The Arabidopsis Thaliana VKOR/LTO1Is Involved In The Mechanism Of Photoprotection

When absorption by chlorophylls exceeds the capacity for energy utilization in photosynthesis, it will lead to photoinhibition or photodamage. To avoid photoinhibition, photosynthetic organisms have developed various photoprotective mechanisms including energy dissipation depending on the xanthophyll cycle and the D1protein of the PSII reaction center turnover. Violaxanthin de-epoxidase (VDE) is an important enzyme in xanthophyll cycle. VDE is localized in the thylakoid lumen and the formation of disulfide bond is essential to the activity of VDE. Research results show that Arabidopsis Vitamin K epoxide reductase (VKOR) is involved in disulfide bonds formation. The redox activity of VKOR is indispensable for the assembly of PSII. Whether VKOR regulated the VDE-mediated xanthophyll cycle and D1protein turnover in the photoprotective pathway is not clear, and it is important significance to further illuminate the photoprotective mechanisms. Here, the wild type (WT), vkor-2mutant and transgenic complementation (vkor-2C) Arabidopsis plants were used as material, and dithiothreitol (DTT), an inhibitor of violaxanthin de-epoxidase (VDE) in xanthophyll cycle and an inhibitor of chloroplast protein synthesis (streptomycin sulphate, SM) were used to investigate the relationship between VKOR gene and xanthophyll cycle and D1turnover under different light irradiance condition. The main results are as follows:(1) VKOR protein deficiency impacted the photosynthetic activity of PSII and aggravates photoinhibition. The maximum efficiency of the PSII photochemistry (Fv/Fm) and the effective quantum yield of PSII (ΦPSII) can reflect the activity of PSII reaction center. Under growth light conditions, Fv/Fm of wild type (WT) was0.810±0.04; however, Fv/Fm of vkor-2was0.53±0.03. When the leaves were exposed to an irradiance of1000μmol·m-2·s-1, vkor-2leaves were more pronounced decrease in Fv/Fm than WT. Changes in the ΦPSII ratio followed a similar trend to the Fv/Fm ratio. Transgenic complementation (vkor-2C) had a similar trend with WT. This result showed that the vkor-2mutant had a severe photoinhibition.(2) The deficiency of VKOR affected energy dissipation depending on the xanthophyll cycle. Under high light condition, plants can dissipation of excess light energy by non-photochemical quenching (NPQ). NPQ is an important excitation energy dissipation mechanism and needs the participation of the xanthophyll cycle. After light irradiance, the NPQ values were lower in the vkor-2mutant than in WT plants. After DTT depressed the xanthophyll cycle, the NPQ significantly decreased. The extent of the reduction in NPQ in the vkor-2mutant was less than in the wild type. vkor-2C ultimately recuperated the characteristic changes described above. These results showed the deficiency of VKOR affected energy dissipation depending on the xanthophyll cycle.(3) VKOR was involved in the regulation the state redox of VDE and affected energy dissipation depending on the xanthophyll cycle. Zeaxanthin (Zea) is involved in dissipation of excess light energy. Epoxide xanthophyll violaxanthin (Vio) is converted via the intermediate antheraxanthin (Ant) to the de-epoxide zeaxanthin (Zea) under the action of VDE. To gain insight into research whether regulated VKOR the activity of VDE. We identified changes in the concentrations of Vio, Ant and Zea using high-performance liquid chromatography (HPLC). The de-epoxidation state of the xanthophyll cycle pigments was described using the de-epoxidation index (DEI)[(Ant+2Zea)/(Vio+Ant+Zea)]. DEI can reflect the activity of VDE. When leaves were exposed to1000μmol·m-2·s-1irradiance, the DEI values were lower in the vkor-2mutant line than in the WT and vkor-2C. The DTT treatment caused significantly decrease in DEI, and the extent of decrease was smaller in the ltol-2mutant line than in WT and vkor-2C plants. Results showed that the deficiency of VKOR affected the activity of VDE, led to the extent of the reduction in NPQ.(4) VKOR protein deficiency accelerated the degradation of D1protein and affected the photoprotective pathway of D1turnover. High light stress can lead to a partial inactivation of PSII reaction center. D1turnover can restore the inactive reaction center. After SM treatment, under growth light or high light condition, the decline in Fv/Fm in the vkor-2mutant was more than the decline in the WT. The result suggested that the degrade D1protein of PSII reaction center in the vkor-2mutant was more than those of WT. To gain insight into the degradation of the D1protein more directly, the quantity of D1protein in the ltol-2mutant, the WT and vkor-2C plants were analyzed by western-blotting. Under growth light, no-SM treatment, D1protein had only about half as much as in wild-type. After SM treatment, D1protein in the PSII centers induced by high light levels was rapidly degraded. There were only trace amounts of D1protein in the lto1-2mutant and the content of D1protein was far less than half WT and vkor-2C.