Nitric oxide and abscisic acid cross talk in guard cells.

Despite recent efforts to elucidate the regulation of stomatal movement, many components within the branched pathways of guard cell abscisic acid (ABA) signaling remain to be identified. Here, we show results supporting the involvement of nitric oxide (NO) as a new component of this signaling pathway. NO is a short life bioactive molecule first described as a toxic compound, but now recognized to be an important signal and effector molecule both in animal and plant cell physiology. Even though NO research in plants is not as advanced as in animals, in the last decade NO was proved to participate in many key physiological processes such as growth, pathogen defense reaction, development, programmed cell death, and stress tolerance (Foissner et al., 2000; Pedroso et al., 2000; Beligni and Lamattina, 2001a). In plants, as in animals, NO was proved to interact with other signaling elements such as cADPR, lipids, cGMP, ion channels, Ca , and others. In addition, much evidence is appearing lately about cross talk between NO and some plant hormones during adaptive responses to adverse conditions (Hausladen and Stamler, 1998; Durner and Klessig, 1999; Jacob et al., 1999; Beligni and Lamattina, 2001b; Wendehenne et al., 2001). Furthermore, it is now becoming clear that a network constituted at many levels is operating during plant responses to stress stimulus (Knight and Knight, 2001). ABA is one of the most studied phytohormones due to its key participation in different physiological events throughout the whole plant cycle. Under drought stress conditions, ABA accumulates in leaf tissue, generating a net loss of guard cell turgor that led to stomatal closure, thus reducing transpirational water loss. ABA-induced stomatal closure involves a net increase in guard cell cytoplasmic Ca concentrations. Furthermore, cADPR, ryanodine receptors, and phospholipases C and D have been also involved as second messengers in this signaling pathway (MacRobbie, 1998; Jacob et al., 1999; Sanders et al., 1999; Schroeder et al., 2001b). Even though NO was recently reported to induce stomatal closure in Vicia fava epidermal strips, there is no information about how NO induces this response (Garcı́a Mata and Lamattina, 2001). In this work, we provide strong evidence on the existence of an NO-mediated action in ABA-dependent stomatal closure. V. fava epidermal strips were treated with increasing concentrations of ABA in the presence of increasing concentrations of the NO releaser sodium nitroprusside (SNP). As expected, both ABA and SNP induced stomatal closure in a dose-dependent manner. However, at the higher concentration assayed (1 and 10 m ABA; 10 and 100 m SNP), the stomatal closure percentage of the combined treatment was significantly lower than in those experiments done with either ABA or SNP alone (Fig. 1). The synergism observed between ABA and NO may reflect two weak signals in the same pathway that augment each other. Thus, small and rapid changes in both ABA and NO concentrations can determine variations in percentages of stomatal closure and probably explain the spatial and temporal heterogeneity in stomatal behavior, as has been already described (Mott and Buckley, 2000). Although this assay presented an evidence of a putative ABA-NO interaction, to test if endogenous NO also takes part of the ABA signaling pathway, we tested the effect of the specific NO scavenger 2-(4carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl3-oxide (c-PTIO) on the ABA-induced stomatal closure. Figure 2A shows that in the presence of c-PTIO, the percentage of open stomata remained constant through all the tested ABA concentrations, showing that the guard cells were not responding to ABA treatment. The difference in the pore width between ABA treatment in the presence or absence of c-PTIO can be clearly observed under an optical microscope (Fig. 2B). In addition, when 200 m SNP was added after the ABA c-PTIO treatment, the stomatal closure was induced again, showing that the c-PTIO-mediated inhibition of ABA-induced stomatal closure is reversible (Fig. 2A, inset). All together, these data suggest that NO might be acting downstream of the ABA-induced signaling cascade. Given the cross talk between ABA and NO, we wanted to know the variations of endogenous NO during the ABA-induced stomatal closure. With that aim, epidermal strips were loaded with the permeable NO sensitive fluorophore 4,5-diaminofluorescein diacetate (DAF-2 DA), which allows the 1 This work was supported by Agencia Nacional de Promoción Cientı́fica y Tecnológica (grant no. PICT 1– 6496 –99 to L.L.), by Consejo Nacional de Investigaciones Cientı́ficas y Técnicas (grant no. PIP 0898/98 to L.L.), and by institutional grants from Universidad Nacional de Mar del Plata (Argentina). * Corresponding author; e-mail [email protected]; fax 54 – 223– 475–3150. www.plantphysiol.org/cgi/doi/10.1104/pp.011020.