Gene Isolation Of A Spotted-leaf Mutant M154 In Rice(Oryza Sativa L.)

The rice spotted-leaf mutants belong to leaf color mutants that are useful for characterization of chlorophyll metabolism.The experimental material in this study was a rice spotted-leaf mutant named M154,which was induced by ethyl methyl sulfone?EMS?from Zhongjian100?ZJ100?.In this study,genetic analysis and gene mapping,measurement of phenotypic traits,determination of biochemical-physiological parameters,light shading experiment,functional complementation,CRISPR/Cas9-mediated mutation,determination of enzymes activity and enzymetic substrate contents,expression analysis of chlorophyll degradation-related genes were conducted to supplementally explain plant chlorophyll degradation pathway.The main results are as follows:1.The reddish-brown spots appeared on the first leaf of M154 14 days after sowing,and appeared on all the leaves 50 days after sowing.Gradually,the lesions mergered and covered the whole leaf leading to even completely withered leaf at the tillering stage,finally resulting in the poor performance of all major agronomic traits especially the length of the third internode.The photosynthetic capacity of M154 decreased significantly,the total chlorophyll content of the mutant and the wild type did not differ significantly at the seedling stage.During the tillering stage,the total chlorophyll content of the mutants decreased significantly,and the photosynthetic parameters decreased significantly,leading to the poor performance of agronomic traits.2.Cell death happened in the leaves of M154.Trypan blue staining showed that there was severe cell death in and around the spots on the mutant leaves.The content of soluble protein?SP?decreased while malondialdehyde?MDA?level increased apparently,and the TUNEL experiment showed that the DNA was fragmented in the mutant.3.ROS accumulation with perturbed ROS scavenging system in M154.The activities of peroxidase?POD?and superoxide dismutase?SOD?are significantly higher than those of the wild type,and the activity of catalase?CAT?was significantly reduced,resulting in a large amount of accumulation of O2-on the mutant leaves,but the H2O2 level was similar between the mutant and wild-type.4.The reddish-spots of M154 were induced by light and mechanical wounding,and the mutant was normal green leaves in the dark.5.The decreased enzyme activity of PAO in M154 led to the apparent accumulation of pheophorbide a?Pheide a?,the potential phototoxic enzyme-specific substrate which is the direct cause for the lesion formation.Compared with the wild-type,the enzyme activity of PAO of M154,the expression of the mutant allele as well as the PAO content had decreased significantly.6.The contents of intermediate metabolites and various related enzyme activities in the chlorophyll degradation pathway of M154 had changed significantly.After dark treatment,the contents of intermediate metabolites and various related enzyme activities had also changed significantly,suggesting that chlorophyllases?CLHs?may play a minor role in the chlorophyll degradation pathway in rice.7.The mutation of M154 was controlled by a single recessive nuclear gene encoding a putative pheophorbide a oxygenase?PAO?.A single base nucleotide substitution was detected in exon 5 of Os03g0146400 or exon 6 of Os MH₀3G0040800,resulting in a substitution from isoleucine to phenylalanine in the amino acid sequences.8.The PAO allele of the wild-type was transferred into M154 calli via Agrobacterium-mediated plant transformation.At the tillering stage,when M154 showed obvious spotted-leaf phenotype,the WT and T0 complementation plants did not show lesion mimic phenotype.The content of chlorphyll of the complementation plants recovered to the wild-type level.The results indicated that the functional complementation was successful and the PAO mutation was responsible for the mutant traits.Cas9-2 was a gensgenic line derived from from CRISPR/Cas9-mediated mutation of PAO in Kitaake.Six base deletions on the 4th exon,resulting in the frameshift mutation,was detected in Cas9-2 which showed a similar and even more severe phenotype compared with M154.The results further comfimed that the mutation of PAO was responsible for the mutant traits.