Physiological Mechanism Of Combined Nitrogen And Potassium Regulating Carbon And Nitrogen Metabolism And Anthocyanin Biosynthesis In Apple Fruits

The apple industry has played an important role in promoting regional economic and social development and increasing farmers’ income.However,due to the strict requirements of the market for large fruits and the significant yield increase effect of nitrogen(N)fertilizer,fruit farmers often overemphasize the application of N fertilizer in production.The phenomenon of excessive and insufficient potassium(K)fertilizer application coexists,and the problem of excessive N fertilizer application is prominent.The resulting N and K nutrition imbalance has become an important factor restricting the production of high-quality apples.Therefore,studying the physiological mechanisms by which N and K combined application improves N utilization efficiency and photosynthetic capacity in apples is of great significance for improving the current status of N and K application in apple orchards and improving fruit quality.Using apple rootstock M9T337 and ‘Fuji’ apple as test materials,15 N and 13 C isotope labeling techniques and transcriptome and metabolomics combined analysis techniques were used to study the effects of different K supply levels,K supply methods,and N and K fertilization on N uptake,assimilation,distribution,photosynthesis,and carbon assimilation and transportation of apples.The main results are as follows:1.Compared with medium K supply treatments,the biomass of each organ,the root-shoot ratio,the root activity and NO3-ion flow rate decreased significantly under deficiency and excess K supply,the net photosynthetic rate(Pn)and photochemical efficiency(Fv/Fm)were lower.Meanwhile,seedlings treated with 6 m M K+ had higher N and C metabolizing enzyme activities and higher nitrate transporter gene expression levels(Md NRT1.1;Md NRT2.1).13 C and 15 N labeling results showed that deficiency and excess K could not only reduce the 15 N absorption and 13 C assimilation accumulation of M9T337 seedlings but also reduce the 15 N distribution ratio in leaves and the 13 C distribution ratio in roots.These results suggest that appropriate K supply promoted nitrate uptake and utilization by improving the C and N metabolizing enzyme activities and increasing the expression of nitrate transport proteins,and optimized the transportation of photosynthetic products from leaves to roots,thereby promoting the growth of apple rootstock M9T337.2.Five K supply treatments were implemented: continuous low K(KL),initial low and then high K(KLH),appropriate and constant K(KAC),initial high and then low K(KHL),and continuous high K(KH).The results showed that compared with KL,KH,KLH and KHL treatments,KAC treatments significantly increased the content of IAA and GA3 in roots and promoted the growth and development of roots;KAC supply also significantly up-regulated the transcription level of N assimilation-related genes(nitrate reductase,glutamine synthetase,glutamate synthase,Md NRT1.1,Md NRT1.2,Md NRT1.5,Md NRT2.4),promoted the NO3-assimilation in leaves,enhanced the activity of C metabolism enzymes such as Rubisco in leaves,improved the Pn,and realized the efficient use of N.The 13 C and 15 N isotope tracing results showed that the KAC treatments promoted the transport of N to the shoot and the transport of photosynthetic products to the root,optimized the distribution of C and N,and coordinated the efficient assimilation and transport of C and N.3.High nitrate-N conditions damaged the root cortex structure,reduced the net NO3-and K+ influx at the surface of roots,thereby resulting in an N/K imbalance in rootstocks;and also hinder N uptake and C assimilation,reduced photosynthesis and photosynthetic nitrogen utilization efficiency,thus inhibited the growth of apple rootstock M9T337 seedlings.Under N10 condition,6 m M K supply level was suitable for rootstock growth and carbon and nitrogen metabolism,while under N30 conditions,the activities of antioxidant enzymes(SOD,POD and APX)and nitrogen metabolism enzymes(NR,GS and GOGAT)of seedlings under10 m M K level are the highest,and the expression of related nitrate uptake and sugar transporter proteins(Md NRT1.1,Md NRT1.2,Md NRT1.5,Md NRT2.1,Md SOT1,Md SOT2,and Md SUT1)is the best,and the photosynthetic nitrogen utilization efficiency is the highest.These results suggest that the negative effects of high nitrate may be related to the N/K imbalance and that reducing N/K in plants by increasing K supply level can effectively alleviate the inhibition of seedling growth,nitrogen absorption and carbon assimilation and transport by high nitrate stress.4.Low K conditions significantly reduced K content in roots and leaves,resulting in N/K imbalance,and allocated more N in leaves to sodium dodecyl sulfate-soluble-insoluble proteins(Nin-SDS),non-protein N(Np),and non-photosynthetic N,resulting in a lower proportion of photosynthetic N.Low K conditions decreased stomatal conductance,the maximum carboxylation rate(Vmax),and photosynthesis and increased cell wall thickness,stomatal limitation(SL),mesophyll limitation(MCL),and biochemical limitation(BL).By setting different N supplies,lowering N levels under low K conditions increased the proportion of water-soluble protein(Nw)and sodium dodecyl sulfate-soluble proteins(Ns)by balancing N/K and increased the proportion of carboxylation N and electron transfer N.This increased Vmax and gm,which reduced MCL and BL and alleviated the low K limitation of photosynthetic carbon fixation in apple rootstocks.5.High N destroyed the N–K balance in the fruit,resulting in a significant reduction in fruit redness and anthocyanin content,and reduced fruit quality.The results of C and N isotope tracing showed that high N increased the ability of fruit to absorb and regulate 15 N,and decreased the 13 C distribution to fruit.K application promoted the transportation of photosynthetic products to the fruit.Anthocyanin-targeted metabonomics and transcriptome analysis revealed that high N downregulated the expression of structural genes related to the anthocyanin synthesis pathway(Md PAL,Md4 CL,Md F3 H,Md ANS,and Md UFGT)and their regulators(Md MYBs and Mdb HLHs),and also decreased some metabolites contents.K promoted the transport capacity of C to fruits and also enhancing the expression of key structural genes(Md PAL,Md4 CL,Md CHS,Md CHI,Md F3 H,Md DFR,Md ANS,and Md UFGT)in the anthocyanin synthesis pathway and promoting anthocyanin accumulation.Targeted metabolism analysis of anthocyanins showed that Cyanidin-3-O-glucoside,Cyanidin-3-O-(6-O-malonyl-beta-D-glucoside),cyanidin-3-O-rutinoside,cyanidin-3-O-sophoricoside,pelargonidin-3-O-galactoside and peonidin-3-O-glucoside may be the key anthocyanosides of N–K affecting apple peel coloration.