Metabolic Flux Engineering of Cembratrien-ol Production in Both the Glandular Trichome and Leaf Mesophyll in Nicotiana tabacum

Cembratrien-ol synthase (CBTS) catalyzes the first step in cembranoid biosynthesis, producing cembratrien-ols in plant trichomes. In our previous study, microarray transcriptomes between leaves with trichomes and leaves without trichomes showed that an NtCBTS2 gene was expressed exclusively and abundantly in trichomes. Here, two NtCBTS2 isogenes (NtCBTS2a and NtCBTS2b), derived from a diploid genome donor, Nicotiana sylvestris, were identified from N. tabacum. Both genes were expressed primarily in trichomes, with relatively decreased transcription in flowers and stems, and faint expression in roots, and no expression was detected in leaves lacking trichomes. To demonstrate the feasibility of producing natural product cembratrien-ols in tobacco mesophylls, the mesophylls of 35S:NtCBTS2b transgenic tobacco plants were used in the analysis, suggesting that constitutive expression of NtCBTS2b led to the cembratrien-ol production in mesophylls. Overexpression of NtCBTS2b using either Cauliflower mosaic virus (CaMV) 35S or trichome-specific Cyt P450 oxygenase (CYP) promoters greatly increased aphid resistance by promoting the accumulation of CBT-ols, increased the secretory cell growth in glandular trichomes and increased the levels of various physiological measures, including sugar esters, gibberellins, and cembranoid production. Meanwhile, specifically overexpressing NtCBTS2b in glandular trichomes could most efficiently promote aphid resistance in tobacco plants. Notably, our results indicate the feasibility of utilizing bio-engineering to produce large amounts of CBT-ols, and modify significantly the composition of naturally produced CBT-ols and CBT-diols, thereby promoting aphid resistance in plants.