Silencing CAFFEOYL SHIKIMATE ESTERASE Affects Lignification and Improves Saccharification in Poplar

Abstract

Caffeoyl shikimate esterase (CSE) was recently shown to play an essential role in lignin biosynthesis in Arabidopsis (Arabidopsis thaliana) and later in Medicago truncatula However, the general function of this enzyme was recently questioned by the apparent lack of CSE activity in lignifying tissues of different plant species. Here, we show that down-regulation of CSE in hybrid poplar (Populus tremula × Populus alba) resulted in up to 25% reduced lignin deposition, increased levels of p-hydroxyphenyl units in the lignin polymer, and a relatively higher cellulose content. The transgenic trees were morphologically indistinguishable from the wild type. Ultra-high-performance liquid chromatography-mass spectrometry-based phenolic profiling revealed a reduced abundance of several oligolignols containing guaiacyl and syringyl units and their corresponding hydroxycinnamaldehyde units, in agreement with the reduced flux toward coniferyl and sinapyl alcohol. These trees accumulated the CSE substrate caffeoyl shikimate along with other compounds belonging to the metabolic classes of benzenoids and hydroxycinnamates. Furthermore, the reduced lignin amount combined with the relative increase in cellulose content in the CSE down-regulated lines resulted in up to 62% more glucose released per plant upon limited saccharification when no pretreatment was applied and by up to 86% and 91% when acid and alkaline pretreatments were used. Our results show that CSE is not only important for the lignification process in poplar but is also a promising target for the development of improved lignocellulosic biomass crops for sugar platform biorefineries.

Figure 1.

Metabolic map of the general phenylpropanoid and monolignol-specific pathways showing the changes in phenolic metabolism upon CSE down-regulation in poplar. Metabolites found to have higher abundance in the hpCSE lines are highlighted in red, while those with decreased abundance are shown in blue. Metabolites belonging to the same class are framed with a box. Solid and dashed arrows represent enzymatic conversions validated by experimental evidence and suggested conversions, respectively (Van Acker et al., 2017). Two successive arrows represent two or more metabolic steps. PAL, PHENYLALANINE AMMONIA LYASE; C4H, CINNAMATE 4-HYDROXYLASE; 4CL, 4-COUMARATE:COENZYME A LIGASE; HCT, p-HYDROXYCINNAMOYL-COENZYME A:QUINATE/SHIKIMATE p-HYDROXYCINNAMOYLTRANSFERASE; C3H, p-COUMARATE 3′-HYDROXYLASE; CSE, CAFFEOYL SHIKIMATE ESTERASE; CCoAOMT, CAFFEOYL-COENZYME A O-METHYLTRANSFERASE; CCR, CINNAMOYL-COENZYME A REDUCTASE; F5H, FERULATE 5-HYDROXYLASE; COMT, CAFFEIC ACID O-METHYLTRANSFERASE; CAD, CINNAMYL ALCOHOL DEHYDROGENASE; HCALDH, HYDROXYCINNAMALDEHYDE DEHYDROGENASE.

Figure 2.

Poplar xylem CSE activity and CSE gene expression. A, CSE activity in crude poplar xylem protein extracts. Total poplar xylem protein was added to reaction buffer containing 100 µm caffeoyl shikimate, and caffeate was measured via UHPLC-MS after 1 and 25 h. Boiled poplar xylem protein was used as a negative control. Error bars indicate se. Differences in caffeic acid abundance were assessed with Student’s t test (**, 0.01 > P > 0.001; n = 4). B, Expression analysis of CSE homologs in different tissues of poplar as determined via RT-quantitative PCR (qPCR). Error bars indicate se (n = 4).

Figure 3.

Residual expression of both CSE genes and growth analyses of poplar trees down-regulated in CSE. A, Residual expression levels of PtxaCSE1 and PtxaCSE2 in the xylem of wild-type (WT) poplar plants and the hpCSE lines determined by RT-qPCR. The expression of each PtxaCSE gene was normalized to that of the wild type. Error bars indicate se. Differences in gene expression were assessed with Student’s t test (*, 0.05 > P > 0.01; **, 0.01 > P > 0.001; and ***, P < 0.001; n = 4). B, Photograph of representative plants grown for 6 months in the greenhouse. C, Fresh and dry weights of stems harvested from wild-type and hpCSE plants at the end of the 6-month growth period. No statistical difference was found among the genotypes. Error bars indicate sd. D, Growth curves of the wild type and the hpCSE lines. Height was monitored every week for a period of 6 months. The average growth in each month was used to plot the graph. Error bars indicate sd. Differences in growth parameters between the wild type and the transgenic lines were assessed with Student’s t test (*, 0.05 > P > 0.01 and **, 0.01 > P > 0.001; wild type, n = 25; hpCSE#1, n = 23; and hpCSE#2, n = 23). The asterisk above the data point represents a statistical difference for line hpCSE#1, whereas asterisks below represent statistical differences for line hpCSE#2.

Figure 4.

Lignin content and composition and cellulose content of stems from hpCSE lines and control plants. A, Total acid-insoluble lignin content determined by the Klason method. Data are expressed as percentage of CWR. B, Cellulose content was determined gravimetrically and is expressed as percentage of CWR. C, Relative levels of releasable H monomers determined by thioacidolysis. D, Relative levels of releasable G and S monomers determined by thioacidolysis. E, S/G ratio determined based on thioacidolysis data. F, Total thioacidolysis yield as determined from the sum of released H, G, and S monomers, expressed in µmol g⁻¹ Klason lignin. For all analyses, data are means ± sd (n = 10), except for thioacidolysis yield, for which se values are shown. Differences in cell wall parameters were assessed with Student’s t test (*, 0.05 > P > 0.01; **, 0.01 > P > 0.001; and ***, P < 0.001). WT, Wild type.

Figure 5.

Structural characterization of lignin by NMR. HSQC spectra are shown for the aromatic regions (A) and the oxygenated aliphatic regions (B) of whole cell walls from stems of the wild type (WT) and the two hpCSE lines, along with a synthetic lignin synthesized biomimetically from p-coumaryl alcohol to help validate the H unit assignments (H-DHP). Integrated values for each monomeric H, G, and S unit and the α-C/H correlation peaks from the major lignin interunit structures A to C are provided. The colors of the substructures shown match those of the corresponding signals in the HSQC spectra (where they are resolved).

Figure 6.

Saccharification efficiency of stem biomass from wild-type (WT) and hpCSE plants. A, C, and E, Cellulose-to-Glc conversion during limited saccharification of stems from the wild type (solid lines) and the hpCSE lines (dashed lines) without (A) and with acidic (C) and alkaline (E) pretreatments. Cellulose-to-Glc conversion was calculated based on the amounts of released Glc and the quantified amount of cellulose. B, D, and F, Relative Glc release per plant after 48 h of saccharification without (B) and with acid (D) and alkaline (F) pretreatments, normalized to the values of the wild type. Error bars indicate se. Significant differences were assessed with Student’s t test (*, 0.05 > P > 0.01; **, 0.01 > P > 0.001; and ***, P < 0.001; n = 10).