Role of mitochondria and chloroplasts during stomatal closure: Subcellular location of superoxide and H2O2 production in guard cells of Arabidopsis thaliana.

Stomatal guard cells are unique in that they have more mitochondria than chloroplasts. Several reports emphasized the importance of mitochondria as the major energy source during stomatal opening. We re-examined their role during stomatal closure. The marked sensitivity of stomata to both menadione (MD) and methyl viologen (MV) demonstrated that both mitochondria and chloroplasts helped to promote stomatal closure in Arabidopsis. As in the case of abscisic acid (ABA), a plant stress hormone, MD and MV induced stomatal closure at micromolar concentration. All three compounds generated superoxide and H2O2, as indicated by fluorescence probes, BES-So-AM and CM-H2DCFDA, respectively. Results from tiron (a superoxide scavenger) and catalase (an H2O2 scavenger) confirmed that both the superoxide and H2O2 were requisites for stomatal closure. Co-localization of the superoxide and H2O2 in mitochondria and chloroplasts using fluorescent probes revealed that exposure to MV initially triggered higher superoxide and H2O2 generation in mitochondria. In contrast, MD elevated superoxide/H2O2 levels in chloroplasts. However, with prolonged exposure, MD and MV induced ROS production in other organelles. We conclude that ROS production in mitochondria and chloroplasts leads to stomatal closure. We propose that stomatal guard cells can be good models for examining inter-organellar interactions.

Phosphorylation of plasma membrane H+-ATPase Thr881 participates in light-induced stomatal opening.

Plasma membrane (PM) H+-ATPase is crucial for light-induced stomatal opening and phosphorylation of a penultimate residue, Thr948 (pen-Thr, numbering according to Arabidopsis AHA1) is required for enzyme activation. In this study, a comprehensive phosphoproteomic analysis using guard cell protoplasts from Vicia faba shows that both red and blue light increase the phosphorylation of Thr881, of PM H+-ATPase. Light-induced stomatal opening and the blue light-induced increase in stomatal conductance are reduced in transgenic Arabidopsis plants expressing mutant AHA1-T881A in aha1-9, whereas the blue light-induced phosphorylation of pen-Thr is unaffected. Auxin and photosynthetically active radiation induce the phosphorylation of both Thr881 and pen-Thr in etiolated seedlings and leaves, respectively. The dephosphorylation of phosphorylated Thr881 and pen-Thr are mediated by type 2 C protein phosphatase clade D isoforms. Taken together, Thr881 phosphorylation, in addition of the pen-Thr phosphorylation, are important for PM H+-ATPase function during physiological responses, such as light-induced stomatal opening in Arabidopsis thaliana.

Light-induced stomatal opening requires phosphorylation of the C-terminal autoinhibitory domain of plasma membrane H+-ATPase.

Plasma membrane H+-ATPase provides the driving force for light-induced stomatal opening. However, the mechanisms underlying the regulation of its activity remain unclear. Here, we show that the phosphorylation of two Thr residues in the C-terminal autoinhibitory domain is crucial for H+-ATPase activation and stomatal opening in Arabidopsis thaliana. Using phosphoproteome analysis, we show that blue light induces the phosphorylation of Thr-881 within the C-terminal region I, in addition to penultimate Thr-948 in AUTOINHIBITED H+-ATPASE 1 (AHA1). Based on site-directed mutagenesis experiments, phosphorylation of both Thr residues is essential for H+ pumping and stomatal opening in response to blue light. Thr-948 phosphorylation is a prerequisite for Thr-881 phosphorylation by blue light. Additionally, red light-driven guard cell photosynthesis induces Thr-881 phosphorylation, possibly contributing to red light-dependent stomatal opening. Our findings provide mechanistic insights into H+-ATPase activation that exploits the ion transport across the plasma membrane and light signalling network in guard cells.

Stomatal development: NRPM proteins in dynamic localization of ERECTA receptor.

Dynamic cellular localization of receptors is key to the perception of their peptide ligands and the activation of downstream signaling pathways. A new study identifies NRPMs as novel regulators of ERECTA receptor localization and stomatal formation downstream of the EPF1/EPF2 peptide ligands and upstream of the YDA MAPK cascade.

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.

Mitochondrial VOLTAGE-DEPENDENT ANION CHANNEL 3 regulates stomatal closure by abscisic acid signaling.

