作物杂志, 2022, 38(6): 124-131 doi: 10.16035/j.issn.1001-7283.2022.06.018

生理生化·植物营养·栽培耕作

硝酸盐缓解油菜铵毒害的生理机制

王雪茹,, 陈海飞, 张振华,

湖南农业大学资源环境学院/南方粮油作物协同创新中心,410128,湖南长沙

Physiological Mechanism of Nitrate Mitigation of Ammonium Toxicity in Rape

Wang Xueru,, Chen Haifei, Zhang Zhenhua,

College of Resource and Environment, Hunan Agricultural University/Southern Regional Collaborative Innovation Center for Grain and Oil Crops in China, Changsha 410128, Hunan, China

通讯作者: 张振华,主要从事作物养分高效研究,E-mail:zhzh1468@163.com

收稿日期: 2021-10-24   修回日期: 2021-11-29   网络出版日期: 2022-04-21

基金资助: 国家自然科学基金(32072664)
湖南省自然科学基金杰出青年基金(2021JJ0004)
国家油菜产业技术体系

Received: 2021-10-24   Revised: 2021-11-29   Online: 2022-04-21

作者简介 About authors

王雪茹,研究方向为植物营养与遗传,E-mail:hunaustuwxr@163.com

摘要

为探究铵态氮条件下增硝营养对油菜铵态氮利用及生长的影响,以品种Bn60为试验材料,以纯硝培养为对照,测定不同氮形态下培养15d油菜的生物量和叶绿素含量。并在5mmol/L NH4+条件下,添加5个不同的NO3-浓度(0.0、0.1、0.3、0.6、1.0mmol/L),处理7d后测定游离NH4+含量及氮素同化酶活性,处理15d后测定生物量、全氮和阳离子含量。结果表明,与硝态氮相比,单一铵态氮导致生长抑制、叶片枯黄,但随着硝酸盐浓度的提高,铵毒害症状逐渐缓解,地上和根系的生物量、全氮量和氮累积量均显著增加。增硝营养显著增强了油菜地上部谷氨酰胺合成酶活性,进而降低游离铵态氮含量,另外K+、Ca2+和Mg2+等阳离子含量均随着硝态氮的增加而显著提高。硝酸盐能增强氮素同化酶的活性,从而降低NH4+含量,同时提高Mg2+等阳离子含量和光合作用,最终缓解铵毒害性状,促进油菜的生长。

关键词: 油菜; 铵毒害; 铵态氮; 硝态氮; 生理机制

Abstract

In order to explore the effects of nitrate nutrition on ammonium nitrogen utilization and growth of rape (Brassica napus L.), the biomass and chlorophyll concentration of rape cultured for 15 days under different nitrogen forms were measured by using Bn60 as experimental material and pure nitrate culture as control. Under the condition of 5mmol/L NH4+, five different NO3- concentrations (0.0, 0.1, 0.3, 0.6 and 1.0mmol/L) were added. After seven days of treatment, the concentration of NH4+ and the activity of nitrogen assimilation enzyme were measured. After 15 days of treatment, the concentration of biomass, total nitrogen and cation groups were measured. The results showed that compared with nitrate nitrogen, single ammonium nitrogen led to growth inhibition and yellow leaves, but with the increase of nitrate concentration, the symptoms of ammonium toxicity gradually relieved, and the shoot and root biomass, total nitrogen and nitrogen accumulation increased significantly. Increased nitrate feeding increased glutamine synthase (GS) activity in rape shoots, which led to a decrease in free ammonium ion concentration. Furthermore, as nitrate nitrogen levels rose, the concentrations of cations such as K+, Ca2+, and Mg2+ rose as well. Nitrate could increase the activity of nitrogen absorbing enzymes, decrease ammonium ion concentrations, increase the contents of Mg2+ and other cations, as well as photosynthesis, mitigate ammonium toxicity and improve rape growth.

