Study On The Function And Mechanism Of The MdNAC104 Transcription Factor In Apple Under Low-Temperature Stress

Apples are one of the top four fruits worldwide,and China is the largest producer and consumer of apples in the world.The apple industry plays a significant role in increasing income for farmers in northern China,adjusting the structure of the agricultural industry,and rural revitalization.In recent years,with the adjustment of the layout of China’s apple industry through "westward movement and northward expansion," apple production has gradually become concentrated in advantageous or characteristic areas such as the Loess Plateau,high-altitude cold areas in southwest China,and Xinjiang.In recent years,these areas have been increasingly affected by low temperatures in winter and cold snaps in spring,and low temperatures have become one of the most important environmental factors limiting the development of the apple industry.NAC transcription factors are a plant-specific family of transcription factors that are widely involved in plant growth,development,and response to abiotic stresses.Currently,there is little research on the function of NAC transcription factors in regulating the low-temperature stress response in apples,and the relevant regulatory mechanisms are not clear.In this study,MdNAC104-overexpressing apple plants were used as materials,and the function of MdNAC104 in regulating cold tolerance was identified via the integrative analyses of multi-omics data,promoter binding assays,and transcriptional regulation analysis.The mechanism by which MdNAC104 responds to low temperatures and regulates cold tolerance in apples was also investigated.The main research results are as follows.First,MdNAC104 was found to enhance the cold tolerance of apple plants by promoting the accumulation of osmotic adjustment substances.The contents of these substances in the leaves,stems,and dormant buds of each plant were detected under freezing stress conditions.The results showed that the contents of soluble sugar,soluble protein,and proline in the leaves and stems of transgenic plants were significantly higher than those in the GL-3 control plants,while the content of malondialdehyde was significantly lower.However,no significant difference was found in the accumulation of osmotic adjustment substances and changes in the organelle structure in the dormant buds.This indicates that MdNAC104 overexpression significantly promotes the accumulation of osmotic adjustment substances in the leaves and stems of apple plants under low-temperature conditions,thereby improving the cold tolerance of apples.Second,MdNAC104 was found to participate in regulating the cold tolerance of apple plants by promoting antioxidant enzyme activity,photosynthetic efficiency,and ABA content.Under low-temperature stress conditions,the overexpression of MdNAC104 was found to alleviate the effects of cold stress on apple plant growth and development,significantly inhibit the accumulation of reactive oxygen species(ROS)in apple leaves under cold stress,and enhance antioxidant enzyme activity to clear excess ROS.Furthermore,measurements of photosynthetic capacity and chlorophyll fluorescence system parameters,such as Pn,Gs,Tr,Fv/Fm,and Y(II),demonstrated that MdNAC104 maintained high photosynthetic efficiency by reducing damage to the PSII system of apple plants under low-temperature stress.In addition,MdNAC104 participated in regulating stomatal closure and affecting ABA accumulation by altering the transcription levels of the Md NCED1 and Md CPK6 genes,thereby enhancing the cold resistance of transgenic plants.After 15 and 30 days of cold stress,there were no significant changes in the contents of lignin,cellulose,hemicellulose,and the microscopic anatomical structure in the stem of transgenic apple plants as compared with the wild type.There was also no significant difference in the contents of JA,CTK,and ACC in the plants.These results indicate that MdNAC104 mainly enhances the cold tolerance of apple plants by increasing their ROS scavenging ability and maintaining efficient photosynthesis ability.Third,transcriptomic and metabolomic analyses revealed that MdNAC104 promotes the biosynthesis of anthocyanins and proline under low-temperature conditions.Via the transcriptomic analysis of MdNAC104-transgenic and GL-3 plants under cold stress conditions,differentially expressed genes were found to be mainly involved in plant hormone signal transduction,starch and sucrose metabolism,plant-pathogen interactions,phenylpropanoid biosynthesis,and flavonoid biosynthesis pathways.Among the 973 metabolites detected,compared to GL-3,42,42,and 39 differential metabolites were respectively identified in the three transgenic lines;these were mainly enriched in secondary metabolite synthesis,arginine and proline metabolism,phenylpropanoid biosynthesis,plant hormones,and flavonoid biosynthesis pathways.Furthermore,key genes involved in the biosynthesis of anthocyanins(Md CHS,Md CHI,Md F3 H,and Md ANS)and proline(Mdami,Mdspe D,and Md ALDH)were found to be significantly upregulated in the transgenic plants,and the corresponding metabolite contents were also significantly increased.This suggests that MdNAC104 may promote the accumulation of anthocyanins and proline under cold stress conditions by upregulating the expression of genes involved in their biosynthesis pathways,thereby enhancing the cold stress tolerance of apple plants.Finally,MdNAC104 was found to positively regulate the cold tolerance of apples through CBF-dependent and CBF-independent pathways.Via promoter binding and transcriptional regulation analysis,it was found that under cold stress,MdNAC104 can directly bind to the promoters of Md CBF1 and Md CBF3 and promote their expression,thus positively regulating the cold tolerance of apple plants through the CBF-COR pathway.On the other hand,several key genes involved in the anthocyanin biosynthesis pathway and antioxidant enzyme coding genes were identified in the transcriptome and metabolome analysis results.Via experiments such as LUC/REN,Y1 H,EMSA,and Ch IP-q PCR,it was found that MdNAC104 can directly activate the expression of key genes in the anthocyanin biosynthesis pathway(Md CHS-b,Md CHI-a,Md F3H-a,and Md ANS-b)and antioxidant enzyme-coding genes(Md FSD2 and Md PRXR1.1),thus promoting anthocyanin accumulation and antioxidant enzyme activity under cold stress,and ultimately enhancing the cold tolerance of apple plants.