Mechanistic insights into phosphoactivation of SLAC1 in guard cell signaling.

Stomata in leaves regulate gas (carbon dioxide and water vapor) exchange and water transpiration between plants and the atmosphere. SLow Anion Channel 1 (SLAC1) mediates anion efflux from guard cells and plays a crucial role in controlling stomatal aperture. It serves as a central hub for multiple signaling pathways in response to environmental stimuli, with its activity regulated through phosphorylation via various plant protein kinases. However, the molecular mechanism underlying SLAC1 phosphoactivation has remained elusive. Through a combination of protein sequence analyses, AlphaFold-based modeling and electrophysiological studies, we unveiled that the highly conserved motifs on the N- and C-terminal segments of SLAC1 form a cytosolic regulatory domain (CRD) that interacts with the transmembrane domain(TMD), thereby maintaining the channel in an autoinhibited state. Mutations in these conserved motifs destabilize the CRD, releasing autoinhibition in SLAC1 and enabling its transition into an activated state. Our further studies demonstrated that SLAC1 activation undergoes an autoinhibition-release process and subsequent structural changes in the pore helices. These findings provide mechanistic insights into the activation mechanism of SLAC1 and shed light on understanding how SLAC1 controls stomatal closure in response to environmental stimuli.

Null mutants of a tomato Rho of plants exhibit enhanced water use efficiency without a penalty to yield.

Improving water use efficiency in crops is a significant challenge as it involves balancing water transpiration and CO2 uptake through stomatal pores. This study investigates the role of SlROP9, a tomato Rho of Plants protein, in guard cells and its impact on plant transpiration. The results reveal that SlROP9 null mutants exhibit reduced stomatal conductance while photosynthetic CO2 assimilation remains largely unaffected. Notably, there is a notable decrease in whole-plant transpiration in the rop9 mutants compared to the wild type, especially during noon hours when the water pressure deficit is high. The elevated stomatal closure observed in rop9 mutants is linked to an increase in reactive oxygen species formation. This is very likely dependent on the respiratory burst oxidase homolog (RBOH) NADPH oxidase and is not influenced by abscisic acid (ABA). Consistently, activated ROP9 can interact with RBOHB in both yeast and plants. In diverse tomato accessions, drought stress represses ROP9 expression, and in Arabidopsis stomatal guard cells, ABA suppresses ROP signaling. Therefore, the phenotype of the rop9 mutants may arise from a disruption in ROP9-regulated RBOH activity. Remarkably, large-scale field experiments demonstrate that the rop9 mutants display improved water use efficiency without compromising fruit yield. These findings provide insights into the role of ROPs in guard cells and their potential as targets for enhancing water use efficiency in crops.

ABA guides stomatal proliferation and patterning through the EPF-SPCH signaling pathway in Arabidopsis thaliana.

Adaptation to dehydration stress requires plants to coordinate environmental and endogenous signals to inhibit stomatal proliferation and modulate their patterning. The stress hormone abscisic acid (ABA) induces stomatal closure and restricts stomatal lineage to promote stress tolerance. Here, we report that mutants with reduced ABA levels, xer-1, xer-2 and aba2-2, developed stomatal clusters. Similarly, the ABA signaling mutant snrk2.2/2.3/2.6, which lacks core ABA signaling kinases, also displayed stomatal clusters. Exposure to ABA or inhibition of ABA catabolism rescued the increased stomatal density and spacing defects observed in xer and aba2-2, suggesting that basal ABA is required for correct stomatal density and spacing. xer-1 and aba2-2 displayed reduced expression of EPF1 and EPF2, and enhanced expression of SPCH and MUTE. Furthermore, ABA suppressed elevated SPCH and MUTE expression in epf2-1 and epf1-1, and partially rescued epf2-1 stomatal index and epf1-1 clustering defects. Genetic analysis demonstrated that XER acts upstream of the EPF2-SPCH pathway to suppress stomatal proliferation, and in parallel with EPF1 to ensure correct stomatal spacing. These results show that basal ABA and functional ABA signaling are required to fine-tune stomatal density and patterning.

