Molecular cloning and functional analysis of spinach SoSMT2 in response to excess nitrate stress

Abstract

Plant sterols are important multifunctional lipids, which are involved in determining membrane properties. Sterol C24 methyltransferase2 (SMT2) governs the pattern of phytosterols synthesized in higher plants. In this study, the spinach sterol C24 methyltransferase2 (SoSMT2) cDNA was cloned by RT-PCR and RACE-PCR. The full length of SoSMT2 consists of 1614bp, including a 278bp 5′-untranslated region (UTR), a 1086bp open reading frame (ORF), and a 250bp 3′-UTR. The SoSMT2 gene encodes a polypeptide of 361 amino acid residues with a predicted molecular mass of 40.47kDa. RT-PCR analysis indicated that the expression of SoSMT2 was induced by excess nitrate in the root and shoot of spinach. The transcript level of SoSMT2 was decreased by NaCl treatment. In contrast, dehydration, and H2O2 treatment increased the transcript levels of SoSMT2. To examine the biological roles of SoSMT2 in stress responses, transgenic Arabidopsis plants that constitutively overexpress SoSMT2 under the control of cauliflower mosaic virus 35S promoter were generated. The germination rate of transgenic seeds was higher than wild type (WT) in MS medium supplemented with 160mM nitrate. When seedlings were transferred to MS liquid medium with 160mM nitrate for 0, 8, 24 and 48h, the transgenic plants showed lower levels of MDA and H2O2 contents, compared with WT plants. Additionally, transgenic plants had relatively higher SOD, CAT and POD activities and soluble sugar contents than WT plants under nitrate stress with the increasing of treatment time. These data suggest that SoSMT2 is involved in nitrate tolerance in spinach.