Background Cembranoids are one kind of diterpenoids with multiple biological activities. The tobacco cembratriene-ol (CBT-ol) and cembratriene-diol (CBT-diol) have high anti-insect and anti-fungal activities, which is attracting great attentions for their potential usage in sustainable agriculture. Cembranoids were supposed to be formed through the 2-C-methyl-d-erythritol-4-phosphate (MEP) pathway, yet the involvement of mevalonate (MVA) pathway in their synthesis remains unclear. Exploring the roles of MVA pathway in cembranoid synthesis could contribute not only to the technical approach but also to the molecular mechanism for cembranoid biosynthesis. Results We constructed vectors to express cembratriene-ol synthase (CBTS1) and its fusion protein (AD-CBTS1) containing an N-terminal GAL4 AD domain as a translation leader in yeast. Eventually, the modified enzyme AD-CBTS1 was successfully expressed, which further resulted in the production of CBT-ol in the yeast strain BY-T20 with enhanced MVA pathway for geranylgeranyl diphosphate (GGPP) production but not in other yeast strains with low GGPP supply. Subsequently, CBT-diol was also synthesized by co-expression of the modified enzyme AD-CBTS1 and BD-CYP450 in the yeast strain BY-T20. Conclusions We demonstrated that yeast is insensitive to the tobacco anti-fungal compound CBT-ol or CBT-diol and could be applied to their biosynthesis. This study further established a feasibility for cembranoid production via the MVA pathway and provided an alternative bio-approach for cembranoid biosynthesis in microbes.
MYB transcription factors play essential roles in regulating plant secondary metabolism and jasmonate (JA) signaling. Putrescine N-methyltransferase (PMT) is a key JA-regulated step in the biosynthesis of nicotine, an alkaloidal compound highly accumulated in Nicotiana spp. Here we report the identification of NtMYB305a in tobacco (Nicotiana tabacum) as a regulatory component of nicotine biosynthesis and demonstrate that it binds to the JA-responsive GAG region, comprised of a G-box, an AT-rich motif, and a GCC-box-like element, in the NtPMT1a promoter. Yeast one-hybrid analysis, EMSA and ChIP assays showed that NtMYB305a binds to the GAG region in vitro and in vivo. Binding specifically occurs at the ∼30 bp AT-rich motif in a G/C-base-independent manner, thus defining the AT-rich motif as previously unknown MYB-binding element. NtMYB305a localized in the nucleus of tobacco cells where it is capable of activating the expression of a 4×GAG-driven GUS reporter in an AT-rich motif-dependent manner. NtMYB305a positively regulates nicotine biosynthesis and the expression of NtPMT and other nicotine pathway genes. NtMYB305a acts synergistically with NtMYC2a to regulate nicotine biosynthesis, but no interaction between these two proteins was detected. This identification of NtMYB305a provides insights into the regulation of nicotine biosynthesis and extends the roles played by MYB transcription factors in plant secondary metabolism.
Basic helix-loop-helix (bHLH) transcription factor MYC2 regulates plant growth and development in many aspects through the jasmonic acid (JA) signaling pathway, while the role of MYC2 in plant carbohydrate metabolism has not been reported. Here, we generated NtMYC2a-overexpressing (NtMYC2a-OE) and RNA-interference-mediated knockdown (NtMYC2a-RI) transgenic plants of tobacco (Nicotiana tabacum L. cv. TN90) to investigate the role of NtMYC2a in carbohydrate metabolism and pollen development. Results showed that NtMYC2a regulates the starch accumulation and the starch-sugar conversion of floral organs, especially in pollen. The RT-qPCR analysis showed that the expression of starch-metabolic-related genes, AGPs, SS2 and BAM1, were regulated by NtMYC2a in the pollen grain, anther wall and ovary of tobacco plants. The process of pollen maturation was accelerated in NtMYC2a-OE plants and was delayed in NtMYC2a-RI plants, but the manipulation of NtMYC2a expression did not abolish the pollen fertility of the transgenic plants. Intriguingly, overexpression of NtMYC2a also enhanced the soluble carbohydrate accumulation in tobacco ovaries. Overall, our results demonstrated that the bHLH transcription factor NtMYC2a plays an important role in regulating the carbohydrate metabolism during pollen maturation in tobacco.
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