Transcriptional deregulation of stress-growth balance in Nicotiana benthamiana biofactories producing insect sex pheromones

Abstract

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.

Tunable control of insect pheromone biosynthesis inNicotiana benthamiana

“…In previous studies we observed that constitutive expression impacts biomass (Juteršek et al, 2022;Mateos-Fernández et al, 2021). Although yields obtained using copper inducible promoters in transient infiltration were lower (12.4 µg Z11-16OH g -1 FW and 4.5 µg Z11-16OAc g -1 FW), than those that we obtained in previous studies using CaMV35s promoters (116.6 µg Z11-16OH g -1 FW and 110.1 µg Z11-16OAc g -1 FW), a lower yield per unit of biomass might be compensated for by high biomass production.…”

“…The economics of scaling-up production are likely to be different for different molecules, however, there is merit in comparing production methods. In previous experiments, we observed that the growth of transgenic lines of N. benthamiana producing the highest yields (per gram fresh weight) of the moth pheromones was negatively affected and photosynthesis-related genes were downregulated (Juteršek et al, 2022;Mateos-Fernández et al, 2021). Inducible gene expression systems are essential tools commonly employed to switch metabolism from growth to production, enabling biomass to accumulate before energy is redirected into the biosynthesis of desired products.…”

“…For example, we recently showed that growth was compromised in transgenic lines N. benthamiana producing the highest yields (per gram fresh weight) of the Lepidopteran sex pheromone components, (Z)-11-hexadecenyl acetate (Z11-16OAc) and (Z)-11-hexadecenol (Z11-16OH) (Mateos-Fernández et al, 2021). Analysis of transcriptional changes revealed stress-like responses, including the downregulation of photosynthesis-related genes (Juteršek et al, 2022). The complex advantages and disadvantages of transient and transgenic approaches make it challenging to determine which strategy will be most cost-effective for large-scale production of a given molecule.…”

Stress Knowledge Map: A knowledge graph resource for systems biology analysis of plant stress responses

“…Although knowledge compiled in SKM is predominately based on Arabidopsis, this use case clearly shows its applicability in other species. Through orthology tools such as PLAZA (Van Bel et al ., 2022), the knowledge graphs in SKM can be translated to other species, as was done for the previous version of CKN to Prunus persica (Foix et al ., 2021), Solanum tuberosum (Ramšak et al ., 2018), and Nicotiana benthamiana (Juteršek et al ., 2022). This way, canonical principles of plant signalling networks can be assessed across species.…”

Tunable control of insect pheromone biosynthesis in Nicotiana benthamiana

“…The resulting transgenics accumulated Z11‐16OH, Z11‐16OAc, and Z11‐16Ald; however, plant growth was compromised in transgenic lines producing the highest yields. A subsequent analysis of transcriptional changes revealed stress‐like responses, including the downregulation of photosynthesis‐related genes (Juteršek et al ., 2022 ). Recent studies have also investigated the production of sex pheromone precursors in field‐grown transgenic Camelina sativa (false flax) (Wang et al ., 2022 ).…”