| When a plant absorbs more light than it can utilize through the photosynthetic electron transport pathway, the excess energy, which is not promptly quenched, can reduce the photosynthetic efficiency and result in photoinhibition, even photooxidative damage. 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. In order to avoid severe photoinhibition, there are several photoprotective mechanisms in higher plants. The irradiance-dependent xanthophyll cycle plays an important role in the protection of plants under environmental stress. It involves interconversions between the three pigments, violaxanthin (V), antheraxanthin (A) and zeaxanthin (Z). The cycle is catalyzed by two enzymes, violaxanthin de-epoxidase (VDE: EC1.10.99.3) and zeaxanthin epoxidase (ZE: EC1.14.13.90). When the energy is excessive, V is converted to Z via A in the presence of ascorbate and an acidic lumen generated by the proton pump. This reaction is thought to occur in the lumen of thylakoids, catalyzed by VDE, and ZE catalyzes the reverse reaction thought to be located in the stromal side. Non-photochemical quenching (NPQ) which depends on the xanthophyll cycle protects the photosynthesis apparatus from inactivation and damage caused by excess excitation energy. Zeaxanthin is the exclusive xanthophyll that accumulates under excess light, by de-epoxidation of existing violaxanthin in the xanthophyll cycle. It is widely thought to play a photoprotective role by dissipation of excessive light energy as heat.In this study, we isolated and characterized zeaxanthin epoxidase gene from tomato using homological clone. The functional analysis showed that expression of the gene was induced by diurnal rhythm, whereas it was unaffected by light intensity and temperature. It is interesting that overexpression of LeZE enhances the sensitivity of tomato PSII photoinhibition to high light and chilling stress. However, the depletion of LeZE was helpful in alleviating the photoinhibition of tomato plants under low temperature and affected the colour of tomato mature flowers. 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 LeZE (Acession numeber: EF581828), contains 2437 bp nucleotides with an open reading frame (ORF) of 2010 bp comprising 669 amino acid residues with the predicted molecular mass of 73 kDa. The deduced amino acid sequence showed high identities with ZE from Solanum tuberosum, Nicotiana plumbaginifolia, Daucus carota subsp. sativus, Vitis vinifera. Amino acid sequence alignment revealed that the plant members contained four acyltransferase domains. The typical domain of the lipocalin family of proteins in blocks I and II, the flavoprotein monooxygenase domain in block III and the phosphopeptide binding domain (Forkhead-associated or FHA domain) in block IV, all of which have been shown to form a catalytically important site in zeaxanthin epoxidase, are absolutely conserved.2. Northern hybridization showed that LeZE constitutively expressed in roots, flowers, leaves, fruits, stems and calyxes of wild type plants. The transcripts were high in the tissues abundant of chlorophyll and the expression level of LeZE in young leaves was obviously higher than that in functional leaves and senescent leaves. LeZE transcript level was similar for extracts from plants treated with high light and chilling stress, and exhibited a diurnal rhythm expression pattern. Furthermore, the LeZE transcript level was affected neither by light intensity nor by temperature. Southern blot analysis showed that LeZE gene was a single copy in tomato genome.3. The full-length LeZE 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 LeZE gene had been recombined into tomato genome and both sense and antisense transgenic tomato plants were obtained. A higher content of V and lower content of A and Z were detected in sense transgenic plants compared with the wild type (WT) tomato plants. The de-epoxidation ratio of xanthophyll cycle pigments (A+Z)/(V+A+Z) of WT was higher than that of sense transgenic plants. Depletion of LeZE in tomato decreased the content of Z and A. But Z accumulated in antisense transgenic plants compared to that of WT plants.4. A recombinant of prokaryotic expression vector pET-LeGPAT 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 hybridization revealed the presence of the strong positive protein signals corresponding to LeZE in sense transgenic plants. Western blot analysis was carried out over the diurnal cycle. Unexpectedly, the LeZE 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 WT than in sense transgenic plants. 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 the WT. At the end of high light stress, Fv/Fm in WT, T1-10 and T1-1 lines decreased about 22.5%, 28.9% and 42.1%, respectively. When tomato plants were transferred to suitable condition of 25℃and a PPFD of 100μmol m-2 s-1, Fv/Fm recovered in both WT and transgenic plants. However, the recovery of Fv/Fm in the WT was faster. After 24 h recovery, Fv/Fm in the WT could recover completely, while recovery of Fv/Fm in the T1-10 and T1-1 lines only reached 93.7% and 94.1%, respectively. Fv/Fm also decreased significantly in transgenic plants during chilling stress (4℃) relative to that in wild type plants. At the end of chilling stress, Fv/Fm in the the WT and transgenic plants of T1-10 and T1-1 decreased about 9.3%, 10.7%, 11.6%, respectively. The recovery of Fv/Fm in the WT was also faster than that in transgenic plants. The Fv/Fm of the WT recovered completely in 12 h, while the Fv/Fm of T1-10 and T1-1 only recovered 98.4% and 98.2%. But at 24 h, the Fv/Fm of the two transgenic lines recovered completely. Under high light stress for 6 and 12 h, the O2 evolution rates of WT and transgenic tomato plants obviously decreased. This decrease was more significant in transgenic plants than in WT. The O2 evolution rates of the WT and transgenic plants also decreased under chilling stress with low irradiance for 6 h and 12 h, but did not significantly decrease compared with that under high light stress.