA-bound receptor conformation. We then transformed Arabidopsis ABA-deficient mutants with the constitutive receptors and monitored suppression of the ABA deficiency phenotype. Our findings suggest that PYL4 and PYL5, monomeric ABA receptors, have differential activity in regulating transpiration and transcription of ABA biosynthesis and stress response genes. Through genetic and metabolic data, we demonstrate that PYR1, but not PYL5, is sufficient to activate the ABA positive feedback mechanism. We propose that ABA signaling - from perception to response - flows differently when triggered by different PYLs, due to tissue and transcription barriers, thus resulting in distinct circuitries.

Accurate and robust methods are needed to monitor crop growth and predict grain yield and quality in breeding programs under variable agrometeorological conditions. Field experiments were conducted during two successive cropping seasons (2021, 2022) at four trial locations (Estonia, Latvia, Lithuania, Norway). The focus was on assessment of grain yield (GY), grain protein content (GPC), and UAV-derived NDVI measured at different plant growth stages. Performance and stability of 16 selected spring wheat genotypes were assessed under two N application rates (75, 150 kg N ha−1) and over contrasted agrometeorological conditions were assessed, and the quantitative relationships between agronomic traits and UAV-derived variables were figured out. None of the traits were subject to a significant (p<0.05) genotype by nitrogen interaction. High-yielding and high-protein genotypes were detected with high WAASB stability, specifically under high and low N rates. This study highlights the significant effect of NDVI analysis on growth stages GS55 and GS75 as key linear predictors, especially in the context of spring wheat GY. However, the effect of these indices depends on the specific growing conditions in the respective locations, thus limiting their universal utility.

Greenhouse gas (GHG) emissions have created a global climate crisis which requires immediate interventions to mitigate the negative effects on all aspects of life on this planet. As current agriculture and land use contributes up to 25% of total GHG emissions, plant scientists take center stage in finding possible solutions for a transition to sustainable agriculture and land use. In this article, the PlantACT! (Plants for climate ACTion!) initiative of plant scientists lays out a road map of how and in which areas plant scientists can contribute to finding immediate, mid-term, and long-term solutions, and what changes are necessary to implement these solutions at the personal, institutional, and funding levels.

Plant transpiration is controlled by stomata, with S- and R-type anion channels playing key roles in guard cell action. Arabidopsis mutants lacking the ALMT12/QUAC1 R-type anion channel function in guard cells show only a partial reduction in R-type channel currents. The molecular nature of these remaining R-type anion currents is still unclear. To further elucidate this, patch clamp, transcript and gas-exchange measurements were performed with wild-type (WT) and different almt mutant plants. The R-type current fraction in the almt12 mutant exhibited the same voltage dependence, susceptibility to ATP block and lacked a chloride permeability as the WT. Therefore, we asked whether the R-type anion currents in the ALMT12/QUAC1-free mutant are caused by additional ALMT isoforms. In WT guard cells, ALMT12, ALMT13 and ALMT14 transcripts were detected, whereas only ALMT13 was found expressed in the almt12 mutant. Substantial R-type anion currents still remained active in the almt12/13 and almt12/14 double mutants as well as the almt12/13/14 triple mutant. In good agreement, CO2 -triggered stomatal closure required the activity of ALMT12 but not ALMT13 or ALMT14. The results suggest that, with the exception of ALMT12, channel species other than ALMTs carry the guard cell R-type anion currents.

Summary Light induces stomatal opening, which is driven by plasma membrane (PM) H+‐ATPase in guard cells. The activation of guard‐cell PM H+‐ATPase is mediated by phosphorylation of the penultimate C‐terminal residue, threonine. The phosphorylation is induced by photosynthesis as well as blue light photoreceptor phototropin. Here, we investigated the effects of cessation of photosynthesis on the phosphorylation level of guard‐cell PM H+‐ATPase in Arabidopsis thaliana. Immunodetection of guard‐cell PM H+‐ATPase, time‐resolved leaf gas‐exchange analyses and stomatal aperture measurements were carried out. We found that light–dark transition of leaves induced dephosphorylation of the penultimate residue at 1 min post‐transition. Gas‐exchange analyses confirmed that the dephosphorylation is accompanied by an increase in the intercellular CO2 concentration, caused by the cessation of photosynthetic CO2 fixation. We discovered that CO2 induces guard‐cell PM H+‐ATPase dephosphorylation as well as stomatal closure. Interestingly, reverse‐genetic analyses using guard‐cell CO2 signal transduction mutants suggested that the dephosphorylation is mediated by a mechanism distinct from the established CO2 signalling pathway. Moreover, type 2C protein phosphatases D6 and D9 were required for the dephosphorylation and promoted stomatal closure upon the light–dark transition. Our results indicate that CO2‐mediated dephosphorylation of guard‐cell PM H+‐ATPase underlies stomatal closure.

Stomatal opening is largely promoted by light-activated plasma membrane-localized proton ATPases (PM H+-ATPases), while their closure is mainly modulated by abscisic acid (ABA) signaling during drought stress. It is unknown whether PM H+-ATPases participate in ABA-induced stomatal closure. We established that BRI1-ASSOCIATED RECEPTOR KINASE 1 (BAK1) interacts with, phosphorylates and activates the major PM Arabidopsis H+-ATPase isoform 2 (AHA2). Detached leaves from aha2-6 single mutant Arabidopsis thaliana plants lost as much water as bak1-4 single and aha2-6 bak1-4 double mutants, with all three mutants losing more water than the wild-type (Columbia-0 [Col-0]). In agreement with these observations, aha2-6, bak1-4, and aha2-6 bak1-4 mutants were less sensitive to ABA-induced stomatal closure than Col-0, whereas the aha2-6 mutation did not affect ABA-inhibited stomatal opening under light conditions. ABA-activated BAK1 phosphorylated AHA2 at Ser-944 in its C-terminus and activated AHA2, leading to rapid H+ efflux, cytoplasmic alkalinization, and reactive oxygen species (ROS) accumulation, to initiate ABA signal transduction and stomatal closure. The phosphorylation-mimicking mutation AHA2S944D driven by its own promoter could largely compensate for the defective phenotypes of water loss, cytoplasmic alkalinization, and ROS accumulation in both aha2-6 and bak1-4 mutants. Our results uncover a crucial role of AHA2 in cytoplasmic alkalinization and ABA-induced stomatal closure during the plant's response to drought stress.

The continuing rise in the atmospheric carbon dioxide (CO 2 ) concentration causes stomatal closing, thus critically affecting transpirational water loss, photosynthesis, and plant growth. However, the primary CO 2 sensor remains unknown. Here, we show that elevated CO 2 triggers interaction of the MAP kinases MPK4/MPK12 with the HT1 protein kinase, thus inhibiting HT1 kinase activity. At low CO 2 , HT1 phosphorylates and activates the downstream negatively regulating CBC1 kinase. Physiologically relevant HT1-mediated phosphorylation sites in CBC1 are identified. In a genetic screen, we identify dominant active HT1 mutants that cause insensitivity to elevated CO 2 . Dominant HT1 mutants abrogate the CO 2 /bicarbonate-induced MPK4/12-HT1 interaction and HT1 inhibition, which may be explained by a structural AlphaFold2- and Gaussian-accelerated dynamics-generated model. Unexpectedly, MAP kinase activity is not required for CO 2 sensor function and CO 2 -triggered HT1 inhibition and stomatal closing. The presented findings reveal that MPK4/12 and HT1 together constitute the long-sought primary stomatal CO 2 /bicarbonate sensor upstream of the CBC1 kinase in plants.