Functional Analysis Of The Chloroplast LeGPAT In Tomato Under Temperature Stress

Low temperature is the major factor limiting the productivity and geographical distribution of chilling-sensitive plant species. It was suggested that membrane was the primary position to be damaged under chilling stress. The tolerance of plants to chilling stress was closely connected with the fatty acid unsaturation of plant membrane lipids. Sn-2 position is occupied mainly by saturated and trans-unsaturated fatty acids, so the content of cis-unsaturated fatty acids at the sn-1 position of Phosphatidylglycerol (PG) determines chilling resistance. The dominant factor that determines the level of cis-unsaturated fatty acids in PG is the substrate selectivity of glycerol-3-phosphate acyltransferase(GPAT: EC2.3.1.15)in chloroplasts, which catalyzes the first step of glycerolipid biosynthesis by transferring the acyl group of acyl-(acyl-carrier protein) (ACP) to the sn-1 position of glycerol-3-phosphate to yield 1-acylglycerol-3-phosphate (lysophosphatidate; LPA). GPAT from chilling-resistant plants prefers oleoyl-ACP (18:1-ACP) to palmitoyl-ACP (16:0-ACP) as a substrate. Thus, a large proportion of oleic acid (18:1) occurs at the sn-1 position of PG in chilling-resistant plants. Under chilling stress oleic acid (18:1) of sn-1 position desaturates further into cis-polyunsaturated fatty acids of linoleic acid (18:2) and linolenic acid (18:3) by acyl-fatty acid desaturase in chloroplast membranes. The enzyme from chilling-sensitive plants hardly distinguishes 18:1-ACP from 16:0-ACP. Fatty acid of sn-1 position remains unchanged, resulting in a low level of cis-unsaturated fatty acids at the sn-1 position of PG, which increase sensitivity of plants to chilling stress. In this study, we isolated and characterized chloroplast glycerol-3-phosphate acyltransferase gene from tomato. The main results are as follows:1. Two degenerate primers were designed to amplify specific 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'fragment of the cDNA was isolated by 5'and 3'RACE. The clone, which named LeGPAT (Acession Numeber:DQ459433), contains 1770 bp nucleotides with an open reading frame (ORF) of 1314 bp comprising 437 amino acid residues with the predicted molecular mass of 48 kDa. The deduced amino acid sequence showed high identities with GPAT from Capsicum annuum, Carthamus tinctorius, Pisum sativum, Spinacia oleracea. Amino acid sequence alignment revealed that the plant members contained four acyltransferase domains. The His and Asp residues in block I, the Gly residue in block III, and the Pro residue in block IV, all of which have shown to form a catalytically important site in this family of acyltransferases, are absolutely conserved.2. p35S-LeGPAT-GFP fusion protein was constructed and transiently expressed in cowpea 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 that LeGPAT subcellular localization on chloroplast.3. Northern hybridization shows that LeGPAT constitutively expressed in stems, petals, fruits and leaves of wild type plants. The transcripts were high in the tissues abundant of chlorophyll. LeGPAT expressed extensively from 4 to 40℃in leaves and the expression of LeGPAT was obviously induced by low temperature and inhibited by high temperature.4. The full-length LeGPAT 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 verified by PCR and Northern hybridization. It was indicated that the LeGPAT had been recombined into tomato genome and both sense and antisense transgenic tomato plants were obtained. A higher content of 18:2 and 18:3 in PG was detected in sense transgenic plants compared with the wild type (WT) tomato plants. The fluidity of thylakoid membrane of sense transgenic plants was higher than WT under low temperature. Depletion of LeGPAT in tomato decreased the content of unsaturated fatty acids (18:2 and 18:3) in PG. But the contents of 16:0, 16:1 (△3- trans, sometimes referred to as high-melting-point fatty acids) and 18:0 increased in antisense transgenic plants compared to that of WT plants.5. A recombinant of prokaryotic expression vector pET-LeGPAT 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 the strong positive protein signals corresponding to LeGPAT in sense transgenic plants.6. Substrate selectivity and enzyme activity of LeGPAT were measured by using purified enzyme fractions of wild type and sense transgenic tomato leaves and protein from the E. coli. cells expressing LeGPAT. Each reaction mixture contained [1-14C]18:1-CoA and [1-14C]16:0-CoA, glycerol-3-phosphate, HEPES-NaOH buffer and BSA. Results showed that LeGPAT exhibited 18:1-selectivity over 16:0 and transgenic plants had higher selectivity (18:1) than wild type plants.7. Although Pn of WT and sense transgenic plants