Desensitization of the perception system for chitin fragments in tomato cells

Abstract

Suspension-cultured tomato (Lycopersicon esculentum) cells react to stimulation by chitin fragments with a rapid, transient alkalinization of the growth medium, but behave refractory to a second treatment with the same stimulus (G. Felix, M. Regenass, T. Boller [1993] Plant J 4: 307-316). We analyzed this phenomenon and found that chitin fragments caused desensitization in a time- and concentration-dependent manner. Partially desensitized cells exhibited a clear shift toward lower sensitivity of the perception system. The ability of chitin oligomers to induce desensitization depended on the degree of polymerization (DP), with DP5 approximately DP4 >> DP3 >> DP2 > DP1. This correlates with the ability of these oligomers to induce the alkalinization response and to compete for the high-affinity binding site on tomato cells and microsomal membranes, indicating that the alkalinization response and the desensitization process are mediated by the same receptor. The dose required for half-maximal desensitization was about 20 times lower than the dose required for half-maximal alkalinization; desensitization could therefore be used as a highly sensitive bioassay for chitin fragments and chitin-related stimuli such as lipochitooligosaccharides (nodulation factors) from Rhizobium leguminosarum. Desensitization was not associated with increased inactivation of the stimulus or with a disappearance of high-affinity binding sites from the cell surface, and thus appears to be caused by an intermediate step in signal transduction.

Figure 1

Induction of extracellular alkalinization in suspension-cultured tomato cells in response to two consecutive stimuli. Solid lines, Extracellular pH in untreated cells (control) or cells treated with CH4 or ergosterol at the concentrations indicated. Shaded circles and dotted lines, Extracellular pH after a second stimulation with 10 nm CH4 (shown in the inset). The pH of the growth medium was 5.1 at the start of the experiment.

Figure 2

Time dependence of desensitization induced by different doses of CH4. ΔpH_max elicited by 10 nm CH4 in cells pretreated for different times with the concentrations of CH4 indicated. ○, Control (no CH4); ▾, 1 pm; ♦, 3 pm; ▵, 10 pm; □, 30 pm; ░⃞, 100 pm; and •, 300 pm.

Figure 3

Dose-response curves for induction of the alkalinization response (ΔpH_max) by CH4 in cells pretreated for 60 min with different concentrations of CH4. ⋄, Control (no CH4); •, 0.03 nm; ▾, 0.3 nm; and ♦, 3 nm.

Figure 4

Dose-response curves for induction of desensitization by different chitin oligomers. Alkalinization (ΔpH_max) in response to 10 nm CH4 was measured in cells pretreated with different amounts of chitin oligomers for 60 min. ▪, CH4; ▵, CH3; ♦, CH2; and ○, CH1. Hatched lines indicate concentrations of the prestimuli that reduce ΔpH_max in response to 10 nm CH4 by 50% (EC₅₀ values for desensitization).

Figure 5

Dose-response curves for induction of an alkalinization response and for induction of desensitization by a purified Nod factor of R. leguminosarum, Nod Rlv-V(Ac;C18:1). ▵, Alkalinization (ΔpH_max) in response to different concentrations of Nod factor. ▴, Effect of 60 min of pretreatment with different concentrations of the Nod factor on alkalinization (ΔpH_max) induced by 10 nm CH4. Hatched lines indicate the EC₅₀ values.

Figure 6

Inactivation of chitin fragments in cell suspensions before and after desensitization. Inactivation was tested in suspensions without pretreatment (○, ⋄) or 60 min after pretreatment with 1 nm CH4 (•, ♦). At time 0, CH4 (10 nm) was added to cell suspensions or the corresponding culture medium freed of cells by filtration. Samples were taken at intervals, and serial dilutions were assayed for the induction of the alkalinization response. Equivalents of CH4 in the samples were determined from a standard curve obtained with untreated CH4. ○, Cell suspension without pretreatment; ⋄, cell-free medium of control cells; •, cell suspension pretreated with 1 nm CH4; ♦, cell-free medium from pretreated cells.

Figure 7

Binding of chitin fragments to intact tomato cells. Cell suspensions were treated with 0.03 nm (♦) or 0.3 nm (▾) CH4 at time 0 as indicated. ○, Control (no CH4). At intervals, 1-mL samples were taken and assayed for binding of the radioligand CH5-Gly-[³⁵S]Met-Boc in the presence of 10 nm unlabeled CH5. The dotted line without symbols indicates binding of radioligand in the presence of 10 μm CH5 (nonspecific binding).