Map-based Cloning And Functional Analysis Of WSL3 And WSL4 Involved In Cuticular Wax Biosythesis In Rice

The aerial parts of plants are covered with cuticular waxes, which form the first barrier between plants and the environment. The cuticular waxes could limit non-stomatal water loss, protect plants against deposits of dust and impose a physical barrier to pathogen infection, and prevent plants forming organ fusion. It is important to research the biosynthesis, secretion and regulation of wax formation. To date, there is little information about wax formation in rice(Oryza sativa L.), a model monocot plant and an important staple food crop. Here, we identified two wax-deficient mutants of rice, wax crystal-sparse leaf 3(wsl3) and wax crystal-sparse leaf 4(wsl4), which exhibit a pleiotropic phenotype that includes reduced epicuticular wax crystals on the leaf surface and altered wax composition. We characterize the two mutants and use map-based cloning to isolate and understand the molecular mechanism of these two genes. The research results are as follows: 1. Phenotype characterizing of the wsl3 and wsl4 mutantsWe isolate two wax deficient mutants, named wsl3 and wsl4 from a mutant population of japonica variety Nipponbare. Both wsl3 and wsl4 mutants exhibits strong water adhesiveness compared to the WT, and the wsl4 mutants shows less plant height and tiller number that WT. TEM analysis shows the leaf cuticle membrane on wsl3 and wsl4 mutant appears thicker than WT. SEM analysis exhibits the epicuticular wax crystal patterns on the leaf surface of the wsl3 and wsl4 mutants are significantly decreased compared with WT. GC-MS show that there is no obviously difference of cutin content between the wsl3 and wsl4 mutants and WT, but the wax composition on the leaf surfaces are significantly reduced on these two mutants relative to WT. Furthermore, the wsl3 and wsl4 cuticle is more permeable than the WT.2. Map-based cloning and functional analysis of WSL3 and WSL4 genesMap-based cloning demonstrated that defects of the mutant site in wsl3 was narrowed to an 83 kb DNA region delimited by the InDel markers M-2396 and M-2404 on chromosome 4. The LOC_Os04g40730 gene encoding a protein homologous to KCR in this region has two adjacent single-nucleotide substitutions at nucleotides 736 and 737(GCâ†'TT) in the first exon of LOC_Os04g40730, causing the 207 th amino acid change from Ala to Phe. Map-based cloning demonstrated that defects the mutant site in wsl4 was narrowed to an 58 kb DNA region delimited by the InDel markers M-6283 and M-6341 on chromosome 3. The LOC_Os03g12030 gene encoding a protein homologous to KCS in this region contains a mutation in nucleotides 1281(Gâ†'A) in the second exon of the Loc_Os03g12030 gene, causing the 312 th amino acid to change from Val to Met. KCR and KCS genes are involved in the wax biosynthesis, further genetic complementation and RNAi of the WSL3 and WSL4 gene demonstrate they are the target genes.3. Expression pattern and subcellular location of WSL3 and WSL4Quantitative PCR and histochemical staining indicated that WSL3 and WSL4 are universally expressed in tissues. Transient assays of fluorescent protein-tagged WSL3 and WSL4 in tobacco protoplasts showed that they both locate in the endoplasmic reticulum, the compartment of fatty acid elongation in cells.4. Complementation of fatty acid elongation of WSL3 in yeastThe growth rate of the yeast ybr159wΔ mutant could be rescued by the WSL3 gene as well as the native yeast KCR YBR159 w gene. Moreover, very long chain fatty acids(VLCFAs) 20:0 and 22:0, or 20:1Δ11 and 22:1Δ13, were detected when WSL3 and Arabidopsis Fatty Acid Elongation 1(FAE1) were co-expressed in a yeast ybr159wΔ mutant strain, indicating that WSL3 affects rice cuticular wax production by participating in VLCFA elongation.