Figure 4.

Co-treatment of Arabidopsis pMAQ2 with γ-Glu-Leu and flg22 enhances activation of Ca²⁺ flux and defense gene expression in comparison to the single treatments (A,B), but boosting effects are lost when elicitors are MES-buffered (C,D). (A) Seedlings were treated with water or an aqueous solution containing 10 nM flg22, 250 µM γ-Glu-Leu or a mixture of 10 nM flg22 and 250 µM γ-Glu-Leu. [Ca²⁺]_cyt curves are pooled from four independent experiments. Error bars represent standard error of the mean. **Significant difference (P < 0.01) according to Two-way ANOVA with Bonferroni post-test. (B) Pools of seedlings were elicited with water or an aqueous solution containing 10 nM flg22 and/or 500 µM γ-Glu-Leu for 1 hour. Expression of defense genes (relative to the reference gene PP2AA3) was determined in two independent experiments with 3–4 seedling pools per treatment and 2 technical replicates per pool. Combined data of both experiments were log-transformed prior to One-way ANOVA with Bonferroni multiple comparison tests for selected columns, as indicated. *,**,***Significant difference (P < 0.05, 0.01, 0.001, respectively), ns: not significant (C) Seedlings were treated with MES (pH 6) or 10 nM flg22, 250 µM γ-Glu-Leu or a mixture of 10 nM flg22 and 250 µM γ-Glu-Leu (in MES buffer, pH 6.0). Experiments were performed four times and [Ca²⁺]_cyt data were pooled. Error bars represent standard error of the mean. (D) Pools of seedlings were elicited with MES (pH 6.0) or a MES solution containing 10 nM flg22 and/or 500 µM γ-Glu-Leu (pH 6.0) for 1 hour. Gene expression data was pooled from two independent experiments and analyzed as described in B above.