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( E )-β-Farnesene (EBF) serves as the primary component of the alarm pheromone used by most aphid pest species. Pyrethrum ( Tanacetum cinerariifolium ) exhibits tissue-specific regulation of EBF accumulation and release, effectively mimicking the aphid alarm signal, deterring aphid attacks while attracting aphid predators. However, cultivated chrysanthemum ( Chrysanthemum morifolium ), a popular and economically significant flower, is highly vulnerable to aphid infestations. In this study, we investigated the high expression of the pyrethrum EBF synthase ( Tc EbFS) gene promoter in the flower head and stem, particularly in the parenchyma cells. Subsequently, we introduced the TcEbFS gene, under the control of its native promoter, into cultivated chrysanthemum. This genetic modification led to increased EBF accumulation in the flower stem and young flower bud, which are the most susceptible tissues to aphid attacks. Analysis revealed that aphids feeding on transgenic chrysanthemum exhibited prolonged probing times and extended salivation durations during the phloem phase, indicating that EBF in the cortex cells hindered their host-location behavior. Interestingly, the heightened emission of EBF was only observed in transgenic chrysanthemum flowers after mechanical damage. Furthermore, we explored the potential of this transgenic chrysanthemum for aphid resistance by comparing the spatial distribution and storage of terpene volatiles in different organs and tissues of pyrethrum and chrysanthemum. This study provides valuable insights into future trials aiming for a more accurate replication of alarm pheromone release in plants. It highlights the complexities of utilizing EBF for aphid resistance in cultivated chrysanthemum and calls for further investigations to enhance our understanding of this defense mechanism., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Li, Hu, Ren, Yu, Luo, Li, Zeng, Wang and Wang.)

Pyrethrins, synthesized in the perennial plant Tanacetum cinerariifolium , are a class of terpene mixtures with high insecticidal activity and low human toxicity, which are widely used in plant-derived pesticides. Numerous studies have identified multiple pyrethrins biosynthesis enzymes, which can be enhanced by exogenous hormones such as methyl jasmonate (MeJA). However, the mechanism by which hormone signaling regulates pyrethrins biosynthesis and the potential involvement of certain transcription factors (TFs) remain unclear. In this study, we found that the expression level of a TF in T. cinerariifolium was significantly increased after treatment with plant hormones (MeJA, abscisic acid). Subsequent analysis identified this TF as a member of the basic region/leucine zipper (bZIP) family and was thus named TcbZIP60 . TcbZIP60 was localized in the nucleus, suggesting that it is involved in the transcription process. The expression profiles of TcbZIP60 were similar to those of pyrethrins synthesis genes in different flower organs and at different flowering stages. Furthermore, TcbZIP60 could directly bind to the E-box/G-box motifs in the promoters of the pyrethrins synthesis genes TcCHS and TcAOC to activate their expression. Transient overexpression of TcbZIP60 increased the expression levels of pyrethrins biosynthesis genes, leading to the significant accumulation of pyrethrins. Silencing of TcbZIP60 significantly downregulated pyrethrins accumulation and the expression of related genes. Overall, our results reveal a novel TF, TcbZIP60, that regulates both the terpenoid and jasmonic acid pathways of pyrethrins biosynthesis in T. cinerariifolium ., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Xu, Zeng, Li, Zhou, Li, Luo, Zheng, Wang, Hu and Wang.)