Cembranoids produced by tobacco glandular trichomes have bioactivities in resistance to insect pests and pathogens. Cembratrien-ol synthase (CBTS) plays a key role in the biosynthesis of cembranoids and directly determines the cembranoid content in tobacco. This study examined the effect of changing CBTS1 expression on tobacco resistance to the insect pest Spodoptera frugiperda and oomycete pathogen Phytophthora nicotianae. The CDS sequence of CBTS1 was cloned into gene overexpression and silencing vectors and introduced into tobacco (Nicotiana tabacum L. cv. TN90) to obtain CBTS1-overexpression plants (CBTS1-OE) and CBTS1-silenced plants (CBTS1-RI). Compared with control plants, the content of cembratrien-ol (CBT-ol) was increased 4.48 times in the CBTS1-OE plants but decreased by 68% in the CBTS1-RI plants, while that of cembratrien-diol (CBT-diol) was increased 3.17 times in the CBTS1-OE plants but decreased by 76% in the CBTS1-RI plants. The S. frugiperda resistance of transgenic tobacco plants was evaluated by in vitro toxicity test, and the results showed that the resistance of CBTS1-OE plants to S. frugiperda was significantly improved but that of CBTS1-RI plants was reduced. The P. nicotianae resistance of transgenic tobacco plants was assessed by the detached leaf assay, and the results showed that the resistance of CBTS1-OE plants to P. nicotianae was enhanced, while that of CBTS1-RI plants was attenuated. Further gene expression analysis showed that overexpression of CBTS1 increased the expression of the pathogen-related gene PR-1a, while silencing of CBTS1 decreased its expression. This study demonstrated that manipulating the expression of CBTS1 could change the cembranoid content in tobacco plants and alter their resistance to both insect pests and oomycete pathogens.

In metabolically engineered plants, the target products are usually uniformly distributed in the whole plant or specific tissues. When engineering tobacco to produce astaxanthin, a ketocarotenoid with strong antioxidant activity and multiple bioactivities, a scattered distribution of astaxanthin-producing regions was observed in a small portion of astaxanthin-producing tobacco plants, which caused mosaic-like red and green spots on the leaves (ASTA-mosaic). A physiological assay showed that the non-astaxanthin green region (Mosaic_G) had relatively higher chlorophyll content and better chloroplast structure than the astaxanthin-producing red region (Mosaic_R). Then, metabolomics, proteomics, and small RNA transcriptomics were employed to analyze the uneven distribution of astaxanthin-producing regions in tobacco leaves. The results of metabolomics and proteomics revealed a decrease in carotenoid metabolism, chlorophyll biosynthesis, and chlorophyll degradation in the Mosaic_G region. Pheophorbide a, an intermediate of chlorophyll degradation, was found to be significantly reduced in the Mosaic_G region, which was accompanied by the attenuation of chlorophyllase and pheophytinase, which catalyze the formation of pheophorbide a in chlorophyll degradation. Reductions in photosynthetic antenna proteins and photosystem-associated proteins were observed in the Mosaic_R region, consistent with the better chloroplast structure of the Mosaic_G region. Small RNA transcriptomics showed that several small RNAs could target chlorophyll-degradative genes, but they were more effective in targeting the astaxanthin biosynthetic genes. This finding was supported by the fact that the Mosaic_G region can remain green up to the senescence of tobacco leaves. This work provides insights into the mechanism of the uneven distribution of astaxanthin-producing regions in tobacco leaves and may contribute to the specialized utilization of tobacco plants for metabolic engineering.

Biosynthesis of squalene in the plant chassis has broad application prospects, and identifying efficient enzymes is of great importance. Here, we analyzed the function of squalene synthase genes WsSQS and WsSQS2 from Withania somnifera for squalene biosynthesis in tobacco. WsSQS and WsSQS2 shared 93.7% amino acid (aa) similarity, with divergent residues related to catalysis, NADPH binding, and membrane anchoring. Heterologous expression of WsSQS and WsSQS2 in tobacco increased squalene content by 2.05-fold and 1.68-fold, respectively, with the OE-WsSQS lines reaching 3.19 μg/g DW and the OE-WsSQS2 lines reaching 2.58 μg/g DW, compared to the control plants. Further transcriptomic assays revealed that overexpression of WsSQS induced broader transcriptional changes in the squalene metabolic pathway than WsSQS2. Specifically, the overexpression of WsSQS up-regulated AACT, HMGS, MVD, IspE, FPPS1, FPPS2, and SQS upstream of squalene biosynthesis and down-regulated GGPPS3 downstream of FPP biosynthesis, which is the direct precursor of squalene biosynthesis, while WsSQS2 exerted a more targeted impact, primarily up-regulating HMGS and the key rate-limiting enzyme gene HMGR in the squalene biosynthesis pathway. These findings are consistent with the high efficiency of WsSQS in squalene biosynthesis in tobacco. In summary, this study provides fundamental molecular and biochemical insights into the utilization of heterologous SQSs for squalene production based on the tobacco chassis.

Squalene, a high-value triterpenoid precursor widely used in pharmaceuticals and vaccine adjuvants, is primarily sourced from shark liver oil—an unsustainable practice that has driven interest in developing plant-based production alternatives. The first committed reaction in triterpenoid biosynthesis is catalyzed by squalene synthase (SQS), yet no SQS gene has been characterized in Amaranthus tricolor, a species recognized for its high squalene content. Here, we cloned and functionally characterized AtrSQS, a novel squalene synthase gene isolated from A. tricolor for the first time. Sequence analysis revealed that AtrSQS contains conserved domains and six characteristic motifs shared among plant SQSs, with high homology to orthologs from dicotyledonous species. To investigate its functional role in squalene biosynthesis, AtrSQS was overexpressed in Nicotiana tabacum under the CaMV 35S promoter. Transgenic lines exhibited significantly increased AtrSQS expression and accumulated squalene up to 6.81 μg/g dry weight, a 4.76-fold increase over wild-type plants. Additionally, the content of downstream product 2,3-oxidosqualene was also significantly elevated in the transgenic lines. Integrated transcriptomic and metabolomic analyses revealed that AtrSQS overexpression upregulated key mevalonate pathway genes (AACT, HMGS, MVD) and FPPS. Meanwhile, it suppressed competitive carotenoid biosynthesis and the production of an SQS-specific inhibitor, indicating a successful redirection of metabolic flux toward squalene production. These findings demonstrate that AtrSQS is crucial for squalene biosynthesis and provide both a valuable genetic resource and mechanistic insights for engineering plant-based squalene production systems.