In Arabidopsis (Arabidopsis thaliana), stomatal closure mediated by abscisic acid (ABA) is redundantly controlled by ABA receptor family proteins (PYRABACTIN RESISTANCE 1 [PYR1]/PYR1-LIKE [PYLs]) and subclass III SUCROSE NONFERMENTING 1 (SNF1)-RELATED PROTEIN KINASES 2 (SnRK2s). Among these proteins, the roles of PYR1, PYL2, and SnRK2.6 are more dominant. A recent discovery showed that ABA-induced accumulation of reactive oxygen species (ROS) in mitochondria promotes stomatal closure. By analyzing stomatal movements in an array of single and higher order mutants, we revealed that the mitochondrial protein VOLTAGE-DEPENDENT ANION CHANNEL 3 (VDAC3) jointly regulates ABA-mediated stomatal closure with a specialized set of PYLs and SnRK2s by affecting cellular and mitochondrial ROS accumulation. VDAC3 interacted with 9 PYLs and all 3 subclass III SnRK2s. Single mutation in VDAC3, PYLs (except PYR1 and PYL2), or SnRK2.2/2.3 had little effect on ABA-mediated stomatal closure. However, knocking out PYR1, PYL1/2/4/8, or SnRK2.2/2.3 in vdac3 mutants resulted in significantly delayed or attenuated ABA-mediated stomatal closure, despite the presence of other PYLs or SnRK2s conferring redundant functions. We found that cellular and mitochondrial accumulation of ROS induced by ABA was altered in vdac3pyl1 mutants. Moreover, H2O2 treatment restored ABA-induced stomatal closure in mutants with decreased stomatal sensitivity to ABA. Our work reveals that VDAC3 ensures redundant control of ABA-mediated stomatal closure by canonical ABA signaling components.

COBL7 is required for stomatal formation via regulation of cellulose deposition in Arabidopsis.

As a key regulator of plant photosynthesis, water use efficiency and immunity, stomata are specialized cellular structures that adopt defined shapes. However, our knowledge about the genetic players of stomatal pore formation and stomatal morphogenesis remains limited. Forward genetic screening, positional cloning, confocal and electron microscopy, physiological and pharmacological assays were employed for isolation and characterization of mutants and genes. We identified a mutant, dsm1, with impaired cytokinesis and deformed stomata. DSM1 is highly expressed in guard mother cells and guard cells, and encodes COBRA-LIKE 7 (COBL7), a plant-specific glycosylphosphatidylinositol (GPI)-anchored protein. COBRA-LIKE 7 and its closest homologue, COBL8, are first enriched on the forming cell plates during cytokinesis, and then their subcellular distribution and abundance change are correlated with the progressive stages of stomatal pore formation. Both COBL7 and COBL8 possess an ability to bind cellulose. Perturbing the expression of COBL7 and COBL8 leads to a decrease in cellulose content and inhibition of stomatal pore development. Moreover, we found that COBL7, COBL8 and CSLD5 have synergistic effects on stomatal development and plant growth. Our findings reveal that COBL7 plays a predominant and functionally redundant role with COBL8 in stomatal formation through regulating cellulose deposition and ventral wall modification in Arabidopsis.

SCAB1 coordinates sequential Ca2+ and ABA signals during osmotic stress induced stomatal closure in Arabidopsis.

Hyperosmotic stress caused by drought is a detrimental threat to plant growth and agricultural productivity due to limited water availability. Stomata are gateways of transpiration and gas exchange, the swift adjustment of stomatal aperture has a strong influence on plant drought resistance. Despite intensive investigations of stomatal closure during drought stress in past decades, little is known about how sequential signals are integrated during complete processes. Here, we discovered that the rapid Ca2+ signaling and subsequent abscisic acid (ABA) signaling contribute to the kinetics of both F-actin reorganizations and stomatal closure in Arabidopsis thaliana, while STOMATAL CLOSURE-RELATED ACTIN BINDING PROTEIN1 (SCAB1) is the molecular switch for this entire process. During the early stage of osmotic shock responses, swift elevated calcium signaling promotes SCAB1 phosphorylation through calcium sensors CALCIUM DEPENDENT PROTEIN KINASE3 (CPK3) and CPK6. The phosphorylation restrained the microfilament binding affinity of SCAB1, which bring about the F-actin disassembly and stomatal closure initiation. As the osmotic stress signal continued, both the kinase activity of CPK3 and the phosphorylation level of SCAB1 attenuated significantly. We further found that ABA signaling is indispensable for these attenuations, which presumably contributed to the actin filament reassembly process as well as completion of stomatal closure. Notably, the dynamic changes of SCAB1 phosphorylation status are crucial for the kinetics of stomatal closure. Taken together, our results support a model in which SCAB1 works as a molecular switch, and directs the microfilament rearrangement through integrating the sequentially generated Ca2+ and ABA signals during osmotic stress induced stomatal closure.

