Analysis Of Early Leaf Senescence Genes OsFd-GOGAT And OsBBS1 In Rice

Rice is the stable food for more than half of the world's population,and the stable and high yield of rice production is closely associated with the national food security of China.During rice production,natural disasters and inappropriate cultivation management can lead to leaf senescence thus decrease the yield.So far,many leaf senescence associate genes(SAGs)have been identified in rice.However,the genetic and molecular mechanisms during the processes of leaf senescence remain largely unknown.The biological function and metabolic pathway analysis of SAGs will help to explicit the molecular regulation network of leaf senescence,which will provide theoretical basis for anti-leaf senescence breeding.In the present study,we isolated two premature leaf senescence mutants from a rice(Oryza sativa L.cv Zhenong 34)M2 population by mutagenesis with ethylmethane sulfonate(EMS),named gogatl(glutamine 2-oxoglutarate aminotransferase1)and bbsl(bilateral blade senescence1).Phenotype observation,map-based cloning,overexpression knockdown(knockout)and gene expression analysis were used to analyze the function of mutant genes.The results showed OsFd-GOGAT played important roles in the leaf senescence and nitrogen metabolism in leaf,and that OsBBS1 participate leaf senescence and abiotic stress in rice.The main results are the following:1.Both mutants showed rapid leaf senescence after flowering,decreased plant height,less tillering number and reduced seed setting rate.gogatl showed rust-colored early leaf senescence.bbsl exhibited withered-yellow early senescence mainly in the leaf tip and blade margin,while the main vein region could retain green for a relative longer period.2.Genetic analysis results showed that both gogatl and bbsl were controlled by a single recessive nucleus gene.The gogatl gene was positional mapped to a 54.1 kb region interval between InDel markers 17-153 and 17-158 on the long arm of chromosome 7.Within the region,a putative gene LOC_Os07846460,encoding a ferredoxin-dependent glutamate synthase,was considered as the optimal candidate.Sequencing analysis revealed that an A to T substitution occurred in the 20th exon of LOC_Os07g46460.The bbsl gene was mapped to a 24.6 kb region between InDel markers 3M42 and 3M49 on chromosome 3.Sequencing results showed a G was inserted in the first exon of LOC_Os03g24930,which encoding a receptor like cytoplasmic kinase,leading to a reading frameshift mutation.3.The detection OsFd-GOGAT mRNA levels of in root,culm,panicle and leaf showed that OsFd-GOGAT was mainly expressed in leaves.Almost no transcript of OsFd-GOGAT could be detected in root.It was notable that OsFd-GOGAT transcript levels in leaves were declining dramatically when the leaves become senescent.Gus staining of OsBBSl promoter driving ?-glucuronidase transgenic lines showed that OsBBS1 expressed in the entire life cycle.In mature leaf,OsBBS1 expressed highest level in the region near the main vein.4.For clarifying the biological function of OsFd-GOGAT and OsBBS1,artificial microRNA(amiRNA)silencing strategy was used for observing the knockdown influence of OsFd-GOGAT.The calluses of OsFd-GOGAT knockdown lines could not regenerate to new plants as all the calluses exhibited severe browning,suggesting that OsFd-GOGAT played the important roles during plant regenerate from callus.OsBBS1 overexpression lines(under the driven of Ubiquitin promotor)and knockout lines(through CRISPR/cas9 strategy)were obtained,respectively.The results showed the overexpression of OsBBS1 could delay the leaf senescence,and the knockout lines exhibited the same early leaf senescence phenotype.These results demonstrated that OsBBS1 was responsible for the early senescence of bbsl mutant.5.Eighteen out of the 20 protein-forming amino acids were detected in the flag leaves and upmost internodes of gogatl mutant and WT were determined to evaluate the effect of reduced OsFd-GOGAT enzyme activity.The results showed amino-acid pool in both organs of gogatl mutant had higher levels than that in WT.In the sap of flag leaf,glutamine contents of gogatl mutant accumulated about 12-fold as much as WT,and contributed the most to the increased amino acid.In the sap of the upmost internode,the amino-acid pool of gogatl mutant was almost eight times as much as that of wild type and asparagine accounted 61.4%of the total amino acid.These results indicated that OsFd-GOGAT played important roles in amino acid metabolism during leaf senescence.6.Three-leaf stage seedlings were treated with 100 mM NaCl for analyzing the expression profile of OsBBS1.The result indicated that the expression of OsBBS1 could be induced by salt stress and expressed the highest level at 8h.The responses of OsBBS1 overexpression line(OE1),Nipponbare(Nip),OsBBSl knockout line(cas-11),zhenong 34(WT)and bbsl were analyzed under 150 mM NaC1 treatment for 7 d.The results showed that cas-11 and bbsl were most sensitive to salt stress,while the stems and several leaves of OE1,Nip and WT still performed green.These results showed the loss-function of OsBBSl could lead to salinity hypersensitive phenotype.