Plant Molecular Biology 39: 221–229, 1999.
© 1999 Kluwer Academic Publishers. Printed in the Netherlands.
Characterization of a pine multigene family containing elicitor-responsive
stilbene synthase genes
, Axel Schwekendiek
, Hans-Jörg Reif
Fachbereich Chemie, Philipps-Universität, Hans-Meerwein-Strasse 35043 Marburg, Germany (
Bayer AG, Institut für Biotechnologie, 5090 Leverkusen, Germany
Received 2 April 1998; accepted in revised form 12 August 1998
Key words: heartwood constituent, phytoalexin, pinosylvin, Pinus sylvestris, protoplasts, stilbene synthase,
Young pine seedlings respond to environmental stress by induced synthesis of pinosylvin, a stilbene phytoalexin.
Heartwood of pine trees is characterized by a high content of pinosylvin. The formation of pinosylvin from
cinnamoyl-CoA and three molecules malonyl-CoA catalysed by pinosylvin synthase is typical of the genus Pinus.
Its enzyme activity not detectable in unstressed seedlings is substantially increased upon application of stimuli like
UV-light or infection with the phytopathogenicfungusBotrytis cinerea. A genomic DNA librarywas screened with
pinosylvin synthase cDNA pSP-54 as a probe. Ten clones were isolated and grouped into ﬁve subclasses according
to the size of their introns. After subcloning into plasmid T7T3, four different members of the ﬁve gene subclasses
were characterized by sequencing.Emphasis was put on isolating various promoters and analyzing and comparing
their responsiveness. The amino acid sequences deduced from genes PST-1, PST-2, PST-3 and PST-5 shared an
overall identity of more than 95%. In gene PST-5, the putative translation start site ATG was replaced by CTG.
While promoter regions near the TATAA box were almost identical PST-1, PST-2 and PST-3, further upstream
sequences differed substantially. Differences in promoter strength were analysed both in transgenic tobacco plants
and by transient expression in tobacco protoplasts. Constructs used contained the bacterial β-glucuronidase under
the control of the promoters of pine genes PST-1, PST-2 and PST-3. Upon treatment with UV light or fungal
elicitor, the promoter of PST-1 showed highest responsiveness and led to tissue-speciﬁc expression 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 pinosylvin formation in the heartwood.
Plants exposed to pathogenes like viruses, fungi or
bacteria react by the increased production of defence-
related proteins. Among the proteins de novo synthe-
sized are enzymes acting in phytoalexin production
[5, 8], pathogenesis-related proteins, and means to
strengthen the cell wall structure . Thus, the basis
ofresistanceliesin both the preformedpassivebarriers
such as cell wall or cuticle which prevent the pathogen
from invading the plant and the additional induced
responsemainlyprovidedbygene activation. The con-
cept of phytoalexins as means of induced resistance
has been proven for a hydroxystilbene by introducing
the capacity of its synthesis into a plant previously not
able to form it .
In pine plants, especially in young seedlings, biotic
stress results in the induced synthesis of the phy-
toalexin pinosylvin, a stilbene derivative [7, 22]. In
trees, pinosylvin is a heartwood constituent. Unlike
most other hydroxystilbenes, pinosylvin lacks a hy-
droxyl group in ring B (Figure 1) and originates by
condensation of unsubstituted cinnamoyl-CoA with
three molecules of malonyl-CoA. Stress-induced stil-
bene synthesis by the activation of stilbene synthase