摘要
蔬菜是人们日常生活中不可或缺的重要食物,其品质的优劣对于人民的身体健康有极大影响。但是,蔬菜又是一种容易积累硝酸盐的植物,再加上近几十年来以高产为目的的盲目施肥更加剧了硝酸盐在蔬菜体内的积累。由于摄入硝酸盐含量较高的蔬菜可能会增加人体患肠胃癌、高铁血红蛋白症等疾病的几率,因此蔬菜硝酸盐积累的相关研究已经广泛开展。本研究试图通过水培和盆栽等试验,研究了影响蔬菜硝酸盐积累的原因,以及调控硝酸盐同化关键酶的机制,并以此提出减缓蔬菜硝酸盐积累的农艺措施。主要研究结果如下:
1、植物体硝酸盐积累机制的研究
采用水培方法研究了43种基因型小白菜在不同硝铵比条件下体内硝酸盐含量的变化情况。结果表明,不同基因型小白菜的硝酸盐含量差异较大,冬妃青梗菜、上海白叶四月蔓和夏优高抗为低硝酸盐含量基因型小白菜,而高雄甜脆小白菜、宝大矮棋青和苏州青为高硝酸盐含量基因型小白菜。硝铵比50/50是最适宜筛选的氮素形态比例,叶片中的硝酸盐含量也是最适宜的筛选指标。
对上述6个基因型小白菜进一步分析结果表明,不同部位硝酸还原酶、根系硝态氮最大吸收速率以及亲和力这三个因素对小白菜各部位的硝酸盐积累都有显著的影响,但在不同部位,这三个因素所起的贡献率不同。叶片硝酸盐含量取决于NR活性、吸收速率和亲和力三方面的影响;叶柄硝酸盐含量与Vmax和Km相关性更大;根系硝酸盐含量则与Vmax极显著相关。进一步研究发现,环境因素CO_2浓度的增加促进了小白菜的吸氮能力,但也显著降低了植株各部位的硝酸盐含量,而且其降低百分率与NR活性的增加百分率呈极显著相关。虽然,CO_2浓度增加也显著提高各部位的生物量,但是硝酸盐降低百分率与鲜重增加百分率相关性并不显著。因此,可认为CO_2浓度升高引起的硝酸盐含量的降低主要是NR活性提高导致的,体现了NR在植株硝酸盐积累过程中的重要地位。
2、NO对植物体内硝酸盐还原酶活性的调控作用
在低氮(1mM)水培条件下,外源NO供体SNP或NONOate处理可以显著促进小白菜根系的NR酶活性,但不影响根系内的NR蛋白含量,而NO清除剂cPTIO处理抑制了NR活性。有趣的是,小白菜NR纯化后的酶活性也能被SNP或NO气体促进,并被cPTIO抑制。这些结果表明NO能在翻译后水平调控NR活性。利用不同的电子供体和电子受体研究了NO对NR各级反应速率的影响,结果表明NO能显著促进NADH:CR、Flavin:NR和MV:NR的活性,而NADH:FR活性未受影响,说明NO可能是通过NO自身与NR蛋白的直接作用激活了血红素及钼中心,推动了电子在这两者间的传递,从而促进了NR活性。
利用番茄进一步研究了不同氮水平条件下NO对NR的调控作用。在低氮条件下,NO供体和淬灭剂处理对NR的影响与小白菜一致;相反的是,在高氮条件下,NO供体处理却明显抑制了NR的活性,而cPTIO处理却促进了NR的活性。粗酶提取液试验发现,在低浓度SNP(10—20 gM)及NO气体(1cm~3)处理下,无论是低硝态氮还是高硝态氮培养的番茄根系NR均被显著促进。但是,随着NO供体处理量的增加,高硝态氮培养的番茄根系NR却被抑制。这些结果均表明NO在翻译后水平对植物的NR活性具有双重调控作用。而在高氮条件下NO是如何抑制NR活性的机制尚不明确,还需进一步研究。
由于NO是许多信号分子(如IAA)的下游信号,而IAA对NR酶活性也具有促进作用。在本研究中,NAA(IAA类似物)能显著提高小白菜根系NO含量及NR活性,但其促进作用能被cPTIO消除。另一方面,NPA(IAA运输抑制剂)显著抑制小白菜根系NO含量及NR活性,但被NO供体SNP活化。这些结果表明NO是IAA调控NR活性的下游信号分子。此外,许多环境因子变化也能影响促进NR活性。以拟南芥为研究材料,我们发现,CO_2浓度增加显著促进了野生型拟南芥根系的NR活性,但是对NOS(一氧化氮合酶)拟南芥突变体的NR活性没有明显的影响;用一氧化氮合酶抑制剂处理也能抑制CO_2浓度增加对NR活性的促进作用;表明一氧化氮合酶产生的NO是CO_2浓度增加促进NR活性的信号分子。综上所述,NO在调控植物NR酶活性中起到重要的枢纽作用。
3、降低蔬菜硝酸盐积累的不同农艺措施研究
作物秸秆和畜禽粪便(CRAM)堆肥发酵可以产生大量的CO_2。采用这种策略,可以显著增加大棚内的CO_2浓度,使5种常见蔬菜的产量增加1倍以上,同时也可显著降低蔬菜中的硝酸盐含量,增加抗坏血酸和可溶性糖含量。这表明,大棚内推广CRAM发酵技术不但可以促进蔬菜产量的增加,也可以提高其品质。此外,根据我们的估算,如果该技术在全国范围内广泛推广,可减少13.6—54.5%的畜禽粪便排放,并有15.5%的秸秆可被有效利用,因而该技术的推广还可以减缓农业固废对环境造成的污染。
以菠菜为研究材料,在空气中施用微量NO气体后发现菠菜产量增加1倍左右,并且可食部位的硝酸盐含量显著降低,抗坏血酸、谷胱甘肽、类黄酮等抗氧化化合物的含量也明显增加。此外,施用NO气体还增加了可溶性糖和可溶性蛋白的含量,提高了蔬菜食用的口感。同时提高CO_2浓度并施用微量NO气体能进一步促进蔬菜产量的增加以及品质改善。因此,在大棚内施用NO气体也是一种提高产量和品质的有效农艺措施。
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 plants
Hydroponic experiments were carried out to study the nitrate accumulation of 43 Chinese cabbage(Brassica chinensis L.)genotypes under different NO_3~-/NH_4~+ 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 NO_3~-/NH_4~+ 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 CO_2 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 CO_2 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 reuglation
Nitrate 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 CO_2 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 CO_2 on NR activity,indicating that the NO generated by NOS should be the downstream signal of CO_2 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 vegetables
Concentrations of CO_2 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 CO_2 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 CO_2(800μL L~(-1)) treatments resulted in higher biomass by 160%and 193%of ambient,respectively,and even gained 246%of ambient with NO/CO_2 combined.Moreover,trace NO,elevated CO_2 or NO/CO_2 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/CO_2 combined conditions can be adopted as a feasible and efficient practice to improve both the yield and and quality of greenhouse vegetable.
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