Homology Cloning And Functional Analysis Of Very-long Fatty Acyl Co-A Reductase In Wheat

A continuous lipophilic layer called the cuticle covers most of the aerial surfaces of terrestrial plants.The cuticle consists primarily of cutin and waxes.Cutin,the main structural component of the cuticle.And the cuticular waxes are both embedded in the cutin matrix?intracuticular?and cover the cuticle proper?epicuticular?.Epicuticular waxes are often present in the form of crystals,which if dense enough impart a whitish bloom to plant surfaces.Cuticular waxes are complex mixtures of lipids,mostly composed of very-long-chain aliphatic molecules,including primary and secondary alcohols,aldehydes,alkanes,ketones,and esters that are derived from saturated very-long-chain fatty acids?VLCFAs?.The cuticular wax plays an important role in the survival of plants,serving as the interface between plants and their biotic and abiotic environment.The primary physiological function of the plant cuticle is to seal the tissue against a relatively dry atmosphere,preventing desiccation by minimizing nonstomatal water loss.Very-Long-Chain Fatty Acyl Coenzyme A Reductase is the key enzyme in the process of very-long-chain fatty acid to generate the fatty alcohols.So,in this study,we cloned the three FAR genes from the hexaploid wheat C.V.Xinong979 at seedling stage,and studied their functions in yeast,tomato and rice,respectively.The results were as follows:1.The leaf cuticular wax of different ploidy wheat at seedling stage were analyzed,primary alcohols were the main components.The absolute content of primary alcohols was6.30-9.16?g·cm^-2,it was accounted for absolute advantage dominance?71%-88%?,as well as small amount of alkanes,fatty aldehyde,fatty acids and ketones.From the case of carbon chain distribution,in all species,C28:0 alcohol accounted for absolute advantage dominance,while in Aegilops tauschii C26:0 alcohol accounted for the absolute dominance,and the results of SEM showed that the wax crystal structures on adaxial and abaxial in all wheat species were in the shape of platelets in seedling stage.Meanwhile,the leaf cuticular wax of two hexaploid wheat materials at different leaf positions were determined.The results showed that the waxy components of each leaf position contained alkanes,fatty alcohols,fatty aldehydes,fatty acids,diketones,and hydroxyl-?-diketones.Alkanol is always the dominance at the whole development process of wheat leaves.In addition,?-diketones and OH-?-diketones have been showed significant differences between Ming 988 and Xinong 979in the flag leaf waxes.2.Three FAR genes encoding fatty acyl-coenzyme A reductase were isolated from hexaploid wheat seedling leaves by RT-PCR,they were named TaFAR6,TaFAR7 and TaFAR8,the open reading frame were 1 497 bp,1 503 bp and 1 479 bp,and encoding 499 aa,501 aa and 493 aa,respectively.These FAR genes are highly related to the alcohol-forming FARs in Arabidopsis,and they have the typical domain of fatty acyl-coenzyme A reductase.The phylogenetic analysis of the FAR proteins showed that,TaFAR6 exhibit highly identity with HvFAR2,while TaFAR7 exhibit highly identity with BdFAR3,and TaFAR8 is highly similar as OsFAR1,OsFAR6 and OsFAR4.Meanwhile,the expression analysis results showed that,TaFAR6 expressed in young leaves,flag leaves,and glumes;TaFAR7 mainly expressed in young leaves,spike and stamens,and TaFAR8 expressed highly in young leaves,flag leaves,sheath and stamens.Meanwhile,the expressions of TaFAR6,TaFAR7 and TaFAR8 were all induced by biological and abiotic stress.The expression of TaFAR6 and TaFAR8 were up-regulated rapidly,while the TaFAR7 was up-regulated slowly.In addition,three proteins TaFAR6,TaFAR7 and TaFAR8,were located on the endoplasmic reticulum,which was consistent with the previous reports.3.To investigate the function of TaFARs we have cloned,we expressed three TaFARs in the yeast?Saccharomyces cerevisiae?,the results showed that the preferred substrate for TaFAR7 was C24:0 and C26:0 fatty acyl-CoA,while TaFAR6 and TaFAR8 were C24:0 fatty acyl-CoA,these results indicated that three TaFAR may be involved in the formation of the very-long-chain primary alcohol.4.Overexpression of TaFARs in tomato showed that the contents of the primary alcohols have dramatically increased in T₁ progeny plants.In leaves,TaFAR6,TaFAR7 and TaFAR8were involved in the biosynthesis of C26:0-OH,C28:0-OH and C30:0-OH.While in fruits,TaFAR6 and TaFAR8 were involved in the synthesis of C24:0-OH,C26:0-OH,C28:0-OH and C30:0-OH.TaFAR7 was involved in the synthesis of C24:0-OH,C26:0-OH,C28:0-OH,C30:0-OH and C32:0-OH.This shows that TaFAR can utilize a wider range of substrates in the tomato fruit epidermis.SEM results revealed that the increase of fatty alcohol content caused waxy crystals accumulation in the epidermis of tomato fruits.The reducing of water loss rate in transgenic tomto fruits showed that the water retention was improved.5.Overexpression of TaFARs in rice showed that the total amounts of the primary alcohols have also dramatically increased in T₁ progeny plants.In leaves of transgenic rice,TaFAR6 and TaFAR7 were mainly involved in the synthesis of C24:0-OH and C26:0-OH,while the TaFAR8 was mainly involved in the synthesis of C24:0-OH.But in sheath,the preferred substrate for TaFAR6 and TaFAR7 were C24:0 and C26:0 fatty acyl-CoA,while TaFAR8 was C28:0 fatty acyl-CoA.The cuticular wax crystals highly significant increased compared with control by scanning electron microscope?SEM?.SEM results also revealed that the increase of fatty alcohol content caused waxy crystals accumulation in the epidermis of rice leaves and sheaths.The drought resistance and water retention analysis of TaFAR transgenic rice showed that its water retention performance was improved.Based on the above results,we draw the conclusion that the TaFARs we have cloned could generate the primary fatty alcohol involved in the biosynthesis of cuticular wax.These results provide important reference for studying the molecular mechanisms of stress tolerance in crops.