Map-based Cloning And Functional Analysis Of PGL10 Gene Regulating The Biosynthesis Of Chlorophyll In Rice

Rice pale-green leaf(pgl)mutants are often characterized to exhibit the abnormal development of chlorophyll(Chl)and chloroplast structure resulting in enhanced accumulation of reactive oxygen species(ROS)and decreased photosynthesis rate due to less development of Chl pigment.Here,we reported cloning,characterization and a novel function of pale-green leaf mutant,pgl10,under high temperature in rice.pgl10 mutants were derived from the ethyl methanesulfonate mutagenesis of indica rice cv.Zhongjiazao-17(YK-17)and further classified into pgl10a and pgl10b on the basis of two different mutations in single recessive gene,PGL10.We carried out:a)phenotypic characterization,b)physio-biochemical characterization,c)detection of cell death occurrence and ROS accumulation,d)evaluation of mutants under heat stress,e)genetic analysis and Mutmap-assisted mapping of the mutation,f)functional complementation via gene complementation assay,CRISPR/Cas9 and overexpression,and g)identified the subcellular localization of PGL10 protein(LOC?Os10g35370)in this study.This study also provides the evolutionary evidence and the role of PGL10 in Chl synthesis and physio-biochemical changes exhibited by pgl10a and pgl10b mutants under heat stress.The main findings obtained in this study are as follow:1-PGL10,which encodes a Protochlorophyllide oxidoreductase(POR)B enzyme,stimulated the biosynthesis of Chl under light conditions.Both mutants,pgl10a and pgl10b,exhibited a lower concentration of Chl pigments and showed less developed and reduced thylakoid ultrastructure due to which the net photosynthetic rate and rice grain yield were significantly reduced.2-pgl10a and pgl10b mutants exhibited more severe Chl degradation and ROS accumulation than the Wild-type(WT),YK-17.In response to the accumulation of ROS species,mutant plants were subjected to high temperature stress(32?)and found that plants tried to maintain their redox balance by producing antioxidants.However,the mechanism behind this resistance development is yet to elucidate.3-The transient expression of fluorescent protein in rice protoplast and epidermal cells of tobacco(Nicotiana benthamiana)leaf was used to determine the subcellular localization of PGL10 protein.Both experiments confirmed the localization of PGL10 in chloroplasts of the cells.4-Quantitative real-time PCR results revealed that PGL10 had significant low expression at 3^rd leaf stage followed by 4^th leaf,2^nd leaf and 1^st leaf in both mutants pgal10a and pgl10b except in 2^nd leaf in pgl10b which showed higher expression than 2^ndleaf of pgl10a.It was observed that the PGL10 expression was significantly lower in mutants'leaves compared with WT.Likewise,the genes involved in Chl synthesis were also downregulated in pgl10a and pgl10b mutants.5-Transmission electron microscopy analysis of pgl10 mutants'leaf tissues at seedling stage showed disorderly small chloroplast ultrastructure.Thylakoids were thin and poorly developed inside chloroplast of pgl10a and pgl10b mutants compared to WT.At high temperature the structure and number of chloroplasts were reduced in both mutants relative to WT.These results indicate the role of PGL10 in the development of chloroplast and thylakoids and ultimately Chl biosynthesis under heat stress conditions.6-RNA-seq analysis showed that the PORB(PGL10)enzyme was involved in light-dependent Chl biosynthesis pathway as it was downregulated during the biosynthesis of chlorophyllide from protochlorophyllide.Total 5,651 differentially expressed genes were identified,out of which 3,463 genes were upregulated whereas 2,188 genes were downregulated in comparison between WT and pgl10a mutant.7-Kingdom-wide phylogenetic analysis showed that PGL10(PORB)was evolved 119.68 million years ago(MYA)from Chlamydomonas(a unicellular green alga micro-organism).More specifically,the species-wide analysis of the POR family revealed that PORB was evolved earlier than PORA in rice species.PORB gene was located on chromosome 10,whereas PORA on chromosome 4 in all species.Overall,our findings suggest that PGL10 is involved in the development of chloroplasts and Chl,and evolved in rice species from unicellular alga about 119.68 MYA.Heat stress downregulates its activity and upregulates the accumulation of ROS and expression of ROS detoxification genes.This study also provides the foundation for further understanding on the mechanisms involved in development of chloroplasts and Chl under heat stress.