Map-based Cloning And Functional Analyses Of Rice Oscrd1 And Ospsf1

Rice is the main food source for more than half of the global population,and it's essential to increase crop yield to meet the increasing food demand.Crop yield is affected by photosynthesis,and plant leaf color changes directly affect photosynthesis.Leaf color related mutants are invaluable materials to study photosynthesis.Therefore,it is of great value by cloning new rice photosynthetic-related genes and further surveying their function.In this study,we obtained two leaf color mutants by screening EMS mutagenized rice pool and studied their function,the main results are as follows.1.Rice mutant m167,with yellow-green leaves throughout the whole developmental stage,carries a single gene recessive mutation in Os CRD1 gene.The m167 mutant shows defficency of chlorophyll,block of chloroplast development and reduced photosynthesis rate,in addition,m167 exhibits decreases in plant height,tilling number,seeding setting rate,and 1000-grain weight.By map-based cloning,we mapped the candidate gene in oscrd1 to be OsCRD1(Oryza sativa dicarboxylate diiron protein),with a G to A point mutation at position 286 in the first exon,changing the amino acid at position 96 from alanine,a non polar amino acid,to threonine,a polar amino acid.OsCRD1,putatively encoding a subunit of Mg Protoporphyrin ? monomethyl ester enzyme,with a single copy in rice genome,mainly expresses in the green parts of plants,and OsCRD sequences are highly conserved in different rice varieties and species.We got OsCRD1 knock-out transgenic rice seedlings by CRISPR/Cas9 directed genome edition.The transgenic rice seedlings were albino,indicating OsCRD1 knockout lead to abnormal leaf color,so we can infer that yellow-green leaves in oscrd1 mutant result from the site mutation in OsCRD1.OsCRD1 is mainly localized in chloroplast and the point mutation in Os CRD1 does not affect its cellular localization.By determination of intermediates in chlorophyll biosynthesis,compared to WT,Mg protoporphyrin ? monomethyl ester were increased by 44% in oscrd1 mutant.However,Pchlide was decreased by about 30% in oscrd1 mutant.Therefore,we speculate that OsCRD1 might involve in chlorophyll biosynthesis by catalyzing Magnesium protoporphyrin ? monomethyl ester to pchlide.2.Rice mutant ht15-4(Oryza sativa photosynthesis factor1),obtained by EMS mutagenesis,is light yellow in leaf color during the whole growth period.Genetic analysis shows that the lower chlorophyll content of ht15-4 mutant is controlled by a recessive mutation site in nucleus.We mapped the mutation site in ospsf1 mutant to be a point mutation from T to G at the position 284 of Os PSF1,with an amino acid changing from leucine to arginine.OsPSF1 encodes a putative ankyrin domain containing protein.The light yellow leaves of ospsf1 mutant changes into wild type green leaves when wild type OsPSF1 gene is expressed in ospsf1 mutant.The complementation experiment implies that the site mutation in OsPSF1 coding sequence lead to pale yellow phenotype in ospsf1 mutant.At normal conditions,the net photosynthesis rate displays no difference between OsPSF1 overexpression transgenic rice lines(OX1,2,3)and wild type.After methyl viologen treatment,the net photosynthesis rate in transgenic plants(OX1.2 and 3)is much higher than that of the wild type.This indicates that overexpression of OsPSF1 gene in rice can improve the photosynthetic rate under oxidative stress.