Gene Mapping, Cloning And Functional Analysis Of An Early Senescence Mutant M144 In Rice (Oryza Sativa L.)

Leaf senescence is an important part of rice growth and development,and early senescence of rice will seriously affect its final yield.NAC transcription factor was involved in the regulation of aging process.It is of great significance to clarify the function of rice NAC transcription factors for revealing the molecular mechanism of rice aging and improving rice production.In this study,we identified a premature senescence mutant M144 with the background of indica rice Zhongjian 100.The results are as follows:(1)The early senescence phenotype appeared from the tillering stage of the mutant.With the growth of the plant,the senescence degree deepened,accompanied by dwarf and delayed heading.The length of each internode of mutant M144 was shortened.The examination of main agronomic traits showed the plant height,number of panicles,seed-setting rate and 1000-grain weight were significantly reduced in M144.Transmission electron microscopy showed that chloroplasts development was abnormal in the mutant leaves,and detection of photosynthetic pigments suggested chlorophyll content decreased in the mutant.In physiological and biochemical aspects,we observed that M144 had reactive oxygen species(ROS)accumulation,more DNA fragmentation,decreased soluble protein,increased malondialdehyde(MDA)content,and decreased ROS scavenging enzyme activity.By analyzing the expression of senescence and photosynthetic related genes between Zhongjian 100 and M144,we found that in the mutant,the expression of senescence induced genes was up-regulated,while that of photosynthetic related genes was down regulated.(2)The premature senescence phenotype of M144 is controlled by a single recessive gene,OsNAC109,which is a constitutively expressed transcription factor.Genetic analysis showed that the mutation of M144 was recessive.Through map based cloning,the mutation gene of M144 was mapped in a 39 kb genome region of chromosome 9,in which only Os09g0552800 had a single nucleotide substitution at 770 bp site,generating the premature termination of gene translation.Os09g0552800 was predicted to encode a NAC transcription factor,and was named OsNAC109.Complementary verification and gene knockout assay further proved that the mutation of OsNAC109 resulted in M144 early aging.The expression pattern analysis of OsNAC109 showed that the gene was constitutively expressed in rice and inhibited by senescence.The subcellular localization and transcriptional activation experiments demonstrated that OsNAC109 is indeed a transcription factor,which possess a nuclear localization signal at the N-terminal of amino acid sequence and a transcriptional activation domain at the C-terminal.(3)Through transcriptome analysis and interaction assays analysis between transcription factors and DNA,it was found that OsNAC109 specifically identified elements in promoters to regulate downstream senescence related genes.Some downstream gene promoters,including OsSAMS1,OsNAP,OsNYC3,OsEATB,OsZFP185,OsMPS,OsGA2ox3 and OsAMTR1,that might regulated by OsNAC109,were identified by yeast one-hybrid assay and the transcriptome analysis of the wild type and the mutant M144.Dual-luciferase assay was used to determine the regulation of OsNAC109,and the result revealed that OsNAC109 positively regulated the expression of OsSAMS1 and OsNYC3,and negatively regulated the expression of OsNAP,which was consistent with the expression detection of these genes in the wild type and M144.According to the recognition sequence of homologous protein AtNAC57,some fragments were selected from downstream promoters such as OsNAP,OsNYC3,OsSAMS1,OsEATB,OsAMTR1 and OsZFP185,and subsequently analyzed by EMSA.The results showed that OsNAC109 could bind to the promoter fragments,which containing relatively conserved CNTCSSNNSCAVG element,to achieve the regulation to downstream genes.