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. At the end of chilling stress, O 2 content in leaves of T1-1, T1-10 and WT plants increased for about 82.1 %, 76.8% and 66.5 % of initial values, respectively, and H2O2 content of T1-1, T1-10 and WT increased for about 93.4%, 87.8 % and 80.3.6 % of initial values, respectively. The level of peroxide of membrane lipids was enlarged and the MDA contents of T1-1, T1-10 and WT plants increased to 151.1%, 147.7% and 131.5%, respectively.6. Afte long-term acclimation to high light, the high light (HL)-grown plants had high level of O2 evolution rates and Chla/b ratio compared to that in low light (LL)-grown plants. However, the total Chl content was lower that in LL-grown plants. But there was no remarkable difference between WT and sense transgenic plants. It is likely that the combination of higher photosynthetic capacity and a smaller light-harvesting antenna rendered NPQ less important in the HL-grown plants. Uder high light (1980μmol m-2 s-1) in summer midday, Fv/Fm,ΦPSII and MDA content were virtually identical in both types of leaves, but there was high level of total ascorbate content in transgenic lines. Furthermore, activitiy of superoxide dismutase in transgenic plants is higher than that in WT. There was no difference between the activity of ascorbate peroxidase in WT and transgenic palnts.7. Antisense-mediated depletion of LeZE could not affecte NPQ under light stress. Z accumulated in antisense transgenic tomato plants, but V 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 high level before and afte high light and low temperature sress. Although both NPQ of WT and antisense transgenic plants increased markedly under chilling stress in the low irradiance and high light stress, there was no difference between WT and antisense transgenic plants. It was suggested that Z does not play any specific role in direct energy dissipation in PSII.8. The O2 evolution rates and Fv/Fm of WT and antisense transgenic tomato plants decreased under high light stress and there were no evident differences. The O2 evolution rates of WT and antisense transgenic tomato plants decreased under chilling stress in the low irradiance for 12 h. The decrease was more obvious in the wild type than in antisense transgenic plants. After 12 h stress, the O2 evolution rates in wild type, antisense transgenic lines (-)1 and (-)5 decreased to about 42.3 %, 60.8 % and 58.7 % of initial values, respectively. Fv/Fm decreased in both WT and transgenic plants under chilling stress in the low irradiance, with wild types showing the greater decrease. At the end of low temperature stress for12 h, Fv/Fm in wild type, (-)1 and (-)5 lines decreased about 10.5 %, 7.1 % and 7.9 %, respectively.The oxidizable P700 decreased significantly both in WT and sense transgenic plants under chilling stress in the low irradiance, the decrease of P700 was more obvious in WT than in antisense transgenic plants.The SOD and APX activities of WT and antisense transgenic plants increased during first 6 h of chilling stress and then decreased, and there were no evident differences. But both O 2 and H2O2 contents increased more markedly in WT plants than in antisense transgenic plants. At the end of chilling stress, O 2 content in (-)1, (-)5 and WT plant leaves increased for about 37.6%, 42.5% and 82.7% of initial values, respectively, and H2O2 content of (-)1, (-)5 and WT increased for about 39.5%, 39.5% and 84.8% of initial values, respectively. The MDA contents of WT and antisense transgenic tomato plants decreased under chilling stress in the low irradiance. The increase was more obvious in the wild type than in antisense transgenic plants. After 12 h stress, the MDA contents in wild type, antisense transgenic lines (-)1 and (-)5 increased to about 131.5 %, 110.8 % and 115.7% of initial values, respectively. The mature flowers of tomato which are normally bright yellow have amuch paler colour in the LeZE antisense transformants. This was associated with the accumulation of zeaxanthin in the corolla at the expense of the epoxyxanthophylls, e.g. violaxanthin and neoxanthin.In conclusion, we demonstrated that the expression of LeZE was not induced by light and temperature but was regulated by the diurnal rhythm. Overexpression of LeZE decreased the level of de-epoxidation and the thermal dissipation capacity under high light and chilling stress. The sensitivity of PSII photoinhibition to high light and chilling stress was therefore enhanced. But the sense transgenic plants can acclimate to long-term growth in HL in the field. When acclimated to HL, no difference in growth rate or photosynthesis were observed relative to the wild type. The depletion of LeZE could not affect NPQ under light stress but was helpful in alleviating the photoinhibition of tomato plants under low temperature.
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