During drought, the plant hormone abscisic acid (ABA) triggers stomatal closure, thus reducing water loss. Using infrared thermography, we isolated two allelic Arabidopsis mutants ( ost1-1 and ost1-2 ) impaired in the ability to limit their transpiration upon drought. These recessive ost1 mutations disrupted ABA induction of stomatal closure as well as ABA inhibition of light-induced stomatal opening. By contrast, the ost1 mutations did not affect stomatal regulation by light or CO 2 , suggesting that OST1 is involved specifically in ABA signaling. The OST1 gene was isolated by positional cloning and was found to be expressed in stomatal guard cells and vascular tissue. In-gel assays indicated that OST1 is an ABAactivated protein kinase related to the Vicia faba ABA-activated protein kinase (AAPK). Reactive oxygen species (ROS) were shown recently to be an essential intermediate in guard cell ABA signaling. ABA-induced ROS production was disrupted in ost1 guard cells, whereas applied H 2 O 2 or calcium elicited the same degree of stomatal closure in ost1 as in the wild type. These results suggest that OST1 acts in the interval between ABA perception and ROS production. The relative positions of ost1 and the other ABA-insensitive mutations in the ABA signaling network ( abi1-1 , abi2-1 , and gca2 ) are discussed.

SummaryThe Arabidopsis ABI1 and ABI2 genes encode two protein serine/threonine phosphatases 2C (PP2C). These genes have been originally identi®ed by the dominant mutations abi1±1 and abi2±1, which reduce the plant's responsiveness to the hormone abscisic acid (ABA). However, recessive mutants of ABI1 were recently shown to be supersensitive to ABA, which demonstrated that the ABI1 phosphatase is a negative regulator of ABA signalling. We report here the isolation and characterisation of the ®rst reduction-of-function allele of ABI2, abi2±1R1. The in vitro phosphatase activity of the abi2±1R1 protein is approximately 100-fold lower than that of the wild-type ABI2 protein. Abi2±1R1 plants displayed a wildtype ABA sensitivity. However, doubly mutant plants combining the abi2±1R1 allele and a loss-offunction allele at the ABI1 locus were more responsive to ABA than each of the parental single mutants. These data indicate that the wild-type ABI2 phosphatase is a negative regulator of ABA signalling, and that the ABI1 and ABI2 phosphatases have overlapping roles in controlling ABA action. Measurements of PP2C activity in plant extracts showed that the phosphatase activity of ABI1 and ABI2 increases in response to ABA. These results suggest that ABI1 and ABI2 act in a negative feedback regulatory loop of the ABA signalling pathway.

(J.M.K.) Signaling events during abscisic acid (ABA) or methyl jasmonate (MJ)-induced stomatal closure were examined in Arabidopsis wild type, ABA-insensitive (ost1-2), and MJ-insensitive mutants ( jar1-1) in order to examine a crosstalk between ABA and MJ signal transduction. Some of the experiments were performed on epidermal strips of Pisum sativum. Stomata of jar1-1 mutant plants are insensitive to MJ but are able to close in response to ABA. However, their sensitivity to ABA is less than that of wildtype plants. Reciprocally, the stomata of ost1-2 are insensitive to ABA but are able to close in response to MJ to a lesser extent compared to wild-type plants. Both MJ and ABA promote H 2 O 2 production in wild-type guard cells, while exogenous application of diphenylene iodonium (DPI) chloride, an inhibitor of NAD(P)H oxidases, results in the suppression of ABA-and MJ-induced stomatal closure. ABA elevates H 2 O 2 production in wild-type and jar1-1 guard cells but not in ost1-2, whereas MJ induces H 2 O 2 production in both wild-type and ost1-2 guard cells, but not in jar1-1. MJ-induced stomatal closing is suppressed in the NAD(P)H oxidase double mutant atrbohD/F and in the outward potassium channel mutant gork1. Furthermore, MJ induces alkalization in guard cell cytosol, and MJ-induced stomatal closing is inhibited by butyrate. Analyses of the kinetics of cytosolic pH changes and reactive oxygen species (ROS) production show that the alkalization of cytoplasm precedes ROS production during the stomatal response to both ABA and MJ. Our results further indicate that JAR1, as OST1, functions upstream of ROS produced by NAD(P)H oxidases and that the cytoplasmic alkalization precedes ROS production during MJ or ABA signal transduction in guard cells.

The Arabidopsis (Arabidopsis thaliana) Heavy Metal Associated3 (AtHMA3) protein belongs to the P 1B-2 subgroup of the P-type ATPase family, which is involved in heavy metal transport. In a previous study, we have shown, using heterologous expression in the yeast Saccharomyces cerevisiae, that in the presence of toxic metals, AtHMA3 was able to phenotypically complement the cadmium/lead (Cd/Pb)-hypersensitive strain ycf1 but not the zinc (Zn)-hypersensitive strain zrc1. In this study, we demonstrate that AtHMA3 in planta is located in the vacuolar membrane, with a high expression level in guard cells, hydathodes, vascular tissues, and the root apex. Confocal imaging in the presence of the Zn/Cd fluorescent probe BTC-5N revealed that AtHMA3 participates in the vacuolar storage of Cd. A T-DNA insertional mutant was found more sensitive to Zn and Cd. Conversely, ectopic overexpression of AtHMA3 improved plant tolerance to Cd, cobalt, Pb, and Zn; Cd accumulation increased by about 2-to 3-fold in plants overexpressing AtHMA3 compared with wild-type plants. Thus, AtHMA3 likely plays a role in the detoxification of biological (Zn) and nonbiological (Cd, cobalt, and Pb) heavy metals by participating in their vacuolar sequestration, an original function for a P 1B-2 ATPase in a multicellular eukaryote.

