Uncovering the core genetic programs governing plant guard cell biology.

Drought stress triggers abscisic acid (ABA) signaling in guard cells and induces stomatal closure to prevent water loss in land plants. Stomatal movement is accompanied by reorganization of the cytoskeleton. Cortical microtubules disassemble in response to ABA, which is required for stomatal closure. However, how ABA signaling regulates microtubule disassembly is unclear, and the microtubule-associated proteins (MAPs) involved in this process remain to be identified. In this study, we show that OPEN STOMATA 1 (OST1), a central component in ABA signaling, mediates microtubule disassembly during ABA-induced stomatal closure in Arabidopsis thaliana. We identified the MAP SPIRAL1 (SPR1) as the substrate of OST1. OST1 interacts with and phosphorylates SPR1 at Ser6, which promotes the disassociation of SPR1 from microtubules and facilitates microtubule disassembly. Compared with the wild type, the spr1 mutant exhibited significantly greater water loss and reduced ABA responses, including stomatal closure and microtubule disassembly in guard cells. These phenotypes were restored by introducing the phosphorylated active form of SPR1. Our findings demonstrate that SPR1 positively regulates microtubule disassembly during ABA-induced stomatal closure, which depends on OST1-mediated phosphorylation. These findings reveal a specific connection between a core component of ABA signaling and MAPs.

Stomatal movements, which regulate gas exchange in plants, involve pronounced changes in the shape and volume of the guard cell. To test whether the changes are regulated by actin filaments, we visualized microfilaments in mature guard cells and examined the effects of actin antagonists on stomatal movements. Immunolocalization on fixed cells and microinjection of fluorescein isothiocyanate-phalloidin into living guard cells of Commelina communis L. showed that cortical microfilaments were radially distributed, fanning out from the stomatal pore site, resembling the known pattern of microtubules. Treatment of epidermal peels with phalloidin prior to stabilizing microfilaments with m-maleimidobenzoyl N-hydroxysuccimimide caused dense packing of radial microfilaments and an accumulation of actin around many organelles. Both stomatal closing induced by abscisic acid and opening under light were inhibited. Treatment of guard cells with cytochalasin D abolished the radial pattern of microfilaments; generated sparse, poorly oriented arrays; and caused partial opening of dark-closed stomata. These results suggest that microfilaments participate in stomatal aperture regulation.

Analysis of abscisic acid responsive proteins in Brassica napus guard cells by multiplexed isobaric tagging

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.

Stomatal regulation: Role of H2S-induced persulfidation in ABA signaling

Roles of intracellular hydrogen peroxide accumulation in abscisic acid signaling in Arabidopsis guard cells

In this review we concentrate on guard cell metabolism and CO2 sensing. Although a matter of some controversy, it is generally accepted that the Calvin cycle plays a minor role in stomatal movements. Recent data emphasise the importance of guard cell starch degradation and of carbon import from the guard cell apoplast in promoting and maintaining stomatal opening. Chloroplast maltose and glucose transporters appear to be crucial to the export of carbon from both guard and mesophyll cells. The way guard cells sense CO2 remains an unresolved question. However, a better understanding of the cellular events downstream from CO2 sensing is emerging. We now recognise that there are common as well as unique steps in abscisic acid (ABA) and CO2 signalling pathways. For example, while ABA and CO2 both trigger increases in cytoplasmic free calcium, unlike ABA, CO2 does not promote a cytoplasmic pH change. Future advances in this area are likely to result from the increased use of techniques and resources, such as, reverse genetics, novel mutants, confocal imaging, and microarray analyses of the guard cell transcriptome.

