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

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.

Gene regulatory networks for lignin biosynthesis in switchgrass (Panicum virgatum)

“…Switchgrass whole tillers were ground in liquid nitrogen, and washed in organic solvent to prepare the alcohol insoluble residue (AIR) as described previously (Willis et al, 2016). Cell walls were obtained after removing starch from the AIR using Novazyme specific enzymes.…”

Development and use of a switchgrass (Panicum virgatum L.) transformation pipeline by the BioEnergy Science Center to evaluate plants for reduced cell wall recalcitrance

“…[ 42 , 43 ]) and the need to silence expression of multiple family members to achieve a modified cell wall. There are also examples where silencing of a saccharide biosynthesis gene led to altered saccharide or lignin content [ 44 , 45 ]. Thus, it is possible that silencing genes involved in the synthesis of one polymer may be compensated for by those synthesizing a related or alternative wall polymer.…”

Suberin and hemicellulose in sugarcane cell wall architecture and crop digestibility: A biotechnological perspective

“…Suppressed expression of OsUAM1 reduced 6%–44% of the Araf content and 25%–80% of ferulic acid and p ‐coumaric acid amounts in rice (Konishi et al., ). On the same way, PvUAM1 silencing in switchgrass reduced the levels of Araf and increased cellulose and lignin content as a compensation mechanism, maintaining saccharification levels (Willis et al., ). In wheat ( Triticum aestivum ), TaGT43_2 and TaGT47_2 are homologs of A. thaliana IRX9 and IRX10 ( IRREGULAR XYLEM9 and 10 , respectively) and promote biosynthesis of arabinoxylans (Lovegrove et al., ).…”