Functional characterization of two acyltransferases from Populus trichocarpa capable of synthesizing benzyl benzoate and salicyl benzoate, potential intermediates in salicinoid phenolic glycoside biosynthesis.

Salicinoids are phenolic glycosides (PGs) characteristic of the Salicaceae and are known defenses against insect herbivory. Common examples are salicin, salicortin, tremuloidin, and tremulacin, which accumulate to high concentrations in the leaves and bark of willows and poplars. Although their biosynthetic pathway is not known, recent work has suggested that benzyl benzoate may be a potential biosynthetic intermediate. Two candidate genes, named PtACT47 and PtACT49, encoding BAHD-type acyl transferases were identified and are predicted to produce such benzylated secondary metabolites. Herein described are the cDNA cloning, heterologous expression and in vitro functional characterization of these two BAHD acyltransferases. Recombinant PtACT47 exhibited low substrate selectivity and could utilize acetyl-CoA, benzoyl-CoA, and cinnamoyl-CoA as acyl donors with a variety of alcohols as acyl acceptors. This enzyme showed the greatest Km/Kcat ratio (45.8 nM(-1) s(-1)) and lowest Km values (45.1 µM) with benzoyl-CoA and salicyl alcohol, and was named benzoyl-CoA: salicyl alcohol O-benzoyltransferase (PtSABT). Recombinant PtACT49 utilized a narrower range of substrates, including benzoyl-CoA and acetyl-CoA and a limited number of alcohols. Its highest Km/Kcat (31.8 nM(-1) s(-1)) and lowest Km (55.3 µM) were observed for benzoyl-CoA and benzyl alcohol, and it was named benzoyl-CoA: benzyl alcohol O-benzoyltransferase (PtBEBT). Both enzymes were also capable of synthesizing plant volatile alcohol esters, such as hexenyl benzoate, at trace levels. Although the activities demonstrated are consistent with roles in salicinoid biosynthesis, direct tests of this hypothesis using transgenic poplar must still be performed.

Effects of leaching on fungal growth and decay of western redcedar.

We tested the effect of leaching on the concentration of western red cedar (WRC; Thuja plicata Donn ex D. Don) heartwood extractives that are known to exhibit antimicrobial activity and correlated this with fungal growth and decay. We assessed the extractive tolerance of the following fungal species: Acanthophysium lividocaeruleum, Coniophora puteana, Heterobasidion annosum, Pachnocybe ferruginea, Phellinus sulphurascens, and Phellinus weirii by measuring their growth rate (mm/day) on media with or without WRC leachate. These data were correlated with the ability of the fungal species to grow on and decay leached versus nonleached WRC. We used an ergosterol assay to estimate growth and a standard soil-block test to assess decay. We estimated that leaching reduced the concentration of 5 major extractives: (-)-plicatic acid, beta-thujaplicin, gamma-thujaplicin, beta-thujaplicinol, and thujic acid by approximately 80%. Phellinus sulphurascens exhibited the lowest extractive-tolerance in vitro, grew poorly on and caused minimal decay in nonleached WRC, but it grew well on and decayed pine and leached WRC. Coniophora puteana, H. annosum, and P. weirii displayed moderate to high tolerance to leachate, grew on and caused decay in nonleached as well as leached WRC, but their growth and decay were always greatest on leached WRC and pine, suggesting that leaching enhances decay by these fungi. Acanthophysium lividocaeruleum and Pachnocybe ferruginea exhibited high extractive-tolerance. Whereas A. lividocaeruleum clearly caused decay on all types of wood, no decay was observed with Pachnocybe ferruginea, which grew very slowly in the different wood species, and it may or may not be able to decay wood.