Studies on the role of ZjSOD1 gene from Ziziphus jujuba in response to abiotic stresses

Abstract:【Objective】Superoxide dismutases (SODs) are involved in protecting plants against diverse biotic and abiotic stresses. In this study, a novel Fe- SOD gene, named ZjSOD1, was cloned fromZiziphus jujuba Mill. Hupingzao and its function was studied. The expression of ZjSOD1 of jujube plantlets generated from tissue culture was studied under different abiotic stress treatments.【Methods】The ORF sequences of the ZjSOD1 cDNA was cloned by polymerase chain reaction (PCR). The expres- sion of the ZjSOD1 under salt, and drought stresses was analyzed by quantitative RT-PCR. To character- ize the function of the ZjSOD1 gene in plant development, coding sequence of ZjSOD1 was inserted in- to the plant expression vectors PEZR(K)-LNY driven by CaMV 35S promoter and introduced into Ara- bidopsis. Subsequently, seeds of the wild type and homozygotes of T3 generation of the ZjSOD1 overex- pression transgenic lines of Arabidopsis were sowed on MS agar media with or without NaCl, PEG6000 and H2O2, respectively. The seed germination rates, the cotyledon greening rate and the growth rates of the primary roots of the wild type and ZjSOD1 lines were monitored under different abiotic stresses. The levels of proline, malondialdehyde (MDA), electrolyte leakage and the specific activities of various antioxidant enzymes were detected in the leaf tissues for evaluating the extent of cellular damage of  both the ZjSOD1 transgenic lines and the wild type during salt, PEG6000 and H2O2 treatments. In addi- tion, we analyzed the expression levels of antioxidant enzymes (APX1, GPX3 and CAT1) in the ROS scavenging system, drought/high salinity-responsive genes (RD29A and ERD15) and SOS2 (involved in SOS signaling pathway) were employed as markers for monitoring ROS signaling and stress-response pathways in Arabidopsis by quantitative RT-PCR【. Results】The ZjSOD1 contained a 699 bp of open reading frame (ORF) encoding a protein of 232 amino acids (including 22 acidic amino acids and 24 ba- sic amino acids) with a calculated molecular mass of 25.597 1 ku and an isoelectric point of 8.59. Zj- SOD1 protein, which belongs to Fe-SOD family, and contained a C-terminal conservative SOD func- tional domain. The transcript levels of the ZjSOD1 accumulated gradually in response to salt stress and reached the maximum expression with 100 mmol·L-1 NaCl for 24 h. Similarly the regulation of the Zj- SOD at transcript level was also analyzed in response to osmotic stress with PEG6000 (simulating drought stress). In particular, the ZjSOD1 expression level was increased by approximately 7-fold with 0.5 MPa PEG6000 treatment for 24 h. The overexpression of the ZjSOD1 in Arabidopsis led to a drought- and salt-sensitive phenotype of the transgenic plants, resulting in an increase in plant tolerance to H2O2 stress. In the presence of NaCl or PEG6000, the rates of seed germination and cotyledon green- ing in the ZjSOD1 overexpression transgenic plants were lower than those in the wild type, and the roots of the ZjSOD1 overexpression transgenic plantlets were shorter than those of the wild type. In the presence of 150 mmol · L- 1 NaCl, the germination rate of the wild type reached to approximately 90% , but the ZjSOD1 overexpression transgenic seeds reached to only about 60% 6 days after the treatments. Likewise, in the presence of 0.5 MPa PEG6000, about 85% of the wild type, whereas only 65% of theZjSOD1 overexpression transgenic seeds germinated 6 days after the treatments. However, In the presence of H2O2, the rates of seed germination and cotyledon greening in the ZjSOD1 overexpression transgenic plants were higher than those in the wild type, and the roots of the ZjSOD transgenicplantlets were longer than those of the wild type. The levels of SOD, CAT, POD activity, proline contents, MDA contents and electrolyte leakage in the ZjSOD1 overexpression transgenic plantlets were remarkably al- tered under NaCl, PEG6000 or H2O2 stresses. Furthermore, the expression of the genes related to reac- tive oxygen species (ROS), salt and drought signaling pathway were altered in the ZjSOD1 transgenic plants.【Conclusion】These data suggest that the ZjSOD1 was involved in plant response to oxidative, drought and salt stresses. The ZjSOD1 may act as a positive regulator activating a subset of ROS-related genes in response to ROS signaling, but it may be as a negative regulator in response to salt/osmotic stress. Importantly, the over-expression of the ZjSOD1 in crops may be useful for promoting the resis- tance to accumulation of ROS.