1Department of Forestry, University of Aberdeen, MacRobert Building, Aberdeen AB24 5UA, UK; 2Department of Plant Biology, University of Helsinki, Viikki, Helsinki, Finland

Background and objectives
Elicitors induce a range of defence responses in plants, including phytoalexin and lignin biosynthesis, and accumulation of pathogenesis-related proteins. Phenylpropanoid metabolism, which leads to biosynthesis of lignins, flavonoids and stilbenes, has been much studied in many angiosperm plants. On challenge with pathogens, enzyme activity increases rapidly, providing a method for the study of resistance mechanisms, and gene regulation. De novo transcription of genes coding for phenylalanine ammonia lyase (PAL) and other enzymes of the pathway can also be induced following application of elicitors to plant tissues. Because of the importance of lignin and stilbenes in the defence of conifers against pathogens, eliciting of the pathway was selected for this study. This work aims to develop methods for studying early stages of the defence response to root pathogens in conifers. The objectives of the study were to determine the effects of elicitor preparations from Heterobasidion annosum, cause of root and butt rot of conifers, on expression of phenylpropanoid genes in Sitka spruce (Picea sitchensis).

Materials and methods
Washed H. annosum mycelium, from 14-21 day old liquid cultures grown in Norkrans medium, was re-suspended in approx. 20 ml medium, homogenised and autoclaved for 120 mm. The supernatant from autoclaving was diluted in Norkrans medium to a concentration of 2.5 mg fresh weight mycelium/ml and applied to root axes and hypocotyls of aseptically-germinated spruce seedlings. Alternatively, 50 Ál aliquots of preparation were added aseptically to Sitka spruce cell suspensions growing in 50 ml medium. Tissues were also treated with salicylic and jasmonic acids.

Enzymes of the phenylpropanoid pathway were estimated enzymically using published methods. A probe for Northern blotting was amplified by PCR from genomic DNA of Sitka spruce. RNA isolation, northern blotting and hybridisation were carried out as described previously [1].

Results and conclusions
Rapid increases in PAL in roots were detectable enzymically 8 h after treatment, peaking at approx. 48 h. No increases were found in seedlings treated with H. annosum conidiospores (5x106/ml), or solutions of jasmonic acid or salicylic acid at 100 and 1000 pg/ml. Constitutive levels of PAL activity were high. Use of the pal probe, however, indicated that levels of transcripts were similar in all treatments. A 0.3 kb PCR fragment was sequenced and proved to be a partial pal gene. Only a single pal gene was found in this work.

In cell cultures, levels of several phenylpropanoid pathway enzymes were measured, including PAL, O-methyltransferase, cinnamyl alcohol dehydrogenase, 4-coumarate:coenzyme A ligase, chalcone synthase and stilbene synthase. Use of cell cultures gave more reproducible results, presumably resulting from a more even distribution of the elicitor to the target host cells.

Rapid increases in phenylpropanoid metabolism following elicitor application indicate that this response is an important early component of defence in spruce. Failure to detect increases in pal transcripts using molecular techniques may be a consequence of the presence of more than one gene encoding for PAL. The probe may have been constructed using a pal gene encoding for lignification in development, rather than a gene specifically associated with defence. The partial pal sequence makes it possible to determine the complete sequence and to determine whether P. sitchensis has several pal genes with different functions or whether it has a single gene, as in Pinus taeda.

1. Kervinen T, Peltonen S, Utriamen M et al., 1997. Plant Science 123, 143-150.