Gene Cloning And Mechanism Analyzing Of Photorespiratory Mutant Pr2 In Arabidopsis Thaliana

Photorespiration is the biochemical process that photosynthetic organisms absorb O₂ and consume organic matter under light conditions.Photorespiration which is always companied with photosynthesis is a complex metabolic process that takesplace in the chloroplasts,peroxisomes,mitochondria and cytoplas m,forming a closed recirculation flow pathway.It was believed that reduced p hotorespiraroty metabolic flux would improve the net photosynthetic rate and therefore enhance crop products,while it is not available by the evidence in recent studies.Large numbers of studieshave shown that photorespiration is necessary for plant photosynthesis,development and growth in aerobic conditions.Photorespiration is also involved in the synthesis of one-carbon units and glutathione,and the nitrogen metabolism process andanti-oxidative redox system in plants.In this study,Arabidopsis Columbia?Col-0?was used as the experimental materia with random mutagenesis by EMS.Typical photorespiratory phenotype is chlorotic,the leaf-yellow and even lethal in the normal environment,but recover in high CO₂environment.The mutant pr2 was selected by this principle.Phenotype and physiological analysis showed that pr2 is chlorotic,leaf-yellow and curly phenotype in normal environment,but the phenotypic can be recovered under high CO₂ conditions.The pr2 and Ler were crossed and the F₂ population with photorespiration phenotype was choosed for map-based cloning of pr2 gene.The result showed that mutation site located in the 0.98 Mb-1.95 Mb of chromosomes 3.The whole gene sequencing results showed that the mutant gene was Fts Hi5?Filamentation temperature-sensitive H,the''i''indicates proteolytic inactivation?.When the FtsHi5 genome was reintroduced into the mutant pr2,the transgenic plants showed similar phenotype with Col-0 in the normal environment.Using FtsHi5Pro::GUS transgenic plants,histochemical analysis showed that FtsHi5 mainly expressed in the development fruit pods and flowers.q RT-PCR confirmed the results from another side.Moreover,FtsHi5 expression was induced by light.35S::FtsHi5-GFP fluorescence analysis showed that FtsHi5 was localized in chloroplast envelope.Dexamethasone-induced Fts Hi5-RNAi transgenic plant experiment showed that reduced FtsHi5 expression weakened leaf color in a dose-dependent manner.The 35S::FtsHi5 transgenic plants showed variegated phenotypes compared with Col-0.qRT-PCR analysis showed that the expression of FtsHi5 was not increased,but decreased in transgenic plants.Physiological and biochemical analysis showed that pr2 mutant had higher Ser contents and ROS levels compared with Col-0 in normal condition.The activity of POD and content of GSH was lower in pr2 mutant..In summary,a photorespiratory mutant pr2 was screened.The whole gene sequencing and complementary experiment suggested that the mutated gene is FtsHi5.FtsHi5 is localized in the chloroplast envelope.The supression of FtsHi5 expression in RNAi transgenic plants changed the leave color.The levels of ROS in pr2 mutant and RNAi-FtsHi5 transgenic plants were significantly higher than that in wild type.The mechanism of how FtsHi5 affects photorespiration remains unknown.