Isoprenoid biosynthesis regulation in poplars by methylerythritol phosphate and mevalonic acid pathways

Abstract

It is critical to develop plant isoprenoid production when dealing with human-demanded industries such as flavoring, aroma, pigment, pharmaceuticals, and biomass used for biofuels. The methylerythritol phosphate (MEP) and mevalonic acid (MVA) plant pathways contribute to the dynamic production of isoprenoid compounds. Still, the cross-talk between MVA and MEP in isoprenoid biosynthesis is not quite recognized. Regarding the rate-limiting steps in the MEP pathway through catalyzing 1-deoxy-D-xylulose5-phosphate synthase and 1-deoxy-D-xylulose5-phosphate reductoisomerase (DXR) and also the rate-limiting step in the MVA pathway through catalyzing 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR), the characterization and function of HMGR from Populus trichocarpa (PtHMGR) were analyzed. The results indicated that PtHMGR overexpressors (OEs) displayed various MEP and MVA-related gene expressions compared to NT poplars. The overexpression of PtDXR upregulated MEP-related genes and downregulated MVA-related genes. The overexpression of PtDXR and PtHMGR affected the isoprenoid production involved in both MVA and MEP pathways. Here, results illustrated that the PtHMGR and PtDXR play significant roles in regulating MEP and MVA-related genes and derived isoprenoids. This study clarifies cross-talk between MVA and MEP pathways. It demonstrates the key functions of HMGR and DXR in this cross-talk, which significantly contribute to regulate isoprenoid biosynthesis in poplars.

Keywords: DXR; HMGR; isoprenoid biosynthesis; methylerythritol phosphate pathway; mevalonic acid pathway; poplar.

Figure 1

MVA and MEP-related gene expression analyses in PtHMGR-and PtDXR-OEs poplars. (A) Mean comparison of relative expressions of MVA related genes affected by PtHMGR-and PtDXR-OEs. (B) Mean comparison of relative expressions of MEP related genes affected by PtHMGR-and PtDXR-OEs; PtActin was used as an internal reference in all repeats; “ns” means not significant, *p < 0.05, ^**p < 0.01, ^***p < 0.001, ^****p < 0.0001; Three independent replications were performed in each experiment.

Figure 2

Evaluation of the effect of the overexpression of PtHMGR on isoprenoid production and ABA. The qRT-PCR analyses of the expression levels of ABA synthesis-related genes and HPLC-MS/MS analyses of the contents of ABA, β-carotene, lycopene, and lutein in PtHMGR-OEs. The expression levels of the ABA synthesis-related ZEP and NCED genes family are shown. Bars represent mean ± SD (n = 3); Stars reveal significant differences, ^**p < 0.01, ^***p < 0.001; Experiments were performed in triplicates.

Figure 3

Analyzing the effect of the overexpression of PtHMGR on MVA and MEP-derivatives. HPLC-MS/MS analyses of the contents of GA₃ (A), IPA (B), TZR (C), DCS (D), and CS (E) in PtHMGR-OEs compared to NT poplars. Bars represent mean ± SD (n = 3); Stars reveal significant differences, **p < 0.01, ***p < 0.001; Experiments were performed in triplicates.

Figure 4

Assessment of the impact of PtDXR overexpression on isoprenoid and ABA biosynthesis. The qRT-PCR analyses of the expression levels of ABA synthesis-related genes and HPLC-MS/MS analyses of the contents of ABA, β-carotene, lycopene, and lutein in PtDXR-OEs. The expression levels of the ABA synthesis-related ZEP and NCED genes family are indicated. Bars represent mean ± SD (n = 3); Stars reveal significant differences, ^*p < 0.05, ^**p < 0.01, ^***p < 0.001; Experiments were performed in triplicates.

Figure 5

Analyzing the effect of the overexpression of PtDXR on MVA and MEP-derivatives. HPLC-MS/MS analyses of the contents of GA₃ (A), IPA (B), TZR (C), DCS (D), and CS (E) in PtDXR-OEs compared to NT poplars.

Figure 6

Phenotypic changes analyses resulted from the overexpression of PtHMGR and PtDXR effects on MVA and MEP pathways. (A, I), The PtDXR transgenic revealed a higher stem length than PtHMGR-OEs and NT poplars. (A, II), The PtHMGR transgenic presents more stem development than NT poplar. (A, III), NT poplar was used as a control; Scale bar represents 1 cm. (B) The Box and Whisker mean comparison plot of stem lengths revealed significantly higher lengths of PtDXR-OEs than NT poplars compared with PtHMGR-OEs. PtHMGR transgenics also revealed significantly higher lengths than NT poplars. (C,D) The Violin mean comparison plots of ZEP and NCED relative expressions between PtHMGR-and PtDXR-OEs compared with NT poplars. (E) The Box and Whisker mean comparison plot of stem diameters revealed slightly more diameters between PtDXR-OEs and NT poplars. Stars reveal significant differences, ^*p < 0.05, ^**p < 0.01, ^***p < 0.001, ^****p < 0.0001.