Metabolic engineering of terpene biosynthesis in plants using a trichome-specific transcription factor MsYABBY5 from spearmint (Mentha spicata)

Abstract

In many aromatic plants including spearmint (Mentha spicata), the sites of secondary metabolite production are tiny specialized structures called peltate glandular trichomes (PGT). Having high commercial values, these secondary metabolites are exploited largely as flavours, fragrances and pharmaceuticals. But, knowledge about transcription factors (TFs) that regulate secondary metabolism in PGT remains elusive. Understanding the role of TFs in secondary metabolism pathway will aid in metabolic engineering for increased yield of secondary metabolites and also the development of new production techniques for valuable metabolites. Here, we isolated and functionally characterized a novel MsYABBY5 gene that is preferentially expressed in PGT of spearmint. We generated transgenic plants in which MsYABBY5 was either overexpressed or silenced using RNA interference (RNAi). Analysis of the transgenic lines showed that the reduced expression of MsYABBY5 led to increased levels of terpenes and that overexpression decreased terpene levels. Additionally, ectopic expression of MsYABBY5 in Ocimum basilicum and Nicotiana sylvestris decreased secondary metabolite production in them, suggesting that the encoded transcription factor is probably a repressor of secondary metabolism.

Keywords: YABBY; secondary metabolism; spearmint; sweet basil; terpene; transcription factor.

Figure 1

Validation of MsYABBY genes expression pattern in spearmint. (A) Spearmint leaf showing peltate glandular trichome (PGT) on upper leaf surface as visualized under scanning electron microscope. (B) qRT‐PCR analysis of MsYABBY genes in different tissues. PGT, peltate glandular trichome; leaf‐PGT, leaves where PGT were brushed away. The housekeeping gene elongation factor 1 (ef1) was used as control. (C) In situ hybridization: antisense (a) and sense (b) probe detection of MsYABBY5.

Figure 2

Amino acid sequence alignment (A) and phylogenetic tree analysis (B) of MsYABBYs.

Figure 3

Subcellular localization of MsYABBYs in Nicotiana benthamiana. (A) MsYABBY5 showed both nuclear and cytoplasmic expression, while other MsYABBYs were found in nucleus only. (a) MsYABBY5 was localized to both nucleus and cytoplasm. (b) MsYABBY6. (c) MsYABBY2. (d) MsYABBY4. (B) MsYABBY5 protein colocalization with Golgi marker. (C) BFA treatment leads to nuclear localization of MsYABBY5 protein in N. benthamiana. (a) Mock group treated with DMSO. (b) Test group treated with 50 μg/mL BFA for 3 h.

Figure 4

MsYABBY5 promoter analysis and expression pattern. (A) cis‐acting regulatory elements in the 5′UTR (−1116 bp) region of MsYABBY5. (B and C) Trichome‐specific GUS expression pattern observed in Nicotiana benthamiana leaves and stems of plants transformed with pM sYABBY5::GUS.

Figure 5

Localization of MsYABBY5 under its native promoter in sweet basil (A) and tobacco (B). pM sYABBY5:: MsYABBY5‐CFP showed exclusive expression in PGT of plants.

Figure 6

Transcript level of MsYABBY5 and monoterpene production in MsYABBY5 RNAi and overexpression plants. (A) MsYABBY5 transcripts level in RNAi plants. (B) Limonene production in RNAi plants. (C) Carvone production in RNAi plants. (D) MsYABBY5 transcripts level in overexpression plants. (E) Limonene production in overexpression plants. (F) Carvone production in overexpression plants. Gene expression is presented as relative to ef1. Leaves from the second node (2–3 cm) were harvested and used for analysis. Results of terpene production are presented as mean ± SD. *P < 0.05; **P < 0.01.

Figure 7

MsNTT expression and localization. Transcript levels of MsNTT in MsYABBY5 RNAi (A) and overexpression plants (B) Leaves from the second node (2–3 cm) were harvested and used for qPCR analysis. Gene expression was normalized against the house keeping gene ef1. *P < 0.05; **P < 0.01. (C) MsNTT was localized to the chloroplast membrane in Nicotiana benthamiana.

Figure 8

Transactivation of MsWRKY75 by MsYABBY5. (A) Expression pattern of MsWRKY75 in different tissues of wild‐type spearmint. (B) Transcript levels of MsWRKY75 in MsYABBY5 RNAi plants. (C) Purification of recombinant His tagged MsYABBY5 expressed in Escherichia coli. (D) EMSA assay of MsYABBY5 binding ability to MsWRKY75 promoter. Upper panel shows the fragment −909 to −555 of MsWRKY75 promoter region, (E) transactivation of MsWRKY75 by MsYABBY5 in Nicotiana Benthamiana. Results are presented as mean ± SD. **, P < 0.01.

Figure 9

Ectopic expression of MsYABBY5 decreased terpene production in Nicotiana sylvestris and sweet basil. (A) CBT‐diol production in wild‐type and transgenic N. sylvestris overexpressed with MsYABBY5. Volatiles production of total monoterpenes (B), sesquiterpenes (C) and eugenol (D) in wild‐type and transgenic basil overexpressed with MsYABBY5. Leaves from the second node (2–4 cm) were harvested and used for GC–MS analysis. Results of terpenes and phenylpropanoid production are presented as mean ± SD. *P < 0.05; **P < 0.01.