Effect of the repression of NLP function on nitrate-inducible gene expression in Arabidopsis thaliana. Arabidopsis thaliana

Plants take up nitrate in soils and utilize it both for nitrogen assimilation and as a signaling molecule. Thus, an essential role of nitrate in plants is triggering changes in gene expression patterns, including immediate induction of the expression of genes involved in nitrate transport and assimilation as well as several transcription factor genes and genes related with carbon metabolism and cytokinin biosynthesis and response. Recently we identified NIN-like proteins (NLPs) as transcription factors governing nitrate-inducible gene expression in Arabidopsis. Because of their similar DNA-binding property, nine NLP proteins may play redundant roles in controlling nitrate-regulated gene expression in Arabidopsis. The physiological roles of NLPs were therefore assessed by generating transgenic Arabidopsis plants expressing NLP6 fused to the transcriptional repression domain of SUPERMAN (SUPRD), which has an ability to convert transcriptional activators into transcriptional repressors. Our transcriptome analysis revealed that levels of nitrate-inducible expression of nitrate transporter genes (NRT1.1 and NRT2.1), nitrate reduction enzyme genes (NIA1, NIA2 and NIR1), the genes associated with ammonium assimilation (GLT1 for glutamine-2-oxoglutarate aminotransferase and ASN2 for asparagine synthetase), LBD37-39 transcription factor genes and genes encoding homologs of rice nitrate-inducible NIGT1 transcriptional repressor, were reduced in the NLP6-SUPRD lines. Furthermore, levels of nitrate-inducible expression of the genes associated with the oxidative pentose phosphate pathway (G6PD2 and G6PD3 for glucose-6-phosphate dehydrogenases, and 6-phosphogluconate dehydrogenase genes), cytokinin biosynthesis (IPT3) and signal transduction (A-type ARR genes), a gene encoding a Ser/Thr protein kinase associated with a calcineurin B–like calcium sensor (CIPK3) were also found to be largely decreased by reductions in NLP activity. Because induction folds by nitrate and reduction folds by decreases in the NLP activity were well proportional, the NLP activity appeared to be responsible for most of nitrate responsiveness of the genes whose expression occurs immediately after nitrate treatment. Overall design: Overall design: Gene expression in 3.5-day-old seedlings treated with or without 10 mM potassium nitrate was compared between the transgenic lines expressing a chimeric repressor form of NLP6 (NLP6-SUPRD) and the parental control line.

植物可吸收土壤中的硝酸盐，既用于氮同化过程，也可作为信号分子发挥功能。因此，硝酸盐在植物体内的核心作用之一是触发基因表达模式的改变，包括快速诱导硝酸盐转运与同化相关基因、多种转录因子基因，以及碳代谢、细胞分裂素生物合成与响应相关基因的表达。本团队近期鉴定到NIN样蛋白（NIN-like proteins, NLPs）作为调控拟南芥硝酸盐诱导基因表达的转录因子。由于这类蛋白具有相似的DNA结合特性，拟南芥中存在的9种NLP蛋白可能在调控硝酸盐应答基因表达中存在功能冗余。为探究NLPs的生理功能，本研究通过构建融合了SUPERMAN转录抑制结构域（SUPERMAN transcriptional repression domain, SUPRD）的NLP6转基因拟南芥——该结构域可将转录激活因子转化为转录抑制因子——来评估NLPs的生理作用。转录组分析结果显示，在NLP6-SUPRD转基因株系中，硝酸盐转运蛋白基因（NRT1.1与NRT2.1）、硝酸盐还原酶基因（NIA1、NIA2与NIR1）、铵同化相关基因（谷氨酰胺-2-氧戊二酸氨基转移酶编码基因GLT1、天冬酰胺合成酶编码基因ASN2）、LBD37-39转录因子基因，以及水稻硝酸盐诱导型转录抑制因子NIGT1的同源编码基因的硝酸盐诱导表达水平均显著下调。此外，氧化戊糖磷酸途径相关基因（葡萄糖-6-磷酸脱氢酶编码基因G6PD2、G6PD3，以及6-磷酸葡萄糖酸脱氢酶基因）、细胞分裂素生物合成基因（IPT3）与信号转导基因（A型ARR基因），以及与钙调神经磷酸酶B样钙传感器相关的丝氨酸/苏氨酸蛋白激酶编码基因CIPK3的硝酸盐诱导表达水平，也因NLP活性降低而大幅下降。由于硝酸盐诱导的表达倍数与NLP活性降低导致的表达下调倍数呈显著正相关，因此NLP活性似乎主导了绝大多数硝酸盐处理后快速诱导表达的基因的硝酸盐应答能力。整体实验设计：对3.5日龄幼苗施加10 mM硝酸钾处理或不作处理，比较表达NLP6嵌合抑制蛋白（NLP6-SUPRD）的转基因株系与野生型亲本对照株系之间的基因表达差异。