Molecular analysis of chalcone and dihydropinosylvin synthase from Scots pine (Pinus sylvestris), and differential regulation of these and related enzyme activities in stressed plants

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Abstract
Chalcone synthase (CHS) and stilbene synthase (STS) are closely related polyketide synthases which are key enzymes in the biosynthesis of flavonoids and stilbenes. Scots pine (Pinus sylvestris) is an interesting plant for a direct comparison of the enzymes. It not only contains the usual flavonoids, but also an unusual chalcone derivative (pinocembrin), and it synthesizes stilbenes of the pinosylvin type. We analysed a CHS and a STS by molecular cloning and functional expression in Escherichia coli. The CHS was active not only with 4-coumaroyl-CoA (to naringenin chalcone), but also with cinnamoyl-CoA (leading to pinocembrin). The STS was identified as dihydropinosylvin synthase, because it preferred dihydrocinnamoyl-CoA to cinnamoyl-CoA. The protein deviated in 47 positions from the CHS consensus. It had 73.2% identity with the CHS from P. sylvestris and only 65.3% with a STS from peanut (Arachis hypogaea). We also investigated the regulation of both enzyme types in P. sylvestris plantlets exposed to stress. CHS was present in non-stressed plantlets, and induction led to a transient increase with a peak after 16 h. STS type activities were regulated differently and were absent in non-stressed plantlets. Increases were observed after a lag period of at least 6 h, and highest activities were obtained after 30 h. The analysis of the reactions in the plant extracts and the substrate specificity of the cloned STS indicate that the plants contain at least two different types of STS: the cloned dihydropinosylvin synthase and a pinosylvin synthase which preferentially utilizes cinnamoyl-CoA as substrate.