Keywords: Rape; Ammonium toxicity; Ammonium nitrogen; Nitrate nitrogen; Physiological mechanism

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本文引用格式

王雪茹, 陈海飞, 张振华. 硝酸盐缓解油菜铵毒害的生理机制. 作物杂志, 2022, 38(6): 124-131 doi:10.16035/j.issn.1001-7283.2022.06.018

Wang Xueru, Chen Haifei, Zhang Zhenhua. Physiological Mechanism of Nitrate Mitigation of Ammonium Toxicity in Rape. Crops, 2022, 38(6): 124-131 doi:10.16035/j.issn.1001-7283.2022.06.018

氮是植物生长发育不可缺少的大量营养元素,是组成蛋白质、酶和核酸等重要大分子的成分,广泛参与植物生长代谢等诸多生物过程。然而农业生产中氮肥过量和不合理施用,造成氮大量流失,导致土壤环境与水体污染,威胁生物多样性,破坏生态平衡。硝态氮(NO3-)和铵态氮(NH4+)是大多数植物氮的主要来源,占植物对阴离子和阳离子吸收的70%[1]。最近研究[2]表明,随着全球CO2浓度的升高,C3植物中NO3-的还原被抑制,NH4+的同化不受影响,因此增加NH4+的施用对粮食生产至关重要。

低浓度的NH4+能促进大多数植物的生长[3-4],然而高浓度NH4+作为唯一氮源抑制植物根系和地上部的生长。当铵转化为氨基酸和酰胺的总速率低于氨基酸分解代谢、硝酸盐还原、苯基丙类代谢和光呼吸对铵的吸收和细胞产生的速率时,植物组织中铵会积累[5],植株出现根系停止生长、叶片枯黄萎焉等铵毒害症状[6]。研究[7]表明,氨毒害发生是由于细胞内NH4+水平的升高破坏了植物激素稳态,导致光电传递效率下降和活性氧物质爆发、细胞质酸化、膜结构破坏及阴阳离子失衡等问题。其中,谷氨酰胺合成酶(GS)是调控铵同化的关键酶,在植物体中存在2种同工酶,分别是GS1和GS2。最近的研究[8]表明,拟南芥根部GS1的编码基因Gln1;2正调控植株的铵耐受性,但是地上部GS2的编码基因Gln2负调控植株的铵耐受性,说明植物氨毒害发生可能受到多种生理途径的综合影响。

油菜是世界上重要的油料作物,对氮肥的需求量很大且利用率低[9-10]。此外,过量的氮会降低油菜的含油量,因此合理施用氮肥对油菜提质增效至关重要[10-12]。在南方稻油复种的水田中铵态氮含量较高,而油菜等十字花科植物对铵态氮敏感,限制了油菜生长。而硝铵混合营养可缓解植物的铵毒害症状,恢复植物的正常生长,但其作用机理尚不清楚。因此,本研究通过同时添加不同浓度的硝酸盐和5mmol/L铵态氮水培试验,探究硝酸盐缓解油菜铵毒害和提高油菜氮利用效率的生理机制。

1 材料与方法

1.1 试验材料与处理

试验于2020年9月在湖南农业大学第八教学楼进行。采用水培试验,选择油菜品种Bn60为供试材料。将油菜种子播种于塑料盒内铺设的纱布上,用去离子水浸泡。6d后选取长势一致的幼苗移植到55cm×37cm×8cm的水培盒中,每盆含4L营养液。试验采用完全随机区组设计。每升营养液由5.0mmol/L KNO3、5.0mmol/L Ca(NO3)2·4H2O、2.0mmol/L MgSO4·4H2O、1.0mmol/L KH2PO4、50μmol/L Fe-EDTA、9μmol/L MnCl2·4H2O、0.8μmol/L ZnSO4·7H2O、0.3μmol/L CuSO4·5H2O、0.1μmol/L NaMoO4·2H2O和50μmol/L H3BO3组成。每5d更换1次营养液,适应性培养5d后再处理15d。试验设置5个铵态氮处理,在5mmol/L NH4+的条件下,NO3-浓度分别为0、0.1、0.3、0.6和1.0mmol/L[13],均采用NH4Cl和Ca(NO3)2处理,并设置1盆5mmol/L硝态氮培养油菜进行对照(CK)。为了避免Ca2+浓度不同带来的影响,使用CaCl2将所有处理的Ca2+浓度调整一致,共6盆,每盆15株。每次试验的各个处理设置3次生物学重复,2个平行试验。

