Map-based Cloning And Functional Analysis Of The ATP-binding Cassette Transporter OsABCI7 In Rice

Chloroplast is the main organ of plant photosynthesis.The study of its development mechanism and function is helpful to excavate the photosynthetic potential of crops and improve crop yield.The chlorotic and necrotic leaf 1（cnl1）mutant which was isolated from an ethane methyl sulfonate（EMS）-induced indica rice Zhongjian100 mutant bank was used for the research material.Subsequently,we performed phenotype characterization,physio-biochemical and genetic analysis of cnl1.We also cloned the target gene and performed functional analysis of the target gene.The main results of this study were as follows:1)Under the natural field conditions,the cnl1 mutant exhibited chlorotic and necrotic phenotypes approximately 40 days after sowing on the older leaves.At the later growth period,this phenotype became more severe.Until to the heading stage,all leaves of cnl1 plant were obviously chlorotic and necrotic,and the older leaves even displayed wilted phenotype.Compared to the WT,the chlorophyll contents of cnl1 leaves were significantly decreased,and distinct chloroplast degradation especially thylakoid membrane degradation occurred in cnl1.Furthermore,over-accumulated ROS and disturbed antioxidative system were detected in in cnl1,and the exogenous antioxidant（ascorbic acid）can alleviate the phenotypes of cnl1.2)Genetic analysis and gene mapping revealed that the phenotypes of cnl1 were controlled by a single recessive nuclear gene.And this target gene（LOC＿Os11g29850）is located at the 17,329,608–17,332,992 bp of chromosome 11,encoding an ATP-binding cassette transporter which belongs to the member 7 of the 9^th subgroup（subgroup I）of ATP-binding cassette transporter superfamily,thus termed as OsABCI7.The sequencing results revealed a substitution followed by 6 base-pairs deletion at the junction of the 8^th exon and 8^th intron of LOC＿Os11g29850,resulting a frame shift and premature stop codon.Complementation,overexpression and CRISPR/Cas9 knockout tests demonstrated that the mutation of OsABCI7 was responsible for the phenotypes of cnl1.3)By using the yeast-two-hybrid（Y2H）strategy,we screened out OsHCF222 which interacts with OsABCI7.And the Y2H assay,bimolecular fluorescence complementation assay and co-immunoprecipitation assays demonstrated the interactions of OsABCI7 and OsHCF222 in vivo and vitro,while the mutant OsABCI7 cannot interact with OsHCF222.OsABCI7 was localized at the thylakoid membrane,while OsHCF222 was dually targeted to the chloroplast and ER.In addition,homozygous OsHCF222 knockout plants displayed a leaf pale yellow phenotype,accompanied by excessive accumulation of ROS.Due to the disability of photoautotrophy,homozygous OsHCF222knockout plants were seedling-lethal.OsABCI7 plays an important role in the regulation of chloroplast development especially for the chloroplast development at low temperature and in the seedling greening process.In addition,compared with the wild-type,RNA sequencing results showed that a large number of genes related to chloroplast development in cnl1 were disrupted,suggesting that the function of OsABCI7 may be involved in a variety of chloroplast development regulation networksIn conclusion,our results indicated that lack of OsABCI7 led to the mutant phenotype of cnl1.OsABCI7 interacts with OsHCF222 to form an OsABCI7/OsHCF222 complex targeting to thylakoid membranes in chloroplasts,regulating the balance of antioxidative system on thylakoid membrane to maintain the stability of thylakoid membrane.The formation of OsABCI7/OsHCF222 complex was disturbed in cnl1,which resulted in the breakdown of antioxidative system balance on thylakoid membrane.Subsequently,the ROS were over-accumulated and the normal thylakoid membrane structures were damaged,which induced a series of physiological and biochemical changes,like cell death and chlorophyll degradation,and finally caused the mutant phenotype of cnl1.