Functional Analysis Of Arabidopsis Thaliana VkOR At The Assembly Of Photosystem, Signal Transduction And Redox Regulation In Chloroplasts

Redox regulation in plant chloroplasts plays important role in growth and development, gene expression, cell differentiation and apoptosis, signal transduction and stress resistance. The reversible conversion between thiol and disulfide bond is one of the major means of redox regulation in plant cells as a typical oxidoreduction reactions. The reversible conversion between thiol and disulfide bond is enzymatic reactions catalyzed by thiol oxidoreductase. In chloroplasts, enzymes reduced regulatory disulfides are well studied, while the identities of the thio-oxidizing catalysts are currently not fully known. Identification and functional analysis of thio-oxidizing enzymes will help to fully understand redox regulation of the reversible conversion between thiol and disulfide bond and its role in protein transporting, oxidative folding, the assembly and repair of thylakoid complexes as well as signal transduction in chloroplasts.Recently, vitamin K epoxide reductase (VKOR) is found as a novel oxidoreductase in plants. The Arabidopsis VKOR(AtVKOR) can catalyze formation of disulfide bond in prokaryotic cells. However, the function of AtVKOR gene is not well known in plant cells. The homozygous plants of Arabidopsis VKOR deletion mutant were selected and the transgenic complementary plants were identified. The role of AtVKOR in photosynthesis, signal transduction and oxidoreduction of proteins was clarified by using wild type, transgenic complementary plants and homozygous mutant plants through two-dimensional electrophoresis, western blot, yeast two-hybrid assay and qRT-PCR. The main results are as follows:(1) AtVKOR played an important role in the growth and development of plants.Two homozygous mutant lines of AtVKOR deletion were selected. Growth of homozygous mutant was severely stunted with small and pale green leaves, and blossom was delayed. These results showed that the AtVKOR gene was very important for the growth and development. The AtVKOR gene was transformed to wild type Arabidopsis and the transgenic plants were selected. Transgenic complementary plants were selected after hybridizing between transgenic plants and mutant plants. The phenotype of the transgenic complementary plants and the expression level of the AtVKOR gene were similar with those of wild type indicating that the phenotype of mutant was attributable to the mutation of AtVKOR gene. (2),AtVKOR was localized in the thylakoid membrane. AtVKOR was localized in the chloroplast by using green fluorescence protein (GFP) labeling. In this study, the intact chloroplasts were abstracted and fractionated into stroma and thylakoid fractions. The stromal Rubisco activase and thylakoid D1proteins were selected as the control. Western blot showed that AtVKOR was located in the thylakoid and the founctional domain faced to the thylakoid lumen. The results will be helpful to study the function of AtVKOR and to identify its targets.(3) The quantities of many proteins were affected by AtVKOR. To study the effect of AtVKOR deficiency to the growth and development, the proteomic of wild type and mutnt plants were compared and analyzed. The result showed that eleven proteins were up-regulated containing Ribulose bisphosphate carboxylase subunits and twelve proteins were down-regulated containing the subunit of photosystem Ⅱ (PSⅡ) and some reactive oxygen (ROS) scavenging enzymes. The transcriptional level of PsbPl, encoding a subunit of PSⅡ, was down-regulated about50%and the mRNA of APX1, CAT2and DHAR1, encoding three ROS scavenging enzymes, were reduced about20%. These results suggested that AtVKOR may affect the function of PSⅡ and be involved in ROS metabolism.(4) Deficiency of AtVKOR led to severe defects in photosynthetic activities of PSⅡ and to accelerate the degradation of core protein D1of PSⅡ. The main parameters of chlorophyll fluorescence representing the function of PSⅡ, maximum quantum yield of PSⅡ (Fv/Fm), effective quantum yield of PSⅡ ((?)PSⅡ) were significantly decreased in mutant compairing with those of wild type and complementary plants. And the quantity of D1was only about half as much as in wild-type, but the expression of PsbA encoding D1protein was not affected. The results suggested that deficiency of AtVKOR led to accelerate the degradation of core protein D1. The thylakoid complexes were analyzed and showed that the amount of PSⅡ dimer and supercomplexes decreased significantly. The result showed that the assembly of PSⅡ was significantly affected by the absence of AtVKOR resulting in the decrease of PSⅡ activity.(5)At VKOR was involved in ROS metabolism and ABA signal transduction under stress conditions. The amount of ROS was significantly higher in mutnat plants than those of wild type and complementary plants under high light, salt drought and ABA indicating that AtVKOR was involved in ROS metabolism. AnnAt1can scavenge ROS and the AnnAtl gene is typically up-regulated by ABA and salt, drought stress in wild type and complementary plants. The transcriptional and translational levels of AnnAtl were changed slightly after salt and drought treatments, but the expression of AnnAtl was significantly up-regulated by ABA. The change of transcriptional level of RD29B, another typical ABA responsive gene, was consistent with that of AnnAtl. These results indicated that AtVKOR may be involved in the transduction of ABA under stress conditions.(6) The lumenal proteins interacting with AtVKOR were selected and identified. The functional domain of AtVKOR. catalyzing disulfide bond formation faced to thlakoid lumen demonstrated by its topological structure. Twenty-two luminal proteins may contain disulfide bonds were selected as potential targets. The genes encoding these proteins were constructed into AD vector and the AtVKOR gene was constructed into BD vector as bait. Eight proteins were identified through yeast two-hybrid assay at last. Some proteins were expressed by prokaryotic system and were purified. The AtVKOR could interact with some luminal proteins and imply that AtVKOR can regulate redox state of proteins located in thylakoid lumen in vitro.The functional analysis of AtVKOR provides new evidence for the study of regulation mechanism of chloroplast redox regulation in plant growth and development and mechanism of plants againt abiotic and biotic stress.