RBR-Type E3 Ligases and the Ubiquitin-Conjugating Enzyme UBC26 Regulate Abscisic Acid Receptor Levels and Signaling

Abstract

The turnover of abscisic acid (ABA) signaling core components modulates the plant's response to ABA and is regulated by ubiquitination. We show that Arabidopsis (Arabidopsis thaliana) RING finger ABA-related 1 (RFA1) and RFA4 E3 ubiquitin ligases, members of the RING between RING fingers (RBR)type RSL1/RFA family, are key regulators of ABA receptor stability in root and leaf tissues, targeting ABA receptors for degradation in different subcellular locations. RFA1 is localized both in the nucleus and cytosol, whereas RFA4 shows specific nuclear localization and promotes nuclear degradation of ABA receptors. Therefore, members of the RSL1/RFA family interact with ABA receptors at plasma membrane, cytosol and nucleus, targeting them for degradation via the endosomal/vacuolar RSL1-dependent pathway or 26S proteasome. Additionally, we provide insight into the physiological function of the relatively unexplored plant RBR-type E3 ligases, and through mutagenesis and biochemical assays we identified Cys361 in RFA4 as the putative active site cysteine, which is a distinctive feature of RBR-type E3 ligases. Endogenous levels of PYR1 and PYL4 ABA receptors were higher in the rfa1 rfa4 double mutant than in wild-type plants. UBC26 was identified as the cognate nuclear E2 enzyme that interacts with the RFA4 E3 ligase and forms UBC26-RFA4-Receptor complexes in nuclear speckles. Loss-of-function ubc26 alleles and the rfa1 rfa4 double mutant showed enhanced sensitivity to ABA and accumulation of ABA receptors compared to the wild type. Together our results reveal a sophisticated mechanism by which ABA receptors are targeted by ubiquitin (Ub) at different subcellular locations, in which the complexity of the ABA receptor family is mirrored in the partner RBR-type E3 ligases.

PYL8 ABA receptors of Phoenix dactylifera play a crucial role in response to abiotic stress and are stabilized by ABA (118)

“…Transcriptional and posttranslational mechanisms limit accumulation of ABA receptors in Arabidopsis (Fernandez et al, 2020); but transcriptional downregulation either by ABA or different stressors was not observed in palm receptors. Moreover, ABA treatment in Arabidopsis transgenic lines led to higher accumulation of palm receptors.…”

“…Protein extracts for immunodetection experiments were prepared from Arabidopsis transgenic lines expressing HA-tagged or GFP-tagged ABA receptors or GFP-tagged PP2C/ABA receptors transiently expressed in Nicotiana benthamiana as described by Fernandez et al, (2020).…”

Update on Receptors and Signaling

“…The classical topics in hormone signaling, such as for auxin and abscisic acid, continue to be areas of active research. Fernandez et al (2020) report additional complexity in abscisic acid signaling mediated by regulated proteolysis in different subcellular compartments. Ramos Baéz et al (2020) demonstrate the use of synthetic and heterologous systems to dissect the molecular mechanisms of auxin signaling in maize (Zea mays).…”

Plant Mitophagy in Comparison to Mammals: What Is Still Missing?

“…Instead, the Arabidopsis ARI family, a putative RBR E3-ligase family, has been suggested to play an essential role under stress conditions, and actively participates in several stress-related hormone signaling pathways via the ubiquitin proteasome system [ 50 ]. Recently, an RBR E3-ligase ubiquitin conjugating enzyme 26 (UBC26) has been reported to participate in the phytohormone abscisic acid (ABA) response in Arabidopsis, likely by promoting the degradation of ABA receptors in response to abiotic stress [ 51 ]. However, direct evidence for linking RBR E3-ligases in plant mitophagy is still missing.…”