1.3.8
IS INDUCTION OF BIOCHEMICAL DEFENCE MOLECULES IN CEREAL ROOTS REDUCED THROUGH PROGRAMMED CORTICAL DEATH?

E LILJEROTH, K SANTÉN and T BRYNGELSSON

Department of Plant Breeding Research, The Swedish University of Agricultural Sciences, S-268 31 Svalöv, Sweden

Background and objectives
In cereals, a phenomenon called root cortical death (RCD) is likely to play an important role in colonization by necrotrophic fungi, due to substrate release and perhaps also to a reduced potential for biochemical defence. Cortical root cells die in a certain programmed pattern already seen in young seminal roots [1, 2]. RCD is not influenced by the presence of saprophytic microorganisms or mild abiotic stress factors. Usually the outer cortical cell layers die first, as indicated by nuclear staining, and the part of the cortex having stainable nuclei gradually decreases with increasing age of the root. However, the rate of RCD differs greatly among cereal species. Wheat has a much faster rate of RCD than barley, oats and rye. [2]. How cell death of the root cortex is initiated, regulated and genetically controlled is unknown and needs clarification.

It was earlier demonstrated that the necrotrophic fungus Bipolaris sorokiniana can invade root tissue more easily in species which have a faster rate of RCD, presumably due to higher nutrient availability or to a reduced capacity of root cortex cells to synthesize defence biochemicals [3].

To investigate whether RCD is a genetically programmed process, the fragmentation of nuclear DNA along the roots was studied. Implications for root biochemical defence were analysed by investigating the expression of PR proteins.

Materials and methods
DNA fragmentation was investigated histochemically with the TUNEL (terminal deoxynucletidyl tranferase-mediated dUTP-digoxigenin nick end-labelling) assay, and on extracted DNA using Southern analysis. Expression of PR proteins in roots was analysed qualitatively and quantitatively by ELISA, isoelectric focusing and Western blotting. The proteins were localized in root tissue by immunohistochemical methods.

Results and conclusions
Preliminary results indicate that RCD in cereal grasses is a genetically programmed process that resembles apoptosis, as a gradually increased degree of DNA fragmentation in nuclei was observed along the roots of these species using the TUNEL assay. TUNEL-positive nuclei were observed earlier and in higher frequencies in wheat than in barley root cortex cells, confirming earlier results with vital staining methods.

Several PR proteins were induced in the roots of these cereals after infection with different fungal species. It appears that induction of PR proteins takes place predominantly in the inner cortical cell layers, while the outer cell layers presumably have lost their ability to produce PR proteins, probably due to an early induction of cell death. This conclusion was also supported by the observation that inoculation of regions close to the root tip (0-2 days old) resulted in higher PR-protein concentrations in root tissue than inoculation of 6-10-day-old root segments. The implications of RCD for biochemical defence in roots, and how it affects fungal growth and disease development, will be discussed.

References
1. Deacon JW, 1987. In Pegg GF, Ayres PG (eds), Fungal Infection of Plants. Cambridge University Press, Cambridge, pp. 285-297.
2. Liljeroth E, 1995. New Phytologist 130, 495-501.
3. Liljeroth E, Franzon-Almgren I, Gunnarsson T, 1996. Journal of Phytopathology 144, 301-307.