Mechanism Study Of Apple Hexose Transporter MdHT1.2 On Sugar Transportation In Root And Response To Drought Stress

Sugar is the material basis for plant growth and development,and is also an important regulating substance for plants to resist adversity stress.Fruit quality is closely related to soil organic matter content,and as an important component of soil organic matter,glucose from plant bodies or microbial decomposition plays an important role in stabilizing soil structure and increasing soil fertility.Root application of glucose in apple orchards promotes plant biomass accumulation,increases stress resistance and improves fruit quality.However,it is not clear how inter-root glucose affects plant growth and development,and whether apple roots can utilize glucose directly from rhizosphere uptake.Root uptake of soil glucose is mainly dependent on the plasma membrane localized hexose transporter（STP/HT）.To this end,this thesis identifies and screens the highly expressed hexose transporter gene MdHT1.2in apple roots based on genome-wide analysis,demonstrates that it mediates root glucose transport,and explores the relationship between its mediated root glucose uptake and plant growth and development and fruit quality.The main results of the study are as follows:1.MdHT1.2 is localized in the plasma membrane of apple root epidermal cells and mediates inter-root glucose uptake.Twenty-nine MdHTs were identified from the apple genome using a bioinformatic approach,and MdHT1.2 was screened for glucose-induced up-regulated expression based on RNA-seq data analysis.The promoter tissue-specific expression analysis and in situ hybridization results showed that MdHT1.2 was mainly expressed in root epidermal cells.The subcellular localization results showed that its encoded protein was localized on the cytoplasmic membrane,and the yeast functional complementation assay confirmed its high glucose transport capacity.To further investigate its function,MdHT1.2-overexpressed and-silenced transgenic apple lins and MdHT1.2-overexpressed tomato strains were obtained.¹³Glc and 2-NBDG feeding assays showed that overexpression of MdHT1.2 significantly enhanced glucose uptake by apple and tomato roots,while silencing of MdHT1.2 significantly reduced glucose transport capacity in roots.In addition,sugar content determination revealed that overexpression of MdHT1.2significantly increased soluble sugar content in roots,while silencing MdHT1.2 resulted in an approximately 12%decrease in glucose content in apple roots.These results suggest that MdHT1.2 localized in the plasma membrane of root epidermal cells plays an important role in mediating inter-root glucose uptake and sugar accumulation in the root system.2.MdHT1.2-mediated rhizosphere glucose uptake alters assimilated carbon allocation,regulating plant growth and fruit sugar accumulation.The transgenic apple plants grown in the soil substrate with glucose content of 4.4 g.kg^-1 DW showed growth differences,which were reflected in significant increases in apple plant height,primary root length and root fresh weight of apple plants overexpressing MdHT1.2,while apple plants with silent MdHT1.2 decreased significantly.Transgenic tomatoes were phenotypically consistent with transgenic apples overexpressing MdHT1.2.Glucose feeding experiments showed that MdHT1.2-mediated rhizosphere glucose uptake promoted aboveground and underground growth of apple plants,but significantly reduced the root/shoot ratio of dry weight.It was demonstrated by ¹³C pulse labeling experiments that MdHT1.2-mediated rhizosphere glucose uptake reduced the distribution of assimilated carbon to the root.The transgenic tomato grafting assay showed that MdHT1.2-mediated rhizosphere glucose uptake significantly enhanced the distribution of assimilated carbon to fruits and promoted the accumulation of soluble sugars in fruits.3.MdHT1.2-mediated rhizosphere glucose uptake improved root growth and soluble sugar accumulation under drought stress,and enhanced plant drought resistance.Analysis of drought RNA-seq results showed that MdHT1.2 was significantly upregulated by drought.The results of 16-day short-term drought treatment of transgenic apple plants showed that overexpression of MdHT1.2 significantly enhanced the drought resistance of plants,while the drought resistance of plants silent MdHT1.2 decreased.The 2-NBDG and ¹³Glc feeding experiments showed that drought significantly promoted glucose absorption capacity and soluble sugar accumulation in the root of MdHT1.2-overexpressed transgenic apple plants.In order to explore the relationship between MdHT1.2-mediated root glucose uptake and drought tolerance,glucose was added to the sand in which apple plants were grown under sand culture conditions,and it was found that,glucose treatment significantly enhanced the drought resistance of overexpressed MdHT1.2 apple plants,promoted the accumulation of glucose and sucrose in root,and significantly improved root growth under drought stress,while glucose treatment did not enhance the drought resistance of MdHT1.2-silent apple plants.In addition,both genetic and WT apple plants grown in glucose-free sand were not drought-resistant.These results showed that drought stress promoted MdHT1.2-mediated rhizosphere glucose transport,regulated root growth and soluble sugar content,thus affected plant drought tolerance.4.MdHT1.2 was regulated by the transcription factor MdDOF3 under drought stress,which affected the drought resistance of plants.In order to explore the transcription factors that regulate the expression of MdHT1.2 under drought stress,four transcription factors that may bind to the MdHT1.2 promoter and are induced by drought were screened by ATAC-seq and RNA-seq data analysis,and one of the transcription factors,MdDOF3,positively regulated the expression of MdHT1.2 by LUC assay,and the transcriptional regulatory relationship between MdDOF3 and MdHT1.2 was proved by Y1H and GUS activity assays.In addition,the GUS activity assay found that the tandem repeats of the cis-acting element"AAAG"in the MdHT1.2 promoter region significantly promoted the binding ability of MdDOF3 to the MdHT1.2 promoter.Transgenic apple plants overexpressing MdDOF3 in roots were obtained,and short-term drought stress treatment found that overexpression of MdDOF3 in roots significantly promoted the absorption of glucose in roots under drought stress,increased the accumulation of soluble sugar in the root,and enhanced the drought resistance of plants.However,overexpression of MdDOF3 in MdHT1.2-silent transgenic apple roots did not enhance the drought resistance of MdHT1.2-silent transgenic apple lins.These results showed that MdDOF3 enhanced plant drought resistance in a MdHT1.2-dependent manner.