| Photosynthesis is important for plant growth and survival. On a daily as well as seasonal basis most plants receive more sunlight than they could actually use for photosynthesis. If the excessive light energy which has been absorbed by photosynthetic apparatus can not be dissipated rapidly, it may reduce the photosynthetic efficiency and result in photoinhibition, even photooxidative damage to the photosynthetic reaction center. The environmental stresses such as low and high temperatures, drought and salinity enhance photoinhibition. Photoinhibition occurs in the field in plants exposed to conditions of high light. The combination of low temperature with irradiance also has the potential to induce chronic photoinhibition of PSII. This is partly because lower temperature generally reduces the rates of biological reactions particularly carbon dioxide reduction and photorespiration, and therefore limits the sinks for the absorbed excitation energy. Among the short-term mechanisms, the xanthophyll cycle has been conceived to play a key role in photoprotection. The xanthophyll cycle exists in the thylakoid membranes of all higher plants, ferns, mosses and algae. Its pigments have been demonstrated to be associated with all light-harvesting components, including LHCI. It comprises intercoversions of three carotenoid pigments, violaxanthin, antheraxanthin and zeaxanthin, which catalysed by two enzyme, violaxanthin de-epoxidase (VDE, EC:1.10.99.3) and zeaxanthin epoxidase (ZE, EC:1.14.13.90). When light energy is excessive, violaxanthin is catalysed to zeaxanthin via antheraxanthin catalysed by violaxanthin de-epoxidase (VDE). However, when light stress disappeared, epoxidation of zeaxanthin to antheraxanthin and violaxanthin is catalysed by another enzyme, zeaxanthin epoxidase (ZE). The xanthophyll cycle and the associated non-photochemical quenching (NPQ) exert their protective role by thermal dissipation of excess light energy. Zeaxanthin is thought to be the main photoprotector in chloroplasts. Many studies show zeaxanthin can directly quench the 1Chl* state and some reactive oxygen species. So the photosynthetic apparatus can be protected from photooxidation. However, there are other researches which support that the xanthophyll pigments protect the thylakoid membrane by an indirect process. They can allosterically regulate the quenching process inside the LHCII. Zeaxanthin is a allosteric activator of this process and violaxanthin is a inhibitor.In this study, we isolated and characterized violaxanthin de-epoxidase gene from tomato using homological clone. The functional analysis showed that expression of the gene was induced by diurnal rhythm, light intensity and temperature. It is interesting that overexpression of LeVDE increased the level of de-epoxidation and thermal dissipation capacity. It is suggested that the overexpression of LeVDE could alleviate photoinhibition of PSII and PSI under high light and chilling stress. However, the suppression of LeVDE enhanced the photoinhibition of tomato plants under low temperature. 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 was named LeVDE (Acession numeber: FJ648424), contains 1670bp nucleotides with an open reading frame (ORF) of 1437bp, comprising 478 amino acid residues with the predicted molecular mass of 53 kDa. The deduced amino acid sequence showed high identities with VDE from Arabidopsis thaliana, Nicotiana tabacum,Oryza sativa, Spinacia oleracea, Lactuca sativa. Amino acid sequence alignment revealed that the plant members contained the previously defined regions. The cysteine-rich domain in block I, the lipocalin signature domain in block II and the C-terminal glutamate-rich domain in block III, all of which have been shown to form a catalytically essential site in VDE, are absolutely conserved. The cysteine-rich domain in block I was suggested to be the active site of VDE. The lipocalin signature domain could be a possible binding site for violaxanthin and MGDG which was the substrate for VDE. At the C-terminal region, the glutamate-rich domain in block III was thought to be involved in binding of VDE to the thylakoid membrane.2. Northern blot analysis showed that LeVDE constitutively expressed in roots, leaves, fruits, stems and calyxes of wild type (WT) plants. The transcripts were high in the tissues abundant of chlorophyll. LeVDE transcript level was similar for extracts from plants treated with high light and low light condition, and exhibited a diurnal rhythm expression pattern. Furthermore, the LeVDE transcript level was inhibited by both light intensity and low temperature. Southern blot analysis showed that LeVDE gene was a single copy in tomato genome.3. The full-length LeVDE 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 blot. It was indicated that the LeVDE gene had been recombined into tomato genome and both sense and antisense transgenic tomato plants were obtained. A lower content of V and higher content of A and Z were detected in sense transgenic plants compared with WT plants. The de-epoxidation ratio of xanthophyll cycle pigments (A+Z)/(V+A+Z) of sense transgenic plants was higher than that of WT. Suppression of LeVDE in tomato decreased the content of Z and A. But V accumulated in antisense transgenic plants compared to that of WT plants.4. A recombinant of prokaryotic expression vector pET-LeVDE was constructed and transformed to E.Coli BL21 to express. 