A U-box E3 ubiquitin ligase OsPUB67 is positively involved in drought tolerance in rice.

KEY MESSAGE: OsPUB67, a U-box E3 ubiquitin ligase, may interact with two drought tolerance negative regulators (OsRZFP34 and OsDIS1) and improve drought tolerance by enhancing the reactive oxygen scavenging ability and stomatal closure. E3 ubiquitin ligases are major components of the ubiquitination cascade and contribute to the biotic and abiotic stress response in plants. In the present study, we show that a rice drought responsive gene, OsPUB67, encoding the U-box E3 ubiquitin ligase was significantly induced by drought, salt, cold, JA, and ABA, and was expressed in nuclei, cytoplasm, and membrane systems. This distribution of expression suggests a significant role for OsPUB67 in a wide range of biological processes and abiotic stress response. Over-expression of OsPUB67 improved drought stress tolerance by enhancing the reactive oxygen scavenging ability and stomatal closure. Bimolecular fluorescence complementation assays revealed that a few E2s interacted with OsPUB67 with unique functional implications in different cell components. Further evidence showed that several E3 ubiquitin ligases interacted with OsPUB67, especially OsRZFP34 and OsDIS1, which are negative regulators of drought tolerance. This interaction on the stomata implied OsPUB67 might function as a heterodimeric ubiquitination complex in response to drought stress. Comprehensive transcriptome analysis revealed OsPUB67 participated in regulating genes involved in the abiotic stress response and transcriptional regulation in an ABA-dependent manner. Our findings revealed OsPUB67 mediated a multilayered complex drought stress tolerance mechanism.