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Monolignol acyltransferase for lignin p-hydroxybenzoylation in Populus

Abstract

Plant lignification exhibits notable plasticity. Lignin in many species, including Populus spp., has long been known to be decorated with p-hydroxybenzoates. However, the molecular basis for such structural modification remains undetermined. Here, we report the identification and characterization of a Populus BAHD family acyltransferase that catalyses monolignol p-hydroxybenzoylation, thus controlling the formation of p-hydroxybenzoylated lignin structures. We reveal that Populus acyltransferase PHBMT1 kinetically preferentially uses p-hydroxybenzoyl-CoA to acylate syringyl lignin monomer sinapyl alcohol in vitro. Consistently, disrupting PHBMT1 in Populus via CRISPR–Cas9 gene editing nearly completely depletes p-hydroxybenzoates of stem lignin; conversely, overexpression of PHBMT1 enhances stem lignin p-hydroxybenzoylation, suggesting PHBMT1 functions as a prime monolignol p-hydroxybenzoyltransferase in planta. Altering lignin p-hydroxybenzoylation substantially changes the lignin solvent dissolution rate, indicative of its structural significance on lignin physiochemical properties. Identification of monolignol p-hydroxybenzoyltransferase offers a valuable tool for tailoring lignin structure and physiochemical properties and for engineering the industrially important platform chemical in woody biomass.

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Fig. 1: UV-HPLC profiles of the PtrPHBMT1(PtACT54)-catalysed reactions.
Fig. 2: Phylogeny of PtrPHBMT1(PtACT54) with function-known alcohol acyltransferases from different species.
Fig. 3: Analysis of wall-bound phenolics from PHBMT1 knockout and overexpression hybrid aspens.
Fig. 4: Determination of p-hydroxybenzoate in lignin fraction and its effect on lignin dissolution in acetyl bromide solution.
Fig. 5: 2D HSQC NMR spectra of lignin fraction from hybrid aspen transgenic lines.
Fig. 6: Aliphatic subregions of 2D HSQC NMR spectra of lignin fractions from hybrid aspen.

Data availability

The data supporting the findings of this study are available within the paper and/or its Supplementary Information. Other publicly available datasets: BAHD family proteins, NCBI (https://www.ncbi.nlm.nih.gov/); the PtrPHBMT1 homologues in Populus, Phytozome V12 (https://phytozome.jgi.doe.gov/pz/portal.html); the gene chip dataset, PlaNet (http://www.gene2function.de); gene co-expression data; Phytozome v.12.1 (https://phytozome.jgi.doe.gov/pz/portal.html#!info?alias=Org_Ptrichocarpa).

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Acknowledgements

This work was initiated with the support of United States Department of Energy (DOE)-USDA joint Plant Feedstock Genomics Program (FWP no. Bo-135, 2006) and the Laboratory Directed Research and Development Program (LDRD-07-047) of Brookhaven National Laboratory (to C.J.L) for poplar BAHD enzyme functional screening. Generation of poplar knockout lines and chemical analyses were partly supported by the Joint BioEnergy Institute, one of the Bioenergy Research Centers of the US DOE, Office of Science, Office of Biological and Environmental Research, through contract DE-AC02-05CH11231 between Lawrence Berkeley National Laboratory and the US DOE. Enzyme characterization, transgenic poplar generation and chemical analysis were also partially supported by the DOE, Office of Science, Office of Basic Energy Sciences, specifically the Physical Biosciences program of the Chemical Sciences, Geosciences and Biosciences Division under contract no. DE-SC0012704 (to C.-J.L.). P.-Y.L. and Y.T. acknowledge research grants from the Japan Society for the Promotion of Science (grant no. JP20H03044) for NMR analysis and a part of the study was conducted using the facilities in the DASH/FBAS of RISH, Kyoto University and the NMR spectrometer at the JURC of ICR, Kyoto University.

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C.-J.L., Y.Z. and X.Y. conceived the research plan and designed the experiments. X.Y. cloned the genes and conducted initial functional screening. Y.Z. conducted enzymatic kinetic analysis, designed CRISPR–Cas9 gene editing strategy and generated and analysed knockout transgenic lines. K.Z. generated overexpression lines. Y.T. and P-Y.L. conducted NMR analysis. C.-J.L., Y.Z., X.Y. and Y.T. analysed and interpreted data. C.-J.L., Y.Z. and X.Y. wrote the manuscript; all the authors edited the paper.

Corresponding author

Correspondence to Chang-Jun Liu.

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The authors declare no competing interests.

