Isolation And Functional Analysis Of Zeaxanthin Epoxidase Gene In Tomato

The radiant energy absorbed by the photosynthetic apparatus will finally be partially converted into steady chemical energy via photochemical processes. When light that is absorbed by the plant in excess of what can not be utilized through the photosynthetic electron transport pathway, the excess energy, which is not promptly quenched, can reduce the photosynthetic efficiency and result in photooxidative damage and photoinhibition sequentially. Plants have formed many kinds of photoprotective mechanismes, which could minimize the damage caused by excess light energy. The irradiance-dependent xanthophyll cycle plays an important role in the protection of plants under enviromental stress. Xanthophyll cycle involves the interconversions between the three pigments, violaxanthin (V), antheraxanthin (A) and zeaxanthin (Z). When the energy is excessive, V is convered 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 violaxanthin de-epoxidase, and ZE catalyzes the reverse reaction thought to be located in the stromal side. The cycle is catalyzed by two key enzymes, violaxanthin de-epoxidase (VDE) and zeaxanthin epoxidase (ZE).It is significant to make clear the function and regulation mechanism of xanthophyll cycle. The most direct and available method is to investigate the factors which regulate the key enzymes VDE and ZE of xanthophyll cycle by sense and antisense RNA technology.In this reseach, a full length cDNA of ZE gene was cloned from tomato using RT-PCR. The functional analysis showed that overexpression of LeZE decreased level of de-epoxidation and thermal dissipation capacity under irradiance stress. The photoinhibition of PSII was aggravated. The main results are as follows:1. Two specific primers were designed to amplify specific DNA fragment using cDNA prepared from tomato leaves according to the ZE sequences from other plant species. The gene contains 2225bp, an open reading frame (ORF) of 2010 bp comprising 669 amino acid residues. Hereafter, this cDNA is designated as LeZE, encoding chloroplast zeaxanthin epoxidase of tomato.2. The deduced amino acid sequence of LeZE showed high identities with other plant ZE from potato, tobacco, carrot, grape, chrysanthemum and apricot, which were 96.1%, 89.7%, 75%, 74%, 72.5% and 71.2%, respectively. But it had low identities with rice, which was only 66.6%.3. Northern blot analysis of different organs such as the leaves, petals, stems, roots and fruits in tomato indicated that LeZE was mainly expressed in the leaves. Transcripts of the gene were less in the fruits and stems, but transcripts were hardly detected in the roots and petals. Northern blot analysis also indicated that the expression of LeZE was not induced by light intensity and temperature. LeZE transcript levels were similar for extracts from plants under the high light and chilling stress in the low irradiance, exhibited a diurnal rhythm.4. A recombinant of prokaryotic expression vector pET-LeZE 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. Western hybridization revealed the LeZE protein level remained constant in leaves. Moreover, there was no different between leaves expose to chilling stress under low irradiance and high light. 5. The full-length LeZE cDNA was subcloned into the expression vector pBI121 downstream of the 35S-CaMV promoter to form sense 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 had been recombined into tomato genome and sense transgenic tomato plants were obtained.Northern and western blot analysis confirmed that the sense LeZE was transferred into the tomato genome and overexpressed.WT and transgenic plants were exposed to chilling stress under low irradiance and high light. WT plants exhibited substantially faster de-epoxidation kinetics as shown by A+Z formation. In WT and transgenic plants, the de-epoxidation ratio of xanthophyll cycle pigments (A+Z)/(V+A+Z) rapidly increased. This is consistent with the changes of NPQ during stress. Fv/Fm decreased in both WT and transgenic plants under high light stress, with transgenic plants showing the greater decrease. Fv/Fm also decreased obviously in transgenic plants during chilling stress (4℃) relative to that in wild type plants. These results showed that overexpression of LeZE decreased thermal dissipation capacity under irradiance stress. The photoinhibition of PSII was aggravated. Under high light stress for 6 and 12 h, the O2 evolution rates of WT and transgenic tomato plants significantly decreased. This decrease was more obvious in transgenic plants than that in the wild type. On the other hand, under chilling stress with low irradiance for 6 and 12 h, the O2 evolution rates of WT and transgenic tomato plants also decreased, but not significantly compared with that under high light stress.Newly synthesized ZE active protein correlates with RNA abundance. Therefore, overexpress LeZE in tomato may increase amount of newly synthesized ZE active protein and the relative activity of ZE may increased accordingly. And the increase of LeZE resulted in a decrease of de-epoxidation level compareed with wild type plant under excessive light energy. So, the photoinhibition of PSII was aggravated and excessive light sensitivity of tomato was enhanced.