The Mechanism Of High Nitrogen Regulating Carbon And Nitrogen Metabolism In Apple Fruit And Nitrogen Regulation Techniques

In apple(Malus domestica Borkh.)production,the excessive application of nitrogen(N)fertilizer is common for fruit farmers in pursuit of high yields and large fruits.Excessive N application leads to not only a lower N fertilizer utilization and environmental pollution problem,but also the decline of fruit quality,which further affects the economic benefits and the green and high-quality development of apple industry.Therefore,it is of great significance to study the physiological mechanism of apple quality decline due to excessive N application to change the status quo of N fertilizer application in apple orchards,reduce N fertilizer input and improve fruit quality.To this end,the effects of high N on primary and secondary metabolic pathways,C-N nutrition and fruit quality were investigated by using ¹⁵N and ¹³C isotope double-labeling techniques combined with transcriptomic,proteomic and metabolomic association analysis using‘Fuji'apples as test material.In addition,the effects of three N regulation techniques on C,N metabolism and fruit quality of apple were studied by nitrification inhibitor(3,4-dimethylpyrazole phosphate,DMPP),nitrate reductase(NR)inhibitor(sodium tungstate)and exogenous abscisic acid(ABA)at the later stage of apple growth.The main results are as follows:1.Research on C and N metabolism pathways and fruit quality of apple fruits under high N supply through multi-omics joint analysis technology:Compared to control(CK),the soluble sugar and total flavonoid contents of apple fruits under high N(HN)treatment decreased by 16.1%and 19.0%,respectively,resulting in poorer intrinsic quality.Moreover,high N increased the total N and decreased total C and C/N ratio of apple fruits.The ¹⁵N and¹³C isotope labeling results showed that high N increased the ¹⁵N absorption and regulation ability of the fruit,and reduced the distribution of ¹³C from the vegetative organs to the fruit.Transcriptomic,proteomic and metabolomic analysis showed that the differential genes,proteins and metabolites were mainly involved in“carbohydrate metabolism”,“amino acid metabolism”and“biosynthesis of secondary metabolites”under HN treatment.The joint analysis of multi-omics revealed C and N metabolic regulatory network of apple fruits under high N:High N inhibited the accumulation of carbohydrates(sucrose,glucose,and trehalose)and flavonoids(rhamnetin-3-O-rutinoside,rutin,and trihydroxyisoflavone-7-O-galactoside)in fruits,and more C skeletons were used to synthesize amino acids and their derivatives(especially low C/N ratio,e.g.,arginine)to be transferred to N metabolism.This study revealed new insights for the decline in soluble sugar and flavonoids caused by high N,and key genes(MD07G1172700,MD05G1222800,MD16G1227200,MD01G1174400,and MD02G1207200)and key proteins(PFK,gap N,and HK)were obtained.2.Effects of nitrification inhibitor DMPP on C and N metabolism and fruit quality in apple:During the late apple fruit expansion stage,soil application of DMPP decreased the abundance of ammonia-oxidizing bacteria(AOB)amo A,increased the concentrations of NH₄⁺-N in the soil,and decreased NO₃^--N concentrations and its vertical migration.Compared with the control,DMPP reduced ¹⁵N utilization and loss rates to varying degrees and increased ¹⁵N residual and recovery rates.Results of ¹³C and ¹⁵N double isotope labeling revealed that DMPP improved the distribution of ¹³C from vegetative organs to the fruits,reduced the capacity to absorb and regulate fruit with respect to ¹⁵N,and decreased ¹⁵N accumulation in the fruits and whole plant.DMPP regulated the balance between vegetative and reproductive growth,and increased the contents of anthocyanin and soluble sugar in the fruits.The comprehensive analysis revealed that the application of 1 mg·kg^-1DMPP at the later stage of fruit expansion effectively reduced the N losses and alleviated inherent quality degradation caused by excessive N absorption by the tree.3.Effects of NR inhibitor sodium tungstate(Na₂WO₄)on C and N metabolism and fruit quality of apple:The results showed that the spraying of 0.5-1.0 mmol·L^-1Na₂WO₄significantly inhibited the growth of seedling shoot,and the inhibition of root was not obvious in the pot experiment.The growth of seedling root was significantly inhibited when the concentration of Na₂WO₄reached 1.5 mmol·L^-1.The NR activity of each treatment was negatively correlated with the concentration of Na₂WO₄in the same period.The content of nitrate N in leaves showed the tendency of first rising and then decreasing with the extension of processing time,and the content of nitrate N was positively correlated with Na₂WO₄concentration in the same period.Spraying Na₂WO₄reduced the ¹⁵N absorption of each organ,and the higher concentration of Na₂WO₄,the more obvious effect of inhibiting N absorption in the seedlings.The ¹³C accumulation on the ground tended to increase and then decrease as the concentration of Na₂WO₄increased and T₂treatment(1.0 mmol·L^-1Na₂WO₄)reached the highest;the ¹³C accumulation of whole plant was represented in a similar law.The field experiments showed that the application of Na₂WO₄reduced the N content and total N and enhanced the total C content of fruits at maturity stage.Meanwhile,the contents of anthocyanin,soluble solids,soluble sugar,sugar acid ratio,and total flavonoids in fruits were increased to varying degrees.T₂treatment had the best effect.In summary,T₂treatment(1.0mmol·L^-1Na₂WO₄)can effectively reduce the reduction and accumulation of N,improve the accumulation of C in fruits,and is conducive to the improvement of apple quality.4.Effects of exogenous ABA on C,N distribution and fruit quality of‘Fuji'apple:Exogenous ABA-treated fruits up-regulated the expression levels of ABA synthesis and receptor genes in the late stage of apple development(135 days after blooming).Compared with the control,ABA content in peel increased by 13.0%-43.2%and 12.6%-42.0%in 2017and 2018,and the ABA content in flesh increased by 13.4%-57.7%and 12.8%-55.5%respectively.50-100 mg·L^-1ABA significantly increased the expression of anthocyanin synthesis genes and transcription factors and increased the content of anthocyanin in the peel.The anthocyanin content of 100 mg·L^-1ABA treatment reached the highest level,which increased by 28.7%and 39.9%respectively compared with control in 2017 and 2018.The results of ¹³C and ¹⁵N double isotope labeling showed that exogenous ABA coordinated the C-N nutrient of apple fruit in the late stage of the development,reduced the accumulation of¹⁵N and increased the accumulation of ¹³C in fruit.100 mg·L^-1ABA treatment increased the contents of soluble sugar and total flavonoids in flesh and improved the internal quality of fruits.Comprehensive analysis showed that the application of 100 mg·L^-1ABA effectively improved the problem of poor coloring caused by high fruit N in the late stage of apple development and was beneficial to the accumulation of C in fruit and the improvement of fruit quality.