Stomatal patchiness and cellular computing.

Control of carbon dioxide and water vapor exchange between a leaf's interior and the surrounding air is accomplished by variations in the turgor pressures in the small epidermal and guard cells that cover the leaf's surface. These pressures respond to changes in light intensity and wavelength, temperature, CO2 concentration, and air humidity. The dynamical equations that describe such processes are formally identical to those that define computation in a two-layer, adaptive, cellular nonlinear network. This exact identification suggests that leaf gas-exchange processes can be understood as analog computation and that exploiting the output of two-layer, adaptive, cellular nonlinear networks might provide new tools in applied plant research.

Distinct guard cell-specific remodeling of chromatin accessibility during abscisic acid- and CO2-dependent stomatal regulation.

In plants, epidermal guard cells integrate and respond to numerous environmental signals to control stomatal pore apertures, thereby regulating gas exchange. Chromatin structure controls transcription factor (TF) access to the genome, but whether large-scale chromatin remodeling occurs in guard cells during stomatal movements, and in response to the hormone abscisic acid (ABA) in general, remains unknown. Here, we isolate guard cell nuclei from Arabidopsis thaliana plants to examine whether the physiological signals, ABA and CO2 (carbon dioxide), regulate guard cell chromatin during stomatal movements. Our cell type-specific analyses uncover patterns of chromatin accessibility specific to guard cells and define cis-regulatory sequences supporting guard cell-specific gene expression. We find that ABA triggers extensive and dynamic chromatin remodeling in guard cells, roots, and mesophyll cells with clear patterns of cell type specificity. DNA motif analyses uncover binding sites for distinct TFs enriched in ABA-induced and ABA-repressed chromatin. We identify the Abscisic Acid Response Element (ABRE) Binding Factor (ABF) bZIP-type TFs that are required for ABA-triggered chromatin opening in guard cells and roots and implicate the inhibition of a clade of bHLH-type TFs in controlling ABA-repressed chromatin. Moreover, we demonstrate that ABA and CO2 induce distinct programs of chromatin remodeling, whereby elevated atmospheric CO2 had only minimal impact on chromatin dynamics. We provide insight into the control of guard cell chromatin dynamics and propose that ABA-induced chromatin remodeling primes the genome for abiotic stress resistance.

Plant biochemistry influences tropospheric ozone formation, destruction, deposition, and response.

Tropospheric ozone (O3) is among the most damaging air pollutant to plants. Plants alter the atmospheric O3 concentration in two distinct ways: (i) by the emission of volatile organic compounds (VOCs) that are precursors of O3; and (ii) by dry deposition, which includes diffusion of O3 into vegetation through stomata and destruction by nonstomatal pathways. Isoprene, monoterpenes, and higher terpenoids are emitted by plants in quantities that alter tropospheric O3. Deposition of O3 into vegetation is related to stomatal conductance, leaf structural traits, and the detoxification capacity of the apoplast. The biochemical fate of O3 once it enters leaves and reacts with aqueous surfaces is largely unknown, but new techniques for the tracking and identification of initial products have the potential to open the black box.

Arabidopsis SNARE SYP132 impacts on PIP2;1 trafficking and function in salinity stress.

In plants so-called plasma membrane intrinsic proteins (PIPs) are major water channels governing plant water status. Membrane trafficking contributes to functional regulation of major PIPs and is crucial for abiotic stress resilience. Arabidopsis PIP2;1 is rapidly internalised from the plasma membrane in response to high salinity to regulate osmotic water transport, but knowledge of the underlying mechanisms is fragmentary. Here we show that PIP2;1 occurs in complex with SYNTAXIN OF PLANTS 132 (SYP132) together with the plasma membrane H+-ATPase AHA1 as evidenced through in vivo and in vitro analysis. SYP132 is a multifaceted vesicle trafficking protein, known to interact with AHA1 and promote endocytosis to impact growth and pathogen defence. Tracking native proteins in immunoblot analysis, we found that salinity stress enhances SYP132 interactions with PIP2;1 and PIP2;2 isoforms to promote redistribution of the water channels away from the plasma membrane. Concurrently, AHA1 binding within the SYP132-complex was significantly reduced under salinity stress and increased the density of AHA1 proteins at the plasma membrane in leaf tissue. Manipulating SYP132 function in Arabidopsis thaliana enhanced resilience to salinity stress and analysis in heterologous systems suggested that the SNARE influences PIP2;1 osmotic water permeability. We propose therefore that SYP132 coordinates AHA1 and PIP2;1 abundance at the plasma membrane and influences leaf hydraulics to regulate plant responses to abiotic stress signals.