Singlet oxygen induces cell wall thickening and stomatal density reducing by transcriptome reprogramming.

Singlet oxygen (1O2) has a very short half-life of 10-5 s; however, it is a strong oxidant that causes growth arrest and necrotic lesions on plants. Its signaling pathway remains largely unknown. The Arabidopsis flu (fluorescent) mutant accumulates a high level of 1O2 and shows drastic changes in nuclear gene expression. Only two plastid proteins, EX1 (executer 1) and EX2 (executer 2), have been identified in the singlet oxygen signaling. Here, we found that the transcription factor abscisic acid insensitive 4 (ABI4) binds the promoters of genes responsive to 1O2-signals. Inactivation of the ABI4 protein in the flu/abi4 double mutant was sufficient to compromise the changes of almost all 1O2-responsive-genes and rescued the lethal phenotype of flu grown under light/dark cycles, similar to the flu/ex1/ex2 triple mutant. In addition to cell death, we reported for the first time that 1O2 also induces cell wall thickening and stomatal development defect. Contrastingly, no apparent growth arrest was observed for the flu mutant under normal light/dim light cycles, but the cell wall thickening (doubled) and stomatal density reduction (by two-thirds) still occurred. These results offer a new idea for breeding stress tolerant plants.

Light-gated channelrhodopsin sparks proton-induced calcium release in guard cells.

Although there has been long-standing recognition that stimuli-induced cytosolic pH alterations coincide with changes in calcium ion (Ca2+) levels, the interdependence between protons (H+) and Ca2+ remains poorly understood. We addressed this topic using the light-gated channelrhodopsin HcKCR2 from the pseudofungus Hyphochytrium catenoides, which operates as a H+ conductive, Ca2+ impermeable ion channel on the plasma membrane of plant cells. Light activation of HcKCR2 in Arabidopsis guard cells evokes a transient cytoplasmic acidification that sparks Ca2+ release from the endoplasmic reticulum. A H+-induced cytosolic Ca2+ signal results in membrane depolarization through the activation of Ca2+-dependent SLAC1/SLAH3 anion channels, which enabled us to remotely control stomatal movement. Our study suggests a H+-induced Ca2+ release mechanism in plant cells and establishes HcKCR2 as a tool to dissect the molecular basis of plant intracellular pH and Ca2+ signaling.

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.

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.

REGULATOR OF FLOWERING AND STRESS manipulates stomatal density and size in Brachypodium.

Stomata are crucial for gas exchange and water evaporation, and environmental stimuli influence their density (SD) and size (SS). Although genes and mechanisms underlying stomatal development have been elucidated, stress-responsive regulators of SD and SS are less well-known. Previous studies have shown that the stress-inducible Brachypodium RFS (REGULATOR OF FLOWERING AND STRESS, BdRFS) gene affects heading time and enhances drought tolerance by reducing leaf water loss. Here, we report that overexpression lines (OXs) of BdRFS have reduced SD and increased SS, regardless of soil water status. Furthermore, biomass and plant water content of OXs were significantly increased compared to wild type. CRISPR/Cas9-mediated BdRFS knockout mutant (KO) exhibited the opposite stomatal characteristics and biomass changes. Reverse transcription-quantitative polymerase chain reaction analysis revealed that expression of BdICE1 was reversely altered in OXs and KO, pointing to a potential cause for the observed changes in stomatal phenotypes. Stomatal and transcriptional changes were not observed in the Arabidopsis rfs double mutant. Taken together, RFS is a novel regulator of SD and SS and is a promising candidate for genetic engineering of climate-resilient crops.

Experimental validation of the mechanism of stomatal development diversification.

