Fine-tuning of nutrient uptake and response is indispensable for maintenance of nutrient homeostasis in plants, but the details of underlying mechanisms remain to be elucidated. NITRATE-INDUCIBLE GARP-TYPE TRANSCRIPTIONAL REPRESSOR 1 (NIGT1) family proteins are plant-specific transcriptional repressors that function as an important hub in the nutrient signaling network associated with the acquisition and use of nitrogen and phosphorus. Here, by yeast two-hybrid assays, bimolecular fluorescence complementation assays, and biochemical analysis with recombinant proteins, we show that Arabidopsis NIGT1 family proteins form a dimer via the interaction mediated by a coiled-coil domain (CCD) in their N-terminal regions. Electrophoretic mobility shift assays defined that the NIGT1 dimer binds to two different motifs, 5'-GAATATTC-3' and 5'-GATTC-N₃₈-GAATC-3', in target gene promoters. Unlike the dimer of wild-type NIGT1 family proteins, a mutant variant that could not dimerize due to amino acid substitutions within the CCD had lower specificity and affinity to DNA, thereby losing the ability to precisely regulate the expression of target genes. Thus, expressing the wild-type and mutant NIGT1 proteins in the nigt1 quadruple mutant differently modified NIGT1-regulated gene expression and responses towards nitrate and phosphate. These results suggest that the CCD-mediated dimerization confers dual mode DNA recognition to NIGT1 family proteins, which is necessary to make proper controls of their target genes and nutrient responses. Intriguingly, two 5'-GATTC-3' sequences are present in face-to-face orientation within the 5'-GATTC-N₃₈-GAATC-3' sequence or its complementary one, while two 5'-ATTC-3' sequences are present in back-to-back orientation within the 5'-GAATATTC-3' or its complementary one. This finding suggests a unique mode of DNA binding by NIGT1 family proteins and may provide a hint as to why target sequences for some transcription factors cannot be clearly determined.

养分吸收和响应的微调对于维持植物中的养分稳态是必不可少的，但是其潜在机制的细节尚待阐明。硝酸盐诱导型GARP型转录阻遏物1（NIGT1）家族蛋白是植物特异性的转录阻遏物，在与氮和磷的获取和使用相关的营养信号网络中起着重要的枢纽作用。在这里，通过酵母双杂交测定，双分子荧光互补测定和重组蛋白的生化分析，我们显示拟南芥NIGT1家族蛋白通过在其N末端区域中由卷曲螺旋结构域（CCD）介导的相互作用形成二聚体。电泳迁移率变动分析定义了NIGT1二聚体可结合两个不同的基序5'-GAATATTC-3'和5'₃₈ -GAATC-3'，在靶基因启动子中。与野生型NIGT1家族蛋白的二聚体不同，由于CCD内的氨基酸取代而无法二聚的突变体变体对DNA的特异性和亲和力较低，从而失去了精确调控靶基因表达的能力。因此，在nigt1四倍体突变体中表达野生型和突变体NIGT1蛋白可以不同地修饰NIGT1调控的基因表达以及对硝酸盐和磷酸盐的反应。这些结果表明，CCD介导的二聚化赋予NIGT1家族蛋白以双模式DNA识别，这对于正确控制其靶基因和营养反应是必需的。有趣的是，在5'-GATTC-N中以面对面的方向存在两个5'-GATTC-3'序列₃₈ -GAATC-3'序列或其互补序列，而两个5'-ATTC-3'序列以背对背的方向存在于5'-GAATATTC-3'或其互补序列中。这一发现提示了NIGT1家族蛋白与DNA结合的独特模式，并可能提示为什么不能明确确定某些转录因子的靶序列。