Young guard cells function dynamically despite low mechanical anisotropy but gain efficiency during stomatal maturation in Arabidopsis thaliana.

Stomata are pores at the leaf surface that enable gas exchange and transpiration. The signaling pathways that regulate the differentiation of stomatal guard cells and the mechanisms of stomatal pore formation have been characterized in Arabidopsis thaliana. However, the process by which stomatal complexes develop after pore formation into fully mature complexes is poorly understood. We tracked the morphogenesis of young stomatal complexes over time to establish characteristic geometric milestones along the path of stomatal maturation. Using 3D-nanoindentation coupled with finite element modeling of young and mature stomata, we found that despite having thicker cell walls than young guard cells, mature guard cells are more energy efficient with respect to stomatal opening, potentially attributable to the increased mechanical anisotropy of their cell walls and smaller changes in turgor pressure between the closed and open states. Comparing geometric changes in young and mature guard cells of wild-type and cellulose-deficient plants revealed that although cellulose is required for normal stomatal maturation, mechanical anisotropy appears to be achieved by the collective influence of cellulose and additional wall components. Together, these data elucidate the dynamic geometric and biomechanical mechanisms underlying the development process of stomatal maturation.

Climate-resilient crops: Lessons from xerophytes.

Developing climate-resilient crops is critical for future food security and sustainable agriculture under current climate scenarios. Of specific importance are drought and soil salinity. Tolerance traits to these stresses are highly complex, and the progress in improving crop tolerance is too slow to cope with the growing demand in food production unless a major paradigm shift in crop breeding occurs. In this work, we combined bioinformatics and physiological approaches to compare some of the key traits that may differentiate between xerophytes (naturally drought-tolerant plants) and mesophytes (to which the majority of the crops belong). We show that both xerophytes and salt-tolerant mesophytes have a much larger number of copies in key gene families conferring some of the key traits related to plant osmotic adjustment, abscisic acid (ABA) sensing and signalling, and stomata development. We show that drought and salt-tolerant species have (i) higher reliance on Na for osmotic adjustment via more diversified and efficient operation of Na+ /H+ tonoplast exchangers (NHXs) and vacuolar H+ - pyrophosphatase (VPPases); (ii) fewer and faster stomata; (iii) intrinsically lower ABA content; (iv) altered structure of pyrabactin resistance/pyrabactin resistance-like (PYR/PYL) ABA receptors; and (v) higher number of gene copies for protein phosphatase 2C (PP2C) and sucrose non-fermenting 1 (SNF1)-related protein kinase 2/open stomata 1 (SnRK2/OST1) ABA signalling components. We also show that the past trends in crop breeding for Na+ exclusion to improve salinity stress tolerance are counterproductive and compromise their drought tolerance. Incorporating these genetic insights into breeding practices could pave the way for more drought-tolerant and salt-resistant crops, securing agricultural yields in an era of climate unpredictability.

SCARECROW is deployed in distinct contexts during rice and maize leaf development.

The flexible deployment of developmental regulators is an increasingly appreciated aspect of plant development and evolution. The GRAS transcription factor SCARECROW (SCR) regulates the development of the endodermis in Arabidopsis and maize roots, but during leaf development it regulates the development of distinct cell types; bundle-sheath in Arabidopsis and mesophyll in maize. In rice, SCR is implicated in stomatal patterning, but it is unknown whether this function is additional to a role in inner leaf patterning. Here, we demonstrate that two duplicated SCR genes function redundantly in rice. Contrary to previous reports, we show that these genes are necessary for stomatal development, with stomata virtually absent from leaves that are initiated after germination of mutants. The stomatal regulator OsMUTE is downregulated in Osscr1;Osscr2 mutants, indicating that OsSCR acts early in stomatal development. Notably, Osscr1;Osscr2 mutants do not exhibit the inner leaf patterning perturbations seen in Zmscr1;Zmscr1h mutants, and Zmscr1;Zmscr1h mutants do not exhibit major perturbations in stomatal patterning. Taken together, these results indicate that SCR was deployed in different developmental contexts after the divergence of rice and maize around 50 million years ago.

