DataSheet_2_Genome-wide identification of NAC transcription factors and regulation of monoterpenoid indole alkaloid biosynthesis in Catharanthus roseus.docx

NAC transcription factors (TFs) are crucial to growth and defense responses in plants. Though NACs have been characterized for their role in several plants, comprehensive information regarding their role in Catharanthus roseus, a perennial ornamental plant, is lacking. Homology modelling was employed to identify and characterize NACs in C. roseus. In-vitro propagation of C. roseus plants was carried out using cell suspension and nodal culture and were elicited with two auxin-antagonists, 5-fluoro Indole Acetic Acid (5-F-IAA) and α-(phenyl ethyl-2-oxo)-Indole-Acetic-Acid (PEO-IAA) for the enhanced production of monoterpenoid indole alkaloids (MIAs) namely catharanthine, vindoline, and vinblastine. Analyses revealed the presence of 47 putative CrNAC genes in the C. roseus genome, primarily localized in the nucleus. Phylogenetic analysis categorized these CrNACs into eight clusters, demonstrating the highest synteny with corresponding genes in Camptotheca acuminata. Additionally, at least one defense or hormone-responsive cis-acting element was identified in the promoter region of all the putative CrNACs. Of the two elicitors, 5-F-IAA was effective at 200 µM to elicit a 3.07-fold increase in catharanthine, 2.76-fold in vindoline, and 2.4-fold in vinblastine production in nodal culture. While a relatively lower increase in MIAs was recorded in suspension culture. Validation of RNA-Seq by qRT-PCR showed upregulated expression of stress-related genes (CrNAC-07 and CrNAC-24), and downregulated expression of growth-related gene (CrNAC-25) in elicited nodal culture of C. roseus. Additionally, the expression of genes involved in the biosynthesis of MIAs was significantly upregulated upon elicitation. The current study provides the first report on the role of CrNACs in regulating the biosynthesis of MIAs.

NAC转录因子（NAC transcription factors, TFs）对植物的生长与防御响应至关重要。尽管已有多项研究对多种植物中的NAC转录因子功能进行了解析，但关于其在长春花（Catharanthus roseus，一种多年生观赏植物）中的功能的全面信息仍较为匮乏。本研究通过同源建模（homology modelling）技术对长春花中的NAC转录因子进行了鉴定与功能表征。研究采用细胞悬浮培养与茎节培养对长春花植株进行离体繁殖（in-vitro propagation），并使用两种生长素拮抗剂（auxin-antagonists）——5-氟吲哚乙酸（5-fluoro Indole Acetic Acid, 5-F-IAA）与α-(苯乙基-2-氧代)-吲哚乙酸（α-(phenyl ethyl-2-oxo)-Indole-Acetic-Acid, PEO-IAA）——进行诱导处理，以提升单萜吲哚生物碱（monoterpenoid indole alkaloids, MIAs）的产量，这类生物碱包括长春质碱（catharanthine）、文多灵（vindoline）与长春碱（vinblastine）。基因组分析显示，长春花基因组中存在47个推定的CrNAC基因，这些基因主要定位于细胞核内。系统发育分析（phylogenetic analysis）将这些CrNAC基因划分为8个进化簇，且它们与喜树（Camptotheca acuminata）中的同源基因表现出最高的共线性（synteny）。此外，所有推定的CrNAC基因的启动子区域（promoter region）均检测到至少1个防御或激素响应顺式作用元件（cis-acting element）。在两种诱导剂中，5-F-IAA在浓度为200 µM时可有效诱导茎节培养体系中的长春质碱、文多灵与长春碱产量分别提升3.07倍、2.76倍与2.4倍；而悬浮培养体系中的单萜吲哚生物碱产量提升幅度相对较低。通过实时定量PCR（quantitative real-time polymerase chain reaction, qRT-PCR）对RNA测序（RNA sequencing, RNA-Seq）数据进行验证，结果显示，在经诱导处理的长春花茎节培养体系中，胁迫相关基因（CrNAC-07与CrNAC-24）的表达显著上调，而生长相关基因（CrNAC-25）的表达则显著下调。此外，参与单萜吲哚生物碱生物合成的相关基因在诱导处理后其表达量也显著上调。本研究首次报道了CrNAC基因在调控单萜吲哚生物碱生物合成中的功能。