AtHMA4 is an Arabidopsis thaliana P1B-ATPase which transports Zn and Cd. Here, we demonstrate that AtHMA4 is localized at the plasma membrane and expressed in tissues surrounding the root vascular vessels. The ectopic overexpression of AtHMA4 improved the root growth in the presence of toxic concentrations of Zn, Cd and Co. A null mutant exhibited a lower translocation of Zn and Cd from the roots to shoot. In contrast, the AtHMA4 overexpressing lines displayed an increase in the zinc and cadmium shoot content. Altogether, these results strongly indicate that AtHMA4 plays a role in metal loading in the xylem.

Stomatal pores located on the plant epidermis regulate CO(2) uptake for photosynthesis and the loss of water by transpiration. The opening and closing of the pore is mediated by turgor-driven volume changes of two surrounding guard cells. These highly specialized cells integrate internal signals and environmental stimuli to modulate stomatal aperture for plant survival under diverse conditions. Modulation of transcription and mRNA processing play important roles in controlling guard-cell activity, although the details of these levels of regulation remain mostly unknown. Here we report the characterization of AtMYB60, a R2R3-MYB gene of Arabidopsis, as the first transcription factor involved in the regulation of stomatal movements. AtMYB60 is specifically expressed in guard cells, and its expression is negatively modulated during drought. A null mutation in AtMYB60 results in the constitutive reduction of stomatal opening and in decreased wilting under water stress conditions. Transcript levels of a limited number of genes are altered in the mutant, and many of these genes are involved in the plant response to stress. Our data indicate that AtMYB60 is a transcriptional modulator of physiological responses in guard cells and open new possibilities to engineering stomatal activity to help plants survive desiccation.

SummaryBecause plant wilting has been described as a consequence of cadmium (Cd 2þ ) toxicity, we investigate Cd 2þ effects on plant water losses, gas exchanges and stomatal behaviour in Arabidopsis thaliana L. Effects of 1-week Cd 2þ application in hydroponic condition (CdCl 2 10-100 mM) were analyzed. A 10-lM Cd 2þ concentration had no significant effect on the plant-water relationship and carbon assimilation. At higher Cd 2þ concentrations, a Cd 2þ -dependent decrease in leaf conductance and CO 2 uptake was observed despite the photosynthetic apparatus appeared not to be affected as probed by fluorescence measurements. In epidermal strip bioassays, nanomolar Cd 2þ concentrations reduced stomatal opening under light in A. thaliana, Vicia faba and Commelina communis. Application of 5 mM ABA limited the root-to-shoot translocation of cadmium. However, the Cd 2þ -induced stomatal closure was likely ABA-independent, since a 5-day treatment with 50 lM Cd 2þ did not affect the plant relative water content. Additionally, a similar Cd 2þ -induced stomatal closure was observed in the ABA insensitive mutant abi1-1. Interestingly, this mutant displayed a higher transpiration rate than the wild type but did not accumulate more Cd 2þ , arguing that Cd 2þ uptake is not dependent only on the transpiration flow. Application of putative calcium channels inhibitors suppressed the inhibitory effect of Cd 2þ in epidermal strip experiments, suggesting that Cd 2þ could enter the guard cell through calcium channels. Patch-clamp studies with V. faba guard cell protoplasts showed that plasma membrane K þ channels were insensitive to external Cd 2þ application whereas Ca 2þ channels were found permeable to Cd 2þ . In conclusion, we propose that Cd 2þ affects guard cell regulation in an ABA-independent manner by entering the cytosol via Ca 2þ channels.Abbreviations: ABA, Abscissic acid; DMSO, dimethyl sulfoxide; DW, dry weight; FW, fresh weight; NPPB, 5-nitro-2,3-phenylpropylaminobenzoic acid; RWC, relative water content.

Microscopic pores present in the epidermis of plant aerial organs, called stomata, allow gas exchanges between the inner photosynthetic tissue and the atmosphere. Regulation of stomatal aperture, preventing excess transpirational vapor loss, relies on turgor changes of two highly differentiated epidermal cells surrounding the pore, the guard cells. Increased guard cell turgor due to increased solute accumulation results in stomatal opening, whereas decreased guard cell turgor due to decreased solute accumulation results in stomatal closing. Here we provide direct evidence, based on reverse genetics approaches, that the Arabidopsis GORK Shaker gene encodes the major voltage-gated outwardly rectifying K+ channel of the guard cell membrane. Expression of GORK dominant negative mutant polypeptides in transgenic Arabidopsis was found to strongly reduce outwardly rectifying K+ channel activity in the guard cell membrane, and disruption of the GORK gene (T-DNA insertion knockout mutant) fully suppressed this activity. Bioassays on epidermal peels revealed that disruption of GORK activity resulted in impaired stomatal closure in response to darkness or the stress hormone azobenzenearsonate. Transpiration measurements on excised rosettes and intact plants (grown in hydroponic conditions or submitted to water stress) revealed that absence of GORK activity resulted in increased water consumption. The whole set of data indicates that GORK is likely to play a crucial role in adaptation to drought in fluctuating environments