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 blot revealed the presence of the strong positive protein signals corresponding to LeVDE in sense transgenic plants. Western blot analysis was carried out over the diurnal cycle. Unexpectedly, the LeVDE protein level remained constant in leaves. Moreover, there was no difference between leaves exposed to high light and chilling stress.5. Although both NPQ and (A+Z)/(V+A+Z) of WT and sense transgenic plants increased markedly under chilling stress in the low irradiance (4℃, 100μmol m-2 s-1) and high light stress (1200μmol m-2 s-1), the increase of NPQ and (A+Z)/(V+A+Z) was more obvious in sense transgenic plants than in WT. Fv/Fm decreased in both WT and transgenic plants under high light stress, but the decrease of Fv/Fm in transgenic plants was more significant than that in WT. At the end of high light stress, Fv/Fm in WT, T1-10 and T1-7 lines decreased by 32.1%, 13.4 % and 11.2 %, respectively. When tomato plants were transferred to suitable condition of 25℃and a PFD of 100μmol m-2 s-1, Fv/Fm recovered in both WT and transgenic plants. However, the recovery of Fv/Fm in the transgenic plants was faster. Fv/Fm also decreased significantly in transgenic plants during chilling stress (4℃) relative to that in WT plants. At the end of chilling stress, Fv/Fm in the the WT and transgenic plants of T1-10 and T1-7 decreased about 12.1 %, 8.3 % and 7.1 %, respectively. The recovery of Fv/Fm in transgenic plants was also faster than that in WT. Under high light stress for 12 h, the Pn of WT and transgenic tomato plants obviously decreased. This decrease was more significant in WT than in transgenic plants.The oxidizable P700 decreased significantly both in WT and sense transgenic plants under chilling stress in the low irradiance, and the decrease was more significant in WT than in transgenic plants. At the end of chilling stress, O2-|- content in leaves of T1-7, T1-10 and WT plants increased for about 52.3 %,59.8% and 81.1 % of initial values, respectively, and H2O2 content of T1-7, T1-10 and WT increased for about 40.1%,42.3 % and 61.3 % of initial values, respectively. The level of peroxide of membrane lipids was enlarged and the MDA contents of T1-7, T1-10 and WT plants increased to 115.1%, 121.7% and 140.5%, respectively.7. Antisense-mediated suppression of LeVDE affected NPQ under light stress. V accumulated in antisense transgenic tomato plants, but Z and A content was very low. The de-epoxidation ratio of xanthophyll cycle pigments (A+Z)/(V+A+Z) in antisense transgenic plants sustained a very low level before and after high light and low temperature stress. Although both NPQ of WT and antisense transgenic plants increased markedly under chilling stress in the low irradiance and high light stress, the increase was more significant in WT than in transgenic plants. It was suggested that the suppression of LeVDE decreased the energy dissipation in PSII. 8. Fv/Fm decreased in both WT and antisense transgenic plants under chilling stress in the low irradiance, and WT showed the greater decrease. At the end of low temperature stress for 12 h, Fv/Fm in (-)2, (-)9 and WT decreased about 20.1%,17.9%,12.1%, respectively. The oxidizable P700 decreased significantly both in WT and antisense transgenic plants under chilling stress in the low irradiance, and the decrease of P700 was more obvious in WT than in antisense transgenic plants. O2-|- contents increased more markedly in antisense transgenic plants than in WT plants. At the end of chilling stress, O-|- content in (-)2, (-)9 and WT plant leaves increased for about 79.1%,76.4% and 62.7%, respectively. The MDA contents of WT and antisense transgenic tomato plants increased under chilling stress in the low irradiance. The increase was more obvious in antisense transgenic plants than in the wild type. After 12 h stress, the MDA contents in WT, antisense transgenic lines (-)2 and (-)9 increased to about 138.5 %, 159.8 % and 161.7%, respectively.In conclusion, we demonstrated that the expression of LeVDE was induced by light, temperature and diurnal rhythm and it was partially inhibited by chilling temperature. Overexpression of LeVDE increased the level of de-epoxidation and the thermal dissipation capacity under high light and chilling stress. The sensitivity of PSII and PSI photoinhibition to high light and chilling stress was therefore alleviated. The suppression of LeVDE affected NPQ under light stress and enchanced the photoinhibition of tomato plants under low temperature.
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