A Pinus strobus transcription factor PsbHLH1 activates the production of pinosylvin stilbenoids in transgenic Pinus koraiensis calli and tobacco leaves.

Transcription factors (TFs) play an important role in regulating the biosynthesis of secondary metabolites. In Pinus strobus, the level of methylated derivatives of pinosylvin is significantly increased upon pine wood nematode (PWN) infection, and these compounds are highly toxic to PWNs. In a previous study, we found that the expression of a basic helix-loop-helix TF gene, PsbHLH1, strongly increased in P. strobus plants after infection with PWNs. In this study, we elucidated the regulatory role of the PsbHLH1 gene in the production of methylated derivatives of pinosylvin such as pinosylvin monomethyl ether (PME) and dihydropinoylvin monomethyl ether (DPME). When PsbHLH1 was overexpressed in Pinus koraiensis calli, the production of PME and DPME was significantly increased. Overexpression of the stilbene synthase (PsSTS) and pinosylvin methyl transferase (PsPMT) genes, known as key enzymes for the biosynthesis of methylated pinosylvins, did not change PME or DPME production. Moreover, PME and DPME were not produced in tobacco leaves when the PsSTS and PsPMT genes were transiently coexpressed. However, the transient expression of three genes, PsSTS, PsPMT, and PsbHLH1, resulted in the production of PME and DPME in tobacco leaves. These results prove that PsbHLH1 is an important TF for the pinosylvin stilbene biosynthesis in pine plants and plays a regulatory role in the engineered production of PME and DPME in tobacco plants.

Production of Nematicidal Pinosylvin Stilbenes in Cell Suspension Cultures of Pinus koraiensis by Fungal Elicitation.

Pinosylvin stilbenes are natural phenolic compounds found in the Pinaceae family and act as phytoalexins. Some pinosylvin stilbenes have strong nematicidal activity against pine wood nematodes (PWNs: Bursaphelenchus xylophilus). Here, we established the efficient production of two pinosylvin stilbenes, dihydropinosylvin monomethylether (DPME) and pinosylvin monomethylether (PME), by cell suspension culture of Pinus koraiensis after fungal elicitation. DPME and PME were found in small amounts (less than 40 µg/g DW) in the stem bark and leaves of P. koraiensis plants. Cell suspension cultures were established from the cultures of calli derived from mature zygotic embryos of P. koraiensis in 1/2 Litvay medium containing 2.2 µM 2,4-D and 2.2 µM BA. Two types of fungal elicitors, fungal cell extract (CE) and fungal medium filtrate (MF), were prepared from three species of fungi (Penicillium chrysogenum, P. pinophilum, and P. roquefortii). CE and MF treatments strongly stimulated the production of PME and DPME in cultured cells. The production of PME in suspension cells of P. chrysogenum, P. pinophilum, and P. roquefortii MF treatments after 3 days was 5734 µg/g DW, 4051 µg/g DW, and 6724 µg/g DW, respectively. Pinosylvin synthase (PkSTS) and pinosylvin O-methyltransferase (PkPMT) are key genes in DPME and PME biosynthesis. qPCR analysis revealed that the expression of the PkSTS and PkPMT in cultured cells was highly enhanced after fungal elicitor treatment. The cell extracts after MF treatment resulted in 92.5 ± 7.8% immobilization of the adult PWNs and 63.7 ± 3.5% immobilization of the juvenile PWNs within 24 h. However, control cell extracts without MF treatment showed 11.3 ± 1.4% nematicidal activity against adult PWNs. Our results suggest that pinosylvin stilbenes can be produced from the cell culture of P. koraiensis after fungal elicitor treatment and can be used as nematicidal compounds against PWNs.