Map-based Cloning And Functional Analysis Of Two Genes Controlling Yellow-green Leaf In Rice

Leaf color is one of the most common mutations in rice(Oryza sativa).In the basic research,leaf-color mutants are the ideal materials for investigating development-related processes such as photosynthesis,photomorphogenesis and mechanism of deseaseresistantce.By using leaf-color mutants,lots of new genes have been cloned,and been explored for their interactions and mechanism of regulation in photosynthesis,which provides theoretical basis for breeding of high photosynthetic efficiency.1 Map-based cloning and functional analysis of the gene controlling the yellow-green leaf phenotype of 637 ys mutantPhytyl residues are the common side chains of chlorophyll(Chl)and tocopherols.Geranylgeranyl reductase(GGR)is responsible for phytyl biosynthesis.The lightharvesting like protein LIL3 was suggested to be required for stability of GGR and protochlorophyllide oxidoreductase in Arabidopsis(Arabidopsis thaliana).In our previous study,we obtained a yellow-green leaf mutant 502 ys,in rice,which was resulted from the single nucleotide mutation of Os CHL P encoding Os GGR and accumulated majority of Chls with unsaturated geranylgeraniol side chains.In this study,we isolated another yellow-green leaf mutant,637 ys,in rice,which exhibited similar phenotype to 502 ys.The 637 ys mutant accumulated majority of Chls with unsaturated geranylgeraniol side chains as well.The development of chloroplasts was suppressed,and the major agronomic traits,especially No.of productive panicles per plant and of spikelets per panicle,dramatically decreased in 637 ys.Map-based cloning and complementation experiment demonstrated that a point mutation on Os LIL3 gene accounted for the mutant phenotype of 637 ys.Os LIL3 is mainly expressed in green tissues and its encoded protein is targeted to the chloroplast.637 ys and the chl p mutant,502 ys,exhibited similar characteristics,such as Chl compositions,sensitivity to temperature and light intensity,deficiency of tocopherols in leaves and grains,and alteration in expression of Os LIL3,Os CHL P and 18 genes involved in photosynthesis and Chl biosynthesis.Furthermore,the 637 ys 502ys(lil3 chl p)double mutant exclusively accumulated geranylgeranyl Chl and exhibited lethality at the three-leaf stage.Taken together,we demonstrated that Os LIL3 is of extreme importance to the function of Os GGR,and that the complete replacement of phytyl side chain of chlorophyll by geranylgeranyl chain could be fatal to plant survival in rice.2 Fine mapping and functional analysis of the gene controlling the yellow-green leaf phenotype of 501 ys mutantFerredoxins(Fds)are small iron-sulfur proteins that mediate electron transfer in a wide range of metabolic reactions.Besides Fds,there is a type of Fd-like proteins designated as Fd C,which have conserved elements [2Fe-2S] clusters of Fds,but contain a significant Cterminal extension.So far,functions of Fd C proteins still remain unfamiliar,because only two Fd C genes of Arabidopsis have been identified in higher plants.In previous study,we obtained a yellow-green leaf mutant 501 ys in rice,and located 501 ys in the 120 kb region between In Del markers C3 and C6 with 0.31 c M on the long arm of chromosome 3.The 501 ys mutant exhibited yellow-green leaf phenotype and reduced chlorophyll level.In this study,we performed fine mapping for 501 ys mutant,and further narrowed down the 501 ys locus in the 39.1 kb region between In Del markers C4 and C5 with 0.08 c M and 0.23 c M,respectively.The sequencing results showed that,in 501 ys mutant,a single nucleotide mutation occurred in LOC_Os03g48040 gene,which resulted in an amino acid change in its encoded protein.The encoded protein shows high identity with Arabidopsis Fd C2(AT1G32550)and therefore the gene was designated as Os Fd C2.Expression pattern results showed that Os Fd C2 is expressed most abundantly in leaves and subcellular localization assay indicated that its encoded protein is targeted to the chloroplast.The mutant phenotype was rescued by introduction of the wild-type Os Fd C2 gene.Therefore,we demonstrated that mutation of this gene caused yellow-green leaf phenotype in rice.In total,first,we succeeded to identify Os LIL3 gene through a map-based cloning approach,and confirmed that the yellow-green leaf phenotype of 637 ys was resulted from the mutation on Os LIL3.Furthermore,we demonstrated that Os LIL3 is of extreme importance to the function of Os GGR,and that the complete replacement of phytyl side chain of chlorophyll by geranylgeranyl chain could be fatal to plant survival in rice.Second,we successfully identified Fd C2 gene via map-based cloning approach,and demonstrated that mutation of this gene caused yellow-green leaf phenotype of 501 ys mutant in rice.