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.

In land plants, stomatal pores on leaf surfaces developed to control gas exchange between leaf and surrounding air, but also to enable nutrient uptake and leaf cooling. Traits such as stomatal density (SD), guard cell size, stomatal distribution between the upper (adaxial) and lower (abaxial) leaf surfaces (stomatal ratio), and stomatal aperture width exhibit notable variation across different genotypes and environments. These traits influence leaf photosynthesis, water loss, growth, productivity, and pathogen susceptibility. Here, we studied different stomatal traits of spring wheat flag leaves and their relationship with grain yield in field experiments during 2022-2023. Significant genotypic variation among adaxial and abaxial SDs and stomatal ratios was detected, whereas stomatal conductance was mostly affected by annual differences in weather. A strong negative relationship between adaxial stomatal density and grain yield was detected under all conditions, when abiotic factors (water stress or nutrient limitation) resulted in yield losses, whereas under favourable conditions, there was no significant relationship between adaxial stomatal density and grain yield. The effects of leaf surface-specific traits on yield are often overlooked in physiological and breeding experiments. Our results indicate that higher-than-optimal adaxial SD values may result in wheat yield losses under stresses imposed by future climate conditions. ### Competing Interest Statement The authors have declared no competing interest.

Crop yield is at increasing risk due to water scarcity and climate change. Agrochemicals can activate hormone receptors to regulate transpiration and modulate transcription and address water deficits. Structure-guided optimization of multiple abscisic acid (ABA) receptor-agonist interactions is necessary to activate the entire PYRABACTIN RESISTANCE 1 (PYR1)/PYR1-LIKE (PYL)/REGULATORY COMPONENTS OF ABA RECEPTORS (RCAR) receptor family. The new agonist iCB, produced through scaffold-merging led by X-ray structure, activates subfamilies II and III at low-nM concentrations and subfamily I receptors at higher-nM concentrations. Structural analysis of opabactin and iCB ternary complexes reveals selectivity-determining residues, making the PYL1/PYL4/PYL8 subfamilies sensitive to specific agonists and highlighting the differential sensitivity of receptor subfamilies to agonists across plant species. iCB may activate most eudicots' PYL8-like receptors, in contrast to opabactin, due to limited steric constraints. This enables iCB to activate PYL8-like receptors with a bulkier Leu residue in the 3' tunnel, such as AtPYL8, SlPYL8, and VviPYL8. In contrast, opabactin activation is limited to receptors with Val at this position, for example, TaPYL8. Therefore, iCB extends its action to more ABA receptors than CB, iSB09, and opabactin, exhibits higher affinity than ABA for dimeric receptors, and can protect tomato plants against drought. In addition to regulating stomatal conductance and lowering water consumption, iCB protects photosystem II and improves photosynthesis following prolonged water deficit. Moreover, iCB induces an ABA-like transcriptional response, upregulates the osmolyte synthesis, and can be hyperpotentiated when combined with the expression of a customized receptor. Our results provide structural insights for optimizing agonist design and aiding plants in managing water deficits.

See also the Commentary on this article by Chater, 243 : 503–505.

The balance between the CO2 entry for photosynthesis and transpiration water loss is crucial for plant growth, and ABA signaling can affect this equilibrium. To test how ABA balances plant growth and environmental adaptation, we performed molecular genetics studies in the biotech crop Nicotiana benthamiana under well-watered or drought conditions. Studies on ABA signaling in crops are complicated by the multigenic nature of the PYR/PYL/RCAR ABA receptor family and its functional redundancy, which is particularly challenging in polyploid plants. We have generated a pentuple pyl mutant in the allotetraploid Nicotiana benthamiana through CRISPR/Cas9 gene editing. The pentuple mutant is impaired in 2 NbPYL1-like and 3 NbPYL8-like receptors, affecting the regulation of transpiration and several ABA-dependent transcriptional processes. RNA-seq and metabolite analysis revealed that the synthesis of galactinol, an essential precursor for the osmoprotective raffinose family of oligosaccharides, is ABA-dependent and impaired in the mutant under osmotic stress. In contrast, our results show that, under well-watered conditions, partial inactivation of ABA signaling leads to higher CO2 entry and photosynthesis in the mutant than in WT. Photosynthesis analyses revealed an increased CO2 diffusion capacity mediated by higher stomatal and mesophyll conductances, and higher substomatal CO2 concentration in the pentuple mutant. RNA-seq analyses revealed that genes associated with cell wall loosening (e.g., expansins) and porosity were strongly downregulated by ABA in WT. In summary, a partial relief of the ABA control on transpiration mediated by ABA receptors positively affects photosynthesis when water is not limited, at the expense of reduced water use efficiency.

