Isolation And Functional Identification Of Wax Related Gene MdCER1 In Apple

Terrestrial plants evolved from aquatic organisms about 450 million years ago.For adapting to dry terrestrial environment,aquatic organisms require to develop a diffusion membrane.Thereupon plants evolved the cuticle,one of the greatest and most innovative evolutions in the history of plants.The cuticle is composed of cutin and cuticular wax,which plays a vital role in plant development and plant-environment interactions,including protecting plants from drying,adapting to unpredictable temperatures,resisting UV radiation,and invasion of pathogens and insects.The cuticular wax of apple is closely related to the adversity of growth and development and the quality of postharvest storage.The cuticular wax of apple can resist external damage caused by fungi and insects,and prevent evaporation of internal tissue and improve postharvest storage quality.Researches about cuticular wax of apple would be meaningful.In recent years,more and more regulatory mechanisms related to wax synthesis have been reported,such as Drought Hypersensitive(DHS),DEWAX,eceriferum(cer)related genes,and MYB transcription factors,such as MYB106,MYB16,MYB30 and MYB96,etc.Wax plays an important role in the process of biotic and abiotic stresses,however,these wax related genes are mainly studied in Arabidopsis and rice.Few reports were identified in apples.Wax is essential for the quality and resistance of apple.Therefore,it is valuable to study the function of wax related genes in apple.In this study,‘Royal Gala' apple was used,and a gene with the highest homology of Arabidopsis AtCER1,MDP0000461409,was found in Rosaceae genome database and named as MdCER1.We analyzed the sequence of MdCER1 protein and the relationship between the cis-acting elements of upstream promoter and abscisic acid(ABA)and drought on MdCER1,observed the changes of cuticular wax,and explored the relationship between cuticular wax accumulation and the response of ABA signals and drought resistance in Arabidopsis strains ectopically expressing MdCER1.The main findings are as follows:1.Isolation of MdCER1 and sequence analysisThe open reading frame of MdCER1 sequence was 1,872 bp,which encodes 623 amino acids.The phylogenetic tree analysis found that the MdCER1 in apple was closely related to the PbCER1 in white pear.Moreover,the 3D structures of MdCER1 and AtCER1 can overlap,and both contain a fatty acid hydroxylase superfamily and a Wax C-terminal domain.Analysis of the cis-acting element of the MdCER1 promoter revealed that the MdCER1 promoter sequence contained the ABA response element ABRE,and the drought-induced MYB binding site MBS,indicating that MdCER1 might be associated with ABA and drought resistance.2.MdCER1 regulates the synthesis of cuticular waxQuantitative real-time PCR(qRT-PCR)analysis of different apple tissues revealed that MdCER1 was expressed in roots,stems,leaves,flowers and fruits,and with the highest expression in leaves.Scanning electron microscope(SEM)showed that leaves of Arabidopsis strains ectopically expressing MdCER1 increased the cuticular wax,compared with that of empty vector transformed Arabidopsis,and extraction of total wax content of Arabidopsis leaves also confirmed this result.Changes of cuticular wax often affect the permeability of the cuticle.Compared to ectopic expression of MdCER1 in Arabidopsis,leaf water loss rate of Arabidopsis transferred into empty vector was faster,and leaves of Arabidopsis transferred into empty vector were strongly stained with toluidine blue,indicating that MdCER1 significantly reduced the epidermal permeability of Arabidopsis leaves.3.Transcription of MdCER1 responds to ABAqRT-PCR showed that MdCER1 was able to respond to ABA signals,and the response was stronger followed by the treating time.In medium-treated Arabidopsis experiments,10 ?mol/L and 30 ?mol/L ABA treatments resulted in shorter root length in Arabidopsis strains ectopically expressing MdCER1 compared with that of empty vector transformed Arabidopsis,indicating that MdCER1 is sensitive to ABA.4.MdCER1 enhances plant drought resistanceqRT-PCR showed that MdCER1 can respond to drought,and it was found that compared with the control,root length of ectopic expression of MdCER1 in Arabidopsis were longer on 4% Polyethylene glycol(PEG)and 6% PEG medium than that of empty vector transformed Arabidopsis.Using the seedlings in the soil for drought experiments,it was found that Arabidopsis strains ectopically expressing MdCER1 grew well under water-deficient conditions compared with empty vector transformed Arabidopsis,and these results indicated that MdCER1 can enhance plant drought resistance.