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JXB Advance Access originally published online on March 13, 2009
Journal of Experimental Botany 2009 60(6):1663-1678; doi:10.1093/jxb/erp034
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© 2009 The Author(s).
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. This paper is available online free of all access charges (see http://jxb.oxfordjournals.org/open_access.html for further details)

RESEARCH PAPER

Characterization of leaf apoplastic peroxidases and metabolites in Vigna unguiculata in response to toxic manganese supply and silicon

Hendrik Führs1, Stefanie Götze1, André Specht1, Alexander Erban2, Sébastien Gallien3, Dimitri Heintz4, Alain Van Dorsselaer3, Joachim Kopka2, Hans-Peter Braun5 and Walter J. Horst1,*

1Institute of Plant Nutrition, Faculty of Natural Sciences, Leibniz University Hannover, Herrenhäuser Str. 2, D-30419 Hannover, Germany
2Max Planck Institute of Molecular Plant Physiology, Am Mühlenberg 1, D-14476 Potsdam-Golm, Germany
3Laboratoire de Spectrométrie de Masse Bio-Organique, IPHC-DSA, ULP, CNRS, UMR7178 ; 25 rue Becquerel, F-67087 Strasbourg, France
4Institut de Biologie Moléculaire des Plantes (IBMP) CNRS-UPR2357,ULP, F-67083 Strasbourg, France
5Institute of Plant Genetics, Faculty of Natural Sciences, Leibniz University Hannover, Herrenhäuser Str. 2, D-30419 Hannover, Germany

* To whom correspondence should be addressed: E-mail: horst{at}pflern.uni-hannover.de

Previous work suggested that the apoplastic phenol composition and its interaction with apoplastic class III peroxidases (PODs) are decisive in the development or avoidance of manganese (Mn) toxicity in cowpea (Vigna unguiculata L.). This study characterizes apoplastic PODs with particular emphasis on the activities of specific isoenzymes and their modulation by phenols in the Mn-sensitive cowpea cultivar TVu 91 as affected by Mn and silicon (Si) supply. Si reduced Mn-induced toxicity symptoms without affecting the Mn uptake. Blue Native-PAGE combined with Nano-LC-MS/MS allowed identification of a range of POD isoenzymes in the apoplastic washing fluid (AWF). In Si-treated plants Mn-mediated induction of POD activity was delayed. Four POD isoenzymes eluted from the BN gels catalysed both H2O2-consuming and H2O2-producing activity with pH optima at 6.5 and 5.5, respectively. Four phenols enhanced NADH-peroxidase activity of these isoenzymes in the presence of Mn2+ (p-coumaric=vanillic>>benzoic>ferulic acid). p-Coumaric acid-enhanced NADH-peroxidase activity was inhibited by ferulic acid (50%) and five other phenols (50–90%). An independent component analysis (ICA) of the total and apoplastic GC-MS-based metabolome profile showed that Mn, Si supply, and the AWF fraction (AWFH2O, AWFNaCl) significantly changed the metabolite composition. Extracting non-polar metabolites from the AWF allowed the identification of phenols. Predominantly NADH-peroxidase activity-inhibiting ferulic acid appeared to be down-regulated in Mn-sensitive (+Mn, –Si) and up-regulated in Mn-tolerant (+Si) leaf tissue. The results presented here support the previously hypothesized role of apoplastic NADH-peroxidase and its activity-modulating phenols in Mn toxicity and Si-enhanced Mn tolerance.

Key words: BN-PAGE, cowpea, leaf apoplast, metabolome, manganese toxicity, phenolics, proteome

Received 7 November 2008; Accepted 26 January 2009


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