Gene Cloning And Functional Analysis Of Pale Green Leaf Mutant Pgl21 In Rice

Leaf color mutants are ideal materials for researching chloroplast development,chlorophyll synthesis and photosynthesis in plants.Although many leaf color related genes have been reported in rice,the leaf color regulatory network is still not complete due to the complex genetic regulation.In this study,a pale green leaf mutant pgl21 was isolated from EMS mutant library of Nipponbare,and phenotypic identification,genetic analysis,gene cloning and functional analysis of mutants were carried out.The main results are as follows:1.The mutant pgl21 showed pale green leaves during the entire growth stage.The contents of total chlorophyll,chlorophyll a,chlorophyll b,total carotenoids,carotene and lutein in pgl21 were significantly lower than those in the wild type.The expression of most genes related with chlorophyll synthesis and photosynthesis in pgl21 decreased significantly.In addition,the seed setting rate,thousand-grain weight and grain number per panicle of pgl21 decreased significantly.2.It was found that the maximum photosynthetic efficiency(Fv/Fm)and the electron transfer rate of pgl21 was significantly lower than that of wild type though the chlorophyll fluorescence parameters assay,which means the ability of photosynthesis was weaker than that of wild type.3.The results of transmission electron microscopic observation of chloroplasts showed that pgl21 had abnormal chloroplast structure,no obvious thylakoid stacking structure,and contained many plastid globules.The integrity of chloroplast structure in pgl21 was destroyed.4.Genetic analysis showed that the mutant phenotypes of pgl21 was controlled by a single recessive nuclear gene.LOC＿Os02g43500,annotated as ORANGE(OR),was preliminarily determined as the candidate gene of pgl21 by gene mapping and Mut Map resequencing.5.OR has 1 homolog,LOC＿Os02g33149,in rice.OR and its homolog were knocked out in the background of Nipponbare,and 2 single knockout lines of OR(KO-2 and KO-4)and 1 double knockout line(KO-3)were obtained.The contents of related pigments in KO-2 and KO-4 were similar to those in pgl21,which were significantly lower than wild type.At the same time,the integrity of chloroplast structure of knockout lines was destroyed observed by transmission electron microscope,which was similar to that of pgl21.6.RT-q PCR and GUS analysis indicated that the expression of OR was higher in rice seedlings,mature leaves,internodes and stems,but lower in young panicles,roots and leaf sheaths.7.Lost of function of OR in pgl21 increased the expression of the homologous gene,while the double knockout line KO-3 showed severe albino and death,indicating that OR and its homolog may play a cooperative role and have functional redundancy.8.The expression of OR gene decreased while the expression of the homologous gene increased during dark germination.After light treatment,the expression of both genes upregulated and then gradually returned to normal.These results indicated that OR and its homolog may play important roles in the process of chloroplast differentiation under light condition.