Abstract
Abscisic acid (ABA) plays a vital role in responses to abiotic stresses that allow plants to cope with environmental challenges. In this study, we analyzed ABA receptors of subfamily III as the potential targets of Cytosolic ABA Receptor Kinase 1 (CARK1). We previously found that CARK1 phosphorylated the subfamily III member RCAR11 at a distinct threonine residue (T78). Our study now shows the physical interaction of CARK1 with the receptors RCAR12/13/14 in vitro and in vivo. The catalytically inactive form CARK1-N204A did not interact with the receptors. Phosphorylation of these ABA receptors in vitro occurred at a serine/threonine amino acid residue corresponding to T78 in RCAR11, which is located in the loop of β3 within a conserved site. Further analysis revealed that the phosphorylation of RCAR11T78 could increase the sensitivity of the pyr1pyl1pyl2pyl4 quadruple mutant (1124) to ABA, including the inhibition of root elongation and increasing drought tolerance. The analysis of CARK1:1124 complementation and the expression of ABA-related genes indicated that CARK1 could rescue the insensitivity of 1124 to ABA. Our results indicate that CARK1 tends to phosphorylate subfamily III ABA receptors, and the phosphosites RCAR11T78, RCAR12T105, RCAR13T101, and RCAR14S81 are the major sites involved in the activation of the ABA response pathway.
Highlights
The phytohormone abscisic acid (ABA) plays an important role in plant growth and developmental processes as well as in plant responses to environmental stresses, including salinity and drought (Finkelstein et al, 2002; Yoshida et al, 2002; Cutler et al, 2010; Fujita et al, 2011)
A 0.1 μg aliquot of both input proteins Cytosolic ABA Receptor Kinase 1 (CARK1)-KD and CARK1-KDN204A was used. (B) Bimolecular fluorescence complementation (BiFC) assay validates the interactions of RCAR12, RCAR13, and RCAR14 with CARK1, but not with mutant CARK1N204A, in the leaves of N. benthamiana. nYFP was fused to the full-length CARK1 and mutant CARK1N204A to form CARK1–nYFP and CARK1N204A–nYFP, and cYFP was fused to RCAR12, RCAR13, and RCAR14 to form cYFP
Unlike the monomeric state of Abscisic acid (ABA) receptor subfamilies I and II (RCAR1–RCAR10), the members of subfamily III are dimeric receptors, except for RCAR13 that exists in a state of monomer–dimer shift (Nishimura et al, 2009; Dupeux et al, 2011; Hao et al, 2011).The dimeric receptors inhibit the activity of phosphatase 2C (PP2C) in an ABA-dependent manner and possess less sensitivity to ABA than do the monomeric receptors (Ma et al, 2009;Tischer et al, 2017)
Summary
The phytohormone abscisic acid (ABA) plays an important role in plant growth and developmental processes as well as in plant responses to environmental stresses, including salinity and drought (Finkelstein et al, 2002; Yoshida et al, 2002; Cutler et al, 2010; Fujita et al, 2011). When binding to ABA, pyrabactin resistance 1 (PYR1)/PYR1-like (PYL)/regulatory components of ABA receptors (RCAR) (hereafter referred to as RCARs) interact with and inhibit clade A protein phosphatase 2C (PP2C). The quadruple mutant pyr1pyl1pyl2pyl (abbreviated as 1124) which is deficient in the three dimeric receptors (RCAR11, RCAR12, and RCAR14) and a monomeric receptor (RCAR10) shows a strong ABA insensitivity, including ABA-mediated inhibition of germination, root growth, and ABA-induced stomatal closure, providing evidence for the importance of the dimeric receptors in Arabidopsis (Park et al, 2009; Nishimura et al, 2010; Gonzalez-Guzman et al, 2012). The quadruple mutant still responds to ABA to some extent, indicating that all of the ABA receptors function in ABA signaling (Gonzalez-Guzman et al, 2012)
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