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.

Climate change-associated rise in VPD (atmospheric vapor pressure deficit) results in increased plant transpiration and reduced stomatal conductance, photosynthesis, biomass, and yield. High VPD-induced stomatal closure of Arabidopsis is an active process regulated via the kinase SnRK2.6 (OPEN STOMATA 1, OST1). Here, we performed gas exchange, leaf water potential and rosette growth measurements to study, whether (1) high VPD-induced stomatal closure is detected in plants carrying loss-of-function mutations in OST1 (ost1-3) when they are grown at reduced soil water content or measured at increased air temperature; (2) ost1-3 plants expressing OST1 construct with no ABA-activation domain, but intact ABA-independent activation, show stronger stomatal VPD response compared with ost1-3 plants; and (3) rosette area and biomass of ost1-3 are more affected by growth at high VPD compared with Col-0. The stomata of well-watered ost1-3 plants were insensitive to high VPD regardless of air temperature, but in deficit-irrigated ost1-3, leaf water potential decreased the most and stomata closed at high VPD. Differences between VPD-induced stomatal closures of ost1-3 plants and ost1-3 plants expressing OST1 with no ABA-activation domain point at gradual VPD-induced ABA-independent activation of OST1. High VPD conditions led to similar reductions in rosette area and specific leaf area of well-watered Col-0 and ost1-3 plants. Rosette dry mass was unaffected by high VPD. Our results show that OST1 loss-of-function plants display conditional stomatal closure and no extra sensitivity of rosette area growth compared with Col-0 wildtype under high VPD conditions.

Strategies to activate abscisic acid (ABA) receptors and boost ABA signaling by small molecules that act as ABA receptor agonists are promising biotechnological tools to enhance plant drought tolerance. Protein structures of crop ABA receptors might require modifications to improve recognition of chemical ligands, which in turn can be optimized by structural information. Through structure-based targeted design, we have combined chemical and genetic approaches to generate an ABA receptor agonist molecule (iSB09) and engineer a CsPYL1 ABA receptor, named CsPYL15m, which efficiently binds iSB09. This optimized receptor-agonist pair leads to activation of ABA signaling and marked drought tolerance. No constitutive activation of ABA signaling and hence growth penalty was observed in transformed Arabidopsis thaliana plants. Therefore, conditional and efficient activation of ABA signaling was achieved through a chemical-genetic orthogonal approach based on iterative cycles of ligand and receptor optimization driven by the structure of ternary receptor-ligand-phosphatase complexes.

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.

Environmental conditions and photoperiod length drive the seasonal variability of gas exchange in plants. Still, little is known about trees’ adaptation to climate change, expressed as a delay in decreasing photosynthetic capacity at the end of the growing season. We investigated the effect of elevated air humidity (RH) and sampling period (from July to September) on the variability of net photosynthesis (A n ), dark respiration (R), daytime (g l_day ) and night-time (g l_night ) leaf conductance, an index of leaf chlorophyll content (SPAD), and A n :SPAD ratio in cut shoots of silver birch (Betula pendula Roth). Measurements of cut shoots were conducted in a climate chamber to eliminate the direct effect of field conditions. The A n , A n :SPAD ratio, and g l_day were higher in the humidification treatment (H) than in the control (C) (P < 0.05) in autumn. The R was higher in the control than in the humidification treatment across the study period. The g l_night increased considerably in September in both treatments (P < 0.05) and was significantly correlated with R. Our findings suggest that autumnal leaf conductance and gas exchange in silver birch are considerably influenced by long-term exposure to elevated RH and are probably related to a complex of leaf senescence processes, including nitrogen retranslocation.