1.2 测定项目与方法

1.2.1 生物量

处理15d后每个处理取整株,根部用蒸馏水冲洗3~4次,用滤纸擦干表面的水分,烘干至恒重后于万分之一天平称重。

1.2.2 全氮含量

各供试材料在处理15d后,植株样品按地上、地下部位经105℃杀青0.5h,然后在70℃下烘干至恒重并剪碎。称取0.1g干样置于150mL消煮瓶中,用H2SO4-H2O2消煮至透明清亮,转移至50mL容量瓶中,定溶、过滤后用连续流动分析仪(AA3)测定全氮含量[14]。氮累积量=植株生物量×植株氮含量,氮素吸收效率=氮素累积量/供氮量[15]

1.2.3 植物组织离子组

将处理15d的油菜根系和地上部分烘干磨碎后,利用ICP-MS测定其中金属元素含量。油菜根系、地上部等植物样品烘干后粉碎,自封袋保存备用。称取0.1g样品于消解管中,加入浓硝酸浸泡,待消解完全后将消煮液转移到10mL比色管中定容,稀释至规定倍数,以慢速定量滤纸过滤,滤液放置4℃下保存,用ICP-MS(NexIONTM350X)测定其中金属元素含量,同时做空白。

1.2.4 光合参数

采用LI-6400XT便携式光合系统进行测定植物光合作用。测定过程中,叶室温度控制在22°C,光合量子通量密度(PPFD)为200μmol/(m2·s),CO2浓度设定为500μmol/mol,基本接近培养室内的CO2浓度,测定时间为9:00-15:00。试验材料处理15d,选取第3或第4片叶片,将叶片放入叶室并夹好,闭合叶室,待植物适应人工光源和光合作用参数值稳定后记录数据[16]

1.2.5 叶绿素含量

将处理15d的油菜取0.15g叶片,剪碎后置于10mL刻度试管中,加入1:1(V:V)的丙酮:乙醇溶液10mL,塞好瓶盖,室温下暗处提取48h(叶片完全变白),取上清液测定663、645和652nm处的吸光值。并计算总叶绿素含量:总叶绿素含量(mg/g)=D652×V÷(34.5×W),式中,V为提取液的总体积,W为样品的叶面积或鲜重[16]

1.2.6 NH4+含量

采用靛酚蓝比色法测定NH4+含量。处理7d的油菜幼苗用蒸馏水冲洗3~4次,用滤纸擦干表面的水分,称重0.1g后装入5mL的离心管,放入液氮冷冻片刻于组织研磨仪上打碎,用5mL 0.1%的稀盐酸提取30min,过滤后吸取上清液200μL,用0.1%的稀盐酸稀释至4mL,依次加入0.5mL的苯酚溶液(苯酚5g,亚硝基铁氰化钠50mg定容至500mL)和NaClO碱性溶液(NaOH 5g,Na2HPO4 3.53g,Na3PO4 15.9g,量取NaClO[ω(NaClO)=5.25%,即含有效氯5%的漂白剂溶液]溶液5mL,溶解后转移至500mL容量瓶后定容),摇匀后37℃水浴30min,在625nm波长处进行比色[17]

1.2.7 谷氨酰胺合成酶(GS)活性

处理7d的油菜分地上部和根部取样,用蒸馏水冲洗3~4次,用滤纸擦干表面的水分,称取0.1g,采用GS试剂盒(苏州科铭生物技术有限公司)测定GS活性。

1.3 数据处理

采用Excel和DPS软件进行统计分析及单因素方差分析,LSD法进行显著性检验(P<0.05),文中图采用GraphPad Prism 8软件绘制。

2 结果与分析

2.1 铵毒害对油菜生长的影响

与正常培养相比,油菜在5mmol/L NH4+条件下的株型明显变小,侧根较少,并且根长更长(图1a)。在5mmol/L NH4+条件下培养的油菜叶片出现了明显的黄化,叶片数量相较正常培养更少,总叶面积也有明显的减小(图1b)。地上部正常培养生物量是NH4+培养的2.87倍,地下部生物量正常培养是NH4+培养的4.13倍(图1c)。叶绿素含量正常培养是NH4+培养的1.86倍(图1d)。说明5mmol/L NH4+条件对油菜具有明显的毒害作用,抑制了油菜的生长,导致油菜发育缓慢,叶片黄化、侧根减少,生物量和叶绿素含量都有极显著的降低。

