Inhibition Of Stomatal Opening Research Articles

ROS of Distinct Sources and Salicylic Acid Separate Elevated CO2-Mediated Stomatal Movements in Arabidopsis.

Elevated CO2 (eCO2) often reduces leaf stomatal aperture and density thus impacts plant physiology and productivity. We have previously demonstrated that the Arabidopsis BIG protein distinguishes between the processes of eCO2-induced stomatal closure and eCO2-inhibited stomatal opening. However, the mechanistic basis of this action is not fully understood. Here we show that eCO2-elicited reactive oxygen species (ROS) production in big mutants was compromised in stomatal closure induction but not in stomatal opening inhibition. Pharmacological and genetic studies show that ROS generated by both NADPH oxidases and cell wall peroxidases contribute to eCO2-induced stomatal closure, whereas inhibition of light-induced stomatal opening by eCO2 may rely on the ROS derived from NADPH oxidases but not from cell wall peroxidases. As with JA and ABA, SA is required for eCO2-induced ROS generation and stomatal closure. In contrast, none of these three signals has a significant role in eCO2-inhibited stomatal opening, unveiling the distinct roles of plant hormonal signaling pathways in the induction of stomatal closure and the inhibition of stomatal opening by eCO2. In conclusion, this study adds SA to a list of plant hormones that together with ROS from distinct sources distinguish two branches of eCO2-mediated stomatal movements.

Identification and Characterization of Compounds that Affect Stomatal Movements.

Regulation of stomatal aperture is essential for plant growth and survival in response to environmental stimuli. Opening of stomata induces uptake of CO2 for photosynthesis and transpiration, which enhances uptake of nutrients from roots. Light is the most important stimulus for stomatal opening. Under drought stress, the plant hormone ABA induces stomatal closure to prevent water loss. However, the molecular mechanisms of stomatal movements are not fully understood. In this study, we screened chemical libraries to identify compounds that affect stomatal movements in Commelina benghalensis and characterize the underlying molecular mechanisms. We identified nine stomatal closing compounds (SCL1-SCL9) that suppress light-induced stomatal opening by >50%, and two compounds (temsirolimus and CP-100356) that induce stomatal opening in the dark. Further investigations revealed that SCL1 and SCL2 had no effect on autophosphorylation of phototropin or the activity of the inward-rectifying plasma membrane (PM) K+ channel, KAT1, but suppressed blue light-induced phosphorylation of the penultimate residue, threonine, in PM H+-ATPase, which is a key enzyme for stomatal opening. SCL1 and SCL2 had no effect on ABA-dependent responses, including seed germination and expression of ABA-induced genes. These results suggest that SCL1 and SCL2 suppress light-induced stomatal opening at least in part by inhibiting blue light-induced activation of PM H+-ATPase, but not by the ABA signaling pathway. Interestingly, spraying leaves onto dicot and monocot plants with SCL1 suppressed wilting of leaves, indicating that inhibition of stomatal opening by these compounds confers tolerance to drought stress in plants.

The BIG protein distinguishes the process of CO2 -induced stomatal closure from the inhibition of stomatal opening by CO2.

Summary We conducted an infrared thermal imaging‐based genetic screen to identify Arabidopsis mutants displaying aberrant stomatal behavior in response to elevated concentrations of CO 2.This approach resulted in the isolation of a novel allele of the Arabidopsis BIG locus (At3g02260) that we have called CO 2 insensitive 1 (cis1). BIG mutants are compromised in elevated CO 2‐induced stomatal closure and bicarbonate activation of S‐type anion channel currents. In contrast with the wild‐type, they fail to exhibit reductions in stomatal density and index when grown in elevated CO 2. However, like the wild‐type, BIG mutants display inhibition of stomatal opening when exposed to elevated CO 2. BIG mutants also display wild‐type stomatal aperture responses to the closure‐inducing stimulus abscisic acid (ABA).Our results indicate that BIG is a signaling component involved in the elevated CO 2‐mediated control of stomatal development. In the control of stomatal aperture by CO 2, BIG is only required in elevated CO 2‐induced closure and not in the inhibition of stomatal opening by this environmental signal. These data show that, at the molecular level, the CO 2‐mediated inhibition of opening and promotion of stomatal closure signaling pathways are separable and BIG represents a distinguishing element in these two CO 2‐mediated responses.