Stomata, formed by a pair of specialized guard cells, are present on the epidermis of most plant leaves. The regulation of stomatal apertures controls the exchange of moisture and gas between plants and the environment, and balances the carbon dioxide (CO2) entry into the plant for photosynthesis with water loss via transpiration. G-protein, a ubiquitous signaling molecule in eukaryotic cells, is an indispensable participator in the mechanism of stomatal movement. It was widely recognized that G-protein α subunit (GPA1) can regulate a majority of signals during stomatal movement, such as Ca2+, reactive oxygen species (ROS), H2O2, ethylene, and so on. On the other hand, guard cell microtubule (MT) dynamics play a comparably critical role in regulating stomatal movement, and MTs rearrangement has been observed in stomatal responses to a variety of stimuli, like light, dark, nitric oxide (NO). Both heterotrimeric G-protein and MTs are essential for abscisic acid (ABA) signaling in guard cells, but whether and how these two signaling components work together is poorly understood.Here, the interplay between MTs and G-protein during ABA-induced stomatal movement was investigated in Arabidopsis thaliana .It was found that the heterotrimeric G-protein α subunit mutant, gpa1 , had a significantly higher water loss ratio than the wild type under the same conditions.Stomatal bioassay analyses revealed that guard cell sensitivity to ABA was attenuated in gpa1 , and the mutant′s ABA-insensitive phenotype could be partially restored by Oryzalin, an MT-specific inhibitor. However, exogenously applied Ca2+ chelator BAPTA-AM together with Oryzalin impaired the inhibition of stomatal opening by ABA. These results suggest that both MTs dynamics and Ca2+ flux may participate in the process of G-protein-mediated regulation of ABA-induced stomatal movement. Confocal microscopic images showed that after ABA treatment, MTs arrays in wild type underwent rapid disruption from radial arrays to disassembled fragments, whereas those in gpa1 largely remained bundled.A similar behavior was observed in BAPTA-AM plus ABA treatment, which is consistent with the stomatal bioassay results. In other words, G protein can control MTs rearrangement by adjusting the Ca2+ level in ABA-induced stomatal movement.In addition, non-invasive micro-test (NMT) demonstrated that the ABA-induced transmembrane Ca2+ flux was abolished in gpa1 guard cells, but Oryzalin enhanced the ABA response; Ca2+ efflux was suppressed dramatically when Oryzalin was added in gpa1. Therefore, our data clearly indicate that it exists a possitive crosstalking among G-protein, MTs and Ca2+ during ABA-induced stomatal movement. In this signal pathway, both MTs and Ca2+ act downstream of G-protein. Intracellular Ca2+ of guard cells relays signal information from G-protein to MTs; in turn, the changes of MTs dynamics in guard cells also have a feedback to Ca2+ flux. Finally the ABA signalling transduction results in stomatal movement, as well as the regulation of transpirational water loss and drought resistance.

Abscisic acid (ABA)- and methyl jasmonate (MeJA)-induced stomatal closure are accompanied by cytosolic alkalization in guard cells. However, it remains to be clarified how the alkalization functions in not only ABA signaling but also MeJA. We investigated cytosolic alkalization in guard cells during ABA-, MeJA- and Ca(2+)-induced stomatal closure of wild type, abi1-1, abi2-1, ost1-2 and coi1 using a pH-sensitive fluorescent dye, BCECF-AM. ABA induced cytosolic alkalization in guard cells of wild-type and coi1 but not in ost1-2 guard cells whereas MeJA elicited cytosolic alkalization in wild-type and ost1-2 guard cells but not in coi1. Neither ABA nor MeJA induced cytosolic alkalization in abi1-1 and abi2-1 guard cells. Exogenous Ca(2+) induced stomatal closure accompanied by cytosolic alkalization in guard cells of wild-type, abi1-1, abi2-1, ost1-2 and coi1 plants. An agent to acidify cytosol, butyrate, suppressed Ca(2+)-induced cytosolic alkalization and ABA-, MeJA- and Ca(2+)-induced cytosolic Ca(2+) oscillation in wild-type guard cells to prevent stomatal closure. These results indicate that cytosolic alkalization and cytosolic Ca(2+) oscillation coordinately function in ABA and MeJA signaling in Arabidopsis guard cells.

Stomatal movement, which regulates gas exchange in plants, is controlled by a variety of environmental factors, including biotic and abiotic stresses. The stress hormone abscisic acid (ABA) initiates a signaling cascade, which leads to increased H2O2 and Ca2+ levels and F-actin reorganization, but the mechanism of, and connection between, these events is unclear. SINE1, an outer nuclear envelope component of a plant Linker of Nucleoskeleton and Cytoskeleton complex, associates with F-actin and is, along with its putative paralog SINE2, expressed in guard cells. Here, we have determined that Arabidopsis (Arabidopsis thaliana) SINE1 and SINE2 play an important role in stomatal opening and closing. Loss of SINE1 or SINE2 results in ABA hyposensitivity and impaired stomatal dynamics but does not affect stomatal closure induced by the bacterial elicitor flg22. The ABA-induced stomatal closure phenotype is, in part, attributed to impairments in Ca2+ and F-actin regulation. Together, the data suggest that SINE1 and SINE2 act downstream of ABA but upstream of Ca2+ and F-actin. While there is a large degree of functional overlap between the two proteins, there are also critical differences. Our study makes an unanticipated connection between stomatal regulation and nuclear envelope-associated proteins, and adds two new players to the increasingly complex system of guard cell regulation.

