Liu AiLian, Zhu Yue, Wang YuHao, Wang TianYu, Zhao ShuPing, Feng Kai, Li LiangJun, Wu Peng
College of Horticulture and Landscape Architecture, Yangzhou, Jiangsu, China.
Joint International Research Laboratory of Agriculture and Agri-Product Safety of Ministry of Education of China, Yangzhou University, Yangzhou, China.
Front Plant Sci. 2023 Mar 21;14:1114345. doi: 10.3389/fpls.2023.1114345. eCollection 2023.
Flavonoids are one of the most important secondary metabolites in plants, and phenylalanine ammonia-lyase (PAL) is the first rate-limiting enzyme for their biosynthesis. However, detailed information on the regulation of PAL in plants is still little. In this study, PAL in E. ferox was identified and functionally analyzed, and its upstream regulatory network was investigated. Through genome-wide identification, we obtained 12 putative PAL genes from E. ferox. Phylogenetic tree and synteny analysis revealed that PAL in E. ferox was expanded and mostly preserved. Subsequently, enzyme activity assays demonstrated that EfPAL1 and EfPAL2 both catalyzed the production of cinnamic acid from phenylalanine only, with EfPAL2 exhibiting a superior enzyme activity. Overexpression of EfPAL1 and EfPAL2 in Arabidopsis thaliana, respectively, both enhanced the biosynthesis of flavonoids. Furthermore, two transcription factors, EfZAT11 and EfHY5, were identified by yeast one-hybrid library assays as binding to the promoter of EfPAL2, and further luciferase (LUC) activity analysis indicated that EfZAT11 promoted the expression of EfPAL2, while EfHY5 repressed the expression of EfPAL2. These results suggested that EfZAT11 and EfHY5 positively and negatively regulate flavonoid biosynthesis, respectively. Subcellular localization revealed that EfZAT11 and EfHY5 were localized in the nucleus. Our findings clarified the key EfPAL1 and EfPAL2 of flavonoid biosynthesis in E. ferox and established the upstream regulatory network of EfPAL2, which would provide novel information for the study of flavonoid biosynthesis mechanism.
类黄酮是植物中最重要的次生代谢产物之一，苯丙氨酸解氨酶（PAL）是其生物合成的首个限速酶。然而，关于植物中PAL调控的详细信息仍然很少。在本研究中，对峨眉野连中的PAL进行了鉴定和功能分析，并研究了其上游调控网络。通过全基因组鉴定，我们从峨眉野连中获得了12个假定的PAL基因。系统发育树和共线性分析表明，峨眉野连中的PAL发生了扩增且大多得以保留。随后，酶活性测定表明，EfPAL1和EfPAL2均仅催化从苯丙氨酸生成肉桂酸，其中EfPAL2表现出更高的酶活性。分别在拟南芥中过表达EfPAL1和EfPAL2，均增强了类黄酮的生物合成。此外，通过酵母单杂交文库分析鉴定出两个转录因子EfZAT11和EfHY5与EfPAL2的启动子结合，进一步的荧光素酶（LUC）活性分析表明，EfZAT11促进EfPAL2的表达，而EfHY5抑制EfPAL2的表达。这些结果表明，EfZAT11和EfHY5分别对类黄酮生物合成起正向和负向调控作用。亚细胞定位显示，EfZAT11和EfHY5定位于细胞核。我们的研究结果阐明了峨眉野连中类黄酮生物合成的关键基因EfPAL1和EfPAL2，并建立了EfPAL2的上游调控网络，这将为类黄酮生物合成机制的研究提供新的信息。