Stomata are the structures responsible for gas exchange in plants. The established framework for stomatal development is based on the model plant Arabidopsis, but diverse patterns of stomatal development have been observed in other plant lineages and species. The molecular mechanisms behind these diversified patterns are still poorly understood. We recently proposed a model for the molecular mechanisms of the diversification of stomatal development based on the genus Callitriche (Plantaginaceae), according to which a temporal shift in the expression of key stomatal transcription factors SPEECHLESS and MUTE leads to changes in the behavior of meristemoids (stomatal precursor cells). In the present study, we genetically manipulated Arabidopsis to test this model. By altering the timing of MUTE expression, we successfully generated Arabidopsis plants with early differentiation or prolonged divisions of meristemoids, as predicted by the model. The epidermal morphology of the generated lines resembled that of species with prolonged or no meristemoid divisions. Thus, the evolutionary process can be reproduced by varying the SPEECHLESS to MUTE transition. We also observed unexpected phenotypes, which indicated the participation of additional factors in the evolution of the patterns observed in nature. This study provides novel experimental insights into the diversification of meristemoid behaviors.

Arabidopsis stomatal lineage cells establish bipolarity and segregate differential signaling capacity to regulate stem cell potential.

Cell polarity combined with asymmetric cell divisions (ACDs) generates cellular diversity. In the Arabidopsis stomatal lineage, a single cortical polarity domain marked by BASL orients ACDs and is segregated to the larger daughter to enforce cell fate. We discovered a second, oppositely positioned polarity domain defined by OCTOPUS-LIKE (OPL) proteins, which forms prior to ACD and is segregated to the smaller (meristemoid) daughter. Genetic and misexpression analyses show that OPLs promote meristemoid-amplifying divisions and delay stomatal fate progression. Polarity mediates OPL segregation into meristemoids but is not required for OPL function. OPL localization and activity are largely independent of other stomatal polarity genes and of the brassinosteroid signaling components associated with OPLs in other contexts. While OPLs are unique to seed plants, ectopic expression in the liverwort Marchantia suppressed epidermal fate progression, suggesting that OPLs engage ancient and broadly conserved pathways to regulate cell division and cell fate.

Altered cell wall hydroxycinnamate composition impacts leaf- and canopy-level CO2 uptake and water use in rice.

Cell wall properties play a major role in determining photosynthetic carbon uptake and water use through their impact on mesophyll conductance (CO2 diffusion from substomatal cavities into photosynthetic mesophyll cells) and leaf hydraulic conductance (water movement from xylem, through leaf tissue, to stomata). Consequently, modification of cell wall (CW) properties might help improve photosynthesis and crop water use efficiency (WUE). We tested this using 2 independent transgenic rice (Oryza sativa) lines overexpressing the rice OsAT10 gene (encoding a "BAHD" CoA acyltransferase), which alters CW hydroxycinnamic acid content (more para-coumaric acid and less ferulic acid). Plants were grown under high and low water levels, and traits related to leaf anatomy, CW composition, gas exchange, hydraulics, plant biomass, and canopy-level water use were measured. Alteration of hydroxycinnamic acid content led to statistically significant decreases in mesophyll CW thickness (-14%) and increased mesophyll conductance (+120%) and photosynthesis (+22%). However, concomitant increases in stomatal conductance negated the increased photosynthesis, resulting in no change in intrinsic WUE (ratio of photosynthesis to stomatal conductance). Leaf hydraulic conductance was also unchanged; however, transgenic plants showed small but statistically significant increases in aboveground biomass (AGB) (+12.5%) and canopy-level WUE (+8.8%; ratio of AGB to water used) and performed better under low water levels than wild-type plants. Our results demonstrate that changes in CW composition, specifically hydroxycinnamic acid content, can increase mesophyll conductance and photosynthesis in C3 cereal crops such as rice. However, attempts to improve photosynthetic WUE will need to enhance mesophyll conductance and photosynthesis while maintaining or decreasing stomatal conductance.

Leaf starch metabolism sets the phase of stomatal rhythm.