图1

图1   铵毒害对油菜生长和叶绿素含量的影响

正常培养(CK)和纯铵培养(5mmol/L NH4+)油菜整体的表型(a)、叶片表型(b)、地上和地下的生物量(c)及叶绿素含量(d)。数据以平均值±标准误表示(n=3)。“**”表示在P < 0.01水平差异显著

Fig.1   Effects of ammonium poisoning on rape growth and chlorophyll content

Phenotype (a), leaf phenotype (b), biomass (c) and chlorophyll content (d) of rape under normal culture (CK) and ammonium toxicity (5mmol/L NH4+). Data represent means ± SE (n=3).

“**”indicates significant difference at P < 0.01 level


2.2 添加外源硝酸盐可缓解油菜铵毒害的症状

随着添加硝酸盐浓度的提高,油菜的铵毒害症状逐渐缓解(图2a)。油菜地上部和根系生物量与表型一致,硝酸盐梯度0.1~1.0mmol/L的生物量相比0.0mmol/L分别上升了2.94%、34.88%、36.76%和43.93%(图2b)。与单一铵态氮条件下的油菜相比,随着硝酸盐的浓度增加,油菜的Pn显著增加,硝酸盐浓度在0.3、0.6、1.0mmol/L时的Pn均显著高于0.0和0.1mmol/L处理(图2c)。此外,添加不同浓度的硝酸盐后,下部叶片的黄化症状逐渐消失,与表型一致的是,油菜叶片叶绿素含量随着硝酸盐浓度的增加而上升,在硝酸盐浓度为1.0mmol/L时达到最高,是0mmol/L的2.6倍(图2d)。说明在铵毒害条件下添加低浓度的硝酸盐可提高油菜的叶绿素含量和Pn,缓解铵毒害对油菜生长的抑制。

图2

图2   添加外源硝酸盐对油菜生长和光合的影响

数据以平均值±标准误表示(n=3)。柱子上字母不同表示在P < 0.05水平差异显著,下同

Fig.2   Effects of exogenous nitrate on growth and photosynthesis of rape

Data represent means±SE (n=3). Bars with the different letters indicate significant difference at P < 0.05 level, the same below


2.3 添加外源硝酸盐对油菜阳离子含量的影响

研究[6]表明,NH4+可以抑制作物对K+、Ca2+和Mg2+等的吸收。通过比较添加不同浓度的硝酸盐生长条件下的油菜阳离子含量,发现地上部K+、Ca2+和Mg2+含量随着硝酸盐浓度的上升而上升,硝酸盐浓度在0.6和1.0mmol/L相比0.0和0.1mmol/L,K+含量上升了30%左右(图3a);1.0mmol/L相比0.0mmol/L硝酸盐处理,Ca2+含量上升了88.9%(图3b),Mg2+含量上升了60.6%(图3c)。而油菜根系阳离子含量的上升虽然不如地上部显著,但是随着硝酸盐浓度的上升,K+、Ca2+和Mg2+的含量也都有明显的上升。而综合地上部与根系的上升趋势来看,阳离子含量的上升与硝酸盐浓度呈现正相关,并且添加浓度达到1.0mmol/L时效果最好。说明添加外源硝酸盐在一定程度上促进油菜对阳离子的吸收,增加油菜的阳离子含量。

图3

图3   外源添加硝酸盐对油菜阳离子含量的影响

Fig.3   Effects of exogenous nitrate nitrogen addition on cationic content in rape