Overexpression of an Arabidopsis cysteine-rich receptor-like protein kinase, CRK5, enhances abscisic acid sensitivity and confers drought tolerance.

Receptor-like kinases (RLKs) have been reported to regulate many developmental and defense process, but only a few members have been functionally characterized. In the present study, our observations suggest that one of the RLKs, a membrane-localized cysteine-rich receptor-like protein kinase, CRK5, is involved in abscisic acid (ABA) signaling in Arabidopsis thaliana Overexpression of CRK5 increases ABA sensitivity in ABA-induced early seedling growth arrest and promotion of stomatal closure and inhibition of stomatal opening. Interestingly, and importantly, overexpression of CRK5 enhances plant drought tolerance without affecting plant growth at the mature stages and plant productivity. Transgenic lines overexpressing a mutated form of CRK5, CRK5 (K372E) with the change of the 372nd conserved amino acid residue from lysine to glutamic acid in its kinase domain, result in wild-type ABA and drought responses, supporting the role of CRK5 in ABA signaling. The loss-of-function mutation of the CRK5 gene does not affect the ABA response, while overexpression of two homologs of CRK5, CRK4 and CRK19, confers ABA responses, suggesting that these CRK members function redundantly. We further showed that WRKY18, WRKY40 and WRKY60 transcription factors repress the expression of CRK5, and that CRK5 likely functions upstream of ABI2 in ABA signaling. These findings help in understanding the complex ABA signaling network.

Overexpression of the MYB37 transcription factor enhances abscisic acid sensitivity, and improves both drought tolerance and seed productivity in Arabidopsis thaliana.

Although a lot of genes have been revealed to participate in abscisic acid (ABA) signaling, many of the additional components involved in ABA signaling remain to be discovered. Here we report that overexpression of MYB37, a R2R3 MYB subgroup 14 transcription factor in Arabidopsis thaliana, confers hypersensitive phenotypes to exogenous ABA in all the major ABA responses, including ABA-induced inhibition of seed germination, cotyledon greening and early seedling growth, and ABA-induced stomatal closure and inhibition of stomatal opening. Interestingly and importantly, MYB37-overexpression improves plant tolerance to drought, enhances growth of mature plants and seed productivity, thought it delays flowering, which suggests that this gene may be used for improving crop adaptability to drought environment and productivity. However, a myb37-1 knockout mutant displays wild-type ABA responses most likely due to a functional redundancy of the multiple MYB members. Real-time PCR analysis shows that upregulation of the MYB37 expression changes expression of a subset of ABA-responsive genes. Together, these findings suggest that the MYB37 transcription factor plays an important, positive role in plant response to ABA and drought stress, and meanwhile, it plays a positive role in the regulation of seed production.Electronic supplementary materialThe online version of this article (doi:10.1007/s11103-015-0411-1) contains supplementary material, which is available to authorized users.

A link between magnesium-chelatase H subunit and sucrose nonfermenting 1 (SNF1)-related protein kinase SnRK2.6/OST1 in Arabidopsis guard cell signalling in response to abscisic acid.

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.

Crucial roles of the pentatricopeptide repeat protein SOAR1 in Arabidopsis response to drought, salt and cold stresses.

