Membrane-bound guaiacol peroxidases from maize (Zea mays L.) roots are regulated by methyl jasmonate, salicylic acid, and pathogen elicitors

Abstract

Plant peroxidases are involved in numerous cellular processes in plant development and stress responses. Four plasma membrane-bound peroxidases have been identified and characterized in maize (Zea mays L.) roots. In the present study, maize seedlings were treated with different stresses and signal compounds, and a functional analysis of these membrane-bound class III peroxidases (pmPOX1, pmPOX2a, pmPOX2b, and pmPOX3) was carried out. Total guaiacol peroxidase activities from soluble and microsomal fractions of maize roots were compared and showed weak changes. By contrast, total plasma membrane and washed plasma membrane peroxidase activities, representing peripheral and integral membrane proteins, revealed strong changes after all of the stresses applied. A proteomic approach using 2D-PAGE analysis showed that pmPOX3 was the most abundant class III peroxidase at plasma membranes of control plants, followed by pmPOX2a >pmPOX2b >pmPOX1. The molecular mass (63 kDa) and the isoelectric point (9.5) of the pmPOX2a monomer were identified for the first time. The protein levels of all four enzymes changed in response to multiple stresses. While pmPOX2b was the only membrane peroxidase down-regulated by wounding, all four enzymes were differentially but strongly stimulated by methyl jasmonate, salicylic acid, and elicitors (Fusarium graminearum and Fusarium culmorum extracts, and chitosan) indicating their function in pathogen defence. Oxidative stress applied as H(2)O(2) treatment up-regulated pmPOX2b >pmPOX2a, while pmPOX3 was down-regulated. Treatment with the phosphatase inhibitor chantharidin resulted in distinct responses.

Fig. 1.

Effects of stress treatments on total soluble, microsomal, PM-bound, and wPM-bound protein yields from 5-d-old maize roots. Maize seedlings were exposed to different stressors as indicated. Each stress treatment was analysed using 2–3 independent experiments, i.e. each experiment with approximately 900 plant seedlings. Total protein yields were determined in triplicate in each experiment. Relative yields were calculated in comparison with direct controls that were handled in parallel. Shown are means ±SE (n=6–9; controls n=30). Values that were significantly different from the controls in t test analysis were marked at P < 0.001 (*) and P < 0.05 (**). H₂O₂ (2 mM); wounding (cutting); methyl-jasmonate (50 μM); salicylic acid (0.5 mM); F. graminearum extract (250 ng protein ml⁻¹); F. culmorum extract (125 ng protein ml⁻¹); chitosan (20 μg ml⁻¹); cantharidin (20 μM); FW, fresh weight. For treatment times see Table 2. Soluble enzymes (grey bars), membrane-bound (microsomes) (dotted bars); PM-bound, checked bars; wPM-bound, solid bars.

Fig. 2.

Effects of stress treatments on total microsomal and soluble guaiacol peroxidase activities from 5-d-old maize roots. Maize seedlings were exposed to different stressors as indicated. Each treatment was analysed using 2–3 independent experiments. Total class III peroxidase activities were determined in triplicate in each experiment. Relative peroxidase activities were calculated in comparison with direct controls that were handled in parallel. Shown are means ±SE (n=6–9; controls n=30). Class III peroxidase activities were measured in the presence of 8.26 mM guaiacol and 8.8 mM H₂O₂. Values that were significantly different from the controls in t test analysis were marked at P < 0.001 (*) and P < 0.05 (**). H₂O₂ (2 mM); wounding (cutting); methyl-jasmonate (50 μM); salicylic acid (0.5 mM); F. graminearum extract (250 ng protein ml⁻¹); F. culmorum extract (125 ng protein ml⁻¹); chitosan (20 μg ml⁻¹); cantharidin (20 μM); FW, fresh weight. For treatment times see Table 2. (Soluble enzymes, grey bars; membrane-bound enzymes (microsomes), dotted bars.

Fig. 3.

Effects of stress treatments on total PM-bound and wPM-bound guaiacol peroxidase activities from 5-d-old maize roots. Maize seedlings were exposed to different stressors as indicated. Each treatment was analysed using 2–3 independent repetitions. Plasma membranes were isolated representing peripheral and integral PM-bound proteins. By contrast, thoroughly washed PM vesicles contain mainly integral PM-bound proteins, and thus represent the total activity of the four pmPOX of the plant PM. Relative peroxidase activities were determined in triplicate for each independent experiment. Shown are means ±SE (n=6–9; controls n=30; further details see Fig. 2). Class III peroxidase activities were measured in presence of the peroxidase substrates guaiacol and H₂O₂. Values that were significantly different from the controls in t test analysis were marked at levels of P < 0.001 (*) and P < 0.05 (**). PM-bound, checked bars; wPM-bound enzymes, solid bars.

Fig. 4.

2D-PAGE analysis of pmPOX after treatment with salicylic acid. Shown are exemplary gels for a control (A) and salicylic acid treatment (B). Solubilizates of washed PM were separated by isoelectric focusing in the first dimension with two different overlapping ampholyte gradients (A1, B1, pH range 10–8; A2, B2, pH range 8–3) to allow optimal separation of pmPOX, followed by a modified non-reducing gradient SDS-PAGE in the second dimension. Thus, oligomers and haem proteins remain intact. Protein spots were stained with guaiacol and H₂O₂. As described earlier (Mika et al., 2008), under these conditions this detects the haem groups of class III peroxidases, i.e. visualizing the protein abundance of the pmPOX (for details see the Materials and methods). pmPOX1, pmPOX2a, pmPOX2b, and pmPOX3 are visible. All four pmPOX are up-regulated in salicylic acid-treated plants.