| Low temperature is one of the major environmental factors that affect the growth, yieldand quality of crops severely. Under chilling stress, imbalances in metabolic processes maylead to increased accumulation of ROS, and it will attack the biomacromolecule such asprotein, nucleic acid, lipids etc and induce oxidative damage to cell and tissues if they couldnot be scavenge efficiently. Therefore, plants have evolved enzymatic and non-enzymaticantioxidant defense systems to protect themselves against ROS. The antioxidant systeminclude several low molecular weight antioxidants involved in non-enzymatic scavenging,several enzymes, as well as enzymes which involved in the AsA-Glu cycle, such as ascorbateperoxidase (APX), monodehydroascorbate reductase (MDHAR), glutathione reductase (GR).Among the AsA-Glu cycle, APX has been considered as key enzyme detoxifying H2O2inchloroplasts. It is very impotant to investigate the relationship between tAPX and stressfultolerance in plants.In the present study, we isolated and characterized thylakoidal ascorbate peroxidase gene,LetAPX, from Lycopersicon esculentum cv. Zhongshu6. Sequence comparison, expressionanalysis and further functional analysis on the transgenic plants were analyzed in this work.The main results are as follows:(1) We isolated a tAPX gene, designated LetAPX, from tomato. The LetAPX encodes aprotein of421amino acids. The deduced amino acid sequence showed high identities withtAPX from Nicotiana tabacum, Cucurbita moschata Duch and Mesembryanthemumcrystallinum Linn. Amino acid sequence alignment revealed that the isolated cDNA containedthe previously defined two domains. The domain I is thought to APX active site signature.The domain II is the proximal heme-ligand motif.(2) p35S-LetAPX-GFP fusion protein was constructed and transiently expressed inArabidopsis protoplasts derived from leaf tissue. It was observed with confocal microscopy that the green fluorescence was clearly associated with chloroplasts and colocalized with thered autofluorescence of chloroplasts, demonstrating the LetAPX subcellular localization onchloroplast.(3) Northern hybridization showed that the expression of LetAPX was obviously inducedby several kinds of stresses and changed with the treatment.(4) The full-length LetAPX cDNA was subcloned into the expression vector pBI121downstream of the35S-CaMV promoter to form sense and antisense constructs. Theconstructs were first introduced into Agrobacterium tumefaciens LBA4404by the freezingtransformation method and the transgenic tomato plants were verified by PCR and northernhybridization. It was indicated that the LetAPX gene had been recombined into tomatogenome and both sense and antisense transgenic tomato plants were obtained. Compared toWT plants, sense transgenic plants showed higher APX activity, lower content of H2O2.However, lower APX activity and the higher H2O2content were detected in antisensetransgenic plants.(5) A recombinant of prokaryotic expression vector pET-LetAPX was constructed andtransformed to E.Coli. BL21. The strong induced fusion protein bands were collected intophosphate buffer (PBS) solution and were used to immunize mice to obtain antiserum. Thevalue of antibody reaches1:500. Western blot analysis revealed the presence of the strongpositive protein signals corresponding to LetAPX in transgenic plants. The results showedthat the expression of LetAPX at protein level was induced by stresses and changed with thetreatment.(6) The growth analysis of WT and transgenic tomato indicated that under normalconditions there is no difference in the growth state of all plants. However, under chillingstress, the growth of sense lines was obviously higher than that of WT plants. These resultsshowed that overexpression of LetAPX enhanced the tolerance to low temperature.(7) Although net photosynthetic rate (Pn) and maximal photochemical efficiency of PSII(Fv/Fm) in WT and transgenic plants decreased markedly under chilling stress in the lowirradiance (4℃,100μmol m-2s-1), the decrease of Pn and Fv/Fm was more slower in sensetransgenic plants and more obvious in antisense transgenic plants compare with WT. The results indicated that overexpression of LetAPX alleviated the photoinhibition of PSII. Underchilling stress with low irradiance, the oxidizable P700decreased significantly in all plants.When tomato plants were under optimal conditions of25℃and a PFD of100μmol m-2s-1for24h, the oxidizable P700could recover faster in sense transgenic plants compare with WT.After treatment at4℃, the level of malondialdehyde (MDA) increased in all plants, and theincrease was obvious in the antisense plants compared to WT plants.(8) Under chilling stress, the level of AsA increased in all plants. There is no differencein the AsA content between WT and sense transgenic plants. However, the content of AsA inantisense lines was obviously higher than WT plants. The results indicated that AsA as one ofmost abundant water-soluble redox compound and can compensate for the loss of APX.(9) Expression of several known stress-responsive and antioxidative genes in WT andantisense lines was analyzed by the fluorescence quantitative PCR technology. After chillingtreatment for6h, expression of genes we have studied was higher in WT plants comparedwith transgenic plants. Interestingly, after12h chilling, the expression of some genes washigher in transgenic plants than that of WT plants.(10) Under the transmission electron microscope, after chilling stress for12h, comparedwith WT plants, the cell membrane system of antisense plants damaged severely, starchgrains increased distinctively and the chloroplast seemed to be globular. However, sensetransgenic plants damaged slightly. The results that suggested that tAPX can play a key rolein alleviating the damage of leaf cell ultrastructure induced by low temperature. |
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