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.For a wide range of plant species, formation of brown spots is part of a characteristic development of Mn toxicity symptoms in older leaves. The subsequent development of chlorosis and necrosis and finally leaf shedding occurs before a reduction in vegetative growth on the whole plant level (Horst, 1988; El-Jaoul and Cox, 1998). Analysis of the Mninduced formation of brown spots revealed the presence of oxidized Mn and oxidized phenols, especially in the cell wall of the epidermis layer (Horiguchi, 1987;Wissemeier and Horst, 1992). The formation of visible Mn toxicity symptoms is accompanied by the spatial formation of callose in the area of brown spots (Wissemeier and Horst, 1987). The physiological role of callose formation in response to toxic Mn levels in the tissue is unknown, but its detection serves as an additional sensitive parameter for Mn-induced injury of the leaf tissue. In cowpea (Vigna unguiculata), the leaf apoplast has been proposed to be the most important compartment for the defense of Mn stress (Horst et al., 1999 (Horst, 1988). This hypothesis is supported by a close relationship between the Mn-induced formation of brown spots, activation of constitutive apoplastic POD, and Mn-induced release of POD into the apoplast (Fecht-Christoffers et al., 2003). Among the existing information about the physiological functions of POD in plant tissue, its activation in response to a broad range of biotic and abiotic factors plays a particularly important role (Greppin et al., 1986;Obinger et al., 1996). POD activity is often used as a physiological marker for plant stress response as part of a complex cascade of reactions with an apparent lack of specificity. However, some specific relationships between horseradis...

The apoplast is considered the leaf compartment decisive for manganese (Mn) toxicity and tolerance in cowpea (Vigna unguiculata). Particularly apoplastic peroxidases (PODs) were proposed to be key enzymes in Mn toxicity-induced processes. The presented work focuses on the characterization of the role of hydrogen peroxide (H 2 O 2 )-producing (NADH peroxidase) and H 2 O 2 -consuming peroxidase (guaiacol POD) in the apoplastic washing fluid (AWF) of leaves for early stages of Mn toxicity and genotypic differences in Mn tolerance of cowpea. Leaf AWF of the Mn-sensitive cultivar (cv) TVu 91 but not of the Mntolerant cv 1987 showed an increase of guaiacol-POD and NADH-peroxidase activities at elevated AWF Mn concentrations. two-dimensional resolutions of AWF proteins revealed that cv TVu 91 expressed more and additional proteins at high Mn treatment, whereas Mn-tolerant cv TVu 1987 remained nearly unaffected. In both cultivars, NADH-peroxidase activity and accompanied H 2 O 2 formation rate in vitro were significantly affected by Mn 21 , p-coumaric acid, and metabolites occurring in the AWF. The total phenol concentration in the AWF was indicative of advanced stages of Mn toxicity but was rather unrelated to early stages of Mn toxicity and genotypic differences in Mn tolerance. The NADH oxidation by AWF PODs was significantly delayed or enhanced in the presence of the protein-free AWF from cv TVu 1987 or cv TVu 91, respectively. High-performance liquid chromatography analysis of AWF indicates the presence of phenols in cv TVu 1987 not observed in cv TVu 91. We conclude from our studies that the H 2 O 2 -producing NADH peroxidase and its modulation by stimulating or inhibiting phenolic compounds in the leaf apoplast play a major role for Mn toxicity and Mn tolerance in cowpea.

Excessive manganese (Mn) supply induced the formation of brown spots on leaves as typical Mn toxicity symptoms in cowpea (Vigna unguiculata L. Walp.) grown in hydroponics. Differences in Mn resistance between cv. TVu 91 (Mn‐sensitive) and cv. TVu 1987 (Mn‐tolerant) expressed in the density of brown spots in older leaves were due to higher Mn tissue tolerance. Apoplastic water‐soluble peroxidase (POD) in the apoplastic washing fluid (AWF) was enhanced by increasing Mn leaf content and generally significantly higher in leaves of cv. TVu 91 than in cv. TVu 1987. Electrophoresis of AWF revealed the presence of several water‐soluble POD isoenzymes. At toxic Mn supply, the activities of these and additional POD isoenzymes increased more in the Mn‐sensitive cultivar. Levels of ascorbic acid in the apoplast and cytoplasm of the Mn‐sensitive cv. TVu 91 decreased with increasing leaf Mn contents, whereas Mn‐tolerant cv. TVu 1987 was not affected. Mn treatment lead to a stimulation of the enzymes of the ascorbic acid regeneration system (monodehydroascorbic acid reductase and glutathione reductase) in both cultivars, but the activation of glutathione reductase was clearly more enhanced in the Mn‐tolerant cultivar TVu 1987. The results provide circumstantial evidence that apoplastic ascorbate and peroxidases are involved in the expression of Mn toxicity and genotypic Mn tolerance.

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In cowpea (Vigna unguiculata), the development of manganese (Mn) toxicity is considered to be accompanied by the formation of reactive oxygen species, oxidized Mn, and phenoxy radicals in the leaf apoplast. Ascorbic acid (AA) is a common antioxidant in plants, and the oxidation of AA, particularly in the leaf apoplast, contributes to the first line of defence against several biotic and abiotic stress factors. The objective of the present study was to contribute to a better understanding of the role of AA in Mn leaf‐tissue tolerance of cowpea and common bean (Phaseolus vulgaris). Five cowpea cultivars (cvs.) differed greatly in Mn tolerance, which was expressed in differences in numbers of brown spots on leaves and in peroxidase (POD) activity in the apoplastic washing fluid (AWF). In a Mn‐sensitive cv., after 3 d of Mn treatment, brown spots were formed, and POD activities were increased, accompanied by a release of proteins into the apoplast. In the AWF, the concentration of AA and the ratio of AA : (AA+DHA) decreased already after 1 d, and to only 2% after 3 d. In the leaf tissue, the ratio was nearly unaffected, and the total AA+DHA content in the leaf tissue was even increased with advanced expression of Mn toxicity. The application of AA solutions in the range of 5–10 μM via the petiole slightly enhanced Mn tolerance as indicated by the reduction of brown spots (however inconsistently) and POD activity (consistently) in the AWF. Common bean cultivars differing in ozone tolerance, which has been reported to be due to a high AA availability in the leaf apoplast, were studied for their Mn tolerance. Clear differences in Mn tolerance between the cultivars existed, however, these differences were not related to their ozone tolerance. From these results, we conclude that the maintenance of sufficient AA levels in the leaf apoplast contribute to Mn tolerance, but does not fully explain genotypic differences in Mn tolerance in cowpea and common bean.