1.9.26
DEFENCE-RELATED ACTIVATION OF PHENYLALANINE AMMONIA-LYASE IN BARLEY

S PELTONEN, T KERVINEN and R KARJALAINEN

University of Helsinki, Department of Plant Biology, Section of Plant Pathology, PO Box 28, FIN-00014 University of Helsinki, Finland

Background and objectives
Phenylalanine ammonia-lyase (PAL) catalyses the first reaction in the plant phenylpropanoid pathway leading to a number of potentially protective compounds such as phytoalexins, a variety of flavonoids and UV-protectants, lignin as well as salicylic acid. In many plants, the increase in PAL activity has been shown to be a direct response of host plants to attempted penetration by the pathogen. PAL activity may also be induced by elicitors present in cell walls or culture filtrates of both phytopathogenic and non-pathogenic micro-organisms, and by structurally unrelated abiotic elicitors or environmental factors such as light [1]. The importance of PAL has been demonstrated in dicotyledons using transgenic plants but, so far, little is known about the precise role of PAL in monocotyledonous plants although lignification has been considered as an important disease resistance mechanism in cereals. Increased PAL activity has been found to be associated with resistance of barley to powdery mildew but the induction has often been regarded to be more associated with the general defence responses of plants [2]. The objectives of this study were to evaluate the induction patterns of PAL in different barley-pathogen/non-pathogen systems, to define the pathogen-derived components that cause the activation of PAL and to assess the corresponding expression of pal genes in barley.

Results and conclusions
Fungal infection caused by a necrotrophic pathogen Bipolaris sorokiniana-induced PAL activity in barley leaves typically showing two adjacent peaks, the first representing the plants' general defence reactions to attempted penetration by the pathogen while the second peak was suggested to be more associated with resistance reactions, e.g. with attempts to limit the further spread of the pathogen. The inclusion of the later peak for PAL activity was clearly evident only in plants with increased resistance to fungal infection. Treatment of barley leaves with an abiotic elicitor, mercuric chloride, also caused a strong induction of PAL. The resistance of plants to non-pathogens was not connected with activation of PAL, neither did wounding induce PAL. Non-pathogens were, however, able to induce PAL when inoculated through the wounds of barley leaves. The crude fungal mycelium preparation, purified chitin, chitosan and glucan were all able to strongly induce PAL in barley cell suspension cultures while endo-1,4-6xylanase purified from the culture filtrates of B. sorokiniana was only weakly active. At least five different cDNA clones showing high homology with different rice pal genes could be cloned from barley root cDNA library, and four of them were further characterised. Using gene-specific pal probes it was shown that the isolated pal genes exhibited differential expression in barley. Three pal genes were strongly and constitutively present in barley roots while the expression was lower in sprouts and more mature leaves. Two pal genes showed strong, early induction after inoculation with B. sorokiniana or treatment with mercuric chloride while the induction of the third pal gene was later. One pal gene showed specific inducibility only by light in etiolated barley leaves. The results suggested that the pal genes might be responsible for the activation of different branches in the phenylpropanoid biosynthesis of barley. Furthermore, it is suggested that the contact of fungal cell wall components whether through penetration or wounds rather than the plant cell wall degrading enzymes (xylanase) secreted by the fungus function as recognisable components that cause barley to induce PAL activity.

References
1. Dixon RA, Paiva NL, 1995. Plant Cell 7, 1085-1097.
2. Shiraishi T, Yamada T, Nicholson RL, Kunoh H. 1995. Physiological and Molecular Plant Pathology 46, 153-162.