Cell type-specific dynamics underlie cellular growth variability in plants.

Coordination of growth, patterning and differentiation is required for shaping organs in multicellular organisms. In plants, cell growth is controlled by positional information, yet the behavior of individual cells is often highly heterogeneous. The origin of this variability is still unclear. Using time-lapse imaging, we determined the source and relevance of cellular growth variability in developing organs of Arabidopsis thaliana. We show that growth is more heterogeneous in the leaf blade than in the midrib and petiole, correlating with higher local differences in growth rates between neighboring cells in the blade. This local growth variability coincides with developing stomata. Stomatal lineages follow a specific, time-dependent growth program that is different from that of their surroundings. Quantification of cellular dynamics in the leaves of a mutant lacking stomata, as well as analysis of floral organs, supports the idea that growth variability is mainly driven by stomata differentiation. Thus, the cell-autonomous behavior of specialized cells is the main source of local growth variability in otherwise homogeneously growing tissue. Those growth differences are buffered by the immediate neighbors of stomata and trichomes to achieve robust organ shapes.

Spider mite herbivory induces an abscisic acid-driven stomatal defense.

Arthropod herbivory poses a serious threat to crop yield, prompting plants to employ intricate defense mechanisms against pest feeding. The generalist pest two-spotted spider mite (Tetranychus urticae) inflicts rapid damage and remains challenging due to its broad target range. In this study, we explored the Arabidopsis (Arabidopsis thaliana) response to T. urticae infestation, revealing the induction of abscisic acid (ABA), a hormone typically associated with abiotic stress adaptation, and stomatal closure during water stress. Leveraging a FRET-based ABA biosensor (nlsABACUS2-400n), we observed elevated ABA levels in various leaf cell types post-mite feeding. While ABA's role in pest resistance or susceptibility has been debated, an ABA-deficient mutant exhibited increased mite infestation alongside intact canonical biotic stress signaling, indicating an independent function of ABA in mite defense. We established that ABA-triggered stomatal closure effectively hinders mite feeding and minimizes leaf cell damage through genetic and pharmacological interventions targeting ABA levels, ABA signaling, stomatal aperture, and density. This study underscores the critical interplay between biotic and abiotic stresses in plants, highlighting how the vulnerability to mite infestation arising from open stomata, crucial for transpiration and photosynthesis, reinforces the intricate relationship between these stress types.

Regulation of stomatal development by epidermal, subepidermal and long-distance signals.

Plant leaves consist of three layers, including epidermis, mesophyll and vascular tissues. Their development is meticulously orchestrated. Stomata are the specified structures on the epidermis for uptake of carbon dioxide (CO2) while release of water vapour and oxygen (O2), and thus play essential roles in regulation of plant photosynthesis and water use efficiency. To function efficiently, stomatal formation must coordinate with the development of other epidermal cell types, such as pavement cell and trichome, and tissues of other layers, such as mesophyll and leaf vein. This review summarizes the regulation of stomatal development in three dimensions (3D). In the epidermis, specific stomatal transcription factors determine cell fate transitions and also activate a ligand-receptor- MITOGEN-ACTIVATED PROTEIN KINASE (MAPK) signaling for ensuring proper stomatal density and patterning. This forms the core regulation network of stomatal development, which integrates various environmental cues and phytohormone signals to modulate stomatal production. Under the epidermis, mesophyll, endodermis of hypocotyl and inflorescence stem, and veins in grasses secrete mobile signals to influence stomatal formation in the epidermis. In addition, long-distance signals which may include phytohormones, RNAs, peptides and proteins originated from other plant organs modulate stomatal development, enabling plants to systematically adapt to the ever changing environment.

