Ubiquitination is involved in a variety of biological processes, but the exact role of ubiquitination in abiotic responses is not clearly understood in higher plants. Here, we investigated Rma1H1, a hot pepper (Capsicum annuum) homolog of a human RING membrane-anchor 1 E3 ubiquitin (Ub) ligase. Bacterially expressed Rma1H1 displayed E3 Ub ligase activity in vitro. Rma1H1 was rapidly induced by various abiotic stresses, including dehydration, and its overexpression in transgenic Arabidopsis thaliana plants conferred strongly enhanced tolerance to drought stress. Colocalization experiments with marker proteins revealed that Rma1H1 resides in the endoplasmic reticulum (ER) membrane. Overexpression of Rma1H1 in Arabidopsis inhibited trafficking of an aquaporin isoform PIP2;1 from the ER to the plasma membrane and reduced PIP2;1 levels in protoplasts and transgenic plants. This Rma1H1-induced reduction of PIP2;1 was inhibited by MG132, an inhibitor of the 26S proteasome. Furthermore, Rma1H1 interacted with PIP2;1 in vitro and ubiquitinated it in vivo. Similar to Rma1H1, Rma1, an Arabidopsis homolog of Rma1H1, localized to the ER, and its overexpression reduced the PIP2;1 protein level and inhibited trafficking of PIP2;1 from the ER to the plasma membrane in protoplasts. In addition, reduced expression of Rma homologs resulted in the increased level of PIP2;1 in protoplasts. We propose that Rma1H1 and Rma1 play a critical role in the downregulation of plasma membrane aquaporin levels by inhibiting aquaporin trafficking to the plasma membrane and subsequent proteasomal degradation as a response to dehydration in transgenic Arabidopsis plants.

Ubiquitination is involved in diverse cellular processes in higher plants. In this report, we describe Arabidopsis thaliana PUB22 and PUB23, two homologous U-box–containing E3 ubiquitin (Ub) ligases. The PUB22 and PUB23 genes were rapidly and coordinately induced by abiotic stresses but not by abscisic acid. PUB22- and PUB23-overexpressing transgenic plants were hypersensitive to drought stress. By contrast, loss-of-function pub22 and pub23 mutant plants were significantly more drought-tolerant, and a pub22 pub23 double mutant displayed even greater drought tolerance. These results indicate that PUB22 and PUB23 function as negative regulators in the water stress response. Yeast two-hybrid, in vitro pull-down, and in vivo coimmunoprecipitation experiments revealed that PUB22 and PUB23 physically interacted with RPN12a, a subunit of the 19S regulatory particle (RP) in the 26S proteasome. Bacterially expressed RPN12a was effectively ubiquitinated in a PUB-dependent fashion. RPN12a was highly ubiquitinated in 35S:PUB22 plants, but not in pub22 pub23 double mutant plants, consistent with RPN12a being a substrate of PUB22 and PUB23 in vivo. In water-stressed wild-type and PUB-overexpressing plants, a significant amount of RPN12a was dissociated from the 19S RP and appeared to be associated with small-molecular-mass protein complexes in cytosolic fractions, where PUB22 and PUB23 are localized. Overall, our results suggest that PUB22 and PUB23 coordinately control a drought signaling pathway by ubiquitinating cytosolic RPN12a in Arabidopsis.

