Figure 2.

flg22 pretreatment does not enhance other AtPep1-triggered responses. A, Medium alkalinization assay. Cell cultures were either pretreated with 1 µm flg22 or without any peptide (control) for 16 h and then treated with 1 µm of the indicated elicitor or mock treated (control). Bars represent mean pH shift values of five biological replicates. Error bars show se of the mean. B, Measurement of cytosolic calcium concentrations. Leaf discs were pretreated with 1 µm flg22 or without any peptide (control) for 16 h and then treated with the indicated elicitor (1 µm) or mock treated (control). Graphs represent normalized mean values of 12 biological replicates. Error bars show se of the mean. C and D, MAPK phosphorylation. Leaf discs were pretreated with 1 µm flg22 (+) or without any peptide (−) for 16 h and then treated for 5 min with the indicated concentrations of AtPep1 or a mock treatment (0 nm; C) and treated with 1 µm AtPep1 for the indicated period of time (D). MAPK phosphorylation was detected by immunoblotting using an antiphospho-p44/42-MAPK antibody detecting the pTE-pY motif of MPK6 and MPK3. The immunoblot was reprobed with anti-actin antibody to determine equal loading. E, Induction of marker gene transcription. Leaf discs were pretreated with 1 µm flg22 or without any peptide (control) for 16 h and then directly flash frozen (0) or treated with 1 µm AtPep1 for 30 min before freezing (Pep1). Transcript levels of indicated genes were first normalized to the reference gene UBQ10 before calculating the mean of three biological replicates. Error bars show se of the mean. F, Ethylene production. Leaf strips of Col-0 plants were pretreated with either 1 µm flg22 or 1 µm AtPep1 or without any peptide (control) for 16 h and then treated (elicitation treatment) with either 1 µm flg22, 1 µm AtPep1, or without any peptide (control) for 4 h. Columns represent averages of detected ethylene values of six biological replicates. Error bars show se of the mean. MNE, Mean normalized expression.