Molecular Mechanisms Of MdMYB88 And MdMYB124 In Response To Cold And Drought Stresses In Apple

Apple is one of the most important cultivated fruits in the world,which has a high economic quality.Loess Plateau is the biggest apple production region in China.Nevertheless,cold and drought stress often attack this region and influence the yield and quality of apple.Consequently,it is crucial to identify stress critical genes to enhance cold and drought tolerance of apple.Our RNA-seq analysisrevealed that MdMYB88 and MdMYB124 are responsive to cold and drought stresses in ‘Golden delicious'(Malusxdomestica Borkh.cv.Golden Delicious)andMalus prunifolia(Malus prunifolia Borkh).In this study,we investigated their molecular mechanismsin response to cold and drought stresses.The main results are the following:1.MdMYB88 and MdMYB124 can enhance the cold and freezing tolerance of apple.The mRNA level of MdMYB88 and MdMYB124 was up-regulared under cold stress.Tissue specific expression results revealed that MdMYB88 and MdMYB124 were highly expressed in roots and stems and weakly expressed in leaves,flowers,and fruits.After chilling treatment,the shoot length of MdMYB88/124 RNAi(RNA interference)transgenic apple plants were shorter than the non-transgenic plants.MdMYB88 and MdMYB124 positively regulated the expression of MdCCA1(CIRCADIAN CLOCK ASSOCIATED 1)?MdCBFs(C-REPEAT/DRE BINDING FACTOR)?MdCOR47(COLD-REGULATED 47),the key genes responsive to cold stress.After freezing treatment,MdMYB88 and MdMYB124 overexpression transgenic apples showed higher survival rate and integrity of the membrane system than the non-transgenic and RNAi transgenic apple plants.Chilling stress usingMdMYB88 and MdMYB124 transgenic apple plants grown in pot revealed that MdMYB88 and MdMYB124 promoted anthocyanin accumulation in apple plants under cold stress.RT-qPCR results indicated that MdMYB88 and MdMYB124 positively regulated expression of MdF3H(FLAVANONE 3-HYDROXYLASE),MdCHS(CHALCONE SYNTHASE)and MdANS(ANTHOCYANIDIN SYNTHASE),key genes in anthocyanin biosynthesis.In addition,MdMYB88 and MdMYB124 can promote the activities of antioxidant enzymes thus promote the elimination of reactive oxygen.In addition,we found that MdMYB88 and MdMYB124 regulated stomatal development of apple.MdMYB88/124 RNAi plant showed clustered stomata.After cold stress,apple stomata were closed,but MdMYB88 or MdMYB124 were not involve in this process.2.MdMYB88 and MdMYB124 positively regulate key genes in cold stress response.We explored about 1000 target genes of MdMYB88 and MdMYB124 by ChIP-seq methods.By EMSA and ChIP-qPCR,we verified that MdMYB88 directly bound to the promoter of MdCCA1 and MdCSP3(COLD SHOCK DOMAIN PROTEIN 3).Dual luciferase assay revealed that MdMYB88 activated MdCCA1 which then induce the activity of MdCBFs,but MdCSP3 did not activate MdCBFs.Therefore,MdMYB88 and MdMYB124 enhance cold tolerance of apple plants by CBF-dependent and CBF-independent ways.3.MdMYB88andMdMYB124 can enhance the tolerance of apple under drought stress.The mRNA level of MdMYB88 and MdMYB124 were induced under drought stress.Dehydration experiment revealed that MdMYB88 and MdMYB124 reduced water loss of apple plants.Natural drought experiment showed that survival rate of MdMYB88 and MdMYB124 overexpression transgenic apples were higher than non-transgenic GL-3 plants while that of MdMYB88/124 RNAi transgenic apple was lower than GL-3.Furthermore,the photosynthetic capacity of MdMYB88 and MdMYB124 overexpression transgenic apples were greater than GL-3 while that of MdMYB88/124 RNAi transgenic apple plants was lower than GL-3.4.MdMYB88 and MdMYB124 can promote the biosynthesis of ABA.When treated with5 ?M ABA(abscisic acid)for 2 h,the stomata opening of MdMYB88 and MdMYB124 overexpression transgenic apples was smaller than that of GL-3 while the stomata aperture of MdMYB88/124 RNAi transgenic apple was bigger than GL-3,suggestingMdMYB88 and MdMYB124 overexpression transgenic apples were sensitive to ABA while MdMYB88/124 RNAi transgenic apple was insensitive to ABA.RT-qPCR results indicated that MdMYB88 and MdMYB124 positively regulated MdNCED3(NINE-CIS-EPOXYCAROTENOID DIOXYGENASE 3)in response to drought,the key gene of ABA biosynthesis.ChIP-qPCR and EMSA results verified that MdMYB88 directly bound to promoter of MdNCED3.5.MdMYB88 and MdMYB124 interact with MdHYL1 and MdSE.By yeast two hybrid(Y2H)screen and affinity purification mass(AP-MASS),we obtained two interacting protein of MdMYB88 and MdMY124,MdHYL1 and MdSE.By Y2 H,Co-IP and BiFC,we found that MdMYB88 and MdMYB124 interacted with MdHYL1 both in yeast and planta.In addition,MdHYL1 can enhance the transcriptional activity of MdMYB88.However,MdMYB88 and MdMYB124 interacted with MdSE only in planta but not in yeast.Moreover,MdSE reduce the transcription activity of MdMYB88.