Phospholipase D (PLD) is involved in responses to abiotic stress and abscisic acid (ABA) signaling. To investigate the roles of two Arabidopsis (Arabidopsis thaliana) PLDs, PLDα1 and PLDδ, in ABA signaling in guard cells, we analyzed ABA responses in guard cells using Arabidopsis wild type, pldα1 and pldδ single mutants, and a pldα1 pldδ double mutant. ABA-induced stomatal closure was suppressed in the pldα1 pldδ double mutant but not in the pld single mutants. The pldα1 and pldδ mutations reduced ABA-induced phosphatidic acid production in epidermal tissues. Expression of either PLDα1 or PLDδ complemented the double mutant stomatal phenotype. ABA-induced stomatal closure in both pldα1 and pldδ single mutants was inhibited by a PLD inhibitor (1-butanol ), suggesting that both PLDα1 and PLDδ function in ABA-induced stomatal closure. During ABA-induced stomatal closure, wild-type guard cells accumulate reactive oxygen species and nitric oxide and undergo cytosolic alkalization, but these changes are reduced in guard cells of the pldα1 pldδ double mutant. Inward-rectifying K(+) channel currents of guard cells were inhibited by ABA in the wild type but not in the pldα1 pldδ double mutant. ABA inhibited stomatal opening in the wild type and the pldδ mutant but not in the pldα1 mutant. In wild-type rosette leaves, ABA significantly increased PLDδ transcript levels but did not change PLDα1 transcript levels. Furthermore, the pldα1 and pldδ mutations mitigated ABA inhibition of seed germination. These results suggest that PLDα1 and PLDδ cooperate in ABA signaling in guard cells but that their functions do not completely overlap.

Magnesium-chelatase H subunit [CHLH/putative abscisic acid (ABA) receptor ABAR] positively regulates guard cell signalling in response to ABA, but the molecular mechanism remains largely unknown. A member of the sucrose nonfermenting 1 (SNF1)-related protein kinase 2 family, SnRK2.6/open stomata 1 (OST1)/SRK2E, which plays a critical role in ABA signalling in Arabidopsis guard cells, interacts with ABAR/CHLH. Neither mutation nor over-expression of the ABAR gene affects significantly ABA-insensitive phenotypes of stomatal movement in the OST1 knockout mutant allele srk2e. However, OST1 over-expression suppresses ABA-insensitive phenotypes of the ABAR mutant allele cch in stomatal movement. These genetic data support that OST1 functions downstream of ABAR in ABA signalling in guard cells. Consistent with this, ABAR protein is phosphorylated, but independently of the OST1 protein kinase. Two ABAR mutant alleles, cch and rtl1, show ABA insensitivity in ABA-induced reactive oxygen species and nitric oxide production, as well as in ABA-activated phosphorylation of a K(+) inward channel KAT1 in guard cells, which is consistent with that observed in the pyr1 pyl1 pyl2 pyl4 quadruple mutant of the well-characterized ABA receptor PYR/PYL/RCAR family acting upstream of OST1. These findings suggest that ABAR shares, at least in part, downstream signalling components with PYR/PYL/RCAR receptors for ABA in guard cells; though cch and rtl1 show strong ABA-insensitive phenotypes in both ABA-induced stomatal closure and inhibition of stomatal opening, while the pyr1 pyl1 pyl2 pyl4 quadruple mutant shows strong ABA insensitivity only in ABA-induced stomatal closure. These data establish a link between ABAR/CHLH and SnRK2.6/OST1 in guard cell signalling in response to ABA.

Signal crosstalk between jasmonate and ethylene is crucial for a proper maintenance of defense responses and development. Although previous studies reported that both jasmonate and ethylene also function as modulators of stomatal movements, the signal crosstalk mechanism in stomatal guard cells remains unclear. Here, we show that the ethylene signaling inhibits jasmonate signaling as well as abscisic acid (ABA) signaling in guard cells of Arabidopsis thaliana and reveal the signaling crosstalk mechanism. Both an ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) and an ethylene-releasing compound ethephon induced transient stomatal closure, and also inhibited methyl jasmonate (MeJA)-induced stomatal closure as well as ABA-induced stomatal closure. The ethylene inhibition of MeJA-induced stomatal closure was abolished in the ethylene-insensitive mutant etr1-1, whereas MeJA-induced stomatal closure was impaired in the ethylene-overproducing mutant eto1-1. Pretreatment with ACC inhibited MeJA-induced reactive oxygen species (ROS) production as well as ABA-induced ROS production in guard cells but did not suppress ABA activation of OPEN STOMATA 1 (OST1) kinase in guard cell-enriched epidermal peels. The whole-cell patch-clamp analysis revealed that ACC attenuated MeJA and ABA activation of S-type anion channels in guard cell protoplasts. However, MeJA and ABA inhibitions of Kin channels were not affected by ACC pretreatment. These results suggest that ethylene signaling inhibits MeJA signaling and ABA signaling by targeting S-type anion channels and ROS but not OST1 kinase and K+ channels in Arabidopsis guard cells.