decreased markedly under chilling stress in the low irradiance (4℃, 100μmol m-2 s-1), the decrease of Pn was more obvious in WT than in sense transgenic plants. After tomato plants were transferred to a condition of 25℃and a PFD of 600μmol m-2 s-1, Pn of T1-5 and T1-19 recovered completely in 12 h, whereas Pn of wild type plants recovered only 73.2% in 12 h and 86.4% in 24 h. Fv/Fm decreased obviously during chilling stress (4℃) and recovered slowly in wild type plants relative to in sense transgenic plants. Fv/Fm of T1-5 and T1-19 recovered completely in 8 h, while Fv/Fm of WT only recovered 95.2%. The oxidizable P700 decreased significantly both in WT and sense transgenic plants under chilling stress in the low irradiance and there were no evident differences. When tomato plants were transferred to a suitable condition of 25℃and a PFD of 100μmol m-2 s-1, the oxidizable P700 of sense transgenic plants recovered more quickly than WT. After 24 h recovery, the oxidizable P700 could recover 98.5%, 99.4% and 85.3% in T1-5, T1-19 and WT, respectively. After treatment at 4℃for 12 h, the relative electrolytic leakage of T1-5 and T1-19 increased to 21.3% and 19.3%, whereas 24.4% in WT. Both NPQ and the de-epoxidized ratio of the xanthophylls cycle, (A+Z)/(V+A+Z), increased in WT as well as in sense transgenic plants at chilling temperature. NPQ and (A+Z)/(V+A+Z) of sense transgenic plants markedly increased relative to that of WT during chilling stress. The chloroplastic SOD and APX activities of WT plants increased during first 6 h of chilling stress and then decreased, whereas the SOD and APX activities of sense transgenic plants increased during first 9 h of chilling stress and then slightly decreased. After 6 h chilling stress, Chloroplast SOD and APX activities of transgenic plants were higher than that of WT. The contents of O 2 and H2O2 in WT increased after 6 h chilling stress, while the contents of O 2 and H2O2 of sense transgenic plants increased only after 9 h chilling stress. Both O 2 and H2O2 contents increased more markedly in WT plants than in sense transgenic plants. At the end of chilling stress, O 2 content in leaves of T1-5, T1-19 and WT plants increased for about 15.7%, 14.8% and 63.0% of initial values, respectively, and H2O2 content of T1-5, T1-19 and WT increased for about 26.0%, 15.8% and 77.6% of initial values, respectively.8. Antisense-mediated depletion of LeGPAT severely affected tomato male fertility. Examination of scanning electron micrographs of pollen grains revealed that the majority of pollen grains of antisense line were collapsed in morphology. Clear evidence of tapetum developmental defects was detected beginning at the microspore mother cell stage. More arrested pollen grains were found at the vacuolated microspore stage in antisense line (-)12 than in wild type tomato plants. In addition, lipid bodies in antisene line (-)12 were more evident and ER was less than in WT. After germination on culture medium for 60 min, approximately 65% of WT pollen grains germinated, while only 8% of (-)12 germinated. After 120 min, the proportion of germinated pollen grains in WT was nearly 100%, compared to only 20% in (-)12 line. According to the statistical analysis, flower development was arrested in 53.8% (14/26) of the samples from antisense lines, compared to only 7.8% (4/51) in wild type plants. Seeds of wild type developed normally and could reproduce, whereas the progenitive ability of antisense seeds was lost.9. The O2 evolution rates of WT and antisense transgenic tomato plants significantly decreased at 45℃for 6 h and 12 h. The decrease was more obvious in the wild type than in antisense transgenic plants. After 6 h heat stress, the O2 evolution rates in wild type, antisense transgenic lines (-)7 and (-)12 decreased to about 30.5%, 47.4% and 50.9% of initial values, respectively. After 12 h heat stress the O2 evolution rates of wild type, (-)7 and (-)12 lines decreased to 7.2%, 18.5% and 19.4% of initial values, respectively. Fv/Fm decreased in both WT and transgenic plants at 45℃heat stress, with wild types showing the greater decrease. At the end of 12 h heat stress, Fv/Fm in wild type, (-)7 and (-)12 lines decreased about 42.5%, 30.9% and 26.8%, respectively.The functional analysis showed that expression of the gene was induced by low temperature, whereas it was inhibited by heat stress. LeGPAT exhibited 18:1-selectivity over 16:0. The content of LeGPAT was higher in transgenic plants than in wild type tomato. It is interesting that overexpression of chloroplast LeGPAT increased the resistance to chilling stress. However, the depletion of LeGPAT was helpful in improving the thermal tolerance of tomato plants to high temperature and caused a massive arrest in pollen development.