Melo Bruno P, Fraga Otto T, Silva José Cleydson F, Ferreira Dalton O, Brustolini Otávio J B, Carpinetti Paola A, Machado Joao Paulo B, Reis Pedro A B, Fontes Elizabeth P B
National Institute of Science and Technology in Plant-Pest Interactions, Bioagro, Universidade Federal de Viçosa, Viçosa, Brazil.
Departamento de Bioquímica e Biologia Molecular/BIOAGRO, Universidade Federal de Viçosa, Viçosa, Brazil.
Front Plant Sci. 2018 Dec 18;9:1864. doi: 10.3389/fpls.2018.01864. eCollection 2018.
The (NAM, ATAF, and CUC) genes encode transcription factors involved with the control of plant morph-physiology and stress responses. The release of the last soybean genome assembly (Wm82.a2.v1) raised the possibility that new NAC genes would be present in the soybean genome. Here, we interrogated the last version of the soybean genome against a conserved NAC domain structure. Our analysis identified 32 putative novel NAC genes, updating the superfamily to 180 gene members. We also organized the genes in 15 phylogenetic subfamilies, which showed a perfect correlation among sequence conservation, expression profile, and function of orthologous genes and NAC soybean genes. To validate our analyses, we monitored the stress-mediated gene expression profiles of eight new NAC-genes by qRT-PCR and monitored the GmNAC senescence-associated genes by RNA-seq. Among ER stress, osmotic stress and salicylic acid treatment, all the novel tested GmNAC genes responded to at least one type of stress, displaying a complex expression profile under different kinetics and extension of the response. Furthermore, we showed that 40% of the GmNACs were differentially regulated by natural leaf senescence, including eight (8) newly identified GmNACs. The developmental and stress-responsive expression profiles of the novel NAC genes fitted perfectly with their phylogenetic subfamily. Finally, we examined two uncharacterized senescence-associated proteins, GmNAC065 and GmNAC085, and a novel, previously unidentified, NAC protein, GmNAC177, and showed that they are nuclear localized, and except for GmNAC065, they display transactivation activity in yeast. Consistent with a role in leaf senescence, transient expression of GmNAC065 and GmNAC085 induces the appearance of hallmarks of leaf senescence, including chlorophyll loss, leaf yellowing, lipid peroxidation and accumulation of HO. GmNAC177 was clustered to an uncharacterized subfamily but in close proximity to the TIP subfamily. Accordingly, it was rapidly induced by ER stress and by salicylic acid under late kinetic response and promoted cell death . Collectively, our data further substantiated the notion that the GmNAC genes display functional and expression profiles consistent with their phylogenetic relatedness and established a complete framework of the soybean NAC superfamily as a foundation for future analyses.
（NAM、ATAF和CUC）基因编码参与植物形态生理控制和应激反应的转录因子。大豆最新基因组组装版本（Wm82.a2.v1）的发布增加了大豆基因组中存在新NAC基因的可能性。在此，我们根据保守的NAC结构域结构对大豆基因组的最新版本进行了研究。我们的分析鉴定出32个推定的新NAC基因，使该超家族更新为180个基因成员。我们还将这些基因组织成15个系统发育亚家族，这些亚家族在直系同源基因和大豆NAC基因的序列保守性、表达谱和功能之间显示出完美的相关性。为了验证我们的分析，我们通过qRT-PCR监测了8个新NAC基因的应激介导基因表达谱，并通过RNA-seq监测了GmNAC衰老相关基因。在内质网应激、渗透胁迫和水杨酸处理中，所有测试的新GmNAC基因都对至少一种胁迫有反应，在不同的动力学和反应程度下呈现出复杂的表达谱。此外，我们发现40%的GmNACs受自然叶片衰老的差异调节，其中包括8个新鉴定的GmNACs。新NAC基因的发育和应激反应表达谱与其系统发育亚家族完美匹配。最后，我们研究了两种未表征的衰老相关蛋白GmNAC065和GmNAC085，以及一种新的、以前未鉴定的NAC蛋白GmNAC177，结果表明它们定位于细胞核，除GmNAC065外，它们在酵母中显示出反式激活活性。与在叶片衰老中的作用一致，GmNAC065和GmNAC085的瞬时表达诱导了叶片衰老特征的出现，包括叶绿素损失、叶片变黄、脂质过氧化和HO积累。GmNAC177聚类到一个未表征的亚家族，但与TIP亚家族接近。因此，它在后期动力学反应中被内质网应激和水杨酸迅速诱导，并促进细胞死亡。总体而言，我们的数据进一步证实了GmNAC基因的功能和表达谱与其系统发育相关性一致的观点，并建立了大豆NAC超家族的完整框架，为未来的分析奠定了基础。
