Characterization Of Transcription Factors MdMYB88,Md MYB124,and MdMYB3 In Response To Drought And Low Nitrate Stresses In Apple

With its important economic value,apple is one of the four major fruits in the world.The Loess Plateau is the largest and best apple producing area in China,but the precipitation in this area is less and the distribution of annual precipitation is ununiformed.Therefore,drought has become an important restricting factor of apple industry in the Loess Plateau.At the same time,the soil in this area is poor,excessive nitrogen application reduces nitrogen use efficiency and causes a series of harm to the environment and human health.In this study,the biological functions of transcription factors MdMYB88,MdMYB124,and Md MYB3 in apple were analyzed to explore their molecular mechanisms in response to drought and low nitrogen stress.The main results are as follows:1.MdMYB88 and MdMYB124 regulate NO₃^-absorption and transportation under drought stress.PEG was used to simulate drought stress,and NO₃^-concentrations of transgenic and wild-type plants in root,shoot,old leaf,and younger leaf were measured.The results showed that MdMYB88 and MdMYB124 could promote NO₃^-absorption from the soil,transportation from shoot to root,old leaves to younger leaves under drought stress.By analyzing the transcriptome of MdMYB88/124RNAi under drought stress,we found that MdMYB88 and MdMYB124 could regulate the expression of Md NRTs.The results of real-time quantitative RT-q PCR showed that MdMYB88 and MdMYB124 could positively regulate Md NRT1.7,Md NRT1.8,and Md NRT2.4,and negatively regulate Md NRT1.5.2.MdMYB88 and MdMYB124 regulate NO₃^-absorption and transportation under low nitrogen stress.The results showed that MdMYB88 and MdMYB124 could promote the absorption from the soil,transportation of NO₃^-from shoot to root,from old leaves to younger leaves under low nitrogen stress.The results of real-time quantitative RT-q PCR showed that MdMYB88 and MdMYB124 could positively regulate Md NRT1.7,Md NRT1.8,and Md NRT2.4 and negatively regulate Md NRT1.5.Chip-q PCR and EMSA results showed that MdMYB88 and MdMYB124 could directly bind to the promoters of Md NRT1.7,Md NRT1.8,and Md NRT2.4 genes,but not to the promoter of Md NRT1.5,thus promoting NO₃^-usage under low nitrogen stress.The interaction protein Md BT2 between MdMYB88 and MdMYB124 was screened by yeast two-hybrid test.The results showed that MdMYB88 and MdMYB124 interacted with Md BT2 in vitro and in vivo,and Md BT2(BTB and TAZ domain protein 2)could degrade MdMYB88 and MdMYB124 through 26S proteasome under normal nitrogen condition,and further regulate NO₃^-absorption and transportation.3.Md ALI can improve the tolerance of apple in response to drought and low nitrate stress.The Md ALI(alu minum induced protein)was screened by yeast two-hybrid assay.The results of yeast two-hybrid assay,split-Luc,and Co IP showed that MdMYB88 and MdMYB124 interacted with Md BT2 in vitro and in vivo.Meanwhile,the results of Chip-q PCR and EMSA showed that MdMYB88 and MdMYB124 could directly bind to the promoter of Md ALI.The natural water loss assay of Md ALI transgenic plants showed that:Md ALI could slow down the water loss rate of leaves.The short-term drought stress showed that the ion leakage of Md ALI OE transgenic plants was significantly higher than that of wild-type GL-3,while the ion leakage of Md ALI RNAi transgenic plants was significantly lower than that of wild-type GL-3;At the same time,the survival rate of Md ALI OE transgenic plants was higher than that of wild-type GL-3,while the survival rate of Md ALI RNAi transgenic plants was lower than that of wild-type GL-3.Under low-nitrate conditions,the Md ALI OE plants showed better growth with a longer root length,more newly developed leaves,and greater biomass;by contrast,the growth of Md ALI RNAi plants was restricted.4.Md MYB3 can improve the tolerance of apple in response to drought and low nitrate stress.The natural water loss assay showed that Md MYB3 could slow down the water loss rate of apple leaves.The ion leakage and survival rate of Md MYB3 OE transgenic plants were significantly higher than that of wild-type GL-3 under short-term drought stress.The plant height,stem diameter,leaf water content,anthocyanins,and proanthocyanidins of Md MYB3 OE transgenic plants were significantly higher than those of wild-type GL-3 under long-term drought stress.Md MYB3 can promote the accumulation of ROS under drought stress.Meanwhile,the stomatal density,the ABA content in leaves,the hydraulic conductivity of roots and shoots in Md MYB3 OE transgenic plants increased,which could maintain higher photosynthetic rate and water use efficiency.Further analysis showed that Md MYB3 could positively regulate and directly bind to the promoters of Md DFR?Md ANR?Md ERF1B?Md MYBPA1?Md GSTU17 and promote the accumulation of anthocyanins and proanthocyanidins and the activity of glutathione-S-transferase(GST)in apple plants,thus,improve the ability of scavenging ROS.Md MYB3 can positively regulate and directly bind to the promoter of Md WRKY53,and indirectly regulate Md CYP707A4 to increase the accumulation of ABA in apple plants;Md MYB3 can positively regulate and directly bind to Md CAD9 promoter,and indirectly positively regulate Md LAC15 and Md LAC17 genes,which can affect lignin accumulation in apple plants and further improve hydraulic conductivity.Under low-nitrate stress,Md MYB3 OE transgenic lines showed stronger tolerance.Further studies showed that Md MYB3 could directly bind to Md LBD38 promoter and inhibits its expression,thus regulating nitrate absorption and usage.