Functionnal Characterization Of CER17 Gene Involved In Cuticular Wax Biosynthesis In Arabidopsis

The epicuticular wax coating the aerial surface of land plants is a hydrophobic lipid component,which forms a barrier against environmental stresses.It plays key roles in restricting non-stomatal water loss,protecting plants against the attack of pathogen and insect,and ultraviolet radiation.It is extraordinarily useful for crop yield improvement by understanding the genetic mechanism.In this study,we cloned the CER17 gene and analyzed the function of CER17 gene involved in cuticular wax and cutin biosynthesis in Arabidopsis.The Arabidopsis eceriferum17（cer17）mutant was generated using ethyl methanesulfonate,and first identified as a wax-deficient mutant based on its visible semiglossy stem phenotype.Using the postional cloning and allele test,CER17/ADS4 gene was found to encode a predicted ACYL-COENZYME A DESATURASE LIKE4（ADS4）.The entire open reading frame of ADS4 was sequenced to reveal a G-to-A nucleotide change in the cer17-1 mutant,which is predicted to cause a single amino acid substitution from Pro to Leu and lead to lose the VLCFA-CoA desaturase activity in yeast.CER17-YFP was colocalized with the ER membrane marker CD3-959 in Arabidopsis protoplasts and tobacco cells,indicating it was localized to the ER membrane.CER17/ADS4 exhibits highest expression in the upper inflorescence stem,flower,silique and exclusively expression in inflorescence stem epidermal cells which conformed by qRT-PCR and GUS histochemical staining.We obtained two T-DNA insertion alleles of CER17/ADS4,Salk₀43674,and SM₃₂0206,which were designated as cer17-2 and cer17-3,respectively.RT-PCR analysis showed that the CER17/ADS4 transcript abundance in cer17-2 was dramatically reduced,whereas the CER17/ADS4 transcript in cer17-3 was absent.Scanning electron microscopy analysis showed that the wax crystals on distal and basal inflorescence sterm surfaces of cerl 7-1 and cer17-2 were generally less dense and flatter than wild type.The total stem wax deposition and single compound per area on the distal and basal segments of the inflorescence stems of cer17-2 and cer17-3 were significantly increased compared with wild-type levels,especially the amount of saturated primary alcohols.We report n-6 monounsaturated primary alcohols（C26:1,C28:1,and C30:1 homologs）in the cuticular waxes of Arabidopsis（Arabidopsis thaliana）inflorescence stem,a class of wax not previously reported in Arabidopsis.The Arabidopsis cer17 mutant was completely deficient in these monounsaturated alcohols and the phenotype was rescued in the cer17-2 transgenic complementation lines harboring the 35S-CER17::YFP.CER17/ADS4 transgenic lines in wild type harboring the 35S-CER17::YFP construct had much higher amounts of monounsaturated primary alcohols in basal stems than that of wild-type basal stems.These results indicate that the CER17/ADS4 gene is involved in the biosynthesis of very long chain monounsaturated alcohl.Studies of the Arabidopsis cer4-4 mutant and yeast variously expressing CER4（a predicted fatty acyl-CoA reductase）with CER17/ADS4 which produced n-6 monounsaturated primary alcohols,demonstrated CER4’s principal role in synthesis of these monounsaturated alcohols.Besides unsaturated alcohol deficiency,cerl 7 mutants exhibited a thickened and irregular cuticle ultrastructure and increased amounts of cutin monomers.Although unsaturated alcohols were absent throughout the cerl 7 stem,the mutation’s effects on cutin monomers and cuticle ultrastructure were much more severe in distal than basal stems,consistent with observations that the CER17/ADS4 transcript was much more abundant in distal than basal stems.Furthermore,distal but not basal stems of a double mutant deficient for both CER17/ADS4 and LONG-CHAIN ACYL-COA SYNTHETASE1 produced even more cutin monomers and a thicker and more disorganized cuticle ultrastructure and higher cuticle permeability than observed for wild type or either mutant parent,indicating a dramatic genetic interaction on conversion of very long chain acyl-CoA precursors.These results provide evidence that CER17/ADS4 performs n-6 desaturation of very long chain acyl-CoAs in both distal and basal stems and has a major function associated with governing cutin monomer amounts primarily in the distal segments of the inflorescence stem.