Downregulation of a UDP-Arabinomutase Gene in Switchgrass (Panicum virgatum L.) Results in Increased Cell Wall Lignin While Reducing Arabinose-Glycans.

Jonathan D Willis,Maor Bar-Peled,James A Smith,Michael K Udvardi,Mark F Davis,David G J Mann,Jason Backe,C N Stewart,Robert W Sykes,Ji-Yi Zhang,Geoffrey B Turner,Charleson R Poovaiah,Maria J Peña,Mitra Mazarei,Holly L Baxter,Stephen R Decker

doi:10.3389/fpls.2016.01580

Abstract

Background: Switchgrass (Panicum virgatum L.) is a C4 perennial prairie grass and a dedicated feedstock for lignocellulosic biofuels. Saccharification and biofuel yields are inhibited by the plant cell wall’s natural recalcitrance against enzymatic degradation. Plant hemicellulose polysaccharides such as arabinoxylans structurally support and cross-link other cell wall polymers. Grasses predominately have Type II cell walls that are abundant in arabinoxylan, which comprise nearly 25% of aboveground biomass. A primary component of arabinoxylan synthesis is uridine diphosphate (UDP) linked to arabinofuranose (Araf). A family of UDP-arabinopyranose mutase (UAM)/reversible glycosylated polypeptides catalyze the interconversion between UDP-arabinopyranose (UDP-Arap) and UDP-Araf.Results: The expression of a switchgrass arabinoxylan biosynthesis pathway gene, PvUAM1, was decreased via RNAi to investigate its role in cell wall recalcitrance in the feedstock. PvUAM1 encodes a switchgrass homolog of UDP-arabinose mutase, which converts UDP-Arap to UDP-Araf. Southern blot analysis revealed each transgenic line contained between one to at least seven T-DNA insertions, resulting in some cases, a 95% reduction of native PvUAM1 transcript in stem internodes. Transgenic plants had increased pigmentation in vascular tissues at nodes, but were otherwise similar in morphology to the non-transgenic control. Cell wall-associated arabinose was decreased in leaves and stems by over 50%, but there was an increase in cellulose. In addition, there was a commensurate change in arabinose side chain extension. Cell wall lignin composition was altered with a concurrent increase in lignin content and transcript abundance of lignin biosynthetic genes in mature tillers. Enzymatic saccharification efficiency was unchanged in the transgenic plants relative to the control.Conclusion: Plants with attenuated PvUAM1 transcript had increased cellulose and lignin in cell walls. A decrease in cell wall-associated arabinose was expected, which was likely caused by fewer Araf residues in the arabinoxylan. The decrease in arabinoxylan may cause a compensation response to maintain cell wall integrity by increasing cellulose and lignin biosynthesis. In cases in which increased lignin is desired, e.g., feedstocks for carbon fiber production, downregulated UAM1 coupled with altered expression of other arabinoxylan biosynthesis genes might result in even higher production of lignin in biomass.

Highlights

Switchgrass (Panicum virgatum) is a perennial grass species that is considered to be a lignocellulosic bioenergy feedstock with great potential, owing to its wide adaptations to various geographies and temperate climates
The target sequence was amplified by PCR and was cloned into the pCR8 entry vector and Gateway R sub-cloned into the pANIC-8A plant expression vector (Mann et al, 2012b) to yield the pANIC-8A-PvUAM1 construct (Supplementary Figure S2A)
We have identified UDP-arabinopyranose mutase (UAM) in switchgrass and the downregulated PvUAM1 switchgrass plants have altered cell wall sugar content and side chains

Summary

Introduction

Switchgrass (Panicum virgatum) is a perennial grass species that is considered to be a lignocellulosic bioenergy feedstock with great potential, owing to its wide adaptations to various geographies and temperate climates. Hemicelluloses are comprised of non-cellulose cell wall polysaccharides, and share a sugar backbone composed of 1,4-linked β-D-glycoses and include xyloglucan mixed-linkage glucan, xylan, and glucomannan. The Araf residues are attached to the Xylp residues in the backbone predominately at O-3 but occasionally at O-2. The aromatic residues (feruloyl and p-coumaroyl) can be ester-linked to O-5 of terminal or substituted arabinose residues of xylan, whereas the acetate can be attached at O-2, O-3 or both to xylose in the backbone. Saccharification and biofuel yields are inhibited by the plant cell wall’s natural recalcitrance against enzymatic degradation. Plant hemicellulose polysaccharides such as arabinoxylans structurally support and cross-link other cell wall polymers. A family of UDP-arabinopyranose mutase (UAM)/reversible glycosylated polypeptides catalyze the interconversion between UDP-arabinopyranose (UDP-Arap) and UDP-Araf

Methods

Results

Conclusion