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Peer review information Nature Plants thanks Gerald Tuskan, Yihua Zhou and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Preliminary enzymatic activity screening of the recombinant BAHD acyltransferase candidates from P. trichocarpa.

Schema of the postulated monolignol acylation reactions in Populus and preliminary screening of enzymatic activities of the recombinant BAHD acyltransferase candidates from P. trichocarpa in catalysing conjugation of sinapyl alcohol with acetyl-CoA. Only PtACT54 exhibited a detectable activity when the enzymes were incubated with both substrates. The data are presented as means ± S. D. of three independent experiments.

Extended Data Fig. 2 The apparent optimal temperature and pH value of buffer system of PtrPHBMT1(PtACT54)-catalysed reactions.

a, b, The pH preference of PtrPHBMT1(PtACT54) with p-hydroxybenzoyl-CoA (a) or acetyl-CoA (b) and sinapyl alcohol substates. c, d, The optimal temperature for PtrPHBMT1(PtACT54)-catalysed reaction with p-hydroxybenzoyl-CoA (c) or acetyl-CoA (d) and sinapyl alcohol substrates. The data in (a–d) are presented as means ± S. D. of three (b) or four (a, c, d) experimental repeats. In (a, b) the measured activity at pH 5 was set as 100%. In (c and d) the activity at 20 °C was set as 100%.

Extended Data Fig. 3 Kinetic plots of PtrPHBMT1 against thioester donor substrates.

a, A plot of enzymatic activity as a function of p-hydroxybenzoyl-CoA concentration that obeys Michaelis–Menten kinetics. b, A plot of enzymatic activity as a function of benzoyl-CoA concentration that obeys Hill kinetics. c, A plot of enzymatic activity as a function of acetyl-CoA concentration that obeys Hill kinetics. The reactions were conducted at the fixed concentration of sinapyl alcohol with varied concentrations of thioesters in MES buffer (pH 5.5) at 25 °C for 15 min. Each data point represents mean of at least three replicates.

Extended Data Fig. 4 Expression pattern of PtrPHBMT1 in Populus.

a, Relative expression levels of PtrPHBMT1 was determined via RT–qPCR using RNAs from P. trichocarpa leaf, root, stem (> 1 year), shoot apex, and the bark from the young stem (<3 months), the developing xylem scrapped from young stem (<3 months), the debarked young (<3 months) and old wood (>1 year). Data are presented as means ± S. D. of three biological repeats. The relative expression levels were referenced with ubiquitin gene. The expression level in the old wood sample was set as 100%. b, In silico gene expression pattern of PtrPHBMT1. The data were retrieved from PlaNet (http://aranet.sbs.ntu.edu.sg/) using Probset ID PtpAffx.59062.1.A1_at. Data are presented as means ± S. D. of signal values from three individual microarray experiments.

Extended Data Fig. 5 Subcellular localization of PtrPHBMT1.

a, b, Fluorescence distribution of the free GFP(a) and PtrPHBMT1–GFP fusion protein (b) transiently expressed in N. benthamiana leaf cells. c, d, Plasmolysis analysis for GFP (c) and PtrPHBMT1–GFP (d) transiently expressed in N. benthamiana leaf cells. The GFP fluorescence in green and chlorophyll fluorescence in red. Bar = 20 μm. Arrows point out the cell wall region. e, Immunoblots of PHBMT1 in the soluble (S), microsomal (MS), and cell (CW) protein fractions using anti-PHBMT1 antibody. 20 µg proteins were loaded in each lane. M, marker. The experiments in (a–e) were repeated twice independently with similar results.

Extended Data Fig. 6 DNA sequences of PHBMT1 target site and the potential off-target site of its close homologous gene in the obtained CRISPR/Cas9 transgenic lines.

The homozygous, biallelic, and chimeric mutations were determined according to the allelic sequences of the target site compared to Populus PHBMT1 reference sequence. PAM sequence(AGG) is highlighted. The edited events usually happen after -3 position of PAM (g1-8, g1-9, g1-11). The mutations are occurred in the targeted PtxaPHBMT1 gene but not in its homologue Ptxa001G447300.

