Cloning, Expression And Functional Analysis Of The LeGR In Tomato Under Temperature Stresses

Chilling stress is the major environmental stress for agricultural production, severely affecting the growth, yield and quality of crops. Reactive oxygen species (ROS) are produced inevitably in plants in normal cell metabolism such as respiration and photosynthetic electronic transport (Asada, 1999). Abiotic stresses (salinity, drought, extreme temperature, O3 etc.) accelerate the production of ROS. ROS attack biomacromolecules such as protein, nucleic acid, lipids etc. and aggravate oxidative damage to cell and tissues if they could not be scavenged efficiently. The chloroplast is one of the major sites for formation of ROS in higher plant. The fast remove of ROS under environmental stress is essential for protecting photosynthetic apparatus and maintaining photosynthesis. Glutathione (GSH) plays a key role in detoxifing ROS in chloroplast. Under stressful conditions, GSH can be oxidized quickly and finally degrade. Chloroplastic glutathione reductase (GR) reduces oxidized glutathione (GSSG) back to its reduced form GSH and sustains the reduced pool of glutathione in chloroplast. It is very impotant to investigate the relationship between chloroplastic GR and stressful tolerance in plants.In this study, the chloroplastic glutathione reductase gene was isolated and characterized from tomato. The main results are as follows:1. Two degenerate primers were designed to amplify the middle DNA fragment using cDNA prepared from tomato leaves according to the homologous sequences from other plants. The middle fragment of interested cDNA was obtained by RT-PCR. The 5'and 3'fragments of the cDNA was isolated by 5'and 3'RACE. The clone named LeGR (Acession numeber: EU285581 in GenBank) contains 2229 bp nucleotides with an open reading frame (ORF) of 1674 bp comprising 557 amino acid residues with the predicted molecular mass of 59 kDa. The deduced amino acid sequence showed high identities with GR from Nicotiana tabacum, Arabidopsis thaliana, Pisum sativum, Oryza sativa, Brassica juncea. 2. p35S-LeGR-GFP fusion protein and transiently expressed in Arabidopsis protoplasts derived from leaf tissue. It was observed with confocal microscopy that the green fluorescence was clearly associated with chloroplasts and colocalized with the red autofluorescence of chloroplasts, demonstrating the LeGR subcellular localization on chloroplast.3. Northern hybridization showed that the transcript of LeGR was high in the tissues abundant of chlorophyll. The expression of LeGR was obviously induced by temperature stresses and changed with the treatment.4. The LeGR genomic sequence contains 6672 bp nucleotides with 10 exons. Southern blot analysis showed that was only one single copy of LeGR gene in tomato genome.5. The full-length LeGR cDNA was subcloned into the expression vector pBI121 downstream of the 35S-CaMV promoter to form sense and antisense constructs. The constructs were first introduced into Agrobacterium tumefaciens LBA4404 by the freezing transformation method and the transgenic tomato plants were verified by PCR and northern hybridization. It was indicated that the LeGR gene had been recombined into tomato genome and the antisense transgenic tomato plants were obtained. Compared to WT plants, in normal growth conditions, GR activity in transgenic plants decreased by 60%.6. A recombinant of prokaryotic expression vector pET-LeGR was constructed and transformed to E.Coli BL21. The strong induced fusion protein bands were collected into PBS solution and used to immunize white mice to obtain antiserum. The value of antibody reaches 1:500. Western hybridization revealed the presence of a weak positive protein signal corresponding to LeGR in antisense transgenic plants.7. The growth analysis of WT and transgenic tomato indicated that under normal conditions there is no difference in the growth state of all plants. However, under temperature stresses, the growth of antisense lines was suppressed evidently than WT plants. These results showed that the depletion of LeGR enhanced the susceptibility to temperature stresses.8. Although net photosynthetic rate (Pn) and the maximal photochemical efficiency of PSII (Fv/Fm) in WT and transgenic plants decreased markedly under chilling stress in the low irradiance (4℃, 100μmol m^-2 s^-1) or high temperature (40℃), the decrease of Pn and Fv/Fm was more serious in antisense transgenic plants than that in WT, which indicated that the suppression of LeGR aggravated the photoinhibition of PSII. Under chilling stress with low irradiance, the oxidizable P700 decreased significantly in all plants. When tomato plants were under optimal conditions of 25 oC and a PFD of 100μmol m^-2 s^-1 for 24 h, the oxidizable P700 recovered to 70.2%, 72.9% and 69.8% in antisense transgenic lines, respectively. After treatment at 4℃and 40℃for 24 h, the level of hydrogen peroxide (H2O2) and thiobarbituric acid reactive substance (MDA) increased in all plants, and the increase was obvious in the antisense transgenic plants compared to WT plants.9. The decrease in GR activity results in an inhibition of regeneration of GSH but leads to no affect on total glutathione pool size. GR is essential to maintain GSH content and redox status of glutathione. And the inhibited of regeneration of GSH resulting from decrease in GR activity lead to an inhibition of AsA regeneration and a decrease in ascorbate pool size, which consequently resulted in a greater accumulation of H2O2 and an enhanced sensitivity to chilling stress in transgenic plants. Moreover, the excessive accumulation of H2O2 in transgenic plants was, to a certain extent, attributed to the reduced activities of APX. GR, directly and/or indirectly, played an important role in maintaining the efficiency of the H2O2 scavenge metabolism.