Vapour pressure deficit (VPD), the difference between the saturation and actual air vapour pressures, indicates the level of atmospheric drought and evaporative pressure on plants. VPD increases during climate change due to changes in air temperature and relative humidity. Rising VPD induces stomatal closure to counteract the VPD-mediated evaporative water loss from plants. There are important gaps in our understanding of the molecular VPD-sensing and signalling mechanisms in stomatal guard cells. Here, we discuss recent advances, research directions and open questions with respect to the three components that participate in VPD-induced stomatal closure in Arabidopsis, including: (1) abscisic acid (ABA)-dependent and (2) ABA-independent regulation of the protein kinase OPEN STOMATA 1 (OST1), and (3) the passive hydraulic stomatal response. In the ABA-dependent component, two models are proposed: ABA may be rapidly synthesised or its basal levels may be involved in the stomatal VPD response. Further studies on stomatal VPD signalling should clarify: (1) whether OST1 activation above basal activity is needed for VPD responses, (2) which components are involved in ABA-independent regulation of OST1, (3) the role of other potential OST1 targets in VPD signalling, and (4) to which extent OST1 contributes to stomatal VPD sensitivity in other plant species.

Stomatal pores that control plant CO 2 uptake and water loss affect global carbon and water cycles. In the era of increasing atmospheric CO 2 levels and vapor pressure deficit (VPD), it is essential to understand how these stimuli affect stomatal behavior. It is unknown whether stomatal responses to sub-ambient and above-ambient CO 2 levels are governed by the same regulators and whether these responses depend on VPD. We studied stomatal conductance responses in Arabidopsis stomatal signaling mutants under conditions where CO 2 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 CO 2 -induced stomatal closure have different roles in the sub-ambient and above-ambient CO 2 levels. The CO 2 -specific regulators prominently affected sub-ambient CO 2 responses, whereas the lack of guard cell slow-type anion channel SLAC1 more strongly affected the speed of above-ambient CO 2 -induced stomatal closure. Elevated VPD caused lower stomatal conductance in all and faster CO 2 -responsiveness in some studied genotypes and CO 2 -transitions. Our results highlight the importance of experimental set-ups in interpreting stomatal CO 2 - responsiveness, as stomatal movements under different CO 2 concentration ranges are controlled by distinct mechanisms. Sometimes elevated CO 2 and VPD responses also interact. Hence, multi-factor treatments are needed to understand plant behavior under future climate conditions.

Initiation of stomatal closure by various stimuli requires activation of guard cell plasma membrane anion channels, which are defined as rapid (R)- and slow (S)-type. The single-gene loss-of-function mutants of these proteins are well characterized. However, the impact of suppressing both the S- and R-type channels has not been studied. Here, by generating and studying double and triple Arabidopsis thaliana mutants of SLOW ANION CHANNEL1 (SLAC1), SLAC1 HOMOLOG3 (SLAH3), and ALUMINUM-ACTIVATED MALATE TRANSPORTER 12/QUICK-ACTIVATING ANION CHANNEL 1 (QUAC1), we show that impairment of R- and S-type channels gradually increased whole-plant steady-state stomatal conductance. Ozone-induced cell death also increased gradually in higher-order mutants with the highest levels observed in the quac1 slac1 slah3 triple mutant. Strikingly, while single mutants retained stomatal responsiveness to abscisic acid, darkness, reduced air humidity, and elevated CO2, the double mutant lacking SLAC1 and QUAC1 was nearly insensitive to these stimuli, indicating the need for coordinated activation of both R- and S-type anion channels in stomatal closure.

SignificanceWith the continuing increase in global temperatures, plants transpire more water due to the increasing vapor pressure deficit. Stomatal pores in plants close rapidly in response to the rising vapor pressure deficit to counteract water loss. We demonstrate that mutations in the stomatal CO2signaling pathway do not impair the response to an increase in vapor pressure difference (VPD). Osmotic stress causes cytoplasmic Ca2+transients in guard cells. Nevertheless, we show that diverse investigated higher-order calcium-signaling mutants do not affect the VPD response. We reveal that B3 family Raf-like protein kinases and a plasma membrane receptor-like protein GHR1 function in the elusive leaf-to-air VPD-mediated stomatal closure pathway. Notably,ghr1mutant alleles disrupt the classical “wrong-way” stomatal VPD response.

