Expression And Functional Analysis Of The LeLUT1in Tomato Under Chilling Stress

Low temperature is one of the most serious environmental stresses, which affect thegrowth, yield and quality of crops severely. The decrease in photosynthesis induced bychilling stress enhances the amount of excess excitation energy. Under chilling stress, theexcess light energy may lead to increased accumulation of ROS, and it will attack thebiomacromolecule such as protein, nucleic acid, lipids etc and induce oxidative damage tocell and tissues if they could not be scavenge efficiently. In the long term of evolvement,plants have evolved many kinds of antioxidant defense mechanism to protect themselvesagainst ROS. The antioxidant system includes α-carotene-derived lutein, which is structuralcomponents of light-harvesting complex subunits, contribute to the dissipation of excessabsorbed light energy and the protection of plants from photooxidative damage. Numerousstudies attributed the antioxidant function of lutein to singlet oxygen quenching and oxygenradical dissipation in vitro. Thus, the photosynthetic apparatus can be protected fromphotooxidation. So, it is significant to study the relationship between the lutein content andchilling tolerance in plants.In the present study, we isolated and characterized carotenoid ε-ring hydroxylase gene,LeLUT1, from Lycopersicon esculentum cv. Zhongshu6. Sequence comparison, expressionanalysis and further functional analysis on the transgenic tobacco plants were analyzed.In order to investigate the physiological effects and functional mechanism of lutein in thetomato at chilling stress-induced photoinhibition and photooxidation, wild type （WT） andsense transgenic lines （T-5, T-19, T-20） of tobacco were used to determine the lutein contentand growth performance, net photosynthetic rate, chlorophyll fluorescence parameters, theproduction of chloroplastic reactive oxygen species （ROS）, lipid peroxidation, relativeelectrolyte leakage and the content of D1protein. The main results are as follows: （1） We isolated a carotenoid ε-ring hydroxylase gene LeLUT1from tomato. The LeLUT1encodes a protein of548amino acids. The deduced amino acid sequence showed highidentities with LUT1from Cucumis sativus, Vitis vinifera and Ricinus communis. Quantitativereal-time （qRT-PCR） showed that the expression of LeLUT1was obviously induced byseveral kinds of stresses and changed with the treatments. p35S-LeLUT1-GFP fusion proteinwas constructed and transiently expressed in Arabidopsis protoplasts derived from leaf tissue.It was observed with confocal microscopy that the green fluorescence was clearly associatedwith chloroplasts and colocalized with the red autofluorescence of chloroplasts,demonstrating the LeLUT1subcellular localization on chloroplast.（2） The full-length LeLUT1cDNA was subcloned into the expression vector pBI121downstream of the35S-CaMV promoter to form sense constructs. The constructs were firstintroduced into Agrobacterium tumefaciens LBA4404by the freezing transformation methodand the transgenic tobacco plants were verified by PCR, qRT-PCR and western blot. It wasindicated that the LeLUT1gene had been recombined into tobacco genome and both sensetransgenic tobacco plants were obtained. HPLC showed that the content of lutein in thetransgenic tobacco plants were higher than those in the WT plants even under lowtemperature conditions.（3） Compared to WT plants, transgenic plants showed higher APX and CAT activity,lower content of H₂O₂and O₂. The growth analysis of WT and transgenic tobacco indicatedthat under normal conditions there is no difference in the growth state of all plants. However,under chilling stress, the growth of sense lines was higher than that of WT plants. Theseresults showed that overexpression of LeLUT1enhanced the tolerance to low temperature.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 low irradiance(4°C,100μmol m^-2s^-1), the decrease of Pn and Fv/Fm was more slower in transgenic plantscompare with WT. The results indicated that overexpression of LeLUT1alleviated thephotoinhibition of PSII. After treatment at4°C, the level of malondialdehyde （MDA）increased in all plants, and the increase was obvious in the WT plants.（4） The expression patterns of stress-related genes in tobacco in WT and transgene lines were analyzed by the quantitative real-time PCR technology. After chilling treatment for12h,expression of the genes we have studied was higher in transgenic plants compared with WTplants.（5） To investigate the photosynthetic mechanism underlying the enhanced cold resistancein the transgenic lines, the activities of photosynthetic enzymes were examined in WT andtransgenic lines before and after12h chilling stress. Under normal conditions, the activities ofall6enzymes （initial Rubisco, sFBPase, GAPDH, FBP aldolase, SPS and SS） in WT andtransgenic plants were similar. Exposure to cold （4°C） for12h caused down-regulation of theactivities of the analysed6enzymes in all lines, but the decreases were more serious in WTplants.