Effect of manganese toxicity on the proteome of the leaf apoplast in cowpea

Abstract

Excess manganese (Mn) supply causes formation of visible brown depositions in the cell walls of leaves of cowpea (Vigna unguiculata), which consist of oxidized Mn and oxidized phenols. Because oxidation of Mn and phenolic compounds in the leaf apoplast was proposed to be catalyzed by apoplastic peroxidases (PODs), induction of these enzymes by Mn excess was investigated. POD activity increased upon prolonged Mn treatment in the leaf tissue. Simultaneously, a significant increase in the concentration of soluble apoplastic proteins in "apoplastic washing fluid" was observed. The identity of the released proteins was systematically characterized by analysis of the apoplast proteome using two-dimensional gel electrophoresis and liquid chromatography-tandem mass spectrometry. Some of the identified proteins exhibit sequence identity to acidic PODs from other plants. Several other proteins show homologies to pathogenesis-related proteins, e.g. glucanase, chitinase, and thaumatin-like proteins. Because pathogenesis-related-like proteins are known to be induced by various other abiotic and biotic stresses, a specific physiological role of these proteins in response to excess Mn supply remains to be established. The specific role of apoplastic PODs in the response of plants to Mn stress is discussed.

Figure 1.

Effect of treatment duration on the total Mn content in the leaf tissue (A), the Mn concentration in the AWF of leaves (B), and the ratio of water-soluble Mn in the apoplast to total Mn in the leaf tissue (C). Plants of cowpea were treated with 50 μm Mn, whereas control plants received 0.2 μm Mn, continuously. AWF was collected by vacuum-infiltration and centrifugation of leaves. Means of 14 replicates each day are significantly different at P < 0.05 (Tukey) as indicated by different letters. Coefficients of determination of regression analysis are significant as ^***, ^**, and ^* for P < 0.001, 0.01, and 0.05, respectively.

Figure 2.

Relationship between the Mn tissue content and the number of brown spots on the trifoliate leaves of cowpea. Four leaf discs per leaf were incubated in ethanol to remove chlorophyll. The number of brown spots was then determined by direct counting. Means of 14 replicates each day are significantly different at P < 0.05 (Tukey) as indicated by different letters. Coefficients of determination of regression analysis are significant as ^***, ^**, and ^* for P < 0.001, 0.01, and 0.05, respectively.

Figure 3.

Relationship between the Mn and callose contents of leaves. Means of 14 replicates each day are significantly different at P < 0.05 (Tukey) as indicated by different letters. Leaf discs, previously used for counting brown spots, were homogenized in NaOH. The extracted callose was detected by anilin blue staining. Coefficients of determination of regression analysis are significant as ^***, ^**, and ^* for P < 0.001, 0.01, and 0.05, respectively.

Figure 4.

Relationships between the Mn tissue content and the activity of guaiacol-POD (A), the activity of NADH-POD (B), and the concentration of total protein in the AWF of leaves (C). Guaiacol-POD activity was measured in the presence of 20 mm guaiacol and 0.03% (w/w) H₂O₂ (pH 6). NADH-POD was measured in the presence of 0.6 mm NADH, 1.6 mm p-coumaric acid, and 16 mm MnCl₂ (pH 5). Protein concentrations were measured according to Bradford (1976). Means of 14 replicates each day are significantly different at P < 0.05 (Tukey) as indicated by different letters. Coefficients of determination of regression analysis are significant as ^***, ^**, and ^* for P < 0.001, 0.01, and 0.05, respectively.

Figure 5.

One-dimensional (1D) resolution of water-soluble proteins from the leaf apoplast of cowpea. Separation was carried out by BN-PAGE using a 12% to 20% (w/v) gradient gel. PODs were detected by staining with 20 mm Guaiacol+0.01% (w/w) H₂O₂ (A) and total protein with colloidal Coomassie Blue (B). The numbers above the gels indicate duration of the Mn treatment (in days); numbers on the right indicate molecular masses of proteins.

Figure 6.

2D resolution of water-soluble proteins from the leaf apoplast of cowpea by BN/SDS-PAGE. PODs were detected by staining with 20 mm guaiacol + H₂O₂ (i) and total proteins with colloidal Coomassie Blue (ii and second dimension). Plants precultured for 13 d were treated with 50 μm Mn for 5 d (B), whereas control plants received 0.2 μm Mn continuously (A). Numbers on the top of the gels indicate the acrylamide concentration, and the numbers on the left indicate molecular masses of proteins. Marked spots were identified by nano LC-MS/MS.

Figure 7.

2D resolution of water-soluble proteins from the leaf apoplast of cowpea by IEF/SDS-PAGE. Plants precultured for 13 d were treated with 50 μm Mn for 5 d (B), whereas control plants received 0.2 μm Mn continuously (A). Numbers on the top of the gels indicate the pH gradient, and the numbers on the left indicate molecular masses of proteins. Marked spots were identified by nano LC-MS/MS.