A stilbene synthase from Japanese red pine (Pinus densiflora): implications for phytoalexin accumulation and down-regulation of flavonoid biosynthesis

Abstract

Stilbene synthase (STS) and chalcone synthase (CHS) are plant-specific polyketide synthases that play key roles in the stilbenoid and flavonoid biosyntheses, respectively. We have recently isolated from Pinus densiflora three STS cDNAs (PDSTS1, PDSTS2, and PDSTS3) and one CHS cDNA (PDCHSX). We then heterologously expressed these cDNAs in Escherichia coli and characterized their properties. An unusual STS isozyme, PDSTS3, lacks the common C-terminal extension of STS because of a frame-shift mutation and shows the highest pinosylvin-forming activity among the STSs tested. Pinosylvin was shown to be a potent inhibitor of PDCHSX (K(i) = 6 microM) as well as PDSTS2 (K(i) = 13 microM), which presumably maintains the balance between the stilbenoid and flavonoid biosyntheses. PDSTS3 was insensitive to product inhibition. We identified PDSTS3 in the pine seedlings as well as full-length STS. The data provide evidence that PDSTS3 is involved in the potential regulation of the stilbenoid and flavonoid biosynthetic pathways in pine trees.

Figure 1

Pathways for stilbenoid and flavonoid biosyntheses. Stilbene synthase (STS) and chalcone synthase (CHS), respectively, lead to stilbenoid and flavonoid biosynthesis from a cinnamoyl-CoA/p-coumaroyl-CoA with three malonyl-CoAs. PAL, phenylalanine ammonia-lyase; C4H, cinnamate 4-hydroxylase.

Figure 2

A frame-shift mutation of PDSTS3. (A) Nucleotide deletion in PDSTS3 cDNA. The two nucleotide deletions are indicated by the hyphens. (B) Alignment of the deduced amino acid sequences for PDCHSX, PDSTS1, PDSTS2, and PDSTS3. The frameshift mutation in PDSTS3 changes the amino acid sequence from N293 indicated by the arrow to the newly occurred stop codon indicated by the asterisk.

Figure 3

Production of soluble PDSTSs in E. coli. (A) The same amounts of the soluble proteins (3 μg) were separated by SDS/PAGE and stained. Lane 1, PDSTS1; lane 2, PDSTS2; lane 3, PDSTS3. (B) Western blot analysis. The soluble fusion proteins were crossreacted with the S-protein alkaline phosphatase conjugate. The band of PDSTS3 is indicated by the arrow. PDSTS1 and PDSTS2, 60 kDa; PDSTS3, 52 kDa.

Figure 4

Activities of PDCHSX that coexisted with three PDSTSs. The purified recombinant PDCHSX (10 pmol) was mixed with equimolar amounts of the purified recombinant. 1, PDSTS1; 2, PDSTS2; 3, PDSTS3. The activities were measured twice as described in the text.

Figure 5

Occurrence of PDSTS3 in pine. (A) STS and CHS activities in pine extracts preincubated with either 200 μM pinosylvin (PS) or anti-STS antibody. The enzyme assays were performed as described in the text. (B) Immunodetection of naturally occurring STS in pine seedlings.