【作者】 尹晓明；

【导师】 沈其荣；

【作者基本信息】 南京农业大学 ， 植物营养学， 2010， 博士

【摘要】 虽然水稻是喜NH4+作物,但是由于水稻根系的泌氧作用,使水稻实际处于NH4+、N03-混合营养之中。与其他旱地作物一样,水稻根系吸收的NO3-也主要在植株的地上部还原。所以,研究水稻的NO3-营养也很重要。本文利用离子微电极技术研究水培条件下水稻吸收利用NH4+、NO3-的电生理特征,分析了水稻根系吸收NH4+、NO3-过程的细胞膜电位变化以及在不同品种间的差异；分别用NH4+选择性双阻微电极、NO3-选择性双阻微电极分析了NH4+、NO3-在细胞内的区域化分布以及NO3-的再调动；用双阻H+选择微电极分析了水稻吸收NH4+、NO3-对根系质外体pH的影响。用RT-PCR方法分析了水稻NH4+、NO3-转运蛋白和相关同化酶的基因表达,旨在为提高水稻的N利用率提供理论依据。本文首先研究了水稻吸收NH4+的过程中根系细胞膜电位的变化以及在品种间的差异。利用单电极分别测定了两个水稻品种即武育粳3号(粳稻)和扬稻6号(籼稻)幼苗根尖细胞在不同NH4+浓度处理下(0.025 mmol L-1、0.05 mmol L-1、0.1 mmol L-1、0.5 mmol L-1、1 mmol L-1和1.5 mmol L-1)膜电位的变化特征。结果表明：水稻根系吸收NH4+主要引起膜电位的去极化,去极化到一定程度出现部分复极化,有约20%的被测根系还有超极化现象。去极化大小随外界处理液中NH4+浓度的增加而增加,达到一定程度以后趋于平稳,吸收进程符合Michaelis-Menten动力学特征。扬稻6号对NH4+较敏感,产生的平均去极化大小显著高于武育粳3号(p<0.05),表明扬稻6号吸收NH4+的能力比武育粳3号强,这与吸收动力学的结果是一致的。此外,不同pH值(pH 4.0、8.0)减小了相等NH4+浓度处理下膜电位去极化大小,高pH值(pH 8.0)提高了膜电位超极化出现的比例(50%-70%),超极化大小不依赖于供应的NH4+的浓度并且在不同品种之间没有差异。根系RT-PCR结果表明,两个NH4+运输蛋白(OsAMT1;1、OsAMT1;3)、两个NO3-运输蛋白(OsNRT1.1、OsNRT2.1)在不同品种和不同N处理条件下的表达不同：在不同浓度NH4+培养下,除了OsNRT1.1以外其余3个基因的表达都是扬稻6号高于武育粳3号；在NH4++NO3-培养下,OsNRT2.1的表达是扬稻6号高于武育粳3号,而OsNRT1.1的表达是武育粳3号高于扬稻6号,其余两个基因在两个品种间的表达水平差异不显著。电生理和基因表达分析的结果总体表明扬稻6号吸收NH4+的能力比武育粳3号强。不同水稻品种对NO3-的响应程度也不同。利用单电极分别测定了4个水稻品种即农垦57(粳稻)、泗优917(杂粳)、扬稻6号(籼稻)和汕优63(杂籼)幼苗根尖表皮细胞在3种NO3-浓度(0.1 mmol L-1、1 mmol L-1、10 mmol L-1)处理过程中膜电位的变化特征。结果表明：水稻根系吸收NO3-引起膜电位的去极化、复极化和超极化。去极化程度随外界处理液中NO3-浓度的增加而加强,就单位时间膜电位变化大小而言,扬稻6号对外界NO3-较敏感,3种NO3-浓度处理下膜电位去极化值均高于其他3个品种,表现出对NO3-的吸收能力较强；两个杂交品种泗优917和汕优63表现出相似的去极化大小和相似的反应时间,而农垦57对NO3-相对不敏感,膜电位去极化值均低于其他3个品种,表现出对NO3-的吸收能力较弱。同水稻对NH4+的吸收类似,去极化大小随外界处理液中NO3-浓度的增加而增加,达到一定程度以后趋于平稳,吸收进程符合Michaelis-Menten动力学特征。扬稻6号对NO3-较敏感,产生的平均去极化大小显著高于其余品种,这与吸收动力学的结果是一致的。这表明用根系对NO3-响应的细胞膜电位变化来研究水稻对NO3-的吸收是可行的。不同pH值对水稻根系细胞膜电位的影响的结果表明,膜电位的去极化大小因溶液pH值和其中NO3-浓度的不同而有差异：在同一pH值下膜电位的去极化大小随着处理NO3-浓度的升高而增大,而在不同pH值时膜电位的去极化都以pH4.0时最大。吸收动力学的结果表明,扬稻6号对NO3-的吸收能力强于武育粳3号,最大吸收速率Vmax在品种间差异显著(p<0.05),但是表观米氏常数Km在品种间差异不显著,pH4.0促进了根系对NO3-的吸收、而高的pH如8.0抑制了根系对NO3-吸收,原因可能是碱性降低了酶对底物的亲和力,而对Vmax没有影响。了解NH4+在细胞内的区域分布有利于研究NH4+穿膜运输的机制。微电极与溶液中NH4+的浓度呈对数曲线的关系,NH4+选择性微电极与其他类型的电极(如H+、NO3-)最大区别是K+的干扰,在含有72 mmol L-1K+的标定溶液中,电极标定曲线的斜率为48～58 mV,对NH4+的检出限小于10-3 mmol L-1,说明电极对NH4+有较高的选择性,受K+的影响较小。用以测定2.5 mmol L-1 NH4+培养两周的水稻叶片,结果表明叶片细胞中NH4+活度分布在活度高低不同的两个区间内,即分别代表了细胞质和液泡中的测定,水稻叶片细胞质和液泡NH4+的活度分别为2.58～9.37mmol L-1,11.36～25.2 mmol L-1。用以测定不同水稻品种液泡NH4+活度的结果表明,扬稻6号叶片液泡NH4+活度显著高于武育粳3号,说明扬稻6号可利用的NH4+较高、维持了细胞质中的NH4+相对稳定,这在一定程度促进了GS活性而提高了NH4+同化效率。NH4+选择性微电极为研究水稻对NH4+的吸收利用提供了技术支撑。与NH4+在细胞内的区域分布类似,NO3-主要在液泡中积累,液泡NO3-的再调动与利用与N素高效利用关系密切。利用双阻NO3-选择性微电极测定了在外界停止供应NO3-前后,不同水稻品种细胞质和液泡中NO3-活度的变化。结果表明水稻叶片细胞质和液泡中NO3活度存在着明显的不同变化趋势,NO3-的再调动能力在不同水稻品种间存在差异。在停止供应NO3-以后,水稻叶片液泡中的NO3-逐渐降低,而细胞质中的NO3却维持在一个较低的活度而基本稳定；水稻植株组织水平的NO3-随缺N时间延长而呈降低的趋势。扬稻6号液泡中和细胞质中的NO3-活度均高于武育粳3号,且在停止供应NO3-的不同时间段,液泡中NO3-的释放速率也较快。此外,在N饥饿的不同时段,硝酸还原酶活性(Nitrate reduatase activity, NRA)在品种间差异显著(扬稻6号高于武育粳3号),同时RT-PCR的结果表明OsNia2在扬稻6号叶片表达较强、OsNRT2.1在其根系的表达比武育粳3号高2-3倍。这些结果表明在受到NO3-营养胁迫时,水稻先前积累在叶片液泡中的NO3-可以进行再调动,而且扬稻6号在N胁迫下能有效地吸收和利用NO3-。水稻根系吸收NH4+、NO3-对根系质外体pH的影响不同。双阻H+选择微电极的P2值在40～59mV之间,P3值在10-8～10-9 mol L-1,说明电极对H+有较高的选择性可以用来测定。水稻质外体的pH值维持在5.8左右；不同供N条件影响水稻质外体pH值：4.0 mmol L-1NO3-培养下水稻质外体的pH上升了0.2～0.8个单位；2 mmol L-1 NO3-+2 mmol L-1NH4+培养下水稻质外体的pH下降0.7～0.9个单位。就不同品种来说,NO3-以及NO3-+NH4+培养下武育粳3号根质外体的pH都高于扬稻6号,表明不同水稻品种质外体pH的调控能力不同。H+选择性微电极为研究水稻根系质外体pH的调节以及对N的吸收利用的影响提供了新视角。更多还原

