Alleles Causing Resistance to Isoxaben and Flupoxam Highlight the Significance of Transmembrane Domains for CESA Protein Function

Dario T. Bonetta,Zachary Kileeg,Janice L. Strap,Patricia Stronghill,Isaac Shim,Robert Law,Julian G. B. Northey

doi:10.3389/fpls.2018.01152

Abstract

The cellulose synthase (CESA) proteins in Arabidopsis play an essential role in the production of cellulose in the cell walls. Herbicides such as isoxaben and flupoxam specifically target this production process and are prominent cellulose biosynthesis inhibitors (CBIs). Forward genetic screens in Arabidopsis revealed that mutations that can result in varying degrees of resistance to either isoxaben or flupoxam CBI can be attributed to single amino acid substitutions in primary wall CESAs. Missense mutations were almost exclusively present in the predicted transmembrane regions of CESA1, CESA3, and CESA6. Resistance to isoxaben was also conferred by modification to the catalytic residues of CESA3. This resulted in cellulose deficient phenotypes characterized by reduced crystallinity and dwarfism. However, mapping of mutations to the transmembrane regions also lead to growth phenotypes and altered cellulose crystallinity phenotypes. These results provide further genetic evidence supporting the involvement of CESA transmembrane regions in cellulose biosynthesis.

Highlights

Plant cell walls are a complex array of networks that are primarily made up of polysaccharides
Our approach took advantage of two known cellulose biosynthesis inhibitors (CBIs), isoxaben and flupoxam, which were originally developed as pre-emergence herbicides (Hoffman and Vaughn, 1996; Sabba and Vaughn, 1999; Vaughn and Turley, 2001; García-Angulo et al, 2012)
Our analysis indicates that the CESA genes, which encode cellulose synthases, are the primary targets of the two CBIs

Summary

INTRODUCTION

Plant cell walls are a complex array of networks that are primarily made up of polysaccharides. Isoxaben (Heim et al, 1989; Scheible et al, 2001; Desprez et al, 2002; Sethaphong et al, 2013) and quinoxyphen (Harris et al, 2012; Sethaphong et al, 2013) have been used in conjunction with forward genetics in Arabidopsis to select for resistance alleles Related compounds, such as the triazofenamide (Heim et al, 1998; García-Angulo et al, 2012) and its derivative, such as flupoxam (Hoffman and Vaughn, 1996; Sabba and Vaughn, 1999; Vaughn and Turley, 2001), which lead to a similar cellulose-depletion syndrome in plants, have not been tested using fluorescently tagged CESAs. Mutations conferring resistance to isoxaben (ixr) map to the CESA3 (ixr1) or CESA6 (ixr2) gene (Scheible et al, 2001). Hypocotyl lengths were measured using seedlings that had been grown for 5–7 days in the dark on 0.5× MS with or without 10 nM CBI (n ≥ 20) or supplemented with 4.5% sucrose

MATERIALS AND METHODS

RESULTS

Findings

DISCUSSION