Whereas several mitochondrial/chloroplast pentatricopeptide repeat (PPR) proteins have been reported to regulate plant responses to abiotic stresses, no nucleus-localized PPR protein has been found to play role in these processes. In the present experiment, we provide evidence that a cytosol-nucleus dual-localized PPR protein SOAR1, functioning to negatively regulate abscisic acid (ABA) signaling in seed germination and postgermination growth, is a crucial, positive regulator of plant response to abiotic stresses. Downregulation of SOAR1 expression reduces, but upregulation of SOAR1 expression enhances, ABA sensitivity in ABA-induced promotion of stomatal closure and inhibition of stomatal opening, and plant tolerance to multiple, major abiotic stresses including drought, high salinity and low temperature. Interestingly and importantly, the SOAR1-overexpression lines display strong abilities to tolerate drought, salt and cold stresses, with surprisingly high resistance to salt stress in germination and postgermination growth of seeds that are able to potentially germinate in seawater, while no negative effect on plant growth and development was observed. So, the SOAR1 gene is likely useful for improvement of crops by transgenic manipulation to enhance crop productivity in stressful conditions. Further experimental data suggest that SOAR1 likely regulates plant stress responses at least partly by integrating ABA-dependent and independent signaling pathways, which is different from the ABI2/ABI1 type 2C protein phosphatase-mediated ABA signaling. These findings help to understand highly complicated stress and ABA signalling network.Electronic supplementary materialThe online version of this article (doi:10.1007/s11103-015-0327-9) contains supplementary material, which is available to authorized users.

Cochaperonin CPN20 negatively regulates abscisic acid signaling in Arabidopsis

Previous study showed that the magnesium-protoporphyrin IX chelatase H subunit (CHLH/ABAR) positively regulates abscisic acid (ABA) signaling. Here, we investigated the functions of a CHLH/ABAR interaction protein, the chloroplast co-chaperonin 20 (CPN20) in ABA signaling in Arabidopsis thaliana. We showed that down-expression of the CPN20 gene increases, but overexpression of the CPN20 gene reduces, ABA sensitivity in the major ABA responses including ABA-induced seed germination inhibition, postgermination growth arrest, promotion of stomatal closure and inhibition of stomatal opening. Genetic evidence supports that CPN20 functions downstream or at the same node of CHLH/ABAR, but upstream of the WRKY40 transcription factor. The other CPN20 interaction partners CPN10 and CPN60 are not involved in ABA signaling. Our findings show that CPN20 functions negatively in the ABAR-WRKY40 coupled ABA signaling independently of its co-chaperonin role, and provide a new insight into the role of co-chaperones in the regulation of plant responses to environmental cues.Electronic supplementary materialThe online version of this article (doi:10.1007/s11103-013-0082-8) contains supplementary material, which is available to authorized users.

OsPFA-DSP1, a rice protein tyrosine phosphatase, negatively regulates drought stress responses in transgenic tobacco and rice plants

Dephosphorylation plays a pivotal role in regulating plant growth, development and abiotic/biotic stress responses. Here, we characterized a plant and fungi atypical dual-specificity phosphatase (PFA-DSP) subfamily member, OsPFA-DSP1, from rice. OsPFA-DSP1 was determined to be a functional protein tyrosine phosphatase (PTP) in vitro using phosphatase activity assays. Quantitative real-time PCR and GENEVESTIGATOR analysis showed that OsPFA-DSP1 mRNA was induced by drought stress. Transfection of rice protoplasts showed that OsPFA-DSP1 accumulated in both the cytoplasm and nucleus. Ectopic overexpression of OsPFA-DSP1 in tobacco increased sensitivity to drought stress and insensitivity to ABA-induced stomatal closure and inhibition of stomatal opening. Furthermore, overexpression of OsPFA-DSP1 in rice also increased sensitivity to drought stress. These results indicated that OsPFA-DSP1 is a functional PTP and may act as a negative regulator in drought stress responses.

