Abstract
Rice U-box E3 Ub ligases (OsPUBs) are implicated in biotic stress responses. However, their cellular roles in response to abiotic stress are poorly understood. In this study, we performed functional analyses of two homologous OsPUB2 and OsPUB3 in response to cold stress (4°C). OsPUB2 was up-regulated by high salinity, drought, and cold, whereas OsPUB3 was constitutively expressed. A subcellular localization assay revealed that OsPUB2 and OsPUB3 were localized to the exocyst positive organelle (EXPO)-like punctate structures. OsPUB2 was also localized to the nuclei. OsPUB2 and OsPUB3 formed a hetero-dimeric complex as well as homo-dimers in yeast cells and in vitro. OsPUB2/OsPUB3 exhibited self-ubiquitination activities in vitro and were rapidly degraded in the cell-free extracts with apparent half-lives of 150–160 min. This rapid degradation of OsPUB2/OsPUB3 was delayed in the presence of the crude extracts of cold-treated seedlings (apparent half-lives of 200–280 min). Moreover, a hetero-dimeric form of OsPUB2/OsPUB3 was more stable than the homo-dimers. These results suggested that OsPUB2 and OsPUB3 function coordinately in response to cold stress. OsPUB2- and OsPUB3-overexpressing transgenic rice plants showed markedly better tolerance to cold stress than did the wild-type plants in terms of survival rates, chlorophyll content, ion leakage, and expression levels of cold stress-inducible marker genes. Taken together, these results suggested that the two homologous rice U-box E3 Ub ligases OsPUB2 and OsPUB3 are positive regulators of the response to cold stress.
Highlights
Higher plants are constantly subjected to adverse environmental conditions owing to either biotic factors, such as pathogens and herbivores, or abiotic factors, such as extreme temperature, water availability, and high salinity
Reverse transcription polymerase chain reaction analysis showed that transcripts of OsPUB2 and OsPUB3 were detected in all tissues examined, including early seedlings, developing and mature leaves, stems, developing seeds, and panicles, with the expression level of OsPUB3 being higher than that of OsPUB2 (Figure 1B)
Real-time quantitative RT-PCR (qRT-PCR) assay indicated that the amount of OsPUB2 mRNA began to increase at 1 h after exposure to cold stress and was continuously elevated up to 35-fold after 48 h (Figure 1D)
Summary
Higher plants are constantly subjected to adverse environmental conditions owing to either biotic factors, such as pathogens and herbivores, or abiotic factors, such as extreme temperature, water availability, and high salinity. The ubiquitin (Ub)-proteasome system (UPS) regulates the stability and activity of many proteins and influences diverse cellular processes, including signal transduction, cell division, and response to biotic and abiotic stresses, in higher plants (Santner and Estelle, 2010; Lyzenga and Stone, 2012; Stone, 2014; Zhang et al, 2015; Yu et al, 2016). E3 Ub ligases are divided into two groups based on their structures: single-subunit and multi-subunit E3 ligases (Lee and Kim, 2011; Guerra and Callis, 2012; Sharma et al, 2016) The former group consists of RING (for Really Interesting New Gene)/U-box and HECT (for Homology to E6AP Carboxyl Terminus) E3 Ub ligases. The latter group includes SCF (for Skp1-Cullin-F-box) and APC (for Anaphase-Promoting Complex) E3 ligases
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