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.