In leaves of C3 and C4 plants, stomata open during the day to favor CO2 entry for photosynthesis and close at night to prevent inefficient transpiration of water vapor. The circadian clock paces rhythmic stomatal movements throughout the diel (24-h) cycle. Leaf transitory starch is also thought to regulate the diel stomatal movements, yet the underlying mechanisms across time (key moments) and space (relevant leaf tissues) remain elusive. Here, we developed PhenoLeaks, a pipeline to analyze the diel dynamics of transpiration, and used it to screen a series of Arabidopsis (Arabidopsis thaliana) mutants impaired in starch metabolism. We detected a sinusoidal, endogenous rhythm of transpiration that overarches days and nights. We determined that a number of severe mutations in starch metabolism affect the endogenous rhythm through a phase shift, resulting in delayed stomatal movements throughout the daytime and diminished stomatal preopening during the night. Nevertheless, analysis of tissue-specific mutations revealed that neither guard-cell nor mesophyll-cell starch metabolisms are strictly required for normal diel patterns of transpiration. We propose that leaf starch influences the timing of transpiration rhythm through an interplay between the circadian clock and sugars across tissues, while the energetic effect of starch-derived sugars is usually nonlimiting for endogenous stomatal movements.

Integration of Phosphoproteomics and Transcriptome Studies Reveals ABA Signaling Pathways Regulate UV-B Tolerance in Rhododendron chrysanthum Leaves.

The influence of UV-B stress on the growth, development, and metabolism of alpine plants, such as the damage to DNA macromolecules, the decline in photosynthetic rate, and changes in growth, development, and morphology cannot be ignored. As an endogenous signal molecule, ABA demonstrates a wide range of responses to UV-B radiation, low temperature, drought, and other stresses. The typical effect of ABA on leaves is to reduce the loss of transpiration by closing the stomata, which helps plants resist abiotic and biological stress. The Changbai Mountains have a harsh environment, with low temperatures and thin air, so Rhododendron chrysanthum (R. chrysanthum) seedlings growing in the Changbai Mountains can be an important research object. In this study, a combination of physiological, phosphorylated proteomic, and transcriptomic approaches was used to investigate the molecular mechanisms by which abiotic stress leads to the phosphorylation of proteins in the ABA signaling pathway, and thereby mitigates UV-B radiation to R. chrysanthum. The experimental results show that a total of 12,289 differentially expressed genes and 109 differentially phosphorylated proteins were detected after UV-B stress in R. chrysanthum, mainly concentrated in plant hormone signaling pathways. Plants were treated with ABA prior to exposure to UV-B stress, and the results showed that ABA mitigated stomatal changes in plants, thus confirming the key role of endogenous ABA in plant adaptation to UV-B. We present a model that suggests a multifaceted R. chrysanthum response to UV-B stress, providing a theoretical basis for further elaboration of the mechanism of ABA signal transduction regulating stomata to resist UV-B radiation.

CURLY LEAF modulates apoplast liquid water status in Arabidopsis leaves.

The apoplast of plant leaves, the intercellular space between mesophyll cells, is normally largely filled with air with a minimal amount of liquid water in it, which is essential for key physiological processes such as gas exchange to occur. Phytopathogens exploit virulence factors to induce a water-rich environment, or "water-soaked" area, in the apoplast of the infected leaf tissue to promote disease. We propose that plants evolved a "water soaking" pathway, which normally keeps a nonflooded leaf apoplast for plant growth but is disturbed by microbial pathogens to facilitate infection. Investigation of the "water soaking" pathway and leaf water control mechanisms is a fundamental, yet previously overlooked, aspect of plant physiology. To identify key components in the "water soaking" pathway, we performed a genetic screen to isolate Arabidopsis (Arabidopsis thaliana) severe water soaking (sws) mutants that show liquid water overaccumulation in the leaf under high air humidity, a condition required for visible water soaking. Here, we report the sws1 mutant, which displays rapid water soaking upon high humidity treatment due to a loss-of-function mutation in CURLY LEAF (CLF), encoding a histone methyltransferase in the POLYCOMB REPRESSIVE COMPLEX 2 (PRC2). We found that the sws1 (clf) mutant exhibits enhanced abscisic acid (ABA) levels and stomatal closure, which are indispensable for its water soaking phenotype and mediated by CLF's epigenetic regulation of a group of ABA-associated NAM, ATAF, and CUC (NAC) transcription factor genes, NAC019/055/072. The clf mutant showed weakened immunity, which likely also contributes to the water soaking phenotype. In addition, the clf plant supports a substantially higher level of Pseudomonas syringae pathogen-induced water soaking and bacterial multiplication, in an ABA pathway and NAC019/055/072-dependent manner. Collectively, our study sheds light on an important question in plant biology and demonstrates CLF as a key modulator of leaf liquid water status via epigenetic regulation of the ABA pathway and stomatal movement.