Extended Data Fig. 7 Analysis of CRISPR/Cas9-mediated PHBMT1 knockout hybrid aspens.

a, Genotyping of the transgenic lines with selection marker NPT II gene. The experiment was repeated twice independently with the same results. Marker,Thermo Scientific GeneRuler 1 kb DNA ladder. b, Immunoblots with anti-PHBMT1 antibody on the crude proteins from the WT, non-edited control and PHBMT1 knockout transgenic lines. The immunoblots against anti-actin antibody served as the loading control. The original blots are presented in Supplemental Fig. 5a and c. This experiment was repeated twice independently with similar results. c, Growth phenotype of 2-month-old hybrid aspens. Bar = 4 cm. d, Growth phenotype of 6-month-old plants. Bar = 30 cm. e, The basal stem (30 cm) of the 6-month-old WT, non-edited control, and PHBMT1 knockout lines. f, Total acetyl bromide soluble lignin content in the stem of 2-month-old plantlets. Data are presented as means ± S. D. of three biological replicates. Asterisk indicates significant difference compared to the WT, * P < 0.05 (Student’s t-test, with two-tailed distribution, two-sample unequal variance). P = 3.76×10−4 (g1-8), 3.03×10−4 (g1-9), and 0.03 (g1-10). CWR, cell wall residues. g, Stem height of 3- and 5-month-old plants. h, Basal stem diameter of 3- and 5-month-old plants. i, Woody biomass yield of 6-month-old hybrid aspens. j, The calculated wood density of the basal stems of 6-month-old plants. k, l, Total acetyl bromide lignin content (k) and monomeric composition (l) of 6-month-old basal stems. CWR, cell wall residues. G, Guaiacyl monomer. S, Syringyl monomer. The data in (g–l) are presented as means ± S. D. of three biological replicates for WT, Ctrl, g1-9, g1-11 and five biological replicates for g1-8. No statistical differences were found between the knockout and the WT and/or non-edited control (Ctrl) plants.

Extended Data Fig. 8 Analysis of PHBMT1 overexpression hybrid aspens.

a, Growth phenotype of 6-month-old PtrPHBMT1 overexpression aspens (OEs). Bar = 30 cm. b, Immunoblots probed with anti-PHBMT1 antibody on the crude proteins from the WT and PHBMT1 OE transgenic aspens. The immunoblots against anti-actin antibody served as the loading control. The original blots are presented in Supplementary Fig. 5b and d. The experiments were repeated twice independently with similar results. c, d, Stem height (c) and basal stem diameter (d) of the 3- and 5-month-old WT and OE lines. The data in (c, d) are presented as means ± S. D. of four (WT and OE3) and seven (OE1) biological repeats. e, Total acetyl bromide soluble lignin content of 6-month-old stems. The data are presented as means ± S. D. of three biological repeats. f, Woody biomass yield of the 6-month-old hybrid aspens. g, The calculated wood density of the basal stems of 6-month-old plants. The data in (f, g) are presented as means ± S. D. of three (WT and OE3) and four (OE1) biological repeats. No statistical differences were found relative to the WT.

Extended Data Fig. 9 Detection of sinapyl p-hydroxybenzoate (SA-pBA) from poplar lignin fraction via DFRC.

a–c, Extract ion chromatography of the DFRC products from the prepared cellulolytic enzyme lignin of the PHBMT1 overexpression line OE1(a), the WT (b) and the PHBMT1 knockout mutant g1-8 (c), The ions were scanned at m/z = 372 for 4-O-acetylsinapyl-p-O-acetylbenzoate. Note the different ion abundance scale in a–c. d, The full mass spectra of the peak at retention time 39 min in a, b which is absent in c. e, Schematic diagram of 4-O-acetylsinapyl-p-O-acetylbenzoate, the DFRC product of SA-pBA, and its characteristic ions.

Extended Data Fig. 10 Quantification of wall-bound acetate in the poplar WT and transgenic lines.

a, Acetate (AA) content released from the cell wall residues (CWR) of the 2-month-old WT, non-edited control (Ctrl) and PHBMT1 knockout lines. The data are presented as means ± S. D. of four biological repeats. b, Acetate content released from the CWRs of 6-month-old WT, non-edited control and PHBMT1 knockout lines. The data are presented as means ± S. D. of three (WT, Ctrl, g1-9, g1-11) or five (g1-8) biological replicates. c, Acetate content released from the CWRs of 6-month-old WT and PHBMT1 overexpression (OE) lines. The data are presented as means ± S. D. of three biological repeats. d, Acetate content released from the prepared cellulolytic enzyme lignin (CEL) of the 6-month-old WT, PHBMT1 knockout (phbmt1, combination of g1-8, g1-9 and g1-11) and overexpression line (OE1). The data are presented as means ± S. D. of three biological replicates. Asterisk indicates significant difference compared to the WT with P = 0.012 (Student’s t-test, with two-tailed distribution, two-sample unequal variance).

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Zhao, Y., Yu, X., Lam, PY. et al. Monolignol acyltransferase for lignin p-hydroxybenzoylation in Populus. Nat. Plants 7, 1288–1300 (2021). https://doi.org/10.1038/s41477-021-00975-1

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