Common and unique elements of the ABA-regulated transcriptome of Arabidopsis guard cells

BackgroundIn the presence of drought and other desiccating stresses, plants synthesize and redistribute the phytohormone abscisic acid (ABA). ABA promotes plant water conservation by acting on specialized cells in the leaf epidermis, guard cells, which border and regulate the apertures of stomatal pores through which transpirational water loss occurs. Following ABA exposure, solute uptake into guard cells is rapidly inhibited and solute loss is promoted, resulting in inhibition of stomatal opening and promotion of stomatal closure, with consequent plant water conservation. There is a wealth of information on the guard cell signaling mechanisms underlying these rapid ABA responses. To investigate ABA regulation of gene expression in guard cells in a systematic genome-wide manner, we analyzed data from global transcriptomes of guard cells generated with Affymetrix ATH1 microarrays, and compared these results to ABA regulation of gene expression in leaves and other tissues.ResultsThe 1173 ABA-regulated genes of guard cells identified by our study share significant overlap with ABA-regulated genes of other tissues, and are associated with well-defined ABA-related promoter motifs such as ABREs and DREs. However, we also computationally identified a unique cis-acting motif, GTCGG, associated with ABA-induction of gene expression specifically in guard cells. In addition, approximately 300 genes showing ABA-regulation unique to this cell type were newly uncovered by our study. Within the ABA-regulated gene set of guard cells, we found that many of the genes known to encode ion transporters associated with stomatal opening are down-regulated by ABA, providing one mechanism for long-term maintenance of stomatal closure during drought. We also found examples of both negative and positive feedback in the transcriptional regulation by ABA of known ABA-signaling genes, particularly with regard to the PYR/PYL/RCAR class of soluble ABA receptors and their downstream targets, the type 2C protein phosphatases. Our data also provide evidence for cross-talk at the transcriptional level between ABA and another hormonal inhibitor of stomatal opening, methyl jasmonate.ConclusionsOur results engender new insights into the basic cell biology of guard cells, reveal common and unique elements of ABA-regulation of gene expression in guard cells, and set the stage for targeted biotechnological manipulations to improve plant water use efficiency.

Heterotrimeric G-protein regulation of ROS signalling and calcium currents in Arabidopsis guard cells

Heterotrimeric G proteins composed of Gα, Gβ, and Gγ subunits are important signalling agents in both animals and plants. In plants, G proteins modulate numerous responses, including abscisic acid (ABA) and pathogen-associated molecular pattern (PAMP) regulation of guard cell ion channels and stomatal apertures. Previous analyses of mutants deficient in the sole canonical Arabidopsis Gα subunit, GPA1, have shown that Gα-deficient guard cells are impaired in ABA inhibition of K(+) influx channels, and in pH-independent activation of anion efflux channels. ABA-induced Ca(2+) uptake through ROS-activated Ca(2+)-permeable channels in the plasma membrane is another key component of ABA signal transduction in guard cells, but the question of whether these channels are also dependent on Gα for their ABA response has not been evaluated previously. We used two independent Arabidopsis T-DNA null mutant lines, gpa1-3 and gpa1-4, to investigate this issue. We observed that gpa1 mutants are disrupted both in ABA-induced Ca(2+)-channel activation, and in production of reactive oxygen species (ROS) in response to ABA. However, in response to exogenous H(2)O(2) application, I(Ca) channels are activated normally in gpa1 guard cells. In addition, H(2)O(2) inhibition of stomatal opening and promotion of stomatal closure are not disrupted in gpa1 mutant guard cells. These data indicate that absence of GPA1 interrupts ABA signalling between ABA reception and ROS production, with a consequent impairment in Ca(2+)-channel activation.

