Abscisic acid (ABA) is a well-studied regulator of stomatal movement. Hydrogen sulfide (H 2 S), a small signaling gas molecule involved in key physiological processes in mammals, has been recently reported as a new component of the ABA signaling network in stomatal guard cells. In Arabidopsis (Arabidopsis thaliana), H 2 S is enzymatically produced in the cytosol through the activity of L-cysteine desulfhydrase (DES1). In this work, we used DES1 knockout Arabidopsis mutant plants (des1) to study the participation of DES1 in the cross talk between H 2 S and nitric oxide (NO) in the ABA-dependent signaling network in guard cells. The results show that ABA did not close the stomata in isolated epidermal strips of des1 mutants, an effect that was restored by the application of exogenous H 2 S. Quantitative reverse transcription polymerase chain reaction analysis demonstrated that ABA induces DES1 expression in guard cell-enriched RNA extracts from wild-type Arabidopsis plants. Furthermore, stomata from isolated epidermal strips of Arabidopsis ABA receptor mutant pyrabactin-resistant1 (pyr1)/pyrabactin-like1 (pyl1)/pyl2/pyl4 close in response to exogenous H 2 S, suggesting that this gasotransmitter is acting downstream, although acting independently of the ABA receptor cannot be ruled out with this data. However, the Arabidopsis clade-A PROTEIN PHOSPHATASE2C mutant abscisic acid-insensitive1 (abi1-1) does not close the stomata when epidermal strips were treated with H 2 S, suggesting that H 2 S required a functional ABI1. Further studies to unravel the cross talk between H 2 S and NO indicate that (1) H 2 S promotes NO production, (2) DES1 is required for ABA-dependent NO production, and (3) NO is downstream of H 2 S in ABA-induced stomatal closure. Altogether, data indicate that DES1 is a unique component of ABA signaling in guard cells.Abscisic acid (ABA) regulates diverse physiological and developmental processes in plants, among which seed dormancy and stomatal movement are the most studied. Stomata are pores bordered by pairs of specialized cells named guard cells located in the epidermis of the aerial part of most land plants. Due to the waxy cuticle of plants, stomatal pores regulate approximately 90% of all the gas exchange (i.e. the uptake of CO 2 required for photosynthesis and the loss of water vapor during transpiration) between the plant and the environment (Hetherington and Woodward, 2003). Thus, stomatal movement is a key process for the regulation of plant water status and biomass production. In guard cells, ABA induces an increase of intracellular calcium concentrations, which, in turn, induces an efflux of anions that causes membrane depolarization. In this voltage milieu, ion uptake is blocked through the inactivation of inward-rectifying potassium channels, and ion efflux is induced through activation of outward-rectifying potassium channels. This solute relocation drives water out of the guard cells and closes the stomatal pore as a result of a reduction in guard cell turgor (Blatt, 2000;Kim et al...

Phospholipase C (PLC) is well known for its role in animal signaling, where it generates the second messengers, inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG), by hydrolyzing the minor phospholipid, phosphatidylinositol 4,5-bisphosphate (PIP2), upon receptor stimulation. In plants, PLC's role is still unclear, especially because the primary targets of both second messengers are lacking, i.e. the ligand-gated Ca2+ channel and protein kinase C, and because PIP2 levels are extremely low. Nonetheless, the Arabidopsis genome encodes nine PLCs. We used a reversed-genetic approach to explore PLC's function in Arabidopsis, and report here that PLC3 is required for proper root development, seed germination and stomatal opening. Two independent knock-down mutants, plc3-2 and plc3-3, were found to exhibit reduced lateral root densities by 10-20%. Mutant seeds germinated more slowly but were less sensitive to ABA to prevent germination. Guard cells of plc3 were also compromised in ABA-dependent stomatal closure. Promoter-β-glucuronidase (GUS) analyses confirmed PLC3 expression in guard cells and germinating seeds, and revealed that the majority is expressed in vascular tissue, most probably phloem companion cells, in roots, leaves and flowers. In vivo 32Pi labeling revealed that ABA stimulated the formation of PIP2 in germinating seeds and guard cell-enriched leaf peels, which was significantly reduced in plc3 mutants. Overexpression of PLC3 had no effect on root system architecture or seed germination, but increased the plant's tolerance to drought. Our results provide genetic evidence for PLC's involvement in plant development and ABA signaling, and confirm earlier observations that overexpression increases drought tolerance. Potential molecular mechanisms for the above observations are discussed.

Hydrogen sulfide (HS) is an important gaseous signaling molecule in plants that participates in stress responses and development. l-Cys desulfhydrase 1, one of the enzymatic sources of HS in plants, participates in abscisic acid-induced stomatal closure. We combined pharmacological and genetic approaches to elucidate the involvement of HS in stomatal closure and the interplay between HS and other second messengers of the guard cell signaling network, such as hydrogen peroxide (HO) and phospholipase D (PLD)-derived phosphatidic acid in Arabidopsis (). Both NADPH oxidase isoforms, respiratory burst oxidase homolog (RBOH)D and RBOHF, were required for HS-induced stomatal closure. In vivo imaging using the cytosolic ratiometric fluorescent biosensor roGFP2-Orp1 revealed that HS stimulates HO production in Arabidopsis guard cells. Additionally, we observed an interplay between HS and PLD activity in the regulation of reactive oxygen species production and stomatal movement. The PLDα1 and PLDδ isoforms were required for HS-induced stomatal closure, and most of the HS-dependent HO production required the activity of PLDα1. Finally, we showed that HS induced increases in the PLDδ-derived phosphatidic acid levels in guard cells. Our results revealed the involvement of HS in the signaling network that controls stomatal closure, and suggest that HS regulates NADPH oxidase and PLD activity in guard cells.