Map-based Cloning And Functional Analysis Of WSL5-1 Involved In Cuticular Wax Biosynthesis In Rice

The cuticular waxes of plants is the first physical barrier against the invasion from the external environment,which plays an important role in protecting the non-stomatal water loss of plants.The cuticular waxes also protect plants from various biological and non-biological stresses.As an important food crop,it is of great significance to study the cuticular wax associated genes in rice(Oryza sativa)and apply them to breeding to improve the resistance of varieties.In this study,we identified a wax crystal-spare leaf(wsl5-1)mutant by using EMS chemical mutagenesis in japonica variety Zhong Hua11.We isolated and explored the molecular mechanism of this gene using map-based cloning method.The main research results are as follows:1.Phenotype characterizing of the wsl5-1 mutant.The wsl5-1 mutant shows less plant height and senescence phenotype at the heading stage compared to the wild type Zhong Hua 11.SEM analysis exhibits that cuticular wax crystals of the wsl5-1 leaves are significantly reduced compared to the WT.GC-MS analysis show that the wsl5-1 wax components on the leaf surfaces are all decreased,and the total wax content was significantly reduced compared with WT,among some components were significantly reduced.Moreover,the chlorophyll content of the wsl5-1 decreased in all four growth stages,and decreased significantly after the heading stage.The photosynthetic rate is also significantly reduced at the heading stage.Futhermore,analysis of yield traits showed that the seed setting rate and 1000-grain weight of the wsl5-1 are significantly reduced compared to the WT.2.Map-based cloning and functional analysis of WSL5-1 gene.Map-based cloning demonstrated that the mutant site in wsl5-1 was narrowed to a 52 kb DNA region between the In Del markers C4 and C7 on chromosome 4 by Using wsl5-1/TN1to construct the F₂localization population.On the basis of annotation information and sequencing analysis,LOC?Os04g30760 was found a single base mutation(G to A)on the9th exon of the coding region in the fourth ORF,and making the 254^thposition of the encoded protein from alanine(Ala)to threonine(Thr).LOC?Os04g30760 gene encoding a predicted 3-oxoacyl ACP reductase was involved in the wax biosynthesis by the bioinformatics analysis.Further genetic transgene complementation demonstrated LOC?Os04g30760 was the target gene.3.Expression pattern and subcellular localization of WSL5-1.Quantitative PCR and histochemical staining indicated that WSL5-1 gene is universally expressed in tissues.The results showed that the WSL5-1 gene was expressed in chlorenchyma tissues such as culms,leaves,sheaths and panicle of plants.Subcellular localization GFP vector was constructed for WSL5-1,transient assays of fluorescent protein-tagged WSL5-1 in tobacco and protoplasts,which co-localized with the chloroplast marker fusion protein signal.This result indicates that WSL5-1 protein is localized on chloroplasts.4.WSL5-1 gene mutation affects the expression of the wax associated genes and premature leaf senescence.To further explored that the wax biosynthesis pathway was blocked by mutations in the WSL5-1 gene.We measured the expression levels of wax-associated genes,most of genes were down-regulated in the wsl5-1 plants compared with the WT.In addition,in order to clarify how the WSL5-1 mutation regulated premature senescence leaves,we observed the microstructure of chloroplasts to verify defective chloroplast development by TME.Physiological and biochemical experiments showed the significant differences of H₂O₂,MDA content and CAT enzyme activity between the tillering stage and heading stage,and the analysis of senescence-associated genes expression was also up-regulated in the wsl5-1mutant.Meanwhile,the analysis of chlorophyll degradation and senescence-associated genes showed the up-regulated expression in the wsl5-1 mutant and led to accelerate chlorophyll degradation and leaf senescence.5.WSL5-1 gene plays an important role in cold tolerance of rice.In this study,three-leaf-stage seedling of both the WT and wsl5-1 were treated at low temperature,and the result indicated that the wsl5-1 is more sensitive than WT to cold stress.To explore the molecular mechanism by which WSL5-1 gene regulates chilling tolerance in rice,our research showed that three cold tolerance-associated genes was obviously suppressed in the wsl5-1 mutant,the WSL5-1 expression level was similar to that of these cold tolerance-associated genes.In addition,the more H₂O₂accumulated in the wsl5-1 than in the WT,and the expression level of peroxidase gene Os POD1 was always low in wsl5-1 at the chilling stress process.These results indicated that the WSL5-1 gene regulated cold tolerance in the seedling stage.