The RING-H2 finger gene 1 (RHF1) encodes an E3 ubiquitin ligase and participates in drought stress response in Nicotiana tabacum

Abstract

Drought is one of the most important limiting factors for plant growth and development. To identify genes required for drought stress response in tobacco, one highly induced mRNA encoding a RING-H2 Finger gene (RHF1) was isolated by mRNA differential display. The full-length NtRHF1 encodes a protein of 273 amino acids and contains a single C3H2C3-type RING motif in its C-terminal region. NtRHF1 is an ortholog of Arabidopsis SDIR1 (salt- and drought-induced RING finger 1) (73% identity to AtSDIR1). The recombinant NtRHF1 protein purified from E. coli exhibited an in vitro E3 ubiquitin ligase activity. Real-time quantitative PCR analysis indicated that the transcript levels of NtRHF1 were higher in aerial tissues and were markedly up-regulated by drought stress. Overexpression of NtRHF1 enhanced drought tolerance in transgenic tobacco plants while RNA silencing of NtRHF1 reduced drought tolerance. Further expression analysis by real-time PCR indicated that NtRHF1 participates in drought stress response possibly through transcriptional regulation of downstream stress-responsive genes NtLEA5, NtERD10C, NtAREB, and NtCDPK2 in tobacco. Together, these results demonstrated that NtRHF1 plays a positive role in drought stress tolerance possibly through transcriptional regulation of several stress-responsive marker genes in tobacco. This study will facilitate to improve our understanding of molecular and functional properties of plant RING-H2 finger proteins and to provide genetic evidence on the involvement of the RING-H2 E3 ligase in drought stress response in Nicotiana tabacum plants.