Molecular Mechanism Of OsLIL3 In Regulating Chlorophyll Synthesis In Rice

Grain yield of rice,one of the most important crops in the world,mainly comes from photosynthesis of leaves.Chlorophyll(Chl)is an essential pigment involved in photosynthesis in chloroplast.Understanding of the Chl synthesis mechanism is benefit for the improvement of light use efficiency and yield potential in rice.Light-harvesting-like(LIL)protein belongs to light-harvesting complex(LHC),a key component in plant photosynthetic mechanism.In this study,we developed the CRISPR/Cas9 mutant of OsLIL3 gene and investigated its roles in rice Chl synthesis.The main results are as follows;1.We previously reported that mutation of LYL1,encoding a geranylgeranyl reductase(GGR),caused deficiency of Chl biosynthesis in rice.In Arabidopsis,GGR associates with LIL3 and thus contributes to the Chl biosynthesis.However,in yeast two-hybrid system,no such interaction was found between LYL1 and OsLIL3 in rice.2.CRISPR/Cas9 was employed to develop the mutant of OsLIL3 gene in the Nipponbare background.Only one homozygous mutant,oslil3-1,was identified,A 6-bp deletion in the OsLIL3 gene caused three amino acid changes in the oslil3-1 mutant.But all the other mutants were heterozygous.We proposed that knockout of OsLIL3 may lead to death of the seedlings.3.At tillering stage,we measured the Chl contents of the upmost three leaves in wild-type(WT)and oslil3-1 plants.Chl contents of the first(youngest,upper position),second,and third(oldest,lower position)leaf blades in the oslil3-1 mutant were 70.3%,40.5%and 34.5%of those in WT,respectively.These data suggested that mutation of OsLIL3 decreased the synthesis of Chl in rice,especially in the old leaves.4.Ultrastructure of the chloroplast were observed using transmission electron microscope.Compared with WT,the thylakoid stacking was decreased while the plastid spherule were increased in the oslil3-1 mutant.This suggested that mutation of OsLIL3 gene resulted in abnormal chloroplast development.5.Nitro Blue Tetrazolium(NBT)staining showed that the accumulation of reactive oxygen species(ROS)in old leaves was higher than that in young leaves.The mutation of OsLIL3 gene accelerated ROS accumulation,which is one of the reasons for leaf chlorosis in the oslil3-1 mutant.6.Continuous and high light decreased the Chl content of the oslil3-1 mutant,suggested that light caused leaf chlorosis in the oslil3-1 mutant.Western Blot indicated that the protein levels of Lhcal,Lhca2,Lhcb2 and RbcL were declined in the oslil3-1 mutant.We concluded that the mutation of OsLIL3 gene decreased the stability of photosynthesis protein under light conditions.7.qPCR analysis indicated that the expression of OsLIL3 was rapidly induced by light.However,continuous light also suppressed the transcripts of OsLIL3.The expression levels of OsLIL3 in old leaves were lower than those in young leaves.In addition,the OsLIL3 mRNA levels in the top section of leaves were significantly lower than those in the middle section.