Ubiquitin Ligases RGLG1 and RGLG5 Regulate Abscisic Acid Signaling by Controlling the Turnover of Phosphatase PP2CA

Abstract

Abscisic acid (ABA) is an essential hormone for plant development and stress responses. ABA signaling is suppressed by clade A PP2C phosphatases, which function as key repressors of this pathway through inhibiting ABA-activated SnRK2s (SNF1-related protein kinases). Upon ABA perception, the PYR/PYL/RCAR ABA receptors bind to PP2Cs with high affinity and biochemically inhibit their activity. While this mechanism has been extensively studied, how PP2Cs are regulated at the protein level is only starting to be explored. RING DOMAIN LIGASE5 (RGLG5) belongs to a five-member E3 ubiquitin ligase family whose target proteins remain unknown. We report that RGLG5, together with RGLG1, releases the PP2C blockade of ABA signaling by mediating PP2CA protein degradation. ABA promotes the interaction of PP2CA with both E3 ligases, which mediate ubiquitination of PP2CA and are required for ABA-dependent PP2CA turnover. Downregulation of and stabilizes endogenous PP2CA and diminishes ABA-mediated responses. Moreover, the reduced response to ABA in germination assays is suppressed in the (artificial microRNA) triple mutant, supporting a functional link among these loci. Overall, our data indicate that RGLG1 and RGLG5 are important modulators of ABA signaling, and they unveil a mechanism for activation of the ABA pathway by controlling PP2C half-life.

ABA inhibits myristoylation and induces shuttling of the RGLG1 E3 ligase to promote nuclear degradation of PP2CA

“…In order to further investigate the subcellular localization of RGLG1, we generated RGLG1‐GFP and the RGLG1 G2A ‐GFP variant that contains a mutation in the predicted N‐terminal myristoylation site and delivered them into leaf cells of Nicotiana benthamiana by agroinfiltration (Figure a). RGLG1‐GFP was shown to co‐immunoprecipitate with PP2CA when co‐expressed in N. benthamiana (Wu et al ., ), and we provide additional evidence that it is a functional protein in Arabidopsis (Figure S3). Expression of the corresponding fusion proteins was verified by immunoblot analysis using anti‐GFP antibodies (Figure b).…”

“…ABA receptors and ABA are strictly required (in the case of PYR1) or enhance (monomeric receptors) PUB13‐mediated ABI1 ubiquitination (Kong et al ., ). ABA receptors were not required for the in vitro ubiquitination of PP2CA by RGLG1/5; however, under in vivo conditions ABA enhanced the interaction of RGLG1/5 with PP2CA through an unknown mechanism (Wu et al ., ).…”

ABRE‐BINDING FACTORS play a role in the feedback regulation of ABA signaling by mediating rapid ABA induction of ABA co‐receptor genes

“…These observations may be explained by two possibilities: ABI1 and ABI2 are predominantly expressed in roots, or GUS substrate takes a longer time to enter into cotyledons than into roots. Nevertheless, a recent study has also reported that ABA markedly induces PP2CA/AHG3 promoter activity in roots and leaf tissues (Wu et al ., ), suggesting that ABA‐induced expression of group A PP2C genes in roots may be a common feature. These observations have prompted us to speculate that ABA‐induced expression of group A PP2C genes may serve as a desensitization mechanism acting in the roots, thus allowing roots to continue to grow under water stress conditions.…”

Functional analysis of the pepper protein phosphatase, CaAIPP1, and its interacting partner CaAIRF1: Modulation of ABA signalling and the drought stress response

“…The U‐box E3 ligases PUB12 and PUB13 were reported to be involved in degradation of the group A PP2C ABI1 with interacting partners PYR1 and PYL8 only in the presence of ABA (Kong et al, ). Moreover, the E3 ubiquitin ligases RGLG5 and RGLG1 mediate PP2CA ubiquitination and are required for PP2CA turnover in an ABA‐dependent manner (Wu et al, ). In this study, we have demonstrated that the pepper RING‐type E3 ligase CaAIRF1 is involved in ubiquitination and degradation of CaAIPP1, which is homologous to PP2CA.…”