VpPUB24, a novel gene from Chinese grapevine, Vitis pseudoreticulata, targets VpICE1 to enhance cold tolerance

The ubiquitination system plays important roles in the degradation and modification of substrate proteins. In this study, we characterize a putative U-box type E3 ubiquitin ligase gene, VpPUB24 (plant U-box protein 24), from Chinese wild grapevine, Vitis pseudoreticulata accession Baihe-35-1. We show that VpPUB24 is induced by a number of stresses, especially cold treatment. Real-time PCR analysis indicated that the PUB24 transcripts were increased after cold stress in different grapevine species, although the relative expression level was different. In grapevine protoplasts, we found that VpPUB24 was expressed at a low level at 22 °C but accumulated rapidly following cold treatment. A yeast two-hybrid assay revealed that VpPUB24 interacted physically with VpICE1. Further experiments indicated that VpICE1 is targeted for degradation via the 26S proteasome and that the degradation is accelerated by VpHOS1, and not by VpPUB24. Immunoblot analyses indicated that VpPUB24 promotes the accumulation of VpICE1 and suppresses the expression of VpHOS1 to regulate the abundance of VpICE1. Furthermore, VpICE1 promotes transcription of VpPUB24 at low temperatures. We also found that VpPUB24 interacts with VpHOS1 in a yeast two-hybrid assay. Additionally, over-expression of VpPUB24 in Arabidopsis thaliana enhanced cold tolerance. Collectively, our results suggest that VpPUB24 interacts with VpICE1 to play a role in cold stress.