Ubiquitination is a eukaryotic posttranslational protein modification that is mediated by the cascade of E1, E2, and E3 ubiquitin (Ub) ligases and is involved in regulating numerous cellular functions. In this study, we obtained 100 different Arabidopsis (Arabidopsis thaliana) T-DNA insertion mutant plants in which RING E3 Ub ligase genes were suppressed and monitored their phenotypes in the presence of exogenous abscisic acid (ABA), a plant stress hormone. One of these loss-of-function mutants displayed ABA-insensitive phenotypes at the germination stage and was named atairp1 (for Arabidopsis ABA-insensitive RING protein 1). AtAIRP1 encodes a cytosolic protein containing a single C3H2C3-type RING motif with in vitro E3 Ub ligase activity. AtAIRP1 was significantly induced by ABA and drought stress. In contrast to atairp1 mutant plants, AtAIRP1-overexpressing transgenic plants (35S:AtAIRP1-sGFP) were hypersensitive to exogenous ABA in terms of radicle emergence, cotyledon development, root elongation, and stomatal closure. Ectopic expression of AtAIRP1-sGFP in atairp1 effectively rescued the loss-of-function ABA-insensitive phenotype. Both 35S:AtAIRP1-sGFP and atairp1/35S:AtAIRP1-sGFP plants accumulated higher amounts of hydrogen peroxide in response to exogenous ABA than did wild-type and atairp1 mutant plants. AtAIRP1 overexpressors were markedly tolerant to severe drought stress, as opposed to atairp1, which was highly susceptible. The levels of drought stress-related genes and basic leucine zipper transcription factor genes were up-regulated in the 35S:AtAIRP1-sGFP lines relative to wild-type and atairp1 mutant plants in response to ABA. Overall, these results suggest that AtAIRP1, a C3H2C3-type RING E3 Ub ligase, is a positive regulator in the Arabidopsis ABA-dependent drought response.

Ubiquitination is a unique protein degradation system utilized by eukaryotes to efficiently degrade detrimental cellular proteins and control the entire pool of regulatory components. In plants, adaptation in response to various abiotic stresses can be achieved through ubiquitination and the resulting degradation of components specific to these stress signalings. Arabidopsis has more than 1,400 E3 enzymes, indicating E3 ligase acts as a main determinant of substrate specificity. However, as only a minority of E3 ligases related to abiotic stress signaling have been studied in Arabidopsis, the further elucidation of the biological roles and related substrates of newly identified E3 ligases is essential in order to clarify the functional relationship between abiotic stress and E3 ligases. Here, we review the current knowledge and future prospects of the regulatory mechanism and role of several E3 ligases involved in abiotic stress signal transduction in Arabidopsis. As another potential approach to understand how ubiquitination is involved in such signaling, we also briefly introduce factors that regulate the activity of cullin in multisubunit E3 ligase complexes.

AtPUB18 and AtPUB19 are homologous U-box E3 ubiquitin ligases in Arabidopsis (Arabidopsis thaliana). AtPUB19 is a negative regulator of abscisic acid (ABA)-mediated drought responses, whereas the role of AtPUB18 in drought responses is unknown. Here, loss-of-function and overexpression tests identified AtPUB18 as a negative regulator in ABA-mediated stomatal closure and water stress responses. The atpub18-2atpub19-3 double mutant line displayed more sensitivity to ABA and enhanced drought tolerance than each single mutant plant; therefore, AtPUB18 and AtPUB19 are agonistic. Stomatal closure of the atpub18-2atpub19-3 mutant was hypersensitive to hydrogen peroxide (H₂O₂) but not to calcium, suggesting that AtPUB18 and AtPUB19 exert negative effects on the ABA signaling pathway downstream of H₂O₂ and upstream of calcium. AtPUB22 and AtPUB23 are other U-box E3 negative regulators of drought responses. Although atpub22atpub23 was more tolerant to drought stress relative to wild-type plants, its ABA-mediated stomatal movements were highly similar to those of wild-type plants. The atpub18-2atpub19-3atpub22atpub23 quadruple mutant exhibited enhanced tolerance to drought stress as compared with each atpub18-2atpub19-3 and atpub22atpub23 double mutant progeny; however, its stomatal behavior was almost identical to the atpub18-2atpub19-3 double mutant in the presence of ABA, H₂O₂, and calcium. Overexpression of AtPUB18 and AtPUB19 in atpub22atpub23 effectively hindered ABA-dependent stomatal closure, but overexpression of AtPUB22 and AtPUB23 in atpub18-2atpub19-3 did not inhibit ABA-enhanced stomatal closure, highlighting their ABA-independent roles. Overall, these results suggest that AtPUB18 has a linked function with AtPUB19, but is independent from AtPUB22 and AtPUB23, in negative regulation of ABA-mediated drought stress responses.