The Arabidopsis stomatal polarity protein BASL mediates distinct processes before and after cell division to coordinate cell size and fate asymmetries.

In many land plants, asymmetric cell divisions (ACDs) create and pattern differentiated cell types on the leaf surface. In the Arabidopsis stomatal lineage, BREAKING OF ASYMMETRY IN THE STOMATAL LINEAGE (BASL) regulates division plane placement and cell fate enforcement. Polarized subcellular localization of BASL is initiated before ACD and persists for many hours after the division in one of the two daughters. Untangling the respective contributions of polarized BASL before and after division is essential to gain a better understanding of its roles in regulating stomatal lineage ACDs. Here, we combine quantitative imaging and lineage tracking with genetic tools that provide temporally restricted BASL expression. We find that pre-division BASL is required for division orientation, whereas BASL polarity post-division ensures proper cell fate commitment. These genetic manipulations allowed us to uncouple daughter-cell size asymmetry from polarity crescent inheritance, revealing independent effects of these two asymmetries on subsequent cell behavior. Finally, we show that there is coordination between the division frequencies of sister cells produced by ACDs, and this coupling requires BASL as an effector of peptide signaling.

The Egyptian wheat cultivar Gemmeiza-12 is a source of resistance against the fungus Zymoseptoria tritici.

BACKGROUND: Wheat is one of the world's most important cereal crops. However, the fungal pathogen Zymoseptoria tritici can cause disease epidemics, leading to reduced yields. With climate change and development of new agricultural areas with suitable environments, Z. tritici may advance into geographical areas previously unaffected by this pathogen. It is currently unknown how Egyptian wheat will perform in the face of this incoming threat. This project aimed to assess the resistance of Egyptian wheat germplasm to Z. tritici, to identify cultivars with high levels of resistance and characterise the mechanism(s) of resistance present in these cultivars. RESULTS: Eighteen Egyptian wheat cultivars were screened against two Z. tritici model isolates and exhibited a wide spectrum of responses. This ranged from resistance to complete susceptibility to one or both isolates tested. The most highly resistant cultivars from the initial screen were then tested under two environmental conditions against modern UK field isolates. Disease levels under UK-like conditions were higher, however, symptom development on the cultivar Gemmeiza-12 was noticeably slower than on other Egyptian wheats. The robustness of the resistance shown by Gemmeiza-12 was confirmed in experiments mimicking Egyptian environmental conditions, where degree of Z. tritici infection was lower. The Kompetitive allele-specific PCR (KASP) diagnostic assay suggested the presence of an Stb6 resistant allele in several Egyptian wheats including Gemmeiza-12. Infection assays using the IPO323 WT and IPO323ΔAvrStb6 mutant confirmed the presence of Stb6 in several Egyptian cultivars including Gemmeiza-12. Confocal fluorescence microscopy demonstrated that growth of the IPO323 strain is blocked at the point of stomatal penetration on Gemmeiza-12, consistent with previous reports of Stb gene mediated resistance. In addition to this R-gene mediated resistance, IPO323 spores showed lower adherence to leaves of Gemmeiza-12 compared to UK wheat varieties, suggesting other aspects of leaf physiology may also contribute to the resistance phenotype of this cultivar. CONCLUSION: These results indicate that Gemmeiza-12 will be useful in future breeding programs where improved resistance to Z. tritici is a priority.

H2S is involved in drought-mediated stomatal closure through PLDα1 in Arabidopsis.