【Abstract】 The nitrogen (N) available for rice plants is chiefly present in NH4+. However, some NH4+ near rice root surface can be oxidized to NO3- owing to leaked oxygen, and thus rice is in fact under the mixed nutrition of NH4+ and NO3-. Like other anaerobic crops, NO3-absorbed by rice plants is mainly reducted in shoots. Therefore, it is important to study NO3- nutrition of rice plants. Microelectrodes, developed from 1980s-1990s, were used to study NO3- and NH4+ nutrition of rice plants in this thesis.The main results are shown as in follows:Firstly, the thesis studied changes of the plasma membrane potential of rice root tips during the uptake of NH4+ and differences among rice cultivars. The plasma membrane potential of rice root tips of two rice (Oryza Sativa L) cultivars, i.e., Wuyujin 3 (Japonica) and Yangdao 6 (Indica) were monitored using microelectrodes under different concentrations of NH4+(0.025 mmol L-1,0.05 mmol L-1,0.1 mmol L-1,0.5 mmol L-1,1.0 mmol L-1 and 1.5 mmol L-1). NH4+ uptake by rice roots mainly made plasma membrane potential depolarized then partially repolarized and sometimes hyperpolarized. The magnitude of depolarization increased with the increasing of NH4+concentrations outside and went placidly, while the course of NH4+ uptake exhibited Michaelis-Menten kinetics. Yangdao 6 was more sensitive to NH4+ with its significant high average level of depolarization than Wuyujin 3 (p< 0.05). This suggested that Yangdao6 had the stronger capability to absorb NH4+ than Wuyujin 3. The results obtained from microelectrodes experiment were in accord with that of kinetics of NH4+ influx. Besides, the size of membrane potential was reduced when external pH was 4.0 or 8.0 and high pH (8.0, for example) increased proportion of hydepolarization (about 60%-70%) in tested roots. While the size of depolarization was independent of external NH4+ concentration and it showed no significant differences between cultivars. Results of RT-PCR of genes including two OsAMT and two OsNRT in rice roots showed that the expression of most genes in Yangdao 6 was higher than that of in Wuyujing 3 except OsNRT1.1 under the treatment of NH4+, while only the expression of OsNRT2.1 was higher when seedlings were supplied with NH4+ NO3-. Results of electrophysiology and molecular indicated that Yangdao 6 had a strong ability to uptake NH4+.Response of rice to NO3- was also different among rice cultivars. Changes of the membrane potential of