Cloning And Functional Analysis Of RLS3 Controlling Rapid Leaf Senescence In Rice (Oryza Sativa L.)

Leaf is the most important photosynthesis organ for crops. Rapid leaf senescence can cause the reduction of seed set rate and the decrease of grain yield in crops.In this study, we characterized a rls3 mutant, which derived from the ethyl methane sulphonate （EMS）-mutagenized of YIL18 （a Teqing introgression line）. The rls3 mutant exhibited a series of phenotypes related to senescence compared with YIL18, such as shorter plant height and panicle length, red leaves, reduction of chlorophyll content and photosynthetic efficiency, eventually affected grain yield.Transmission electron microscopy （TEM） images displayed that obvious differences in chloroplasts structure were observed between the YIL18 and rls3 mutant at 60 DAS. The chloroplasts fully developed in YIL18, but the array of grana stacks and inter-grana lamellae became slightly disordered in rls3. At 100 DAS, YIL18 chloroplasts had shrunk to the edge of the cell membrane, whereas most of the chloroplasts had already disappeared in the rls3 mutant.To isolate the RLS3 gene, we generated F2 population from a cross between the rls3 mutant and a japonica cultivar Zhonghual7 （ZH17）. Genetic analysis results showed that rapid leaf senescence phenotype of the mutant was controlled by a single dominant gene. We mapped RLS3 on the short arm of chromosome 3 and further delimited RLS3 within 134 kb region. Genomic sequence analysis of the YIL18 and rls3 mutant revealed that a single-nucleotide transition （Gâ†'A） at the splice site of the 10th intron/llth exon in LOC_Os03g38990 led to the cleavage of the first nucleotide （G） in 11th exon, eventually caused the frame-shift mutation and premature termination of translation.To verify whether the rapid leaf senescence phenotype was caused by the frame-shift mutation in the LOC_Os03g38990 gene, we performed a complementation experiment. A construct containing 1,956 bp coding sequence of RLS3 was introduced into the rls3 mutant. The overexpression transgenic lines displayed green leaves, higher plant height, longer panicle length and increased seed set rate. Meanwhile, we generated RNA interference （RNAi） construct to disrupt the expression of the RLS3 gene. Independent RNAi transgenic lines displayed the phenotype similar to the rls3 mutant, such as red leaves, shorter plant height and panicle length, and lower seed set rate. Together, this evidence confirmed that the LOC_Os03g38990 gene corresponded to RLS3 and that the mutation in RLS3 was responsible for the leaves’ rapid senescence phenotype in the rls3 mutant.We examined the expression of RLS3 in the leaf, pulvinus, sheath, stem base and root using qRT-PCR, and the RLS3 was expressed in various tissues, especially in leaf. We further confirmed the tissue-specific expression of RLS3 using RNA in situ hybridization analysis. RLS3 was predominantly expressed in mesophyll cells. The subcellular localization of RLS3 protein showed that the fluorescent signal was localized to chloroplasts. Thus, RLS3 may mainly be functional in chloroplasts.The RLS3 gene encodes a protein containing AAA₁1 domain, which includes the hallmarks of the AAA protein family, Walker A and Walker B motifs. During leaf senescence, RLS3 may be involved in photo-protection and maintain the photosynthetic apparatus through the degradation of photosynthetic damaged protein, further sustaining the normal function of chloroplast.The cloning and functional analysis of RLS3 plays important roles in revealing the molecular genetic mechanisms of leaf senescence and yield traits in rice.