Characterization of the novel salt stress responsive gene in rice Oryzativa L.

Abstract

The goals of this research have two main points; the first is to characterize the novel salt-responsive gene, OsNUC1, in rice, in order to indicate its function as a salt-resistant gene. The other is to elucidate of salt-tolerant mechanism of OsNUC1 and OsP5CS1 (the key regulator gene in proline biosynthesis) in relation with abscisic acid (ABA) signaling pathway. RiceNucleolin genes, OsNUC genes are the members of a small gene family in rice, consisting of two genes, OsNUC1 and OsNUC2, which are located on chromosome 4 and 8 respectively. OsNUC1encodes the protein with three conserved domains of Nucleolins found in other species, which are the acidic glycine rich domain with nuclear localization signal (NLS) at amino-terminal, two RNA recognition motifs (RRM) at the central domain, and glycine-and arginine-rich (GAR) domain at carboxyl terminal. The cDNA clones encoding the polypeptide consisting of all three domains and the one encoding the polypeptide consisting of the central and carboxyl-terminal domains exist in rice genome database. The localization experiment using GFP-Nucleolin fusion protein, performed in the onion epidermal peel, showing that GFP-three-domain Nucleolin1 fusion protein was localized in nucleus only, while the GFP-two-domain Nucleolin1 fusion protein, which was the fusion protein between GFP and central and carboxyl terminal domains of Nuclelin1, was localized in both nucleus and cytoplasm. This suggested that the two-domain Nucleolin1 could be transported to the nucleus without NLS at N-terminal. The OsNUC1 transcripts were found in leaf, root, seed, and flower tissues. The highest expression level was found in flowers and roots. Based on quantitative real-time PCR, salt stress can induce OsNUC1 gene expression up to 9 days after treatment. The salt resistant lines, LPT123-TC171 and FL530-IL showed the higher OsNUC1 gene expression, when compared to their salt-susceptible isogenic lines, LPT123, and KDML105, respectively. This suggests that OsNUC1 may function as a salt resistant gene in rice. The cDNA clone encoding the central and carboxyl terminal domains was over-expressed in transgenic Arabidopsis, and three independent transgenic lines with the different gene expression levels were used to investigate the effect of OsNUC1 gene expression. It was shown that OsNUC1 gene expression can enhance root growth, increase leaf number in both normal and salt-stress conditions. These data confirm the hypothesis that OsNUC1 is involved in salt resistant mechanism in plants. Moreover, it was found that OsNUC1 could increase at least two salt resistant genes, AtSOS1 and AtP5CS1, which supports the role of OsNUC1 in salt resistant function. Exogenous ABA can induce OsNUC1 gene expression in rice, which is consistent with the existence of ABRE cis-element in the promoter region. However, during salt stress, the application of abamineSG, ABA synthesis inhibitor, did not affect OsNUC1 expression, suggesting that ABA may not required for OsNUC1 gene expression during salt stress. The signaling response via ABA was also studied in proline synthesis mechanism. OsP5CS1was reported to be responsible for proline synthesis in rice during salt stress. The topical application of ABA can induce salt resistance and proline accumulation, assuming that exogenous ABA induces OsP5CS1 gene expression. However, the quantitative real-time PCR detecting OsP5CS1 gene expression at various timing after ABA application and salt stress, showed that the up-regulation of OsP5CS1 was not well correlated with the increase in proline accumulation pattern. On the contrary, the proline accumulation pattern was consistent with the gene expression pattern of OsP5CR, another gene in proline synthesis pathway. This suggested that the increase in salt resistance via the topical ABA application was due to OsP5CR function rather than OsP5CS1 function. Moreover, the experiment showing the involvement of CaM1-1 signaling pathway in proline accumulation during salt stress was also performed.