epidermal cells in root tips of four rice (Oryza SativaL.) cultivars, i.e., Nongken 57(Japonica), Siyou 917 (Hybrid Japonica), Yangdao 6 (Indica), and Shanyou 63 (Hybrid Indica) were monitored using microelectrodes under different concentrations of NO3-(0.1 mmol L-1,1 mmol L-1 and 10 mmol L-1, respectively). Uptake of NO3- by rice roots made the membrane potential depolarized and then repolarized, sometimes hyperpolarized. The magnitude of depolarization increased with the increasing of NO3-concentrations outside. Among the four rice cultivars tested, Yangdao6 was most sensitive to NO3- with its higher level of depolarization and least time to reach the maximum membrane potential, while Nongken 57 was less sensitive to NO3- with its lower level of depolarization and most time to reach the maximum membrane potential. The two Hybrid cultivars Siyou917 and Shanyou63 exhibited the similar manner in terms of the two aspects. This suggested that Yangdao 6 has the strongest capability to absorb NO3- while Nongken 57 had the poorest uptake of NO3-. Similar with the uptake of NH4+, the magnitude of depolarization due to uptake of NO3- increased with the increasing of NO3- concentrations outside and went placidly, and the course of NO3- uptake exhibited Michaelis-Menten kinetics. The results obtained in this experiment implied that response of electro-potentials of cell membranes to NO3- could be a feasible way to the study of NO3- nutrition in rice plants. Results of pH effects on membrane potential and NO3- uptake kenetics showed that the size of membrane potential increased with the increasing of NO3- concentration outside at the same pH and reached maxim at pH 4.0. Km and this tendency was higher in Yangdao 6 than Wuyujing 3. A pH of 4.0 promoted NO3- uptake, while a pH 8.0 showed an inhibition on it. This might be due to the enhacement of affinity in enzyme under alkaline, which did not affect Vmax.Working theory and use of double-barreled NH4+ selective microelectrode were introduced in this thesis to study NH4+ compartmentation in cells. A distinguished difference of NH4+ selective microelectrode from other microelectrodes (such as H+ and NO3- microelectrode) was the interference of K+. These microelectrodes showed a typical log linear response to NH4+ from 0.01～100 mmol L-1 in the presence of 72 mmol L-1 K+, with a slope of 48～58 mV and the detecting limit was below 10-3 mmol L-1. Microelectrode made in our laboratory was more sensitive to NH4+ than others reported so far and could be used as NH4+ microelectrode. Intracellular measurement of NH4+ activity in leaf cells of rice plants cultured in 2.5 mmol L-1 NH4+ for two weeks with double-barreled NH4+ selective microelectrode showed that NH4+ activity fell into two main populations, one was in the cytosol with the NH4+concentrations of 2.58-9.37 mmol L-1 and the anther was