Characterization And Genetic Analysis Of LSN5 And Genome-wide Association Studies Of Leaf Senescence In Rice

Leaf senescence,the final stage of leaf development,is a complex and highly regulated process that involves a series of coordinated actions at the cellular,tissue,organ,and organism levels under the control of a highly regulated genetic program.Rice?Oryza sativa L.?is one of the most important cereal crops in the world,leaf senescence regulation has important effect on rice yield and quality.In this study,we obtained an leaf early senescense mutant named lsn5?leaf senescence necrosis 5?which was induced by ethyl methylsulfonate?EMS?mutagenesis of japonica rice nipponbare.Through the phenotype analysis,physiological index determination,and genetic analysis of this mutant,we obtained the following results:1.The most stable and obvious phenotype of the lsn5 mutant is leaf senescence and tip necrosis.From the beginning of the three-leaf stage,the mutant begins to show leaf senescence and tip necrosis phenotype.The senescence phenotype becomes more obvious with the increase of leaf age.At the same time,other agronomic traits of the mutant also changed,mainly manifested in phenotypes such as decreased plant height,reduced number of tillers,delayed heading date,decreased number of secondary branches,and decreased seed setting rate.2.The decrease of chlorophyll content in lsn5 mutants was mainly attributed to the early stage chlorophyll a and carotenoids degradation.Chlorophyll b showed a large amount of degradation in the late stage of senescence.3.The degradation of chlorophyll resulted in the decrease of photosynthetic rate of lsn5 mutant,which was mainly reflected by the decrease of net photosynthetic rate,the increase of intercellular CO₂concentration,and the increase of transpiration rate.4.The decrease of photosynthetic rate,especially at the grain filling stage,will affect the accumulation of nutrients in rice grains.The lsn5 mutant exhibited a significant reduction of grain thickness resulted in a decrease in grain weight and without affecting grain length and width.5.Transmission electron microscope observation of chloroplasts revealed that the thylakoids in the chloroplast of lsn5 were loose and could not form complete grana stacks.At the same time,there was accumulation of osmiophilic globuli and starch granule in the chloroplast,indicating the accumulation of reactive oxygen species.6.Observation of stomata by scanning electron microscopy and surface replica technique impression revealed abnormal stomatal closure in lsn5.The number of stomata in the closed and semi-open state was significantly decreased.7.The accumulation of reactive oxygen species in the lsn5 mutant was also found by staining of physiological indicators,and increasing of electrolyte leakage,indicating that the mutant is more severe than the wild type.8.The activities were decreased of reactive oxygen scavenging-related enzymes in lsn5 mutants.LSN5 mutations altered the expression of genes involved in senescence and reactive oxygen scavenging related genes,indicating that LSN5 is involved in the regulation of leaf senescence.9.Genetic analysis showed that the mutant phenotype was controlled by a pair of recessive nuclear genes.LSN5 was narrowed to a 46.3 kb inteval in the chromosome 5.At the same time,based on re-sequencing of 134 rice landraces,we conducted a genome-wide association study on chlorophyll content at different growth stages to analyze the genetic regulatory mechanisms of leaf senescence.Finally,we identified 3,2 and 1 significant sites related to the chlorophyll content at 40 days after transplanting,10 days after heading,and 20 days after heading respectively.Similarly,1 and 8significant sites associated with leaf growth index and leaf senescence index.