Arabidopsis thaliana lipid phosphate phosphatase 2 is involved in abscisic acid signalling in leaves

Lipid phosphate phosphatases (LPPs, E.C. 3.1.3.4) catalyse the dephosphorylation of diacylglycerol pyrophosphate (DGPP) and phosphatidic acid (PA), which are secondary messengers in abscisic acid (ABA) signalling. In this study, we investigated the effect of ABA on the expression of AtLPP genes as they encode putative ABA-signalling partners. We observed that AtLPP2 expression was down-regulated by ABA and we performed experiments on Atlpp2-2, an AtLPP2 knockout mutant, to determine whether AtLPP2 was involved in ABA signalling. We observed that Atlpp2-2 plantlets contained about twice as much PA as the wild-type Col-0 and exhibited higher PA kinase (PAK) activity than Col-0 plants. In addition, we showed that ABA stimulated diacylglycerol kinase (DGK) activity independently of AtLPP2 activity but that the ABA-stimulation of PAK activity recorded in Col-0 was dependent on AtLPP2. In order to evaluate the involvement of AtLPP2 activity in guard cell function, we measured the ABA sensitivity of Atlpp2-2 stomata. The inhibition of stomatal opening was less sensitive to ABA in Atlpp2-2 than in Col-0. Watered and water-stressed plants of the two genotypes accumulated ABA to the same extent, thus leading us to consider Atlpp2-2 an ABA-signalling mutant. Taken together our observations show that AtLPP2 is a part of ABA signalling and participate to the regulation of stomatal movements.

Arabidopsis calcium-dependent protein kinase CPK10 functions in abscisic acid- and Ca2+-mediated stomatal regulation in response to drought stress.

Plant calcium-dependent protein kinases (CDPKs) may function as calcium sensors and play important roles in the regulation of plant growth and development and in plant responses to biotic and abiotic stresses. The Arabidopsis (Arabidopsis thaliana) genome encodes 34 CDPKs, and most of them have not been functionally characterized. Here, we report the functional characterization of CPK10 in Arabidopsis response to drought stress. The cpk10 mutant, a T-DNA insertion mutant for the Arabidopsis CPK10 gene, showed a much more sensitive phenotype to drought stress compared with wild-type plants, while the CPK10 overexpression lines displayed enhanced tolerance to drought stress. Induction of stomatal closure and inhibition of stomatal opening by abscisic acid (ABA) and Ca(2+) were impaired in the cpk10 mutants. Using yeast two-hybrid methods, a heat shock protein, HSP1, was identified as a CPK10-interacting protein. The interaction between CPK10 and HSP1 was further confirmed by pull-down and bimolecular fluorescence complementation assays. The HSP1 knockout mutant (hsp1) plants showed a similar sensitive phenotype under drought stress as the cpk10 mutant plants and were similarly less sensitive to ABA and Ca(2+) in regulation of stomatal movements. Electrophysiological experiments showed that ABA and Ca(2+) inhibition of the inward K(+) currents in stomatal guard cells were impaired in the cpk10 and hsp1 mutants. All presented data demonstrate that CPK10, possibly by interacting with HSP1, plays important roles in ABA- and Ca(2+)-mediated regulation of stomatal movements.

Sodium‐calcium interactions with growth, water, and photosynthetic parameters in salt‐treated beans

AbstractCalcium (Ca2+) amelioration of the plant's growth response to salinity depends on genetic factors. In this work, supplemental Ca2+ did not improve growth in Phaseolus vulgaris L. cv. Contender under high‐saline conditions and negatively affected several physiological parameters in nonsalinized plants. The response to supplemental Ca2+ was examined using plants grown in 25% modified Hoagland solution at different Na+ : Ca2+ ratios. In control plants (1 mM Ca2+; 1 mM Na+) surplus Ca2+ (4 or 10 mM) was associated with stomatal closure, decrease of hydraulic conductivity, sap flow, leaf specific dry weight, leaf K+ and leaf Mg2+ concentrations, and inhibition of CO2 assimilation. Leaf water content was enhanced, while water‐use efficiency and dry matter were unaffected during the 15 d experimental period. The Ca2+ effect was not cation‐specific since similar results were found in plants supplied with high external Mg2+ or with a combination of Ca2+ and Mg2+. Relative to control plants, salinization (50 and 100 mM NaCl) caused a decrease in dry matter, hydraulic conductivity, sap flow, leaf Mg2+ activity, and inhibition of stomatal opening and CO2 assimilation. However, NaCl (50 and 100 mM NaCl) enhanced leaf K+ concentration and water‐use efficiency. At 100 mM NaCl, leaf water content also significantly increased. Supplemental Ca2+ had no amelioration effect on the salt‐stress response of this bean cultivar. In contrast, the 50 mM–NaCl treatment improved stomatal conductance and CO2‐assimilation rate in plants exposed to the highest Ca2+ concentration (10 mM). Phaseolus vulgaris is classified as a very NaCl‐sensitive species. The similarities in the effects caused by supplemental Ca2+, supplemental Mg2+, and NaCl salinity suggest that P. vulgaris cv. Contender has a high non‐ion‐specific salt sensitivity. On the other hand, the improvement in gas‐exchange parameters in Ca2+‐supplemented plants by high NaCl could be the result of specific Na+‐triggered responses, such as an increase in the concentration of K+ in the leaves.

