E3 ligase AtAIRP5/GARU regulates drought stress response by stimulating SERINE CARBOXYPEPTIDASE-LIKE1 turnover.

Ubiquitination is a major mechanism of eukaryotic posttranslational protein turnover that has been implicated in abscisic acid (ABA)-mediated drought stress response. Here, we isolated T-DNA insertion mutant lines in which ABA-insensitive RING protein 5 (AtAIRP5) was suppressed, resulting in hyposensitive ABA-mediated germination compared to wild-type Arabidopsis (Arabidopsis thaliana) plants. A homology search revealed that AtAIRP5 is identical to gibberellin (GA) receptor RING E3 ubiquitin (Ub) ligase (GARU), which downregulates GA signaling by degrading the GA receptor GID1, and thus AtAIRP5 was renamed AtAIRP5/GARU. The atairp5/garu knockout progeny were impaired in ABA-dependent stomatal closure and were markedly more susceptible to drought stress than wild-type plants, indicating a positive role for AtAIRP5/GARU in the ABA-mediated drought stress response. Yeast two-hybrid, pull-down, target ubiquitination, and in vitro and in planta degradation assays identified serine carboxypeptidase-like1 (AtSCPL1), which belongs to the clade 1A AtSCPL family, as a ubiquitinated target protein of AtAIRP5/GARU. atscpl1 single and atairp5/garu-1 atscpl1-2 double mutant plants were more tolerant to drought stress than wild-type plants in an ABA-dependent manner, suggesting that AtSCPL1 is genetically downstream of AtAIRP5/GARU. After drought treatment, the endogenous ABA levels in atscpl1 and atairp5/garu-1 atscpl1-2 mutant leaves were higher than those in wild-type and atairp5/garu leaves. Overall, our results suggest that AtAIRP5/GARU RING E3 Ub ligase functions as a positive regulator of the ABA-mediated drought response by promoting the degradation of AtSCPL1.

OsPUB41, a U-box E3 ubiquitin ligase, acts as a negative regulator of drought stress response in rice (Oryza Sativa L.).

KEY MESSAGE: OsPUB41 plays a negative role in drought stress response through the mediation of OsUBC25 and interacts with OsCLC6, suggesting a putative substrate. The notable expansion of Plant U-Box E3 ligases (PUB), compared with those in mammals, implies that PUB proteins have evolved to perform plant-specific functions. OsPUB41, a potential ortholog of CMPG1, was recently reported to regulate the cell wall degrading enzyme (CWDE)-induced innate immune response in rice. Here, we characterized the OsPUB41 gene, which encodes a dual-localized cytosolic and nuclear U-box E3 ligase in rice. OsPUB41 expression was specifically induced by dehydration among various abiotic stresses and abscisic acid (ABA) treatments. Furthermore, we revealed that the core U-box motif of OsPUB41 possesses the E3 ligase activity that can be activated by OsUBC25 in rice. The Ubi:RNAi-OsPUB41 knock-down and ospub41 suppression mutant plants exhibited enhanced tolerance to drought stress compared with the wild-type rice plants in terms of transpirational water loss, long-term dehydration response, and chlorophyll content. Moreover, the knock-down or suppression of the OsPUB41 gene did not cause adverse effect on rice yield-related traits. Yeast two-hybrid and an in vitro pull-down analyses revealed that OsCLC6, a chloride channel, is a putative substrate of OsPUB41. Overall, these results suggest that OsPUB41 acts as a negative regulator of dehydration conditions and interacts with OsCLC6, implying that it is a substrate of OsPUB41.

Suppression of DRR1 results in the accumulation of insoluble ubiquitinated proteins, which impairs drought stress tolerance.

Drought stress has detrimental effects on plants. Although the abscisic acid (ABA)-mediated drought response is well established, defensive mechanisms to cope with dehydration-induced proteotoxicity have been rarely studied. DRR1 was identified as an Arabidopsis drought-induced gene encoding an ER-localized RING-type E3 Ub ligase. Suppression of DRR1 markedly reduced tolerance to drought and proteotoxic stress without altering ABA-mediated germination and stomatal movement. Proteotoxicity- and dehydration-induced insoluble ubiquitinated protein accumulation was more obvious in DRR1 loss-of-function plants than in wild-type plants. These results suggest that DRR1 is involved in an ABA-independent drought stress response possibly through the mitigation of dehydration-induced proteotoxic stress.

AtKPNB1, an Arabidopsis importin-ß protein, is downstream of the RING E3 ubiquitin ligase AtAIRP1 in the ABA-mediated drought stress response.

MAIN CONCLUSION: AtKPNB1, an Arabidopsis importin-ß protein, was regulated by AtAIRP1 E3 ubiquitin ligase, which intensified the ABA-mediated drought stress response. As an early step in the abscisic acid (ABA)-mediated drought response, the ABA signal is transduced into the nucleus, and thus the nuclear transport system is crucially involved in the drought stress response. AtKPNB1, an importin-ß protein, which is a core component of nuclear transport, was previously reported to be a negative factor in the ABA-mediated drought stress response (Luo et al. Luo et al., Plant J 75:377-389, 2013). Here, we report that AtAIPR1, an Arabidopsis RING-type E3 ubiquitin (Ub) ligase, interacted with and ubiquitinated AtKPNB1. A null mutation of AtKPNB1 suppressed the ABA-insensitive germination phenotype of atairp1 mutant seedlings as compared to that of the wild-type plants. Furthermore, the ABA-insensitive stomatal closure and drought-susceptible phenotypes of atairp1 were rescued in atairp1atkpnb1 double mutant progeny, indicating that AtKPNB1 functions downstream of AtAIRP1. These data suggest that AtAIRP1 regulates the ABA-mediated drought response in Arabidopsis via ubiquitination of AtKPNB1.

Arabidopsis RING E3 ubiquitin ligase JUL1 participates in ABA-mediated microtubule depolymerization, stomatal closure, and tolerance response to drought stress.

Ubiquitination is a critical post-translational protein modification that has been implicated in diverse cellular processes, including abiotic stress responses, in plants. In the present study, we identified and characterized a T-DNA insertion mutant in the At5g10650 locus. Compared to wild-type Arabidopsis plants, at5g10650 progeny were hyposensitive to ABA at the germination stage. At5g10650 possessed a single C-terminal C3HC4-type Really Interesting New Gene (RING) motif, which was essential for ABA-mediated germination and E3 ligase activity in vitro. At5g10650 was closely associated with microtubules and microtubule-associated proteins in Arabidopsis and tobacco leaf cells. Localization of At5g10650 to the nucleus was frequently observed. Unexpectedly, At5g10650 was identified as JAV1-ASSOCIATED UBIQUITIN LIGASE1 (JUL1), which was recently reported to participate in the jasmonate signaling pathway. The jul1 knockout plants exhibited impaired ABA-promoted stomatal closure. In addition, stomatal closure could not be induced by hydrogen peroxide and calcium in jul1 plants. jul1 guard cells accumulated wild-type levels of H2 O2 after ABA treatment. These findings indicated that JUL1 acts downstream of H2 O2 and calcium in the ABA-mediated stomatal closure pathway. Typical radial arrays of microtubules were maintained in jul1 guard cells after exposure to ABA, H2 O2 , and calcium, which in turn resulted in ABA-hyposensitive stomatal movements. Finally, jul1 plants were markedly more susceptible to drought stress than wild-type plants. Overall, our results suggest that the Arabidopsis RING E3 ligase JUL1 plays a critical role in ABA-mediated microtubule disorganization, stomatal closure, and tolerance to drought stress.