Histochemical and chemical evidence for lignin accumulation during the expression of resistance to leaf rust fungi in wheat

Abstract

Primary leaves of wheat ( Triticum aestivum) were examined after inoculation with avirulent and virulent strains of the leaf rust fungus ( Puccinia recondita f. sp. tritici) at stages when previous work had shown fungal and host cells to be affected by the expression of the Lr20 or Lr28 alleles for resistance. Histochemical stains for lignin were first shown to react in characteristic ways with cell walls in stomata, vascular tissue and mechanical fibres in sections of uninfected leaves. Toluidine blue and two different chlorine-sulphite reagents reacted positively for lignin with walls of hypersensitive mesophyll cells in the Lr28-bearing leaves, but only toluidine blue in the Lr20-bearing leaves. Phloroglucinol did not react positively with these cells. Aniline blue stained guard cells in the infected Lr28-bearing leaves and hypersensitive cells strongly in these leaves but less so in Lr20-bearing leaves, suggesting the presence of callose. Sudan IV failed to indicate suberin in any situation. Lignin was also extracted and measured as a complex with thioglycolic acid (LTGA). LTGA derived from uninfected and infected leaves had a characteristic UV absorption spectrum with a distinctive peak at 280 nm. Lignin was present in high concentration 48 h after inoculation of Lr20-bearing leaves with an avirulent strain, when widespread hypersensitivity had occurred around fungal colonies. Lignin was also detected at moderate concentrations 24 h after inoculation of Lr28-bearing leaves with an avirulent strain, when one or a few cells had undergone hypersensitivity in the presence of haustoria. Enzymatic digestion of these leaves yielded fractions containing hypersensitive cells and the lignin content in these fractions was significantly higher than in comparable fractions from uninfected leaves. Some evidence was obtained that infection also increased the lignin content of stomatal guard cells. The results suggested that lignification, in cells undergoing a hypersensitive response, may play a rote in the highly specific cultivar resistance expressed by wheat to the leaf rust fungus.