Sucrose-non-fermenting-1-related protein kinase-2s (SnRK2s) are critical for plant abiotic stress responses, including abscisic acid (ABA) signaling. Here, we develop a genetically encoded reporter for SnRK2 kinase activity. This sensor, named SNACS, shows an increase in the ratio of yellow to cyan fluorescence emission by OST1/SnRK2.6-mediated phosphorylation of a defined serine residue in SNACS. ABA rapidly increases FRET efficiency in N. benthamiana leaf cells and Arabidopsis guard cells. Interestingly, protein kinase inhibition decreases FRET efficiency in guard cells, providing direct experimental evidence that basal SnRK2 activity prevails in guard cells. Moreover, in contrast to ABA, the stomatal closing stimuli, elevated CO2 and MeJA, did not increase SNACS FRET ratios. These findings and gas exchange analyses of quintuple/sextuple ABA receptor mutants show that stomatal CO2 signaling requires basal ABA and SnRK2 signaling, but not SnRK2 activation. A recent model that CO2 signaling is mediated by PYL4/PYL5 ABA-receptors could not be supported here in two independent labs. We report a potent approach for real-time live-cell investigations of stress signaling.

Land plants are considered monophyletic, descending from a single successful colonization of land by an aquatic algal ancestor. The ability to survive dehydration to the point of desiccation is a key adaptive trait enabling terrestrialization. In extant land plants, desiccation tolerance depends on the action of the hormone abscisic acid (ABA) that acts through a receptor-signal transduction pathway comprising a PYRABACTIN RESISTANCE 1-like (PYL)-PROTEIN PHOSPHATASE 2C (PP2C)-SNF1-RELATED PROTEIN KINASE 2 (SnRK2) module. Early-diverging aeroterrestrial algae mount a dehydration response that is similar to that of land plants, but that does not depend on ABA: Although ABA synthesis is widespread among algal species, ABA-dependent responses are not detected, and algae lack an ABA-binding PYL homolog. This raises the key question of how ABA signaling arose in the earliest land plants. Here, we systematically characterized ABA receptor-like proteins from major land plant lineages, including a protein found in the algal sister lineage of land plants. We found that the algal PYL-homolog encoded by Zygnema circumcarinatum has basal, ligand-independent activity of PP2C repression, suggesting this to be an ancestral function. Similarly, a liverwort receptor possesses basal activity, but it is further activated by ABA. We propose that co-option of ABA to control a preexisting PP2C-SnRK2-dependent desiccation-tolerance pathway enabled transition from an all-or-nothing survival strategy to a hormone-modulated, competitive strategy by enabling continued growth of anatomically diversifying vascular plants in dehydrative conditions, enabling them to exploit their new environment more efficiently.

Stomata are microscopic pores found on the surfaces of leaves that act to control CO 2 uptake and water loss. By integrating information derived from endogenous signals with cues from the surrounding environment, the guard cells, which surround the pore, ‘set’ the stomatal aperture to suit the prevailing conditions. Much research has concentrated on understanding the rapid intracellular changes that result in immediate changes to the stomatal aperture. In this study, we look instead at how stomata acclimate to longer timescale variations in their environment. We show that the closure-inducing signals abscisic acid (ABA), increased CO 2 , decreased relative air humidity and darkness each access a unique gene network made up of clusters (or modules) of common cellular processes. However, within these networks some gene clusters are shared amongst all four stimuli. All stimuli modulate the expression of members of the PYR/PYL/RCAR family of ABA receptors. However, they are modulated differentially in a stimulus-specific manner. Of the six members of the PYR/PYL/RCAR family expressed in guard cells, PYL2 is sufficient for guard cell ABA-induced responses, whereas in the responses to CO 2 , PYL4 and PYL5 are essential. Overall, our work shows the importance of ABA as a central regulator and integrator of long-term changes in stomatal behaviour, including sensitivity, elicited by external signals. Understanding this architecture may aid in breeding crops with improved water and nutrient efficiency.