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Plants integrate signals in the form of light, humidity, temperature, CO2 concentrations, and daily circadian rhythms. In addition, plants encounter pathogens, pests, herbivores, and other stressors. Physiological processes like responding to stimuli, plant growth, and development are usually governed and coordinated by hormones. Because of the highly coordinated and complex nature of hormone signaling networks, different hormones cooperate in a synergistic matter or antagonize each other’s functions. For example, two hormones that are known to be antagonistic are brassinosteroids (BRs) and abscisic acid (ABA) (Zhang et al., 2009).
Although their respective signal transduction pathways have been elucidated from hormone perception to transcriptional regulation, the nature of the BR-ABA interaction during the response to environmental stimuli (e.g., stomatal closure) remains unclear. In their study, Kim et al. (2018) demonstrate that, in contrast to their roles in growth and development, BR and ABA regulate stomatal closure in a synergistic fashion. This synergism is mediated through the newly discovered interaction between the BR-associated CDG1-LIKE1 (CDL1) and ABA-associated OPEN STOMATA1 (OST1) (see figure).
The CDL1-OST1 interaction provides the basis for BR-ABA synergism in stomatal closure. Yeast-two-hybrid (A) and in vitro pull-down (B) assays show interaction between the BR-associated CDL1 and ABA-associated OST1. Because the authors show that CDL1 is preferentially expressed in the leaf guard cells, this protein-protein interaction and eventual transphosphorylation mechanism indicate that BR and ABA cooperate in a synergistic manner in modulating the stomatal closure response. (Adapted from Kim et al. [2018], Figures 4A and 4B.)
An initial clue was found using a GUS reporter line, which exhibited high and preferential CDL1 expression in stomatal guard cells. Consequently, the authors hypothesized that CDL1 could play a role in stomatal responses. Because ABA is known to induce stomatal closure (Munemasa et al., 2015), they tested whether the cdl1 mutant is sensitive to ABA in this manner. Not only was this mutant ABA-insensitive in terms of its stomatal behavior, it also exhibited perturbed ABA-related responses, like reduced reactive oxygen species levels, higher water loss, and decreased leaf temperature. Like ABA, the active form of BR called BL (brassinolide) also regulates stomatal closure (Ha et al., 2016). Similarly, the cdl1 mutant was insensitive to BL. Complementation of the cdl1 mutant with a wild-type allele of CDL1 recovered both ABA and BL sensitivity, indicating that CDL1 is involved in the stomatal response governed by these two hormones. Surprisingly, the function of CDL1 in stomatal closure is independent of BR biosynthesis and downstream signaling components.
ABA-induced stomatal closure depends on OST1 (Mustilli et al., 2002). What Kim et al. (2018) found is that the ost1 mutant is also insensitive to BR-mediated stomatal closure, phenocopying the cdl1 mutant. With these solid phenotypic characterizations on hand, they investigated a possible mechanism by which both CDL1 and OST1 mediate the stomatal response. Using yeast-two-hybrid, in vitro pull-down, and coimmunoprecipitation approaches, they showed that CDL1 interacts with and phosphorylates OST1 (see figure). This interaction is enhanced by BR and ABA treatments.
Exploring further into how CDL1 phosphorylates OST1, possible Ser residues in OST1 were mutated. Based on previous structural studies of the protein, Ser-166, Ser-171, and Ser-175 were mutated, but these did not alter OST1 phosphorylation by CDL1. Instead, the Ser-7 site of OST1 was determined to be the phosphorylation site of CDL1. The OST1 Ser-7 mutant additionally had reduced phosphorylation and exhibited decreased ABA and BR sensitivity, reminiscent of the ost1 and cdl1 mutants. In vitro kinase assays demonstrated that OST1 and CDL1 phosphorylate each other, with ABA/BR treatments enhancing this transphosphorylation mechanism. Overall, the results of this study indicate that the synergistic action of ABA and BR in the guard cells is mediated by the interaction of CDL1 and OST1, reflecting a new model of hormone crosstalk in stomatal movement. This further expands our understanding that hormone signaling is dynamically regulated in a spatio-temporal manner.
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