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 half‐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 [32 P]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.