The Mechanism Of Nitrogen In Regulating Cucumber Low Temperature Tolerance And Leaf Nitrogen Allocation

Cucumber（Cucumis sativus L.）,as a major vegetable crop in protected cultivation,is popular with consumers for its good economic benefit and nutritional value,but it often suffers from low-temperature（LT）stress during winter and spring production in China,which seriously affects its growth and development,yield and quality.Therefore,LT stress has become a major factor limiting the production of the facility cucumber.The effectiveness of rhizosphere nitrogen（N）is reduced under LT stress,and the crop’s ability to absorb,translocate and assimilate N is weakened,and scientific and reasonable application of N fertilizer is especially critical to enhance LT-resistance of cucumber.However,it is unclear that the intrinsic regulatory mechanisms of N on leaf N allocation,integrated growth and photosynthetic characteristics of cucumber under LT.In order to provide theoretical basis and technical support for N fertilizer application to cucumber grown under LT,the nutrient solution for fruiting and seedling stages of cucumber under different temperatures to clarify the effect of N on the comprehensive growth of cucumber were optimized;the mechanisms of the effects of different N concentrations（NC）and ammonium-nitrate N ratios（A-NR）on leaf growth,N allocation,photosynthetic capacity and N accumulation were resolved under LT;further the mechanism of different NCs on N allocation,N uptake and metabolism,photosynthetic system and cell structure of cucumber leaves under LT were analyzed.The main findings are as following:1.N was identified as the most important element affecting the integrated growth of cucumber during the fruiting stage.The formulation of a nutrient solution with large amounts of elements suitable for the fruiting stage of cucumber cultivated in substrate was obtained by comprehensive evaluation and modeling of cucumber yield,quality and fertilizer utilization.The regression model of N,K⁺,Ca²⁺and Mg²⁺on the comprehensive score of cucumber yield and quality was established using quadratic regression analysis with‘Bonai 526’cucumber as the test material.The effects of each factor on the yield and quality comprehensive score of cucumber were found to be N,K⁺,Ca²⁺and Mg²⁺in descending order.the N-K⁺coupling effect was negative,while the N-Ca²⁺coupling effect was positive.Meanwhile,a multi-objective optimization model was developed for the yield and quality comprehensive score combined with N,K and Ca utilization efficiency,and the optimized N,K⁺,Ca²⁺and Mg²⁺concentrations of 14.83 m M,6.89 m M,3.55 m M and 4.17 m M,respectively,were verified by using a genetic algorithm for simulation to find the best.The yield per plant,soluble protein,vitamin C and total soluble sugar contents of cucumber under the optimal nutrient solution formulation were significantly increased by 21.1%,40.9%,53.3%and 11.5%,respectively,and the organic acid and nitrate contents were significantly reduced by 4.7%and 16.8%,respectively,compared with the Yamazaki cucumber special formulation treatment.2.N was identified as the most important element affecting the comprehensive growth of cucumber seedlings.The formulation of a nutrient solution with a large number of elements suitable for cucumber seedlings grown in substrate under low temperature（LT）was obtained by comprehensive evaluation and modeling of dry matter accumulation,distribution rate and chlorophyll in cucumber.The effects of each factor on the integrated score of cucumber were found to be,in descending order,N,Mg²⁺,K⁺and Ca²⁺,with positive effects of N and Ca²⁺on the integrated score,negative effects of K⁺and Mg²⁺on the integrated score,and positive N-Mg²⁺coupling effects,which were greater than the N-K⁺coupling effects.The optimal nutrient solution formulation for cucumber seedlings under LT was obtained by further model search:N,K⁺,Ca²⁺and Mg²⁺concentrations were 14.58-15.37 m M,1.80-2.00 m M,3.99-4.25 m M and 0.74-0.99 m M,respectively,which was most favorable to the integrated growth of cucumber under LT.3.N supply promotes cucumber growth and photosynthetic capacity by regulating leaf N allocation under LT.In this study,the effects of different NCs and A-NRs supplied to grown LT-stressed cucumber at seedling and flowering to early fruiting stages on growth,photosynthesis,N accumulation,and leaf N allocation were assessed.NC,A-NR,and their interactions significantly affected leaf area,A_n,chlorophyll content,and N accumulation at seedling and flowering to early fruiting stages.When the cucumber plants were grown in LT environment,the maximum photochemical efficiency,leaf area,A_n,chlorophyll content,N_a,water-soluble proteins N（N_w）,and N accumulations in various organs were the greatest.The content of sodium dodecyl sulfate（a detergent）soluble protein N（N_s）was minimal when the NCs at the seedling and flowering to early fruiting stages were 10.5 and 28 m M,respectively,and the A-NRs were both 1:1.Correlation analysis showed that leaf area,A_n,chlorophyll content,and N accumulation could be improved by increasing N_a and N_w or reducing N_s.This finding suggested that increasing NC and NH₄⁺proportion in the nutrient solution could increase N_a and N_w in leaves and reduce the content of N_s,thereby improving photosynthetic capacity to promote leaf growth and increasing cucumber LT resistance.4.‘Jingyou No.1’had higher maximum chlorophyll fluorescence intensity,PSII specific activity parameters,quantum yield and driving force under LT stress,with average increase of8.5%,14.2%,18.3%and 42.6%compared with‘Bonai 526’,which was attributed to the high N-metabolizing enzyme activity and related genes expression in‘Jingyou No.1’.In addition,the response of different genotypes of cucumber to N concentration under LT was significantly different.Compared with‘Bonai 526’,NC supply under low temperature was more favorable to promote root growth and biomass accumulation of‘Jingyou No.1’,significantly increasing N metabolism enzyme activity and chlorophyll content,improving quantum yield captured and absorbed per reaction center and electron chain end driving force,promoting PSII primary photochemical reaction mechanism activity and performance,and thus improving LT tolerance of the plants.5.Nitrogen allocation affects the mesophyll structure to coordinate the adaptation of CO₂conductivity and photosynthetic capacity to LT in Cucumis sativus L.Temperature and N supply significantly affected leaf N allocation and anatomical traits,LT reduced N_a and non-protein N(N_np)to cause the decrease of surface area of chloroplast exposed to intercellular air spaces per leaf area（S_c/S）,which increased CO₂ diffusion resistance in liquid phase(r_liq)resulting in decreased mesophyll conductance（g_m）to inhibit photosynthesis.Under LT,different leaf N forms had significantly direct effects on S_c/S,moreover,the effect of S_c/S on the liquid phase conductance was greater than r_liq.The effect of N supply on g_mand photosynthesis of cucumber depending on cultivars,‘Bonai 526’had higher N supply needs to maintain photosynthetic capacity under LT.The high photosynthetic capacity of‘Jinyou No.1’was attributed to high contents of N_a,N_wand N_np,lower N_s content to increase S_c/S,which improved g_m under LT.These findings suggested that synergistic changes in leaf N forms and mesophyll traits enhance cucumber seedlings adaptation to LT by coordinating g_mand photosynthesis.In conclusion,N was identified as the primary element affecting the integrated growth of cucumber by optimizing the nutrient solution of cucumber grown in substrate fruiting stage under normal temperature and seedling stage under LT;under LT stress,increasing NC and A-NR promoted plant growth,photosynthesis and N accumulation by regulating leaf N allocation,and there were significant differences in the response of different genotypes of cucumber to NC,compared with‘Bonai 526’,‘Jinyou No.1’had higher PSII performance,stronger N metabolism and root growth ability,and N supply enhanced cucumber cold tolerance and thus cucumber growth by affecting leaf N forms and mesophyll structure in coordination with g_mand photosynthesis.