Overexpression of 1-deoxy-D-xylulose-5-phosphate reductoisomerase enhances the monoterpene content in Litsea cubeba

Abstract

Monoterpenes are important components of plant essential oils and have long been used as raw materials for spices and food flavorings. Litsea cubeba is an economically aromatic plant species, the fruits of which produce essential oil with monoterpenes as the dominant components. As a branch point of carbon flow in the methyl erythritol phosphate (MEP) biosynthesis pathway, 1-deoxy-D-xylo-5-phosphate reductoisomerase (DXR) is a key rate-limiting enzyme that catalyzes the MEP pathway's second committed step. Therefore, DXR has become an effective regulation target to improve the biosynthesis of plant monoterpenes. In this study, we identified a DXR from L. cubeba, which was highly expressed in fruits, induced by MeJA and repressed by darkness. An enzyme assay showed that recombination LcDXR protein catalyzed DXP with NADPH as the cofactor. Transient overexpression of LcDXR significantly increased the content of monoterpenes in L. cubeba. Furthermore, LcDXR-overexpressing tobaccos were conducted and showed almost 5.9-fold increase in monoterpenes production, including limonene, α-pinene, eucalyptol, linalool, terpineol and camphor. Overexpression of LcDXR activated the metabolic flux of monoterpene biosynthesis through crosstalk and feedback mechanism. In addition, LcDXR-overexpressing tobaccos had no effect on phenotype of transgenic tobaccos. Our results demonstrate that LcDXR is a critical regulator of the monoterpene production in L. cubeba and other plants.

Keywords: DXP reductoisomerase; Litsea cubeba; MEP pathway; Monoterpene.

Figure 1

Sequence analysis of the LcDXR. (a) Maximum-likelihood tree of DXR protein sequences, including LcDXR and other DXRs. The following protein sequences were used for the analysis: CsDXR (XP_028055701), CaDXR (XP_027123133), HbDXR (XP_021669849), PpDXR (XP_007210092), RcDXR (XP_024173969), SlDXR (AAK96063), LtDXR (UOO00992), CkDXR (RWR93981), CcDXR (AOW69227), LlDXR (AHJ57307), PdDXR (XP_008787962). (b) Sequence alignment of DXRs. The black background indicates the NADPH binding domain and the DXP binding domain.

Figure 2

Tissue specific expression patterns of LcDXR. (a) The transcript levels of LcDXR in different tissues of L. cubeba. R: roots; S: stems; L: leaves; FL: flower; F1, fruits at 60 DAF; F2, fruits at 90 DAF; F3, fruits at 120 DAF; F4, fruits at 150 DAF. (b) The expression levels of LcDXR were induced by MeJA in 60 DAF fruits. (c) The expression levels of LcDXR were repressed by darkness in 60 DAF fruits. Error bars represent ± SD from three biological repeats. The different letters above the bars represent the significant difference (P < 0.01).

Figure 3

Enzyme activity of LcDXR. (a) SDS-PAGE analysis of recombination protein with His tag. M: Protein Marker, 1: Insoluble protein before purified, 2: Cell lysate supernatant, 3: Transmission fluid, 4: Washing fluid, 5−8: Elution fluid with protein. (b) Western blot analysis of recombinant LcDXR protein. M: Protein Marker, 1: The inclusion protein, 2: Total protein induced in pET–LcDXR2/Arctic Express, 3: Washing fluid. (c) Enzyme activity assay of recombinant LcDXR. NADPH has a robust ultraviolet absorption peak at A340 and NADP+ does not, while the light absorption value of NADPH at A340 decreases during the catalytic reaction. Data represent the mean ± SEs of three biological replicates.

Figure 4

Transient overexpression of LcDXR in L. cubeba leaves. Leaves were infiltrated with LcDXR under the control of the Cauliflower mosaic virus 35S promoter, and three independent transient overexpression lines (TOE) 1-3 were conducted and grown for 2 d, respectively. (a) Relative expression level of LcDXR in transient overexpressed L. cubeba. (b) The content of monoterpenes in LcDXR transient overexpressed L. cubeba. (c) Relative expression level of key genes involved in MVA and MEP pathway in LcDXR transient overexpressed L. cubeba. Data represent the mean ± SEs of three biological replicates. **, P < 0.01.

Figure 5

Monoterpene production was increased by overexpression of LcDXR in transgenic tobacco. (a) Relative expression of LcDXR in ten independent stable overexpression lines (SOE), respectively. (b) The content of monoterpenes in LcDXR transgenic tobacco. (c) Different compounds of monoterpenes in transgenic tobacco. (d) Relative expression levels of key genes involved in MVA and MEP pathway. Data represent the mean ± SEs of three biological replicates. *, P < 0.05; **, P < 0.01.