Comparison And Mechanism Analysis Of Different Apple Cultivars In Nitrogen Use Efficiency Under Drought Condition

Water and nitrogen?N?are indispensable environmental factors for plant growth,development and yield formation.Drought stress can limit plants growth and development process by affecting the appearance,physiological structures,biochemical characteristics and protein contents.N,one of the essential mineral elements of plants,is the main component of proteins,nucleic acids,phospholipids,chlorophyll,hormones,vitamins and alkaloids,and is involved in various stages of plant growth and development.Apple,because of its high economic value and nutritional value,has become one of the important cash crops,and is widely planted in China and the world.The Northwest Loess Plateau,as the best and largest apple production area in China,encounters the serious problem of water shortage due to its low annual rainfall and uneven distribution within the year.In addition,low vegetation coverage and poor soil lead to excessive application of N fertilizer in farmland to increase yield.Excessive application of N fertilizer lead to a series of adverse effects,such as reducing NUE,increasing N deposition,intensifying greenhouse effect and aggravating crop diseases.Due to the inherent characteristics of different apple cultivars,different responses to water and N levels will appear.Therefore,selection of apple cultivars with high NUE and study the mechanism are crucial to reduce the N fertilizer application in orchards and ensure the healthy and sustainable development of apple industry.In this study,one-year-old seedlings of six apple cultivars,‘Golden Delicious',‘Qinguan',‘Jonagold',‘Honeycrisp',‘Fuji'and‘Pink Lady',grafted onto Malus hupehensis Rehd.rootstock were used to assess the growth indexes,photosynthetic efficiency,mineral element contents,chlorophyll fluorescence parameters,hormones content,enzymes activity,and gene expression under different water and N supply conditions.Malus hupehensis Rehd.seedlings with hydroponics were used to monitor the response of N absorption related genes under different levels of NO₃^--N.The main findings are as follows:1.Growth status were assessed for one-year-old seedlings of six apple cultivars,‘Golden Delicious',‘Qinguan',‘Jonagold',‘Honeycrisp',‘Fuji'and‘Pink Lady',grafted onto Malus hupehensis Rehd.rootstocks under control water with control N?CWCN?,limited water with control N?LWCN?,control water with low N?CWLN?and limited water with low N?LWLN?.The results showed that drought and low N treatment reduced the accumulation of biomass,chlorophyll content,photosynthesis,and the absorption of mineral elements in apple seedlings.The result of biomass accumulation of different apple cultivars was CWCN>CWLN>LWCN>LWLN.Under LWLN condition,the net photosynthetic rate of‘Qinguan',‘Jonagold',‘Honeycrisp',‘Fuji',‘Pink Lady',and‘Golden Delicious'were 7.44,5.14,3.77,3.67,5.30,and 4.48?mol m^-2 s^-1,respectively.Under LWLN condition,rootstock grafted with‘Qinguan'had the highest N concentration(13.28 mg g^-1),whereas rootstock grafted with‘Jonagold'had the lowest(9.38 mg g^-1).In LWLN condition,‘Qinguan'had the highest RGR of all cultivars(9.51 mg d^-1),while‘Honeycrisp'had the lowest(4.66 mg d^-1).Appropriate N fertilizer supply under water deficient condition can maintain the growth of apple seedlings to a certain extent.Under LWLN conditions,‘Qinguan'and‘Pink Lady'had the best tolerance,followed by‘Fuji'and‘Golden Delicious',while‘Honeycrisp'and‘Jonagold'were the worst among all tested cultivars.2.The seedlings of‘Qinguan'and‘Honeycrisp'grafted onto Malus hupehensis Rehd.rootstocks were used to evaluate leaf stomata characters,N metabolism and antioxidant enzymes activity,hormones,N utilization and uptake gene expression under CWCN,CWLN,LWCN,and LWLN conditions,to analyze the reasons for the differences in NUE of different apple cultivars.The results showed that low N treatment decreased the stomatal aperture of‘Qinguan'and‘Honeycrisp'by 15.61%and 17.28%respectively under drought condition.Compared with CWCN condition,the free proline of‘Qinguan'and‘Honeycrisp'increased by 32.21%and 17.36%respectively under LWLN condition.Compared with CWLN,the content of soluble sugar in‘Qinguan'was significantly increased under LWLN,but the increase of‘Honeycrisp'was not significant.In addition,drought stress significantly reduced the activities of N metabolization related enzymes in leaves of‘Qinguan'and‘Honeycrisp',including NR,NiR and GS.Under drought conditions,compared with proper N supplies,GS in rootstock grafted with‘Honeycrisp'under low N treatment decreased by 26.97%,showing significant difference,while that in rootstock grafted with‘Qinguan'decreased by13.47%,showing no significant difference.Under LWLN condition,the content of IAA and ABA in leaves and rootstock grafted with‘Qinguan'were significantly higher than that of‘Honeycrisp'.Under the same or different treatment conditions,the difference of stomatal morphology,enzymes activity,hormones content,and the level of gene expression between‘Qinguan'and‘Honeycrisp'affected the absorption and assimilation of N,and resulted in the difference of N utilization between the two cultivars.Quantitative analysis of N metabolism-related genes in leaves and rootstocks of the two cultivars showed that MdNRT2.4 was significantly induced by low N stress.The low N treatment of MdNRT2.4overexpressed Arabidopsis proved that MdNRT2.4 could promote root growth and maintain plant fresh weight,then improved low N resistance ability.We speculated that the difference of stomatal apertures,chlorophyll fluorescence levels,activy of N metabolism and antioxidant enzymes,and hormone concentrations impacted N uptake and utilization capacity of the two cultivars,which lead to different NUE.3.We identified 73 NITRATE TRANSPORTER 1/PEPTIDE TRANSPORTER family?NPF?genes in apple,and investigated the phylogenetic,chromosome location,gene structure,and conserved domains using bioinformatics analysis.The results showed that apple NPF gene family could be divided into eight subfamilies,namely subfamily I-VIII,which was consistent with Arabidopsis and rice.The genes of apple NPF family contained 2to 7 exons,included three conserved domains,and the genes were unevenly distributed on 17chromosomes.In addition,the genes of apple NPF family showed tissue expression specificity and varied expression under different nitrate concentrations.4.Quantitative analysis of NPF genes in leaves and roots under different NO₃^--N concentrations indicated that MdNPF6.5 showed dual-affinity nitrate transport ability.Overexpression vectors of MdNPF6.5 was constructed and agrobacterium-mediated transformation of apple‘Orin'calli tissue was performed.Overexpression lines of calli tissue were obtained and treated with low N conditions.When compared with the wild type,MdNPF6.5 transgenic apple calli could maintain higher fresh weight,which demonstrated that this gene confers greater capacity for N uptake under those conditions.