Function And Mechanism Analysis Of ’MdRAD23D1-MdPRP6’ Module In Apple Responding To Drought Stress

Apple is one of the four major fruits in the world.China is the largest producer and consumer of apples in the world,and its cultivated area and output rank first in the world.The Loess Plateau is the largest and best apple production area in China.However,the precipitation in this area is less and the annual distribution is uneven,which seriously affects the growth and development,yield and quality of apples.RAD23(RADIATION SENSITIVE23)proteins are essential transporters in the process of ubiquitin protein degradation.They can transport ubiquitin-modified substrate proteins to 26 S proteasome for degradation.Much attention has been paid to the function of these proteins in plant growth and development,but there are few reports on the function of plants in response to drought and other stresses.In our previous study,we isolated and identified the members of the RAD23 family in the apple genome and obtained the transgenic apple plants of MdRAD23D1.On the basis of previous studies,this study identified the biological function of MdRAD23D1 in response to drought stress in apple,and systematically clarified the molecular mechanism of drought resistance.The results laid a theoretical foundation for the analysis of apple drought response network and provided important genetic resources for apple drought resistance molecular breeding.The main results are as follows:1.MdRAD23D1 plays a positive role in apple responding to drought stressBioinformatics analyses showed that MdRAD23D1 had a conserved UBL-UBA domain.In vitro binding assay and multiple protein interaction experiments showed that MdRAD23D1 could also perform transport function in UPS(Ubiquitin proteasome system).Drought stress and ABA treatment could induce the expression of MdRAD23D1.After short-term drought treatment,the MdRAD23D1-Ri apple plants(transgenic apple plants with suppressed expression of MdRAD23D1 via RNA-interference)were more sensitive to drought than WT(wild-type),the results of MdRAD23D1 transgenic apple calli treated with mannitol showed that the transgenic apple calli overexpressed by MdRAD23D1 showed resistance to mannitol treatment,while the transgenic apple calli interfered by MdRAD23D1 showed more sensitive to mannitol treatment.Under long-term drought,the adaptability of MdRAD23D1-Ri apple plants was also poor,and WUE(Water use efficiency)was significantly lower than WT.The above results showed that MdRAD23D1 could improve the resistance of apple to short-term drought stress and the adaptability to long-term drought stress.2.MdRAD23D1 interacts with proline-rich protein MdPRP6 and promotes its degradation under droughtThe protein MdPRP6 interacting with MdRAD23D1 was obtained by screening yeast two-hybrid library,and the interaction between MdRAD23D1 and MdPRP6 was verified by yeast two-hybrid,Split-Luc,Pull-down and Co-IP experiments.In vivo ubiquitin experiments showed that MdPRP6 was modified by ubiquitination and this modification was not affected by MdRAD23D1.In vitro and in vivo degradation experiments showed that MdRAD23D1 could promote the ubiquitin degradation of MdPRP6 protein,and drought accelerated this process.Phenotypic analysis of co-transgenic calli showed that MdRAD23D1 promoting the degradation of MdPRP6 protein could affect the resistance of calli to mannitol treatment.Phenotypic analysis and physiological data determination of MdPRP6 transgenic apple plants under drought treatment showed that MdPRP6 negatively regulated apple resistance to shortterm drought stress and adaptability to long-term drought stress.3.Proline mediates the response of MdPRP6 and MdRAD23D1 to droughtUnder drought stress,the proline content of transgenic materials interfered with MdPRP6 increased significantly,while that of overexpressed materials decreased significantly.The decrease of intracellular proline content weakened the drought resistance of transgenic materials interfered with MdPRP6,while exogenous addition of proline could restore the resistance of MdPRP6 overexpressed materials.Furthermore,studies showed that the gene expression level and enzyme activity of MdP5 CS in MdPRP6 interfered expressed plants under drought were significantly higher than those in WT.After silencing MdP5 CS,the content of proline in MdPRP6-Ri apple plants(transgenic apple plants with suppressed expression of MdPRP6 via RNA-interference)was still higher than that in WT,indicating that proline mediates drought response regulated by MdPRP6,and it may originate from both P5 CS pathway and MdPRP6 degradation.In addition,it was found that proline also mediates the drought response of MdRAD23D1.The content of proline in the overexpressing MdRAD23D1 calli was significantly increased,while the content of proline in the materials of MdRAD23D1 interfering was significantly decreased,and exogenous proline could also restore the drought resistance of materials interfered with MdRAD23D1.Thus,MdRAD23D1 positively regulated and MdPRP6 negatively regulated of apple drought resistance.Under drought condition,MdRAD23D1 promoted the ubiquitin degradation of MdPRP6.MdPRP6 may regulate the accumulation of free proline under drought through P5 CS pathway and self-degradation,which mediates apple response to drought.