Specific perception of subnanomolar concentrations of chitin fragments by tomato cells: Induction of extracellular alkalinization, changes in protein phosphorylation, and establishment of a refractory state

Abstract

Suspension-cultured tomato cells respond to yeast cell wall preparations with a rapid, transient alkalinization of the culture medium. Depending on the dose of the stimulus, the pH starts to increase after a lag period of about 0.5-2 min and reaches a transient maximum, up to 0.6 pH units above the initial value, after 2-4 min. Using this alkalinization response as a rapid and convenient assay, a sensitive perception system for small chitin fragments was revealed in the tomato cells. Chitin oligomers with four or more N-acetylglucosamine residues stimulated the alkalinization response significantly at concentrations below 10 pM and halt-maximally at concentrations of 100 pM. About 10 000-fold higher concentrations of the trimer, N,N′,N″-triacetylchitotriose, were required to elicit similar responses. For up to 8 h after a first treatment with 10 nM of the tetramer, N,N′,N″,N‴-tetraacetyl-chitotetraose, cells did not respond to a second stimulation with any of the chitin fragments. Throughout this refractory period, however, cells remained fully responsive to preparations of fungal xylanase, another stimulus which induces a more permanent alkalinization after a lag phase of more than 2 min. The alkalinization response to these two qualitatively different stimuli was paralleled by the same characteristic changes in the pattern of protein phosphorylation, detected by in vivo pulse-labelling with [32P]phosphate for 30 sec. The onset of the alkalinization and of the changes in protein phosphorylation coincided in both cases, and both phenomena were blocked by the protein kinase inhibitor K-252a. Although the mechanism underlying the extracellular pH increase is unknown, activation of the alkalinization response provides a sensitive and convenient assay to investigate early events in chemoperception of microbial signals by plant cells.