2.4 添加外源硝酸盐对油菜氮同化能力的影响

铵营养条件下,随着硝酸盐处理浓度的增加,地上部和地下部游离铵的含量在硝酸盐浓度0.1mmol/L时增加趋势显著,在0.3和0.6mmol/L时增加趋势不显著,然而硝酸盐浓度为1.0mmol/L时,地上部的铵盐含量比纯铵处理降低了21.9%,根系的铵盐含量比纯铵处理降低了24.8%。表明增加硝酸盐的供应可降低油菜铵盐的含量,减轻氨毒害(图4a)。与单一铵营养相比,增加硝酸盐供应可显著增加GS活性,而GS活性表征氮同化能力的强弱,这一结果与植株体内游离铵含量下降相吻合。结果表明,随着硝酸盐的供应,地上部氮同化能力呈梯度显著增强,与铵处理相比,硝酸盐浓度为0.1、0.3和0.6mmol/L时,GS活性分别增加了1.03、0.66和2.69倍,在硝酸盐浓度达到1.0mmol/L时,GS活性显著提升了3.58倍。而根系的GS活性则没有明显的变化(图4b)。

图4

图4   添加外源硝酸盐对油菜氮同化能力的影响

Fig.4   Effects of exogenous nitrate addition on nitrogen assimilation ability of rape


2.5 添加外源硝酸盐对油菜总氮含量的影响

增施硝酸盐后,油菜的总氮含量、累积量和氮素吸收效率都呈增加的趋势(图5)。地上部硝酸盐浓度为0.3、0.6和1.0mmol/L时的总氮含量、氮累积量和氮素吸收效率都显著高于0.0和0.1mmol/L处理;而根系随着硝酸盐浓度的上升呈现显著的上升趋势,当硝酸盐浓度为0.6和1.0mmol/L时的总氮含量、氮累积量和氮素吸收效率显著高于其他处理。结果表明,添加外源硝酸盐可增加油菜在铵毒害条件下对氮素的吸收效率。

图5

图5   添加外源硝酸盐对油菜总氮含量的影响

Fig.5   Effects of exogenous nitrate addition on total nitrogen content in rape


3 讨论

3.1 添加外源硝酸盐可提高油菜的阳离子含量

高铵胁迫会造成无机阳离子的减少。当NH4+作为唯一氮源时,植物为了生长发育,不得不吸收过多的NH4+,由于其带正电荷,因此影响了其他无机阳离子的吸收。研究[18]发现,相比于NO3-,当NH4+作为氮源时,植物体内的Ca2+和Mg2+含量显著减少。阳离子含量能够影响作物的电化学平衡,而Ca2+和Mg2+是植物体内重要的无机阳离子,参与众多生理生化过程,如细胞信号转导、许多酶的活性以及光合作用等。植物在吸收铵态氮时会以质子留在膜外的方式进入细胞内,使根际的pH降低。前人[19-20]研究指出,不同的铵硝比在施氮总量不变的情况下并不会使蝴蝶兰对氮的吸收能力升高或降低,但铵硝比的升高会抑制蝴蝶兰对K+、Ca2+和Mg2+等的吸收,相对于混合施氮,硝态氮更有利于蝴蝶兰的生长。常笑超等[21]发现比起纯铵培养,添加硝态氮显著促进了毛白杨根氮、磷、钾元素的累积。在本试验中观察到,在铵态氮条件下加入不同浓度的硝酸盐后,K+、Ca2+和Mg2+的含量上升(图3),这与前人的研究结果一致,说明铵态氮可能会抑制油菜对阳离子的吸收,而添加外源硝酸盐能增加油菜的阳离子含量,进而促进植物体内酶活性和光合作用等生理生化过程。

