Biotechnology Research Center, The University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo, 113-8657, Japan.
BMC Plant Biol. 2019 Feb 28;19(1):90. doi: 10.1186/s12870-019-1692-3.
NIN-LIKE PROTEIN (NLP) transcription factors are master regulators of nitrate-inducible gene expression in higher plants. NLP transcription factors contain a nitrate signal-responsive domain in the amino-terminal region, an RWP-RK-type DNA-binding domain in the middle, and a Phox and Bem1 (PB1) domain at the carboxy terminus. Although the PB1 domain of NLP transcription factors appears to mediate protein-protein interactions associated with nitrate-inducible gene expression in higher plants, its precise role in nitrate-inducible gene expression has not previously been characterized.
Yeast two-hybrid assays with the PB1 domain of the Arabidopsis transcription factor NLP7 revealed NLP-NLP interactions that required the core amino acid residues (K867, D909, D911, and E913) within the PB1 domain. Consistent with previous speculation on redundant and overlapping functions between different Arabidopsis NLP transcription factors, NLP-NLP interactions were observed between a variety of combinations of different NLP transcription factors. Furthermore, a mutated form of NLP7 that harbored amino acid substitutions at K867, D909, D911, and E913 required a far higher level of expression than wild-type NLP7 to restore nitrate-responsive gene expression and growth of nlp6 nlp7-1 double mutants. Surprisingly, however, the ability to transactivate nitrate-responsive promoters in protoplast transient expression assays was similar between wild-type and mutant forms of NLP7, suggesting that the PB1 domain was not required for transcription from naked DNA.
Protein-protein interactions mediated by the PB1 domain of NLP transcription factors are necessary for full induction of nitrate-dependent expression of target genes in planta. The PB1 domains of NLP transcription factors may act on gene expression from chromosomal DNA via homo- and hetero-oligomerization in the presence of nitrate.
NIN-LIKE PROTEIN（NLP）转录因子是高等植物硝酸盐诱导基因表达的主要调控因子。NLP 转录因子在其氨基端区域含有一个硝酸盐信号响应域，在中间含有一个 RWP-RK 型 DNA 结合域，在羧基端含有一个 Phox 和 Bem1（PB1）结构域。虽然 NLP 转录因子的 PB1 结构域似乎介导了与高等植物硝酸盐诱导基因表达相关的蛋白质-蛋白质相互作用，但它在硝酸盐诱导基因表达中的精确作用尚未得到表征。
利用拟南芥转录因子 NLP7 的 PB1 结构域进行酵母双杂交试验，揭示了 NLP-NLP 相互作用，这种相互作用需要 PB1 结构域内的核心氨基酸残基（K867、D909、D911 和 E913）。与不同的 NLP 转录因子之间存在冗余和重叠功能的先前推测一致，观察到了各种不同 NLP 转录因子组合之间的 NLP-NLP 相互作用。此外，一种携带 K867、D909、D911 和 E913 氨基酸取代的突变形式的 NLP7 比野生型 NLP7 恢复硝酸盐响应基因表达和 nlp6 nlp7-1 双突变体的生长需要高得多的表达水平。然而，令人惊讶的是，在原生质体瞬时表达试验中，野生型和突变型 NLP7 之间激活硝酸盐响应启动子的能力相似，这表明 PB1 结构域对于裸露 DNA 上的转录不是必需的。
NLP 转录因子的 PB1 结构域介导的蛋白质-蛋白质相互作用对于靶基因在植物体内完全诱导硝酸盐依赖的表达是必要的。在硝酸盐存在的情况下，NLP 转录因子的 PB1 结构域可能通过同源和异源寡聚化作用作用于染色体 DNA 上的基因表达。
