Table_3_Transcriptional deregulation of stress-growth balance in Nicotiana benthamiana biofactories producing insect sex pheromones.xlsx

Plant biofactories are a promising platform for sustainable production of high-value compounds, among which are insect sex pheromones, a green alternative to conventional insecticides in agriculture. Recently, we have constructed transgenic Nicotiana benthamiana plants (“Sexy Plants”, SxP) that successfully produce a blend of moth (Lepidoptera) sex pheromone compounds (Z)-11-hexadecen-1-ol and (Z)-11-hexadecenyl acetate. However, efficient biosynthesis of sex pheromones resulted in growth and developmental penalty, diminishing the potential for commercial use of SxP in biomanufacturing. To gain insight into the underlying molecular responses, we analysed the whole-genome transcriptome and evaluated it in relation to growth and pheromone production in low- and high-producing transgenic plants of v1.0 and v1.2 SxP lines. In our study, high-producing SxPv1.2 plants accumulated the highest amounts of pheromones but still maintained better growth compared to v1.0 high producers. For an in-depth biological interpretation of the transcriptomic data, we have prepared a comprehensive functional N. benthamiana genome annotation as well as gene translations to Arabidopsis thaliana, enabling functional information transfer by using Arabidopsis knowledge networks. Differential gene expression analysis, contrasting pheromone producers to wild-type plants, revealed that while only a few genes were differentially regulated in low-producing plants, high-producing plants exhibited vast transcriptional reprogramming. They showed signs of stress-like response, manifested as downregulation of photosynthesis-related genes and significant differences in expression of hormonal signalling and secondary metabolism-related genes, the latter presumably leading to previously reported volatilome changes. Further network analyses confirmed stress-like response with activation of jasmonic acid and downregulation of gibberellic acid signalling, illuminating the possibility that the observed growth penalty was not solely a consequence of a higher metabolic burden imposed upon constitutive expression of a heterologous biosynthetic pathway, but rather the result of signalling pathway perturbation. Our work presents an example of comprehensive transcriptomic analyses of disadvantageous stress signalling in N. benthamiana biofactory that could be applied to other bioproduction systems.

植物生物工厂（Plant biofactories）是可持续生产高价值化合物的极具前景的平台，其中昆虫性信息素（insect sex pheromones）是农业领域替代传统杀虫剂（conventional insecticides）的绿色方案。此前，我们构建了转基因本氏烟草（Nicotiana benthamiana）材料“性感植株”（Sexy Plants，简称SxP），其可成功合成鳞翅目（Lepidoptera）蛾类性信息素混合物：(Z)-11-十六碳烯-1-醇与(Z)-11-十六碳烯乙酸酯。然而，高效合成性信息素会导致植株生长发育受损，削弱了SxP用于生物制造（biomanufacturing）的商业化应用潜力。为解析背后的分子响应机制，我们对v1.0和v1.2版本SxP株系的低产、高产转基因植株开展了全基因组转录组（whole-genome transcriptome）分析，并结合其生长状况与信息素产量进行关联评估。本研究中，v1.2版本的高产SxP植株积累的信息素含量最高，且相较于v1.0版本的高产植株，其生长表现更优。为深入解析转录组数据的生物学意义，我们完成了一套完整的本氏烟草基因组功能注释，并实现了本氏烟草基因向拟南芥（Arabidopsis thaliana）的同源注释，从而可借助拟南芥的已知知识网络完成功能信息的跨物种迁移。以信息素合成植株与野生型植株为对照的差异基因表达分析（Differential gene expression analysis）显示：低产植株仅少量基因出现表达差异，而高产植株则发生了广泛的转录重编程（transcriptional reprogramming）。高产植株呈现类应激响应特征，具体表现为光合相关基因的表达下调，以及激素信号通路与次生代谢相关基因的表达显著改变——后者大概率导致了此前报道的挥发物组（volatilome）变化。进一步的网络分析证实了该类应激响应：茉莉酸（jasmonic acid）通路被激活，而赤霉素（gibberellic acid）信号通路被抑制，这表明观测到的生长受损并非仅由异源生物合成通路组成型表达带来的代谢负荷过高所致，而是信号通路扰动的结果。本研究为植物生物工厂中的不利应激信号传导提供了一套完整的转录组分析案例，该方案可推广应用至其他生物制造系统。