3.2 添加外源硝酸盐能增强油菜的铵同化能力和氮素利用效率

氮素利用效率由吸收和同化2个途径决定,植物通过增强体内NH4+的同化来实现自我解毒的能力是减轻铵毒害的重要途径[22-23]。植物吸收的铵态氮大部分在根中进行同化,然而仍有部分铵态氮会通过木质部运输至地上部[24-25],地上部的铵态氮则会通过GS同化或以NH3的形式损失[26]。高浓度的NH4+还可抑制作物氮代谢中关键酶活性,如GS和谷氨酸合成酶(GOGAT)[27]。其中GS是植株同化铵态氮的关键酶,铵态氮浓度越高,GS活性越强[28]。一般认为,植物体内存在2种GS,GS1参与植物体内大部分的铵同化,而GS2则定位于叶绿体或质体中,并参与NO3-还原产生的NH4+同化[29]。迟荪琳等[30]认为,小白菜中GS活性与硝酸盐含量呈正相关关系。总的来说,增加氮的吸收和运输以及无机氮向氨基酸的同化可能有助于油菜对氮限制的适应性[31]。本试验中,纯铵条件下,增加硝酸盐供应的含量可显著增加地上部GS活性,而GS活性表征氮同化能力的强弱。本研究表明,随着硝酸盐的供应,地上部氮同化能力成梯度显著增强,而根系则无显著差异(图4),这可能因根系的GS活性在纯铵培养时已经达到顶峰,而添加硝酸盐后诱导地上部的GS2活性增强。

前人[32]研究证明,在铵硝混施的条件下,植物叶片含氮量明显增加。公华锐等[33]的试验结果表明,番茄生长发育各时期,随着铵态氮含量的提高,番茄的干物质量及其氮素吸收量均显著下降。常笑超等[21]发现,硝态氮能促进毛白杨的氮累积。胡国策等[34]发现,铵硝比例为3:1时,茶树叶片含氮量最高。本试验结果表明,在铵态氮条件下加入不同浓度的硝酸盐能够显著提高油菜全氮、氮累积量和氮素吸收效率(图5),表明添加硝酸盐后可以增加油菜对氮素的吸收。前人[35-37]在花椰菜和葡萄等作物中也观察到了一致的结果。

3.3 添加外源硝酸盐促进油菜的生长

硝态氮中加少量铵态氮有利于喜硝作物的生长[38],而铵态氮中加少量硝态氮有利于喜铵作物的生长[39]。有研究[40]表明,当环境中NH4+超过0.1~0.5mmol/L时就会产生铵毒害,具体表现为根系发育受到抑制,出现叶片黄化甚至枯败掉落,植物的长势不良甚至死亡。刘璐等[41]研究发现,在氮素形态不同配比下,铵硝混施的烟草叶绿素含量均显著高于纯硝或纯铵处理。研究[42-43]发现,单一氮形态培养时,氮素的转移和同化要消耗大量能量,将导致蛋白质和糖类的合成量减少,最终使作物减产,不同形态的氮混施更能促进植物的生长。王利超等[44]研究发现,铵硝比为1:1时可以促进烟草生长,使地上部叶面积增大。并且有研究[45]发现,纯铵培养的植物叶面积变小可能导致碳累积减少,因而植物长势变弱,并且其呼吸作用的消耗比纯硝培养的植物更多,进一步使植物的干物质积累量减少,这可能也是为什么纯铵培养的植物相对体积更小。前人[46]发现叶片含氮量越高,植物的光合作用越强。在本试验中,添加外源硝态氮后,对油菜的铵毒害症状有明显的缓解作用(图2a)。这可能由于在铵态氮条件下加入不同浓度的硝酸盐能够显著改善铵态氮导致的地上部分黄化,老叶枯萎脱落的症状也随着硝态氮浓度的上升而逐渐消失,使叶面积大幅上升,促进了油菜的光合作用,并且消耗的碳源减少,增加了干物质的积累,使油菜的生物量显著上升,长势增强。

4 结论

在本研究中,我们发现与正常培养相比,当以铵态氮作为唯一氮源培养会抑制油菜的生长,减小叶面积,降低叶绿素含量。而在铵态氮条件下加入硝酸盐会使油菜的K+、Ca2+和Mg2+含量上升,平衡了渗透势;添加少量硝态氮既能减轻油菜叶片变黄枯萎症状,促进叶片的生长,增强油菜在铵态氮条件下的光合作用,提高地上部氮素同化酶的活性,从而降低NH4+含量,使地上部和根系的生物量、总氮含量、氮累积量、氮素吸收效率和光合速率显著增加,进而促进油菜的正常生长。

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