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. 2016 Oct;14(10):2010-20.
doi: 10.1111/pbi.12560. Epub 2016 Apr 15.

Knockdown of a laccase in Populus deltoides confers altered cell wall chemistry and increased sugar release

Anthony C Bryan  1 , Sara Jawdy  1 , Lee Gunter  1 , Erica Gjersing  2 , Robert Sykes  2 , Maud A W Hinchee  3 , Kimberly A Winkeler  3 , Cassandra M Collins  3 , Nancy Engle  1 , Timothy J Tschaplinski  1 , Xiaohan Yang  1 , Gerald A Tuskan  1 , Wellington Muchero  4 , Jin-Gui Chen  5
Affiliations

Knockdown of a laccase in Populus deltoides confers altered cell wall chemistry and increased sugar release

Anthony C Bryan et al. Plant Biotechnol J. 2016 Oct.

Abstract

Plant laccases are thought to function in the oxidation of monolignols which leads to higher order lignin formation. Only a hand-full of laccases in plants have been functionally evaluated, and as such little is known about the breadth of their impact on cell wall chemistry or structure. Here, we describe a previously uncharacterized laccase from Populus, encoded by locus Potri.008G064000, whose reduced expression resulted in transgenic Populus trees with changes in syringyl/guaiacyl ratios as well as altered sugar release phenotypes. These phenotypes are consistent with plant biomass exhibiting reduced recalcitrance. Interestingly, the transgene effect on recalcitrance is dependent on a mild pretreatment prior to chemical extraction of sugars. Metabolite profiling suggests the transgene modulates phenolics that are associated with the cell wall structure. We propose that this particular laccase has a range of functions related to oxidation of phenolics and conjugation of flavonoids that interact with lignin in the cell wall.

Keywords: Populus; biofuel; cell wall; lignin; recalcitrance; xylose.

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Figures

Figure 1
Figure 1
Phylogenetic analysis of Laccase (LAC) genes from Populus trichocarpa and Arabidopsis thaliana. The six subfamilies, indicated by Roman numerals, were previously described by McCaig et al. (2005) and Arabidopsis LAC genes named accordingly. Populus trichocarpa LAC genes were identified through BLAST from Phytozome using Populus trichocarpa v3.0 release. A box indicates the Populus LAC2 gene described in this analysis. Previously characterized Populus LAC genes are indicated by name. Newly annotated LAC genes in Populus are indicated by +.
Figure 2
Figure 2
Expression of PdLAC2 across Populus deltoides tissue types. Relative fold expression was calculated using ΔΔC t relative to young stem.
Figure 3
Figure 3
Estimated above‐ground biomass of transgenic Populus samples. Above‐ground biomass was estimated using the formula Diameter2 × Height cm (D2H). *Significant compared to the control, P‐value ≤0.01.
Figure 4
Figure 4
Relative gene expression of endogenous PdLAC2 in RNAi transgenic lines. PdLAC2‐1 and PdLAC2‐2 show reduced expression of endogenous PdLAC2 by 50% and 40%, respectively, compared to control plants.
Figure 5
Figure 5
Syringyl/guaiacyl lignin ratio in PdLAC2 RNAi transgenic lines. Both PdLAC2‐1 and PdLAC2‐2 show an increase in S/G ratio compared to control lines. *Significant compared to the control, P‐value < 0.01.
Figure 6
Figure 6
Xylose and glucose release assay of transgenic samples under liquid hot water (LHW) pretreatment and un‐pretreated. Samples were collected with mild LHW pretreatment (a, c, e) and no pretreatment prior to sugar extraction (b, d, f). Rates of xylose release from LHW pretreatment and un‐pretreatment are shown in (a) and (b). Rates of glucose release from LHW pretreatment and un‐pretreatment are shown in (c) and (d). Rates of combined xylose and glucose release are shown in (e) and (f). *Significant compared to the control, P‐value < 0.01.
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