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.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 Role of Hydrogen Peroxide-Producing and Hydrogen Peroxide-Consuming Peroxidases in the Leaf Apoplast of Cowpea in Manganese Tolerance

“…1B). The rapid accumulation of Mn in the AWF (Fecht-Christoffers et al, 2003a) indicates a particular role of free apoplastic Mn for the expression of Mn toxicity in spite of its low contribution to the total Mn content of the leaf. The significantly higher Mn concentration in the leaf AWF of Mn-treated plants of Mn-tolerant cv TVu 1987 without inducing Mn toxicity symptoms (Fig.…”

“…In the Mn-sensitive cv TVu 91, development of toxicity symptoms is accompanied by an enhanced release of proteins into the leaf apoplast (Fecht-Christoffers et al, 2003a). The protein concentration in the AWF is dependent on the severity of Mn toxicity symptoms.…”

“…The 2D resolution from cv TVu 91 showed a high conformity to previously shown results. Therefore, strongly expressed proteins on the gels can be considered identical with proteins identified previously by nanoliquid chromatography-mass spectrometry (MS)/MS (Fecht-Christoffers et al, 2003a), e.g. pathogenesisrelated-like proteins, chitinase, peroxidase, thaumatinlike protein, and glucanase.…”

“…The oxidation of Mn II and phenols is probably mediated by the formation of highly reactive Mn III and phenoxyradicals, which are considered as phytotoxic agents (Horst, 1988;. Particularly the simultaneous enhancement of POD activity and expression of Mn toxicity symptoms (brown spots) suggest a close relationship between PODs and the oxidation of Mn and phenolic compounds (Fecht-Christoffers et al, 2003a, 2003b. However, the activation of PODs by Mn excess in cotton (Gossypium hirsutum) was considered a secondary effect (Sirkar and Amin, 1974), and also from our own work in cowpea the enhanced apoplastic POD activity could be the reason for or the consequence of Mninduced leaf injury.…”

“…Mn and phenolic compounds presumably control the activity of the NADH peroxidase (Elstner and Heupel, 1976;Gross et al, 1977;Halliwell, 1978;Mäder and Fü ssl, 1982;Stich and Ebermann, 1984;Pedreñ o et al, 1987). Proteins purified from the apoplastic washing fluid (AWF) of leaves from the Mnsensitive cowpea cv TVu 91 showed NADH-peroxidase activity, which was strongly enhanced at high leaf Mn concentrations (Fecht-Christoffers et al, 2003a). The stimulation of this H 2 O 2 -producing NADH peroxidase in the leaf apoplast of Mn-treated plants might be the cause for the elevated H 2 O 2 formation of washed intact leaf segments from cowpea showing Mn toxicity symptoms .…”