Genetic And Physiological Studies On Yellow Leaf Mutants In Rice

Rice leaf color mutants are ideal materials for exploiting photosynthetic mechanism and creating germplasm with high photosynthetic efficiency in the breeding.In this study,two mutants namely yl（t）（Yellow leaf（t）,yl（t））and yllt1（Yellow leaf Less Tiller1,yllt1）were investigated in the phenotype,physiological characteristics,genetic mechanism and common metabolic characteristics.The main findings are shown as follows:1.At the seedling stage,yl（t）presented yellow leaves with a significant decrease of53.02%,55.35%and 46.88%in the contents of total chlorophyll,chlorophyll a and chlorophyll b respectively,compared with T98B.When it grew to mature,the chlorophyll content increased.Further genetic analysis showed yl（t）was controlled by a nuclear recessive gene.By employing a F₂ population from yl（t）crossed with Nipponbare（a japonica rice）,yl（t）was fine mapped in a 42 kb region on chromosome 6.Fragment sequencing confirmed that the candidate gene Os HO1（LOC＿Os06g40080.1）happened to mutate at the splicing site of exon2/intron2（AG/GT）with a deletion of"T".Compared with T98B,the transcription of Os HO1 in yl（t）terminates at the 20-22 bp（TAA）on the second intron,thereby causing a reduction of 179bp in the CDS sequence.Inferred by DNAMAN and CD-search,59 amino acid residues at the C-terminus of Os HO1 in yl（t）would be missed to impair the ability of heme binding.In addition,RT-q PCR analysis showed that Os HEMA was up-regulated while Os HO1 as well as Os CAO1 and Os NOL expressed lower in yl（t）.2.The yllt1 was just 77.78%and 60.00%of wild-type T98B in the content of chlorophyll a and chlorophyll b,and had abnormal chloroplasts with few functional thylakoid lamellar at seedling stage.At the vigorously tillering,the tillering number in yllt1was found 21.43%of the wild type.Genetic analysis revealed that the recombination rate between yellow leaf and less tillering was 0.00%,based on a F₂ population from yllt1crossed with T98B,indicating the two traits are under the control of one gene.Moreover,yllt1 was noticed recessively inherited via a nuclear gene.Further,it was fine mapped on Chromosome 11 using linkage analysis,and the LOC＿Os11g05552 that occurred to mutate with verification by sequencing was supposed as a candidate for yllt1.It encodes signal recognition particle 54 k Da protein,chloroplast precursor（cp SRP54）.In yllt1,a deletion of cds.29（C）on exon 1 of cp SRP54 would result in a shifted ORF,and thereby badly damage its protein structure from N-terminus to C-terminus.The RT-q PCR test showed the genes involved in chlorophyll synthesis such as Os CAO1,Os CAO2 and Os NOL were significantly down-regulated in yllt1.Meanwhile,the positive regulator of tillering Os TAC1 was decreased by 50%while the negative regulator ones Os TB1 and Os DLT were increased（even high by 4.5 folds）in expression,compared with the wild type.3.Based on yl（t）and yllt1,the general law of metabolomics among yellow leaf mutants were analyzed.It found that several acid（arginine succinic acid,prolyl glutamic acid）,lipids（lysophospholipids and phosphocholine lipid）and cationic metabolites were decreased by0.11-0.89 times in the mutants.On the contrary,a few of sugars such as sucrose and maltotriose,glycosylated derivatives and flavonoids such as apigenin are generally increased by 1.23-7.96 times.These results provide a basis for understanding the common law of leaf color variation from the metabolome level.This study revealed the genetic mechanism underlying the mutation for yl（t）and yllt1,and the general rules from the level of material metabolism,which laid a foundation for better understanding the mechanisms of the chrolophy biosynthesis.