Loss of Arabidopsis GAUT12/IRX8 causes anther indehiscence and leads to reduced G lignin associated with altered matrix polysaccharide deposition.

Stefan Eberhard,Ron Orlando,Debra Mohnen,Tipton Sholes,Grace E Rothstein,Michael G Hahn,John Glushka,Wolfgang Lukowitz,Utku Avci,Li Tan,Sivakumar Pattathil,Zhangying Hao,Xiang Zhu

doi:10.3389/fpls.2014.00357

Abstract

GAlactUronosylTransferase12 (GAUT12)/IRregular Xylem8 (IRX8) is a putative glycosyltransferase involved in Arabidopsis secondary cell wall biosynthesis. Previous work showed that Arabidopsis irregular xylem8 (irx8) mutants have collapsed xylem due to a reduction in xylan and a lesser reduction in a subfraction of homogalacturonan (HG). We now show that male sterility in the irx8 mutant is due to indehiscent anthers caused by reduced deposition of xylan and lignin in the endothecium cell layer. The reduced lignin content was demonstrated by histochemical lignin staining and pyrolysis Molecular Beam Mass Spectrometry (pyMBMS) and is associated with reduced lignin biosynthesis in irx8 stems. Examination of sequential chemical extracts of stem walls using 2D 13C-1H Heteronuclear Single-Quantum Correlation (HSQC) NMR spectroscopy and antibody-based glycome profiling revealed a reduction in G lignin in the 1 M KOH extract and a concomitant loss of xylan, arabinogalactan and pectin epitopes in the ammonium oxalate, sodium carbonate, and 1 M KOH extracts from the irx8 walls compared with wild-type walls. Immunolabeling of stem sections using the monoclonal antibody CCRC-M138 reactive against an unsubstituted xylopentaose epitope revealed a bi-lamellate pattern in wild-type fiber cells and a collapsed bi-layer in irx8 cells, suggesting that at least in fiber cells, GAUT12 participates in the synthesis of a specific layer or type of xylan or helps to provide an architecture framework required for the native xylan deposition pattern. The results support the hypothesis that GAUT12 functions in the synthesis of a structure required for xylan and lignin deposition during secondary cell wall formation.

Highlights

The plant extracellular matrix consists of a variety of complex carbohydrate polymers with distinct chemical and physical properties
Semi-quantitative RT-PCR analyses revealed that irx8-2 plants contain trace amounts of full-length GAUT12 transcript, whereas irx8-5 plants contain transcripts that are truncated at the insertion site (Supplemental Figure S1L)
GAUT12 IS REQUIRED FOR ANTHER DEHISCENCE Persson et al (2007) have described the irx8 mutants as semisterile having shorter anther filaments, less pollen than the wild type, and no seeds (Persson et al, 2007)

Summary

Introduction

The plant extracellular matrix (i.e., cell wall) consists of a variety of complex carbohydrate polymers with distinct chemical and physical properties. The Arabidopsis mutant irx (Zhong et al, 2005) is defective in both xylan and cellulose deposition, whereas qua (Bouton et al, 2002; Leboeuf et al, 2005; Orfila et al, 2005), parvus-3/gatl (Lao et al, 2003; Shao et al, 2004; Brown et al, 2007; Lee et al, 2007b; Kong et al., 2009), and irx8/gaut mutants (Peña et al, 2007; Persson et al, 2007) are affected in pectin and xylan biosynthesis These complex effects make it difficult to infer primary gene function on the basis of mutant phenotypes alone. Transient expression of YFP-tagged GAUT12 protein showed that it co-localizes with www.frontiersin.org

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