Characterization Of Apple Small Ubiquitin-like Modifier-MdSUMO2 In Response To Abiotic Stress

China is the largest apple producing country in the world.As the main producing area in China,the yield of loess plateau is limited by drought,cold,heat and other abiotic environment conditions.Identification and characterization of the resistance related genes in apple laid the foundation for apple molecular breeding.Small ubiquitin modifier?SUMO?takes part in various development processes and plays important roles in plant defense to biotic and abiotic stress.In this dissertation,we characterized the molecular function of apple MdSUMO2 in abiotic stress tolerance.The main results are listed:1.MdSUMO2s are expressed in various tissues and respond to different abiotic stressesSix paralogues of MdSUMO2 in apple are located on 3 homologous chromosomes which are from the apple genome duplication event billion years ago,we then named them as MdSUMO2A?MdSUMO2A-chr3,MdSUMO2A-chr11?,MdSUMO2B?MdSUMO2B-chr9,MdSUMO2B-chr17?and MdSUMO2C?MdSUMO2C-chr5,MdSUMO2C-chr10?.All MdSUMO2s are located in membrane,nucleus and cytoplasm.MdSUMO2s are expressed in roots,stems,leaves and flowers in Malus fusca,with more abundance of MdSUMO2A and MdSUMO2B than MdSUMO2C.All MdSUMOs—MdSUMO2A,MdSUMO2B and MdSUMO2C respond to high temperature,low temperature,drought and salt stress,indicating the various functions of MdSUMO2s.2.Fine tuning of MdSUMO2s modulates drought stressMdSUMO2A OE?overexpression?showed enhanced drought resistance than GL-3 after3 months drought stress?moderate drought stress,about 20%soil volumetric moisture content?.Drought resistance phenotypes of MdSUMO2A OE includes higher plant height,stronger root system,lower ABA content and more stomata in leaf,efficient photosynthesis capcacity,better shoot and root hydraulic conductivity.MdSUMO2A RNAi?knocking down only MdSUMO2A?did not show significant difference with GL-3.MdSUMO2 RNAi?knocking down all MdSUMO2s?lines showed typical characteristics of xerophytes,including drawf,smaller and thicker leaf,less stomata and higher ABA content.iTRAQ analysis indicated that compared to GL-3,MdSUMO2A OE up-regulated 164 proteins and down-regulated 213 proteins.While MdSUMO2 RNAi up-regulated 543 proteins and down-regulated 622 proteins.Compared to GL-3,MdSUMO2A OE enriched proteins involved in abscisic acid metabolism,abscisic acid catabolism,transition metal ion transport and sesquiterpenoid catabolic process,whereas MdSUMO2 RNAi enriched proteins including negative regulators of RNA catabolic process and innate immune response.Both MdSUMO2RNAi and MdSUMO2A OE maintained higher survival rate under short-term natural drought stress,while MdSUMO2A RNAi did not show difference with GL-3.In addition,MdSUMO2RNAi had lower leaf ion leakage and increased catalase?CAT?and peroxidase?POD?activities compared with GL-3 after drought stress.Consistently,H₂O₂ and O₂^-were accumulated less in MdSUMO2 RNAi than GL-3 after dehydration.Under dehydration conditions,the leaves of MdSUMO2 RNAi lost less water than GL-3,while MdSUMO2A OE leaves lost more.In addition,MdSUMO2 RNAi plants were more sensitive than GL-3 to exogenous ABA treatment.3.Sumoylation of MdDREB2A induced its degradation by MdRNF4MdDREB2A accumulated more in MdSUMO2 RNAi than GL-3 after dehydration condition.By using an E.coli sumoylation system,we found that MdDREB2A was sumoylated at K192 by MdSUMO2A.Transgenic calli carrying both 35S::MdDREB2A and35S::mMdDREB2A?K192R?in apple callus showed enhanced tolerance than wild type to PEG treatment,and transgenic calli carrying 35S::mMdDREB2A?K192R?grew better than35S::MdDREB2A.Y2H and MST results showed MdSUMO2A interacted whith MdRNF4?an E3 ubiqitin ligase?by SIMs?SUMO interacting motif?.Further study showed the sumoylation of MdSUMO2A at K192 induced its degradation by MdRNF4 through 26S proteasome.4.MdSUMO2 RNAi enhanced thermotoleranceMdSUMO2 RNAi transgenic lines showed enhanced thermotolerance than GL-3.Antioxidant enzyme?CAT?was increased in MdSUMO2 RNAi transgenic lines and MdHSP90,MdNCED3,and MdDREB2A were highly expressed under high temperature stress condition in MdSUMO2 RNAi plants.5.MdSUMO2 RNAi enhanced tolerance to N deficiencyRNAi of MdSUMO2s improved tolerance to N deficiency.MdSUMO2 RNAi transgenic lines showed better root system under N deficiency condition.Nitrate reductase?NR?,nitrate transporters?MdNRT1.1/1.2/2.5?and SUMO E3 ligase?MdSIZ1?were up-regulated significantly in root system of MdSUMO2 RNAi under N deficiency stress than GL-3.6.The difference of MdSUMO2A promoters contributes to its expression level under drought stressMdSUMO2A was up-regulated in‘Qinguan'and down-regulated in‘Naganofuji No.2'under drought stress condition.Promoters of MdSUMO2A-chr3 had 36 bp In/Del in‘Naganofuji No.2'but not‘Qinguan',while MdSUMO2A-chr11 did not show difference in sequences between two apple cultivars.Y1H results showed that a Zinc finger transcription factor might bind to the 36 bp In/Del directly.The MdSUMO2A-chr3 promoter showed higher DNA methylation level in‘Qinguan'than‘Naganofuji No.2'under drought stress.