An inducible, modular system for spatio-temporal control of gene expression in stomatal guard cells

Stomata, flanked by pairs of guard cells, are small pores on the leaf surfaces of plants and they function to control gas exchange between plants and the atmosphere. Stomata will open when water is available to allow for the uptake of carbon dioxide for photosynthesis. During periods of drought, stomata will close to reduce desiccation stress. As such, optimal functioning of stomata will impact on water use efficiency by plants. The development of an inducible, modular system for robust and targeted gene expression in stomatal guard cells is reported here. It is shown that application of ethanol vapour to activate the gene expression system did not affect the ability of stomata to respond to ABA in bioassays to determine the promotion of stomatal closure and the inhibition of stomatal opening. The system that has been developed allows for robust spatio-temporal control of gene expression in all cells of the stomatal lineage, thereby enabling molecular engineering of stomatal function as well as studies on stomatal development.

Arabidopsis MPK3, a Key Signalling Intermediate in Stomatal Function

Regulation of stomatal aperture is of critical importance to plants to balance gas exchange and water loss, and also to control ingress of bacterial pathogens. MAP kinase signal transduction pathways are mediators of biotic and abiotic stress, and have been indicted in the control of stomatal movements. Cell-specific antisense was used to down-regulate MPK3 gene expression in Arabidopsis guard cells, resulting in ABA insensitivity during inhibition of stomatal opening, but a normal ABA response in promotion of closure assays. This response is similar to that of the heterotrimeric G protein alpha subunit mutant gpa1, as is the imposition of ABA insensitivity during stomatal closure by butyrate treatment, suggesting that MPK3 and GPA1 are in the same ABA signal transduction pathway and adding further evidence for parallel signalling pathways during ABA-induced closure. By contrast, antisense plants were less sensitive to H2O2 in both promotion of closure and inhibition of opening assays, although H2O2 production in response to ABA was not affected. Regulation of stomatal aperture by PAMPs has recently been shown to be an important plant defense mechanism; since MPK3 is also activated by such pathogen elicitors, we postulate that in addition to a signalling role in guard cell movements, MPK3 is involved in the active prevention of bacterial infection through stomata.

Guard cell‐specific inhibition of Arabidopsis MPK3 expression causes abnormal stomatal responses to abscisic acid and hydrogen peroxide

MAP kinases have been linked to guard cell signalling. Arabidopsis thaliana MAP Kinase 3 (MPK3) is known to be activated by abscisic acid (ABA) and hydrogen peroxide (H(2)O(2)), which also control stomatal movements. We therefore studied the possible role of MPK3 in guard cell signalling through guard cell-specific antisense inhibition of MPK3 expression. Such transgenic plants contained reduced levels of MPK3 mRNA in the guard cells and displayed partial insensitivity to ABA in inhibition of stomatal opening, but responded normally to this hormone in stomatal closure. However, ABA-induced stomatal closure was reduced compared with controls when cytoplasmic alkalinization was prevented with sodium butyrate. MPK3 antisense plants were less sensitive to exogenous H(2)O(2), both in inhibition of stomatal opening and in promotion of stomatal closure, thus MPK3 is required for the signalling of this compound. ABA-induced H(2)O(2) synthesis was normal in these plants, indicating that MPK3 probably acts in signalling downstream of H(2)O(2). These results provide clear evidence for the important role of MPK3 in the perception of ABA and H(2)O(2) in guard cells.

