Catalytic profile of Arabidopsis peroxidases, AtPrx-2, 25 and 71, contributing to stem lignification.

Jun Shigeto,Yuji Tsutsumi,Mariko Nagano,Koki Fujita,Keqiang Wu

doi:10.1371/journal.pone.0105332

Abstract

Lignins are aromatic heteropolymers that arise from oxidative coupling of lignin precursors, including lignin monomers (p-coumaryl, coniferyl, and sinapyl alcohols), oligomers, and polymers. Whereas plant peroxidases have been shown to catalyze oxidative coupling of monolignols, the oxidation activity of well-studied plant peroxidases, such as horseradish peroxidase C (HRP-C) and AtPrx53, are quite low for sinapyl alcohol. This characteristic difference has led to controversy regarding the oxidation mechanism of sinapyl alcohol and lignin oligomers and polymers by plant peroxidases. The present study explored the oxidation activities of three plant peroxidases, AtPrx2, AtPrx25, and AtPrx71, which have been already shown to be involved in lignification in the Arabidopsis stem. Recombinant proteins of these peroxidases (rAtPrxs) were produced in Escherichia coli as inclusion bodies and successfully refolded to yield their active forms. rAtPrx2, rAtPrx25, and rAtPrx71 were found to oxidize two syringyl compounds (2,6-dimethoxyphenol and syringaldazine), which were employed here as model monolignol compounds, with higher specific activities than HRP-C and rAtPrx53. Interestingly, rAtPrx2 and rAtPrx71 oxidized syringyl compounds more efficiently than guaiacol. Moreover, assays with ferrocytochrome c as a substrate showed that AtPrx2, AtPrx25, and AtPrx71 possessed the ability to oxidize large molecules. This characteristic may originate in a protein radical. These results suggest that the plant peroxidases responsible for lignin polymerization are able to directly oxidize all lignin precursors.

Highlights

Lignin is a main component of vascular plant cell walls and possesses a complex and irregular structure
Assays with ferrocytochrome c as a substrate showed that AtPrx2, AtPrx25, and AtPrx71 possessed the ability to oxidize large molecules. This characteristic may originate in a protein radical. These results suggest that the plant peroxidases responsible for lignin polymerization are able to directly oxidize all lignin precursors
Monolignols are supplied to the cell wall and polymerized to fill, together with hemicellulose, the spaces between cellulose microfibrils; this polymerization proceeds through oxidative coupling catalyzed by plant peroxidases [1]

Summary

Introduction

Lignin is a main component of vascular plant cell walls and possesses a complex and irregular structure. There is limited information available regarding the role of individual isoforms Their contribution to lignification have been evaluated in several studies that have demonstrated that the up- or down-regulation of a target peroxidase gene is an effective strategy. This research group has focused on seven Arabidopsis plant peroxidases selected using amino acid similarities to CWPOC as the probe and found that AtPrx or AtPrx deficiency led both decreased total lignin content and altered lignin structure, including cell wall thinning in the stem. AtPrx deficiency led an altered stem lignin structure, the lignin content is not decreased [11] These results provided in vivo evidence that AtPrx-2, 25, and 71 are involved in Arabidopsis stem lignification

Methods

Results

Conclusion