Map-based Cloning And Functional Analysis Of Early Senescence Mutants Es7 And Pls5 In Rice

Two novel early senescence mutants which named es7 and pls5 were isolated from the mutant library.In order to study the molecular mechanism,studies on phenotype analysis,map-based cloning and functional analysis were carried out.The results showed as follows:1.The es7 mutant was isolated from a ⁶⁰Co?-ray treatment rice 93-11mutant library,The upper three leaves of the es7 mutant began to exhibit senescence at the tillering stage about 60 day after sowing.As development progressed,the es7 mutant began to shown more areas of yellowing and senescence at the heading stage.When es7 plants are grown under high CO₂,the senescence phenotype and chlorophyll content are rescued.We examined multiple senescence-related parameters at the heading stage,CAT and T-AOC activity were significantly lower in es7 plants,while the content of H₂O₂ was significantly higher in es7 plants.Based on map-based cloning and PCR sequencing results,a single base substitution from G to A at position 1292 was identified in the second exon of LOC_Os07g46460 in genome,which encodes a putative ferredoxin-dependent glutamate synthase?Fd-GOGAT?.Leading to a deletion of 57bases in the mRNA transcript,and finally variant occurs in the first conserved purF-type glutamine amide transfer domain of Fd-GOGAT.ES7 was expressed constitutively,and the ES7 protein was localized in chloroplast.qRT-PCR analysis indicated that several genes related to nitrogen metabolism were differentially expressed in es7,the content of most amine acids were decreased.And the chlorophyll synthesis-associated genes were significantly down-regulated in es7.In addition,when seedlings are grown under increasing nitrogen concentrations?NH₄NO₃?for 15 days,the contents of chlorophyll a,chlorophyll b and total chlorophyll were significantly lower in es7.Our results demonstrated that ES7 is involved in nitrogen metabolism,effects chlorophyll synthesis,and may also associated with photorespiration,impacting leaf senescence in rice.2.The pls5 mutant was isolated from an EMS treatment rice ZH8015 mutant library.The leaves began to exhibit senescence at the tillering stage about 60 day after sowing,as development progressed,the pls5 mutant began to shown more areas of yellowing and senescence,at the heading stage,almost whole plant was senescence.The content of chlorophyll,photosynthesis rate were all lower in pls5,finally the grain yield was decreased.DAB,Evan staining,TUNEL and the measured of physiological parameters showed that more H₂O₂ accumulated and the enzyme activities were significantly lower in pls5 plants,and the number of death cell increased.Meanwhile,the results of TEM analysis showed that the rate of chloroplast degradation was higher in pls5,and then effected the leaf senescence.The genetic analysis revealed that pls5 senescence phenotype was controlled by a single recessive nuclear gene.PLS5 was isolated by using a map-based cloning approach,the mutation site was located in a 69.3kb region on chromosome 5,contained 12 ORFs.Sequencing analysis showed that there's a deletion of 11 bp in the seventh ORF,and finally resulted in premature stop codon in pls5.Evolutionary analysis revealed that the homology of PLS5 among different species are highly.PLS5 was expressed constitutively,and highly expressed in the green leaves;The PLS5 protein was localized in the cell membrane and nuclear membrane.Expression of Chlorophyll synthesis related genes,chlorophyll degradation related genes,senescence related genes and photosynthesis related genes suggested that,the chlorophyll synthesis was destroyed and the chlorophyll degradation was rapidly.In addition,the leaves of pls5 were more sensitive to high temperature and became more senescence than ZH8015.Our results demonstrated that the senescence phenotype of pls5 was caused by OsPSS2 loss-of-function mutation.The mutation of PLS5 results in rapidly senescence in rice,promote cell death,finally impacts grain yield,and the pls5 mutant were more sensitive to high temperature.The results of our study will help us to understand why and how the PLS5 gene influences leaf senecence.