Mechanisms Of Nitrate Accumulation In Vegetable And Its Agricultural Regulating Strategies

Vegetable is one of the indispensable foods in human's daily diet,and its qualities are closely related to human's health.However,many vegetables are high-nitrate-accumulated plants,and in addition,the nitrate accumulation in vegetables has been even becoming more serious in recent decades due to higher nitrogen fertilizer input in agricultural production with the aim of high yield.High nitrate intake could put a human into the risk of gastrointestinal cancer and methemoglobinaemia.Therefore,the factors affecting the nitrate accumulation in vegetable should be clearly understood in order to develop agronomic practices to minimize the nitate accumulation.In present research,Chinese cabbages,a kind of mian vegetable in Chian, were used to investage the factors affecting the nitrate-accumulation.We also developed some agronomic technologies to alleviate nitrate accumulation in vegetables.1.Factor affecting nitrate accumulation in plantsHydroponic experiments were carried out to study the nitrate accumulation of 43 Chinese cabbage(Brassica chinensis L.)genotypes under different NO₃^-/NH₄⁺ ratio nutrient solution.The results indicated that the discrepancies in nitrate contents among different genotypes were significant,accordingly,Dongfei,Shanghai baiye and Xiayou gaokang as the low-nitrate-accumulation genotypes,and Gaoxiong tiancui,Baoda aiqi,Suzhouqing as the high-nitrate-accumulation genotypes were screened out.Here,the ratio of NO₃^-/NH₄⁺ 50/50 and the nitrate content in leaves can be considered as the best screen solution and index.Investgation on former six low- or high-nitrate-accumulation genotypes,we demonstrated that nitrate reductase(NR),maximum nitrate uptake rate and affinity significantly contribute to the nitrate accumulation,while the contribution rate of each factor on different vegetable part is different.Here we also found that atmospheric CO₂ elevation could improve nitrate uptake but decrease the nitrate concentrations of the vegetables.Futhermore,the percentage of reduced nitrate was significantly correlated to increased NR activity.Therefore,the decreased nitrate concentration in plant by CO₂ elevation should be attributed to the increased NR activity, demostrating the important role of NR in determinating nitrate concentration in vegetables. 2.Role of NO in NR activity reuglationNitrate reductase(NR),a committed enzyme in nitrate assimilation,involves generation of nitric oxide(NO)in plants.Here we show that the NR activity was significantly enhanced by the addition of NO donors SNP and NONOate to the culturing solution,whereas it was decreased by NO scavenger cPTIO.Interestingly,both NO gas and SNP directly enhanced but cPTIO inhibited the NR activities of crude enzyme extracts and purified NR enzyme.The cPTIO terminated the interaction between NR-generated NO and the NR itself.Furthermore,the NR protein content was not affected by the SNP treatment.The investigation of the partial reactions catalyzed by purified NR using various electron donors and acceptors indicated that the heme and molybdenum centers in NR were the two sites activated by NO.The results suggest that the activation of NR activity by NO is regulated at the post-translational level,probably via a direct interaction mechanism.Accordingly,the concentration of nitrate both in leaves and roots was decreased after two weeks of cultivation with SNP.The present study identifies a new mechanism of NR regulation and nitrate assimilation,which provides important new insights into the complex regulation of N-metabolism in plants.Furthermore,tomato(Lycopersicon esculentum Mill cv.Zheza 809)was used to test whether the effects of NO on NR activity are consistent at different nitrate supply levels.NR activity in the roots with low nitrate pre-treatment was significantly enhanced by the addition of SNP or NONOate to the culturing solution,whereas it was decreased by cPTIO.On the contrary,NR activity in the roots with high nitrate pretreatment showed a reverse behavior with the same SNP or NONOate treatments.Both NO gas and SNP directly enhanced the NR activity of crude enzyme extracts of low nitrate treatment,but inhibited it with high nitrate supply.Direct incubation with cPTIO also showed an opposite effect between low and high nitrate pre-incubation treatments.Hence,it is concluded that NO can dually regulate the NR activity in plants,which depend on the status of plant tissue.The NAA also significantly increased the NR activity in Chinese cabbage,but this stimulating effect was reversed by the addition of cPTIO.The mono-incubation of NPA to the nutrient solution decreased NR activity.However,co-incubation of SNP and NPA increased NR activity. These results suggest that NO is the downstream signal of NAA in NR activity stimulation.Here we also found that CO₂ concentration elevated could increase the NR activities of wild type Arabidopsis thaliana,but did not affect that of NO synthase(NOS)mutant(Atnos).Besides, mono-incubation of NOS inhibitor(L-NNA)to the nutrient solution could also vanish the stimulated effect of elevated CO₂ on NR activity,indicating that the NO generated by NOS should be the downstream signal of CO₂ regulating NR activity.Taking all above together,we proposed that NO may act as the cross-talk signal in regulating multiple upstream signals induced NR activity.3.Agricultural practices developed to alleviate nitrate accumultaion in vegetablesConcentrations of CO₂ are commonly suboptimal for plant production in greenhouses.Here, a new strategy based on the use of crop residues and animal manure(CRAM)compost was developed to increase CO₂ concentration throughout the day.Under this strategy,the production of five common vegetable species was more than doubled in the greenhouse.Meanwhile,nitrate concentrations in the vegetables were significantly decreased,while the ascorbic acid and soluble sugar contents were increased,suggesting that the use of CRAM compost also improved vegetable quality.If such technology was adopted throughout China,the amount of manure discharged to water bodies could be decreased by 13.6-54.5%and 15.5%more crop residues could be efficiently utilized.In conclusion,the use of CRAM compost in greenhouses not only increase farmer income through increase vegetable production and quality,but also alleviates environment pollution.In our previous researches,we have demonstrated that NO can significantly increase the NR activity.Therefore,the effects of atmospheric NO treatment on nitrate accumulation in spinach (Spinacia oleracea L.)was investigated.Trace NO(0.2μL L^-1)or elevated CO₂(800μL L^-1) treatments resulted in higher biomass by 160%and 193%of ambient,respectively,and even gained 246%of ambient with NO/CO₂ combined.Moreover,trace NO,elevated CO₂ or NO/CO₂ combined treatment can also reduce the nitrate accumulation,but increase soluble sugar, protein,Vc,glutathione,flavonoids concentrationts,as well as the FRAP value.These results suggest that trace exogenous NO or NO/CO₂ combined conditions can be adopted as a feasible and efficient practice to improve both the yield and and quality of greenhouse vegetable.