Functional Sites Analysis Of Vitamin K Epoxide Reductase On The Photosynthetic Growth And Activity Of Photosystem Ⅱ In Arabidopsis

Vitamin K epoxide reductase(VKOR) is one kind of membrane-fused oxidoreductase, located on the endoplasmic reticulum in higher animal. In plant kingdom, the homologous protein of VKOR is located on the thylakoid membrane of chloroplast. The deficiency of Arabidopsis VKOR causes pleiotropic defects in plant phenotypes, such as severely stunted growth, smaller sized leaves, and delay of flowering. Arabidopsis VKOR also has the activity of oxidoreductase, which is directly related to the thiol/disulfide states of its four pairs of conservative cysteines. Whether the function of AtVKOR in the plant is dependent on the activity of oxidoreductase is unknown. In this investigation, we mutated the cysteines of AtVKOR to alanines, constructed the plant expression vectors and respectively transformed them into vkor mutant lines. The homozymous plants were obtained and used for the analysis of the photosynthetic growth and changes of photosystem II(PSII) activity under different light densities to display the roles of function sites of AtVKOR in Arabidopsis. The main results are as follows:(1) Construction of cysteine-mutanted AtVKORs vectors, transforming them into vkor mutant lines and screening for homozymous transgenic plants. We respectviely alternatived the cysteines(two nonconserved cysteines and four pairs of conservative cysteines) of AtVKOR with alanines in single or double forming, constructed ten plant expression vectors(pBI121-mVKORs) and transformed into vkor muntant lines. Transgenic plants of cysteine-mutanted AtVKORs were selected by kanamycin culture medium and genome PCR, and screened for vkor mutant background through T-DNA primers. At T2 generation, the homozymous transgenic plants were obtained.(2) Effects of the cysteines in AtVKOR on the photosynthetic growth. The At VKORWT can completely recover the phenotype defects in the vkor lines, while mutations of cysteines in At VKOR led to different effects on the plant growth. These conservative cysteines in VKOR domain(Cys109-Cys116, Cys195-Cys198) played much more important roles in plant growth than that in Trx-like domain(Cys293-Cys296, Cys316-Cys331), among them AtVKORC109AC116 A and AtVKORC195AC198 A could not recover the growth of plants, similar to vkor mutant line. For mutation of conservative cysteines in Trx-like domain, only partly phenotype defects in the vkor mutant lines were recovered. Mutating the two nonconservative cysteines(Cys46, Cys230), the transgenic plants remained similar growth phenotype with AtVKORWT plants. The changes of biomass were consistent with the phenotypes observed above. The results further confirmed the indispensability of the conservative cysteines of AtVKOR in photosynthetic growth of plants, suggesting the oxidoreductase activity of AtVKOR is important for its function in plant growth and development.(3) Effects of the cysteines in AtVKOR on the activity of photosystem II(PSII) under different light densities. The maximal quantum yield of PSII(Fv/Fm), an indicator for the efficiency of PSII photochemistry, was determined to test the activity of PSII. No significant difference in the Fv/Fm ratio was observed among the AtVKORC46 A, AtVKORC230 A, AtVKORWT transgenic plants, and WT plants, indicating that AtVKORWT as well as the mutation of the two nonconservative cysteines were all sufficient to restore photosynthetic efficiency under normal light. However, the ratios of Fv/Fm in mutations of conservative cysteines dramatically decreased. Compared to AtVKORWT plant, AtVKORC109AC116 A and AtVKORC195AC198 A plants decreased above 24% and 22%, while the two pairs of cysteines mutations in Trx-like domain decreased above 10% to 13%. High light increased the differences on Fv/Fm among conservative cysteine mutanted VKORs and AtVKORWT plants. A similar trend of actual PSII photochemical efficiency(ΦPSII) was observed in the investigated plants. These low chlorophyll fluorescence parameters in transgenic plants with mutations of conservative cysteine were correlated with the impaired photosynthesis and reduced activities of PSII.(4) Effects of the cysteines in AtVKOR on the D1 protein turnover. The turnover of D1 protein is one of photoprotective processes in PSII under high light stress. Different from AtVKORWT which could restore the deficience in D1 protein turnover, mutations of cysteines in AtVKOR induced different effects on the turnover of D1 protein. Comparing the conditions of normal growth light, high irradiance the decrease extent of the D1 protein; only trace amount of D1 accumulation could be detected in the AtVKORC109AC116 A and AtVKORC195AC198 A plants, as like to that in vkor mutant line; little difference was observed in the level of D1 protein among the transgenic plants with mutant At VKORs of nonconservative cysteine and At VKORWT. The results above suggested that the turnover of D1 protein in the repair of photodamaged were damaged due to the mutations of conservative cysteine in the AtVKOR domain, which are directly related to its oxidoreductase activity.(5) Effects of the cysteines in AtVKOR on the accumulation of ROS. In chloroplasts, the damage of PSII assembly is usually associated with the harmful production of ROS, such as H2O2 and O2·-, and singlet oxygen. The amounts of ROS in plants with mutations of conservative cysteine VKORs were higher than that of AtVKORWT plants or mutations of nonconservative cysteines under growth light. Under high irradiance, the elevated accumulations of H2O2 and O2·- were detected both in plants with mutations of conservative cysteines in VKOR domain and Trx-like domain, keeping similar levels with that of vkor mutant line, increased about 56% of At VKORWT; the levels of H2O2 and O2·- in mutations of nonconservative cysteines were quite closed to that in AtVKORWT plants. The results further indicated that the photoprotection mechanism was impaired in the transgenic plants with mutations of conservative cysteines, which might affect the turnover of D1 protein.All the results showed that the conservative cysteines were the functional sites of AtVKOR in vivo; especially the conservative cysteines in VKOR domain played a vital role in the function of At VKOR in plant. The functions of AtVKOR in assembly of PSII, activity of PSII, turnover of D1 protein, and homeostasis of ROS metabolism are related to the oxidoreductase activity of At VKOR. The results provide new evidences for the study of chloroplast redox regulation mechanism.