MAIN CONCLUSION: PLDα1 promoted H2S production by positively regulating the expression of LCD. Stomatal closure promoted by PLDα1 required the accumulation of H2S under drought stress. Phospholipase Dα1 (PLDα1) acting as one of the signal enzymes can respond to drought stress. It is well known that hydrogen sulfide (H2S) plays an important role in plant responding to biotic or abiotic stress. In this study, the functions and relationship between PLDα1 and H2S in drought stress resistance in Arabidopsis were explored. Our results indicated that drought stress promotes PLDα1 and H2S production by inducing the expression of PLDα1 and LCD genes. PLDα1 and LCD enhanced plant tolerance to drought by regulating membrane lipid peroxidation, proline accumulation, H2O2 content and stomatal closure. Under drought stress, the H2O2 content of PLDα1-deficient mutant (pldα1), L-cysteine desulfhydrase (LCD)-deficient mutant (lcd) was higher than that of ecotype (WT), the stomatal aperture of pldα1 and lcd was larger than that of WT. The transcriptional and translational levels of LCD were lower in pldα1 than that in WT. Exogenous application of the H2S donor NaHS or GYY reduced the stomatal aperture of WT, pldα1, PLDα1-CO, and PLDα1-OE lines, while exogenous application of the H2S scavenger hypotaurine (HT) increased the stomatal aperture. qRT-PCR analysis of stomatal movement-related genes showed that the expression of CAX1, ABCG5, SCAB1, and SLAC1 genes in pldα1 and lcd were down-regulated, while ACA1 and OST1 gene expression was significantly up-regulated. Thus, PLDα1 and LCD are required for stomatal closure to improve drought stress tolerance.

K+ and pH homeostasis in plant cells is controlled by a synchronized K+ /H+ antiport at the plasma and vacuolar membrane.

Stomatal movement involves ion transport across the plasma membrane (PM) and vacuolar membrane (VM) of guard cells. However, the coupling mechanisms of ion transporters in both membranes and their interplay with Ca2+ and pH changes are largely unclear. Here, we investigated transporter networks in tobacco guard cells and mesophyll cells using multiparametric live-cell ion imaging and computational simulations. K+ and anion fluxes at both, PM and VM, affected H+ and Ca2+ , as changes in extracellular KCl or KNO3 concentrations were accompanied by cytosolic and vacuolar pH shifts and changes in [Ca2+ ]cyt and the membrane potential. At both membranes, the K+ transporter networks mediated an antiport of K+ and H+ . By contrast, net transport of anions was accompanied by parallel H+ transport, with differences in transport capacity for chloride and nitrate. Guard and mesophyll cells exhibited similarities in K+ /H+ transport but cell type-specific differences in [H+ ]cyt and pH-dependent [Ca2+ ]cyt signals. Computational cell biology models explained mechanistically the properties of transporter networks and the coupling of transport across the PM and VM. Our integrated approach indicates fundamental principles of coupled ion transport at membrane sandwiches to control H+ /K+ homeostasis and points to transceptor-like Ca2+ /H+ -based ion signaling in plant cells.

Synthesis and import of GDP-l-fucose into the Golgi affect plant-water relations.

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.

Stomatal closure in maize is mediated by subsidiary cells and the PAN2 receptor.

Stomata are epidermal pores that facilitate plant gas exchange. Grasses have fast stomatal movements, likely due to their dumbbell-shaped guard cells and lateral subsidiary cells. Subsidiary cells reciprocally exchange water and ions with guard cells. However, the relative contribution of subsidiary cells during stomatal closure is unresolved. We compared stomatal gas exchange and stomatal aperture dynamics in wild-type and pan1, pan2, and pan1;pan2 Zea mays (L.) (maize) mutants, which have varying percentages of aberrantly formed subsidiary cells. Stomata with 1 or 2 defective subsidiary cells cannot close properly, indicating that subsidiary cells are essential for stomatal function. Even though the percentage of aberrant stomata is similar in pan1 and pan2, pan2 showed a more severe defect in stomatal closure. In pan1, only stomata with abnormal subsidiary cells fail to close normally. In pan2, all stomata have stomatal closure defects, indicating that PAN2 has an additional role in stomatal closure. Maize Pan2 is orthologous to Arabidopsis GUARD CELL HYDROGEN PEROXIDE-RESISANT1 (GHR1), which is also required for stomatal closure. PAN2 acts downstream of Ca2+ in maize to promote stomatal closure. This is in contrast to GHR1, which acts upstream of Ca2+ , and suggests the pathways could be differently wired.

Constitutive activation of ABA receptors in Arabidopsis reveals unique regulatory circuitries.

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.