PYR/PYL/RCAR family members are majorin‐vivoABI1 protein phosphatase 2C‐interacting proteins in Arabidopsis

Abstract

Abscisic acid (ABA) mediates resistance to abiotic stress and controls developmental processes in plants. The group-A PP2Cs, of which ABI1 is the prototypical member, are protein phosphatases that play critical roles as negative regulators very early in ABA signal transduction. Because redundancy is thought to limit the genetic dissection of early ABA signalling, to identify redundant and early ABA signalling proteins, we pursued a proteomics approach. We generated YFP-tagged ABI1 Arabidopsis expression lines and identified in vivo ABI1-interacting proteins by mass-spectrometric analyses of ABI1 complexes. Known ABA signalling components were isolated including SnRK2 protein kinases. We confirm previous studies in yeast and now show that ABI1 interacts with the ABA-signalling kinases OST1, SnRK2.2 and SnRK2.3 in plants. Interestingly, the most robust in planta ABI1-interacting proteins in all LC-MS/MS experiments were nine of the 14 PYR/PYL/RCAR proteins, which were recently reported as ABA-binding signal transduction proteins, providing evidence for in vivo PYR/PYL/RCAR interactions with ABI1 in Arabidopsis. ABI1–PYR1 interaction was stimulated within 5 min of ABA treatment in Arabidopsis. Interestingly, in contrast, PYR1 and SnRK2.3 co-immunoprecipitated equally well in the presence and absence of ABA. To investigate the biological relevance of the PYR/PYLs, we analysed pyr1/pyl1/pyl2/pyl4 quadruple mutant plants and found strong insensitivities in ABA-induced stomatal closure and ABA-inhibition of stomatal opening. These findings demonstrate that ABI1 can interact with several PYR/PYL/RCAR family members in Arabidopsis, that PYR1–ABI1 interaction is rapidly stimulated by ABA in Arabidopsis and indicate new SnRK2 kinase-PYR/PYL/RCAR interactions in an emerging model for PYR/PYL/RCAR-mediated ABA signalling.

Evolutionarily Conserved Regulatory Mechanisms of Abscisic Acid Signaling in Land Plants: Characterization of ABSCISIC ACID INSENSITIVE1-Like Type 2C Protein Phosphatase in the Liverwort Marchantia polymorpha

“…A model presented in recent reports highlighted the role of the ABA receptor(s) in direct regulation of group A PP2Cs (Ma et al, 2009;Park et al, 2009). In this model, the receptor binds and inhibits ABI1 and ABI2 in the presence of ABA, which leads to activation of SnRKs and downstream signaling molecules (Park et al, 2009;Nishimura et al, 2010). Whether or not similar ABA receptor molecule(s) bind to PP2Cs in mosses or liverworts is not clear.…”

The unique mode of action of a divergent member of the ABA-receptor protein family in ABA and stress signaling

“…Although previous studies suggested that many are ABA receptors and have redundancy [2,31], they also showed functional differences [6,27]. In the presence of ABA, PYR1 and PYLs 1-12 reduced the activity of ABI1 and activated SnRK2.6 to induce RD29B-LUC expression in Arabidopsis mesophyll protoplasts [4].…”

“…Although the PYL family proteins have been studied intensively in recent years [6,[28][29][30][31], little information is available for PYL13, which differs from other PYLs in the highly conserved regions of their ABA-binding pocket. To study the function of PYL13, we examined the interaction of PYL13 with clade A PP2Cs and the effects of PYL13 on the PP2Cs both in vitro and in vivo.…”

Structural insights into PYR/PYL/RCAR ABA receptors and PP2Cs

“…Finally, a chemical genetic approach using a synthetic selective ABA agonist, pyrabactin, made it possible to identify a family of soluble ABA receptors, named PYR/PYL/RCAR for PYRABACTIN RESISTANCE1/PYR1-LIKE/ REGULATORY COMPONENTS OF ABA RECEPTORS, respectively [8,9]. In a convergent approach, yeast-two hybrid screenings [9,10] and in planta interaction studies [11] identified these receptors through their capacity to bind to phosphatases type-2C (PP2Cs), which are key negative regulatory components of the pathway involved in the dephosphorylation of certain sucrose non-fermenting 1-related subfamily 2 (SnRK2) kinases [12,13,14].…”