Characterization of a pine multigene family containing elicitor-responsivestilbene synthase genes

Young pine seedlings respond to environmental stress by induced synthesis o f pinosylvin, a stilbene phytoalexin. Heartwood of pine trees is characteri zed by a high content of pinosylvin. The formation of pinosylvin from cinna moyl-CoA and three molecules malonyl-CoA catalysed by pinosylvin synthase i s typical of the genus Pinus. Its enzyme activity not detectable in unstres sed seedlings is substantially increased upon application of stimuli like U V-light or infection with the phytopathogenic fungus Botrytis cinerea. A ge nomic DNA library was screened with pinosylvin synthase cDNA pSP-54 as a pr obe. Ten clones were isolated and grouped into five subclasses according to the size of their introns. After subcloning into plasmid T7T3, four differ ent members of the five gene subclasses were characterized by sequencing. E mphasis was put on isolating various promoters and analyzing and comparing their responsiveness. The amino acid sequences deduced from genes PST-1, PS T-1, PST-3 and PST-5 shared an overall identity of more than 95%. In gene P ST-5, the putative translation start site ATG was replaced by CTG. While pr omoter regions near the TATAA box were almost identical PST-1, PST-2 and PS T-3, further upstream sequences differed substantially. Differences in prom oter strength were analysed both in transgenic tobacco plants and by transi ent expression in tobacco protoplasts. Constructs used contained the bacter ial B-glucuronidase under the control of the promoters of pine genes PST-I, PST-2 and PST-3. Upon treatment with UV light or fungal elicitor, the prom oter of PST-I showed highest responsiveness and led to tissue-specific expr ession in vascular bundles. The data suggest that in pine the gene product of PST-1 is responsible for both the stress response in seedlings and pinos ylvin formation in the heartwood.