The effects of manipulating phospholipase C on guard cell ABA-signalling.

Studies using stably transformed tobacco plants containing very low levels of PI-PLC in their guard cells show that this enzyme plays a role in the events associated with the inhibition of stomatal opening by ABA, but not in the cellular reactions that are responsible for ABA-induced stomatal closure. However, Commelina communis guard cells microinjected with the InsP3 antagonist, heparin, fail to close on addition of ABA. There are three possible explanations for this apparent data mismatch. The differences may be indicative of species-specific signalling pathways, the presence of a PI-PLC isoform(s) that is not down-regulated in these transgenic lines and/or they may reflect differences between short-term (acute) administration of an inhibitor and long-term (chronic) effects of gene manipulation. It is possible that the guard cell is a robust signalling system that is able to adapt or compensate for the chronic loss of PI-PLC, but which is unable to adjust quickly to acute loss of this component. It would be interesting to investigate this possibility further using either transient manipulation of gene expression or through the use of an inducible promoter.

Actin Filaments Modulate Both Stomatal Opening and Inward K+-Channel Activities in Guard Cells of Vicia faba L.

Actin antagonists have previously been shown to alter responses of Commelina communis stomata to physiological stimuli, implicating actin filaments in the control of guard cell volume changes (M. Kim, P.K. Hepler, S.-O. Eun, K.S. Ha, Y. Lee [1995] Plant Physiol 109: 1077-1084). Since K+ channels in the guard cell play an important role in stomatal movements, we examined the possible regulation of K+-channel activities by the state of actin polymerization. Agents affecting actin polymerization altered light-induced stomatal opening and inward K+-channel activities measured by patch clamping in Vicia faba. Cytochalasin D, which induces depolymerization of actin filaments, promoted light-induced stomatal opening and potentiated the inward K+ current in guard cell protoplasts. Phalloidin, a stabilizer of filamentous actin, inhibited both light-induced stomatal opening and inward K+ current. Inward K+-channel activities in outside-out membrane patches showed responses to these agents that support results at the whole-cell current level, suggesting that cytochalasin D facilitates and phalloidin inhibits K+ influx in intact guard cells, thus resulting in enhancement and inhibition of stomatal opening, respectively. To our knowledge, this is the first report that provides evidence that actin filaments may regulate an important physiological process by modulating the activities of ion channels in plant cells.

Substitution of hydrazones for the 4′ carbonyl on abscisic acid rendered it ineffective in a rapid stomatal–opening–inhibition bioassay

A rapid stomatal bioassay to test abscisic acid (ABA)–like inhibition of stomatal opening was elaborated. The assay was sensitive to low concentrations of ABA (>50% inhibition of opening at 0.02 μM (+)‐S‐ABA) and was insensitive to 20 μM (+)‐S‐ABA‐1 ‐(methyl ester), which is consistent with previous work, With the original intent of developing anti‐idiotypic antibodies, we made conjugates of ABA at the 4′carbonyl because of its accessibility and known requirements for other positions of the ABA molecule in siomatal signal transduction. We found that these conjugates lacked ABA‐like activity and did not interfere with (+)‐S‐ABA in the stomatal bioassay, implying the importance of the 4′carbonyl. These observations cast doubt on the feasibility of using anti‐idiolypic antibodies produced through the use of 4′‐substituted ABA for the isolation of a guard‐cell ABA receptor.