Screening And Gene Function Study Of The Rice High Temperature And High Light Sensitive Mutant Ls1

High light and high temperature?HLHT?stress may become more frequent and severe as the climate changes,affecting crop growth and resulting in reduced production.High light is usually accompanied by high temperature.Both high temperature stress and high light stress can lead to excessive ROS accumulation in plants.If ROS are not scavenged in time,they will cause severe oxidative damage to lipids,proteins and DNA.At present,most reports only studied the effect of high temperature or high light on rice.However,high temperature and high light are usually accompanied by each other in the field,and the regulatory mechanism of the response to HLHT stress in rice is rarely reported.In the present study,we screened a rice mutant library using HLHT condition and isolated a HLHT-sensitive mutant,local lesions 1?ls1?.We characterized and cloned LS1 by map-based cloning and genetic complementation.LS1 encodes the A subunit of the RNase H2 complex?RNase H2A?.The mutation of LS1 led to rice genome instability and excessive ROS accumulation in the ls1 mutant under HLHT condition,which finally produced the leaf local lesions phenotype.The main research results of this paper are as follows:1.No differences in phenotype were observed between the wild type and ls1 mutant under the N22condition.However,the ls1 mutant showed obvious local lesions phenotype,when it was transferred to HLHT condition?H32?.The more intense the high light and high light stress,the more pronounced the ls1 mutant phenotype.The damage area in the leaf increased correspondingly.There was no phenotypic difference between wild-type and ls1 plants when a single change in light or temperature was tested.2.The chlorophyll content,leaf width and relative water content of the white sectors in the ls1mutant leaves were significantly lower than those of the wild-type leaves.The bulliform cells and epidermal cells of the white sectors were atrophied in the ls1 mutant leaves.The white sectors of the ls1mutant leaves showed rough surfaces,and the stomata were severely deformed.The inner structure of chloroplast in the mesophyll cells was changed and most chloroplasts were degraded.3.Genetic analysis showed that the mutant ls1 phenotype was caused by a recessive mutation in a single nuclear gene.For map-based cloning,the ls1 mutant was hybridized with indica cultivar TN1 to construct the F₂ segregating population.The target gene was located on the chromosome 11.We found a C-to-T single base-pair substitution in the third exon of the coding region of LOC_Os11g05570,causing an amino acid substitution from alanine?Ala?to valine?Val?in the 89th residue by sequencing.We transformed the gDNA sequence of Nipponbare to mutant ls1,and the phenotypic observation and molecular identification confirmed that LOC_Os11g05570 was indeed the target gene LS1.The chlorophyll content,relative water content and expression level of the target gene in the complementary lines could be restored to the wild-type level.Bioinformatics analysis showed that LS1 encodes A large subunit of RNase H2?RNase H2A?,which is involved in DNA replication and repair.4.Model expression analysis showed that the target gene LS1 was mainly expressed in leaves.Under the H32 condition,the transcription level of LS1 was significantly reduced in the mutant ls1compared to the wild-type.There was no significant difference in LS1 transcription levels between the ls1 mutant and the wild-type under the N22 condition.The LS1 protein level was similar to that of transcription.Subcellular localization revealed that LS1 protein localized to the nucleus.5.TUNEL and comet assays showed that there were higher levels of DNA damage in the ls1mutant compared to the wild-type,which caused excessive ROS accumulation in the ls1 mutant under the H32 condition;Under the N22 condition,there was no significant difference in DNA damage level and ROS content between the mutant ls1 and the wild-type.Application of antioxidant ascorbic acid?AsA?and dimethyl thiourea?DMTU?could significantly ease the local lesions phenotype of the ls1mutant under the high temperature and high light condition.6.It was found that the expression of most genes related to chlorophyll degradation,senescence,and antioxidant in the mutant ls1 were significantly up-regulated under the H32 condition compared to under the N22 condition.