in the vacuole with the NH4+concentrations of 11.36～25.2 mmol L-1. And we also determined tissue NH4+ of rice leaf using a continuous-flow auto analyzer and the concentrations were 11.12 mmol L-1 that was very similar to NH4+ concentrations in vacuoles. This inferred that NH4+ in rice leaves mainly came from vacuoles. Vacuolar NH4+ activities in Yangdao 6 were significantly higher than that of in Wuyujing 3, suggested that Yangdao 6 had more NH4+ to use for keep NH4+ consistant in the cytocle. This may promote the activity of GS and the assimilation of NH4+. Therefore, NH4+ microelectrode offered technical support for the study of the assimilation and utilization of NH4+ in rice plants.There existed closed relationship between NO3- remobilization and N use efficiency. Measurements with double-barreled NO3- selective microelectrode showed that NO3- in vacuoles changed in different patterns from cytocle when N supply was withdrawn. NO3- in vacuole decreased with the increasing time of N starvation, while it kept a relative low level in cytocle. Meanwhile, NO3- concentrations of tissues in both leaves and roots decreased gradually during N starvation. The patterns of NO3- remobilization were different between organs and cultivars, and leaves, for examples, responded more quickly to N starvation than roots and Yangdao 6 released NO3- from vacuoles faster than Wuyujing 3. Becides, NO3- reductase activities were higher in Yangdao 6 than in Wuyujing 3 before and after N starvation. Results of RT-PCR showed that the expression of OsNial existed in almost no difference, while the expression of OsNia2 was higher in leaves of YD6. Also, the expression of OsNRT2.1 in roots of Yangdao 6 was higher. These measurements suggested that NO3- could be remobilized under N sress, and Yangdao 6 responded well to NO3- supply and thus was more efficient in utilizing NO3- than Wuyujing 3.Uptake of different forms of N may affect the apoplast pH in rice roots. The value of P2 and P3 of a H+ microelectrode was in the range of 40～59mV and 10-8～10-9 mol L-1 repectively, indicating the electrode was more sensitive to H+ than to any other ions and could be used to detect the activity of H+ both in and outsite cytosol. The apoplast pH in rice roots sustained about 5.8 before treatment but was significantly affected by the N forms supplied. The apoplast pH, for example, was increasd by 0.2～0.8 unit under 4.0 mmol L-1 NO3- while was decreased by 0.7～0.9 unit under 2 mmol L-1 NO3- plus 2.0 mmol L-1NH4+. As for cultivars, the apoplast pH in Wuyujing 3 was higher than that of in Yangdao 6 at both treatments, inferring that there were different abilities of regulating pH in apoplasts between different rice cultivars. H+ microelectrode provided a new insight to study the regulation of pH in apoplasts and its effect on N uptake and utilization in rice plants.更多还原