Functional Analysis Of The Tomato SlNAC1 Gene Under Heat Stress

With the increasing of greenhouse effect, global temperatures continue to rise, making horticultural production face the problem of high temperature stress. Tomato is an important economic crop, with a world-wide distribution and major consumption. Tomato is thermophilic, but it can’t withstand high temperature. In summer and autumn in south China,the abnormal high temperature severely inhibited the growth and development of tomato.Currently, that how to relieve heat stress has become a top priority, and taking advantage of genetic engineering to cultivate varieties with high temperature resistance is one of the important ways.In this study, the wild type(WT) tomato cultivar(Solanum lycopersicum cv. Zhongshu6) as well as antisense transgenic plants(A1, A4, A6) is used as the material. We observed the growth of experimental plants under heat stress and measured some physiological parameters, including net photosynthetic rate(Pn) in leaves, the maximal efficiency of PSII photochemistry(Fv/Fm), the actual photochemical efficiency( ? PSII), relative electric conductivity(REC), contents of malondialdehyde(MDA), proline, soluble sugar and protein,the accumulation of hydrogen peroxide(H2O2) and superoxide anion radical(O2?-), and antioxidant enzyme activity. In addition, the expression levels of the heat response genes and sucrose phosphate synthase(SPS) were measured. The key results are as follows:(1) We isolated the full length cDNA of SlNAC1 by RT-PCR, and the full length of SlNAC1 is 954 bp and the ORF is 906 bp encoding 302 amino residues. SlNAC1::GFP fusion protein was transiently expressed in onion epidermal cells and we found that it was located in the nucleus by using confocal laser scanning microscope.(2) The full-length ORF of SlNAC1 was cloned and reversely linked into the expression vector pBI121 under the control of the 35S-CaMV promoter to form antisense constructs. The Agrobacterium tumefaciens-mediated leaf disk method was used to generate transgenictomato plants. The antisense lines were determined by PCR and qRT-PCR.(3) After high temperature stress, the growth of both transgenic and WT plants was restrained. However, compared with WT, transgenic plants wilted more seriously. Heat stress reduced the Pn, Fv/Fm and ?PSII in all of the experimental plants, and the reduced extent of transgenic plants was higher than the WT plants.(4) Compared with WT plants, heat stress resulted in more increase of REC and MDA in transgenic plants. Nevertheless, the accumulation of proline, soluble sugar and protein in transgenic plants was less than the WT plants under heat stress.(5) Under heat stress, the content of H2O2 and the production rate of O2?-increased significantly both in WT and transgenic plants, but the content of ROS in transgenic tomato plants was higher than that in WT tomato plants. Under heat stress, the activities of superoxide dismutase(SOD) and ascorbate peroxidase(APX) increased both in transgenic and WT plants, whereas the activity of peroxidase(POD) reduced in all experimental plants.Compared with the WT plants, transgenic plants, showed the lower activities of antioxidant enzymes. At the same time, transgenic plants had lower expression level of SOD and APX than the WT plants which was in accordance with the activities of antioxidant enzymes.(6) Under heat stress conditions, the relative mRNA levels of the Hsp70, Hsp90,sHsp17.4, and sHsp17.6 had a sharp rise in all the plants, and these transcripts accumulated less in transgenic plants compared to those of WT plants. After heat treatment, the expressions of SPS reduced in both transgenic and WT plants, showing a more decrease in transgenic plants.All of these results suggested that suppression of Sl NAC1 decreased the heat resistance of tomato plants. Suppression of SlNAC1 reduced the accumulation of HSPs and the osmoprotectants, thereby reducing the rugulation function in the plant physiological balance.Meanwhile, suppression of SlNAC1 reduced the antioxidant enzyme activity of plants, leading to accumulation of a large amount of ROS which can increase the membrane lipid oxidation and seriously damage photosynthetic appratus. Our findings suggest that SlNAC1 plays a positive role in regulating heat stress in tomato.