ESCRT-I Component VPS23A Affects ABA Signaling by Recognizing ABA Receptors for Endosomal Degradation

Recent discovery of PYR/PYL/RCAR-type abscisic acid （ABA） receptors has become one of most significant advances in plant science in the past decade. In mammals, endosomal sorting acts as an important pathway to downregulate different types of receptors, but its role in plant hormone signaling is poorly understood. Here, we report that an ubiquitin E2-1ike protein, VPS23A, which is a key component of ESCRT-I, negatively regulates ABA signaling. VPS23A has epistatic relationship with PYR/PYL/RCAR-type ABA receptors and disruption of VPS23A enhanced the activity of key kinase OST1 in the ABA signaling pathway under ABA treatment. Moreover, VPS23A interacts with PYR1/PYLs and K63-1inked diubiquitin, and PYL4 possesses K63-1inked ubiquitinated modification in vivo. Further analysis revealed that VPS23A affects the subcellular localization of PYR 1 and the stability of PYL4. Taken together, our results suggest that VPS23A affects PYR1/ PYL4 via vacuole-mediated degradation, providing an advanced understanding of both the turnover of ABA receptors and ESCRTs in plant hormone signaling.

ABA Signaling in Guard Cells Entails a Dynamic Protein-Protein Interaction Relay from the PYL-RCAR Family Receptors to Ion Channels

Plant hormone abscisic acid （ABA） serves as an integrator of environmental stresses such as drought to trig-ger stomatal closure by regulating specific ion channels in guard cells. We previously reported that SLAC1, an outward anion channel required for stomatal closure, was regulated via reversible protein phosphorylation events involving ABA signaling components, including protein phosphatase 2C members and a SnRK2-type kinase （OST1）. In this study, we reconstituted the ABA signaling pathway as a protein-protein interaction relay from the PYL/RCAR-type receptors, to the PP2C-SnRK2 phosphatase-kinase pairs, to the ion channel SLAC1. The ABA receptors interacted with and inhibited PP2C phosphatase activity against the SnRK2-type kinase, releasing active SnRK2 kinase to phosphorylate, and activate the SLAC1 channel, leading to reduced guard cell turgor and stomatal closure. Both yeast two-hybrid and bimolecular fluorescence complementation assays were used to verify the interactions among the components in the pathway. These biochemical assays demonstrated activity modifications of phosphatases and kinases by their interaction partners. The SLAC1 channel activity was used as an endpoint readout for the strength of the signaling pathway, depending on the presence of different combinations of signaling components. Further study using transgenic plants overexpressing one of the ABA receptors demonstrated that changing the relative level of interacting partners would change ABA sensitivity.

Abscisic acid receptors maintain abscisic acid homeostasis by modulating UGT71C5 glycosylation activity

Uridine diphosphate-glucosyltransferases(UGTs)maintain abscisic acid(ABA)homeostasis in Arabidopsis thaliana by converting ABA to abscisic acid-glucose ester(ABA-GE).UGT71C5 plays an important role in the generation of ABA-GE.Abscisic acid receptors are crucial upstream components of the ABA signaling pathway,but how UGTs and ABA receptors function together to modulate ABA levels is unknown.Here,we demonstrated that the ABA receptors RCAR12/13 and UGT71 C5 maintain ABA homeostasis in Arabidopsis following rehydration under drought stress.Biochemical analyses show that UGT71C5 directly interacted with RCAR8/12/13 in yeast cells,and the interactions between UGT-71C5 and RCAR12/13 were enhanced by ABA treatment.Enzyme activity analysis showed that ABA-GE contents were significantly elevated in the presence of RCAR12 or RCAR13,suggesting that these ABA receptors enhance the activity of UGT71C5.Determination of the content of ABA and ABA-GE in Arabidopsis following rehydration under drought stress revealed that ABA-GE contents were significantly higher in Arabidopsis plants overexpressing RCAR12 and RCAR13 than in nontransformed plants and plants overexpressing RCAR11 following rehydration under drought stress.These observations suggest that RCAR12 and RCAR13 enhance the activity of UGT71C5 to glycosylate excess ABA into ABA-GE following rehydration under drought stress,representing a rapid mechanism for regulating plant growth and development.

LJbiquitin--Proteasome System in ABA Signaling： From Perception to Action

Protein post-translational modification （PTM） by ubiquitination has been observed during many aspects of plant growth, development, and stress responses. The ubiquitin-proteasome system precisely regulates phytohormone signaling by affecting protein activity, localization, assembly, and interaction ability. Absci- sic acid （ABA） is a major phytohormone, and plays important roles in plants under normal or stressed growth conditions. The ABA signaling pathway is composed of phosphatases, kinases, transcription fac- tors, and membrane ion channels. It has been reported that multiple ABA signaling transducers are sub- jected to the regulations by ubiquitination. In particular, recent studies have identified different types of E3 ligases that mediate ubiquitination of ABA receptors in different cell compartments. This review focuses on modulation of these components by monoubiquitination or polyubiquitination that occurs in the plasma membrane, endomembranes, and from the cytosol to the nucleus; this implies the existence of retrograde and trafficking processes that are regulated by ubiquitination in ABA signaling. A number of single-unit E3 ligases, components of multi-subunit E3 ligases, E2s, and specific subunits of the 26S proteasome involved in ABA signal regulation are discussed. Dissecting the precise functions of ubiquitination in the ABA pathway may help us understand key factors in the signaling of other phytohormones regulated by ubiqui- tination and other types of PTMs.

Vlodulation of ABA Signaling by Altering VxGФL Vlotif of PP2Cs in Oryza sativa

The abscisic acid （ABA） signaling pathway is regulated by clade A type 2C protein phosphatases （PP2CAs） in plants. In the presence of ABA, PP2Cs release stress/ABA-activated protein kinases by binding to ABA-bound receptors （PYL/RCARs） for activation. Although the wedging tryptophan in PP2Cs is critical in the interaction with PYL/RCARs in Arabidopsis and rice, it remains elusive as to how other interface regions are involved in the interaction. Here, we report the identification of a conserved region on PP2Cs, termed the VxG4ФL motif, which modulates the interaction with PYL/RCARs through its second and fourth residues. The effects of the second and fourth residues on the interaction of OsPP2CS0 with several OsPYIJRCAR proteins were investigated by systematic mutagenesis. One OsPP2C50 mutant, VFGML （＂FM＂） mutant, lowered the affinity to OsPYL/RCAR3 by Ф15-fold in comparison with the wUd-type. Compar- ison of the crystal structures of wild-type OsPP2C50：ABA：OsPYI./RCAR3 with those composed of FM mutant revealed local conformational changes near the VxGФL motif, further supported by hydrogen-deute- rium exchange mass spectrometry. In rice protoplasts, ABA signaling was altered by mutations in the VxGФL motif. Transgenic Arabidopsis plants overexpressing OsPP2C50 and OsPP2C5OFM showed altered ABA sensitivity. Taken together, the VxGФL motif of PP2Cs appears to modulate the affinity of PP2Cs with PYL/RCARs and thus likely to alter the ABA signaling, leading to the differential sensitivity to ABA in planta.