Map-based Cloning And Physiological Analyzing Of Photorespiratory Mutant Pr1 In Arabidopsis Thaliana

Photorespiration is a biological process only second to photosynthesis if considered in term of mass flow in C3 plant, which consumes amount to about 20-30% of net-photosynthesis in moderate conditions. Undoubtedly photorespiration restricts the efficiency of photosynthesis and, ultimately, reduces crop yield. However, extensive research suggests that scavenging or modification of photorespiration cannot effectively improve photosynthesis rate in plants, but reduce photosynthesis rate or even lead to a fatal phenotype, indicating photorespiration relates not only to photosynthesis, but also to many other metabolic pathways in plants. Currently, photorespiration pathway is reported to relate closely to nitrogen assimilation, respiration, amino acid metabolism, one-carbon metabolism, redox signaling and other metabolic processes. Screening photorespiratory mutants and cloning new gene regulating photorespiration will contribute to further understand photorespiration pathway and its relation with other metabolism pathways.In this study, a mutant library was established by treating Arabidopsis Columbia-0(Col-0) seeds with chemical mutagenesis ethyl methanesulfonate(EMS). The photorespiratory mutants were screened by photorespiratory phenotypes, that is, mutants show chlorotic, dwarf leaves or even fatal phenotype in ambient air condition while which can be recovered in high CO2 air condition. A photorespiratory mutant pr1(Photorespiration related 1) was screened and physiological, molecular and genetics analysis about the mutant were carried out. The results are listed as fowling:1. Phenotype and Physiological Analysis: pr1 mutant displayed dwarf and chlorotic leaves compared to Col-0 in ambient air, while the phenotype showed no obvious difference in high CO2 air condition between pr1 and Col-0. The glycine content was accumulated in pr1 mutant in ambient air condition.2. Gene Mapping: The mutant pr1 and Landsberg erecta(Ler) were crossed to construct a collection of F2 which were used to map the mutated gene. Map based cloning showed that the mutated gene located on a region about 17.97 Mb of chromosomes 4. The whole genome sequencing of mutant pr1 showed that the mutated gene was AT4G38380, which encodes the protein of multidrug and toxic compound extrusion and is named MATE1 in this study.3. Complementary Experiment: The proMATE1::MATE1-Flag vector was constructed and then transformed into pr1. The transgenic lines recovered to normal growth compared with the pr1 mutant in ambient air conditions.4. Tissue Expression: The proMATE1::GUS vector was constructed and then transformed into Col-0. Analysis of GUS staining revealed that MATE1 mainly express in new leaves, shoot meristem, roots, anther and legumes. Real-time PCR suggested that expression of MATE1 dramatically increased by 4 h of intensive light.5. Subcellular Localization: The 35S::MATE1-GFP vector was constructed and then transformed into protoplast of Col-0. Analysis of GFP fluorescence indicated that MATE1 was localized to chloroplast envelope.6. The T-DNA insertion mutant of mate1-1(CS300818) was identified and which showed dwarf shape, curled leaves and low fertility.7. The 35S::MATE1 vector was constructed and then transformed into Col-0. The biomasses of transgenic lines were largely improved if compared with Col-0.In summary, a photorespiratory mutant pr1 was screened from EMS-mutated seed library in this study. Map-based cloning and whole genome sequencing analysis and complementary experiment suggested that the mutated gene was MATE1. MATE1 are localized on the chloroplast envelope and mainly expressed in roots and leaves. The T-DNA insertion mutant of mate1-1 shows obvious developmental defects. The overexpressed transgenic MATE1 plants have higher biomass than Col-0. The mechanism of how MATE1 affects photorespiration remains unknown.