Dissecting the rice genes responsible for long time changes of nitrogen supply forms and nitrogen starvation

In comparison with provision of either ammonium or nitrate alone, simultaneously supplying both forms of N results in superior growth and yield for the majority of plants including rice. Using a rice 22K oligo-array, we performed transcriptome analysis to identify genes of rice (Oryza sativa L. ssp. japonica) responsive to change of N-supply forms and N-starvation. Using the supply of ammonium nitrate (one to one molar ratio) as control, the total number of root genes that were equal or more than two fold up- or down- regulated was 445, 324, and 781 by upon supply of either ammonium or nitrate or continuous N starvation, respectively for 96 h. In the shoot the equivalent numbers were much smaller only 32, 58, and 165, respectively. Clustering of the rice genes associated with different environmental stresses revealed substantial organ specificity of the root and shoot to N starvation, and also to the N supply form. Genes encoding transporters for ammonium and nitrate, nitrate reductase, glutamate dehydrogenase, and aspartate amino transferase, showed great response to change of the N supply form, especially to N starvation. Some of the genes involved in chlorophyll metabolism, carbon fixation and assimilation, were enhanced by ammonium supply only, but significantly suppressed by N-starvation. In the shoot there was increased expression of more general stress genes under nitrate when compared to ammonium nutrition. In the root the reverse situation was true with more apparent stress under ammonium nutrition. The microarray approach has revealed new levels of complexity in the response of rice to the form of N supply. Keywords: Rice; root; shoot; nitrogen starvation; nitrogen form; ammonium; nitrate; gene expression Rice seedlings were grown in a hydroponic system in a growth chamber with control of both temperature and light. After normal growth for two weeks and for one week with completely avoiding any N source, the plants at four and a half leaf stage were re-supplied with ammonium nitrate (equal amount of either form of N), or only ammonium (ammonium sulphate and ammonium chloride), or only nitrate (calcium nitrate and magnesium nitrate), and continuing at zero N (N starvation). All the other essential nutrients were the same for all four treatments, except for sulfate and chloride which ranged between 1.1 – 2.6 mM and 0.5 – 2.5 mM, respectively. In Arabidopsis, addition of 3 mM Cl to ‘controls’ did not produce any changes in the expression of genes in an experiment investigating the re-supply of 3 mM nitrate (Scheible et al., 2004). In other microarray experiments, extra addition of 5 mM Cl for Arabidopsis (Wang et al., 2000; Wang et al., 2004) and 1.4 mM sulphate for tomato (Wang et al., 2001) were used to replace nitrate for identifying the N deprivation responsive genes. Therefore, we assume that the relative smaller difference in sulfate and chloride concentrations among the four treatments in the normal supply range for plants would not significantly affect the expression profile of genes responsible for changes in N supply form and starvation in our experiments. For identifying the genes responding to the various N conditions by micro-array analysis, we extracted total RNA respectively from the roots and shoots at the 96 h after initiation of the four respective treatments. We used amplified and labeled cRNA from ammonium nitrate treatment as control, we performed array-hybridization for the cRNA from the treatment of single ammonium form, single nitrate form, or continuous N depletion, respectively. Agilent 60-mer oligonucleotide arrays containing 21,938 unique transcription units (Agilent Technologies, Tokyo, Japan) were used. Two biological replicates of each treatment were performed for microarray analyses.

与单独供应铵态氮或硝态氮相比，同时供给两种形态的氮可使包括水稻在内的多数植物获得更优良的生长与产量。本研究利用水稻22K寡核苷酸芯片（rice 22K oligo-array）开展转录组分析（transcriptome analysis），以鉴定粳稻（Oryza sativa L. ssp. japonica）中响应氮供应形态变化与氮饥饿（N-starvation）的基因。以硝酸铵（摩尔比1:1）供应为对照，处理96小时后，相较于对照，根中上调或下调幅度≥2倍的基因总数分别为：单独供应铵态氮时445个、单独供应硝态氮时324个、持续氮饥饿时781个；而地上部的相应基因数量则显著更少，分别仅为32个、58个与165个。
对与不同环境胁迫相关的水稻基因进行聚类分析后发现，根与地上部对氮饥饿及氮供应形态的响应存在显著的器官特异性。编码铵态氮与硝态氮转运蛋白、硝酸还原酶、谷氨酸脱氢酶以及天冬氨酸氨基转移酶的基因，对氮供应形态的变化，尤其是氮饥饿，表现出强烈的响应。部分参与叶绿素代谢（chlorophyll metabolism）、碳固定与同化（carbon fixation and assimilation）过程的基因仅在铵态氮供应时被上调，却会因氮饥饿受到显著抑制。与铵态氮营养相比，硝态氮供应条件下地上部的一般胁迫相关基因表达量更高；而根部的情况则恰好相反，铵态氮营养下的胁迫响应更为显著。本微阵列研究揭示了水稻对氮供应形态响应的新复杂度。
关键词：水稻；根；地上部；氮饥饿；氮形态；铵态氮；硝态氮；基因表达
水稻幼苗在控温控光的生长箱（growth chamber）内采用水培系统（hydroponic system）培养。先正常生长两周，再置于无氮环境中培养一周，待植株长至四叶一心期时，分别重新供应硝酸铵（两种氮形态含量相等）、仅铵态氮（硫酸铵（ammonium sulphate）与氯化铵（ammonium chloride））、仅硝态氮（硝酸钙（calcium nitrate）与硝酸镁（magnesium nitrate）），以及持续无氮（氮饥饿）处理。除硫酸盐（sulfate）浓度介于1.1–2.6 mM、氯化物（chloride）浓度介于0.5–2.5 mM外，四种处理的其余必需营养元素（essential nutrients）均保持一致。已有研究表明，在拟南芥中，当补充3 mM氯化物至对照体系时，并不会对3 mM硝态氮再供应相关的基因表达产生影响（Scheible等，2004）；其他微阵列研究中，为鉴定拟南芥氮剥夺响应基因，会额外添加5 mM氯化物（Wang等，2000；Wang等，2004），或为番茄额外添加1.4 mM硫酸盐以替代硝态氮（Wang等，2001）。因此我们推测，本实验中四种处理间硫酸盐与氯化物浓度的相对差异处于植物正常供应范围之内，不会显著影响与氮供应形态变化及氮饥饿相关的基因表达谱。
为通过微阵列分析鉴定响应不同氮条件的基因，我们分别在四种处理开始后的96小时，提取根与地上部的总RNA。以硝酸铵处理的扩增标记互补RNA（cRNA）作为对照，分别对单独铵态氮、单独硝态氮以及持续氮耗竭处理的cRNA进行芯片杂交。本研究使用的安捷伦60-mer寡核苷酸芯片（oligonucleotide arrays）包含21938个独特转录单元（安捷伦科技，日本东京）。每种处理均设置两次生物学重复（biological replicates）用于微阵列分析。