Stomatal movements control CO2 uptake for plant photosynthesis and water loss by transpiration, then determine plant productivity and water utilization efficiency. The microtubule dynamics is widely recognized to be essential for guard cell function. However, the molecular mechanisms underlying this process remain largely unknown. WDL3 belongs to the microtubule-associated protein WAVE-DAMPENED 2(WVD2)/WVD2-LIKE (WDL) family, which binds to and stabilizes microtubules against low-temperature and dilution-induced depolymerization. In the presence of WDL3, tubulins assemble into large microtubule bundles in vitro , otherwise only single filament patterns form. It has been intensively investigated that WDL3 participates in COP1(CONSTITUTIVE PHOTOMORPHOGENIC 1)-mediated hypocotyl cell elongation in darkness, but whether and how it modulates the microtubule behaviors during guard cell signaling transduction is still an open question. In this study, we dissected the interplay among WDL3, microtubule dynamics and Ca2+ in ABA-induced stomatal closure. We found that transpirational water loss from detached leaves occurred slowly in the WDL3 RNA interference transgenic line. Stomatal bioassay revealed that guard cell sensitivity to ABA was promoted in the WDL3 RNAi seedlings, and this phenotype could be partially blocked by paclitaxel, a microtubule stabilizing agent. On the other hand, the microtubule-disrupting drug oryzalin, enhanced ABA-triggered stomatal closure even further, and the WDL3 RNAi guard cells were more sensitive to oryzalin treatment. Based on the pharmacological results above, we next tested the effect of WDL3 on cortical microtubules in guard cells directly by Confocal microscopy. The differences in the configurations of microtubule filaments between WDL3 RNAi and wild type (WT) were analyzed after ABA treatment. Microscopic images showed that ABA-induced microtubule disassembly took faster in WDL3 RNAi guard cells than in WT, and the extent of filament bundling decreased significantly in WDL3 RNAi seedlings, as evaluated by the Image J software. This was consistent with the rapid ABA-induced stomatal closing in WDL3 RNAi material . Moreover, we examined the potential role of Ca2+ in this signal pathway. The cytosolic Ca2+ chelator—BAPTA generally alleviated ABA effects in both WT and WDL3 RNAi materials. ABA-induced stomatal closure and microtubule remodeling were much more delayed in WDL3 RNAi seedlings, indicating that Ca2+ acted upstream in the WDL3 mediated-ABA signaling pathway for stomatal movement. In addition, when exogenous ABA was applied, the Ca2+ influx monitored by the non-invasive micro-test technique (NMT) in WDL3 RNAi guard cells was more pronounced than in WT. Taken together, our results suggest that WDL3, probably coordinated with Ca2+, is involved in the precise regulation of microtubule architecture and dynamics, to accurately execute ABA-stimulated signaling transduction during stomatal movement. Thus it contributes to the proper control of leaf transpiration.

Abscisic acid (ABA) signaling in stomatal guard cells is crucial for plants to cope with abiotic stress condition. Pyrabactin is a synthetic agonist of ABA that has a selective affinity to limited isoforms of ABA receptors. Here we investigated the differential utilization of downstream signaling events in guard cell ABA signaling under specific receptor isoforms taking advantage of pyrabactin affinity. Pyrabactin induced stomatal closure as well as ABA, while it did not inhibit stomatal opening. Plasma membrane inwardly rectifying K+ channel was not regulated by pyrabactin, while H+-ATPase activation was negatively regulated by pyrabactin. Pharmacological and molecular genetic evidence supported that reactive oxygen species production occurred differentially between the closure-inducing and opening-inhibiting signals in guard cells. These findings offered a previously unidentified mechanism for ABA signaling events promoting closure induction and opening inhibition of stomata, which were distinct from each other and governed by different ABA receptor isoforms discriminable by their affinity for pyrabactin.