Nitrogen (N) is a key component in plants and their biological macromolecules, having a profound effect on developmental stages, such as germination, vegetative growth, and flowering. However, the mechanism of nitrogen-regulated flowering time remains unclear. In this study, CmNLP7 was isolated from the chrysanthemum cultivar ‘Jinba’ and was characterized. CmNLP7 is a transcription factor localized in the nucleus but has no transcriptional activity. Tissue expression pattern analysis showed that CmNLP7 was mainly transcribed in leaves and roots. Knocking down CmNLP7 through the artificial-miRNA method in chrysanthemum resulted in early flowering under optimal nitrogen (ON) and low nitrogen (LN) conditions; whereas overexpression lines showed delayed flowering under LN conditions. Transcriptome sequencing analysis showed that the nitrate transporters NRT2.5, NPF3.1, and NPF4.6; SBP-like genes SPL7 and SPL12, and flowering integration factor FT were significantly up-regulated in the knockdown lines. Based on the KEGG pathway enrichment analysis, the differentially transcribed genes were enriched in phenylpropanoid biosynthesis and starch and sucrose metabolism pathways, which indicated their alleged function in nitrogen-regulated flowering and development in chrysanthemum. Furthermore CmPP6 as a homolog of the Arabidopsis phosphatase PP6, was verified as an interacting protein of CmNLP7 by yeast two-hybrid, BiFC, pull-down and Biacore in vitro and in vivo, and the knockdown line of CmPP6 (amiR-CmPP6) flowered earlier compared to that of the wild-type chrysanthemum ‘Jinba’. Collectively, these results demonstrated that CmPP6 interacts with CmNLP7 to regulate chrysanthemum flowering, and CmNLP7 could regulate flowering time in response to nitrogen, which lays a foundation for the regulation of flowering and molecular breeding of chrysanthemum through changes in nutrient signaling.

中文翻译：

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CmNLP7 与 CmPP6 相互作用抑制菊花开花时间


氮（N）是植物及其生物大分子的关键成分，对发芽、营养生长和开花等发育阶段具有深远的影响。然而，氮调节开花时间的机制仍不清楚。在本研究中，CmNLP7 从菊花品种“金霸”中分离出来并进行了表征。 CmNLP7是一种定位于细胞核的转录因子，但不具有转录活性。组织表达模式分析表明CmNLP7主要在叶和根中转录。通过人工miRNA方法敲除菊花中的CmNLP7，可在最佳氮（ON）和低氮（LN）条件下提前开花；而过表达株系在液氮条件下表现出延迟开花。转录组测序分析显示硝酸盐转运蛋白NRT2.5、NPF3.1和NPF4.6； SBP 样基因 SPL7 和 SPL12 以及开花整合因子 FT 在敲低品系中显着上调。基于KEGG通路富集分析，差异转录基因在苯丙素生物合成以及淀粉和蔗糖代谢通路中富集，这表明它们在菊花的氮调节开花和发育中具有功能。此外，CmPP6作为拟南芥磷酸酶PP6的同源物，通过酵母双杂交、BiFC、pull-down和Biacore在体外和体内验证为CmNLP7的相互作用蛋白，并且CmPP6的敲低系（amiR-CmPP6）开花与野生型菊花‘Jinba’相比要早。 总的来说，这些结果表明CmPP6与CmNLP7相互作用调节菊花开花，并且CmNLP7可以响应氮调节开花时间，这为通过营养信号变化调控菊花开花和分子育种奠定了基础。