Fig 1.
Structural analysis of acetyl substitutions in Populus xylan.
Xylan was isolated from Populus stems and determined for the presence of acetyl groups at different positions of Xyl residues using 1H NMR spectroscopy. (A) 1H NMR spectrum of Populus xylan showing the resonance signals corresponding to carbohydrate and acetyl groups (left panel) and the enlarged region of acetyl signal peaks attributed to acetylated Xyl residues (right panel). (B) Diagrams of four different types of acetyl substitutions of a xylosyl residue, including Xyl-3Ac-2GlcA (2-O-GlcA-substituted Xyl residue acetylated at O-3), Xyl-2Ac (Xyl residue monoacetylated at O-2), Xyl-3Ac (Xyl residue monoacetylated at O-3), and Xyl-2,3Ac (Xyl residue diacetylated at O-2 and O-3). (C) Integration analysis of the degree of acetyl substitution in Populus xylan. The degree of acetyl substitution in xylan was determined by integration of resonance signals corresponding to each type of acetylated Xyl residues relative to the signals for carbohydrate in the 1H NMR spectra of Populus xylan (B). The data were the average of three independent experiments.
Fig 2.
Phylogenetic analysis of DUF231 genes in P. trichocarpa.
The protein sequences of Arabidopsis DUF231 genes were used to blast search for DUF231 homologs in the genome of P. trichocarpa. Protein sequences from both P. trichocarpa and Arabidopsis were used to construct the phylogenetic tree with the neighbor-joining algorithm. Each DUF231 gene was labeled with its TBL name together with its gene locus identifier. The 0.1 scale denotes 10% change. The 12 Populus DUF231 genes studied in this report were named PtrXOATs and marked with red diamonds.
Fig 3.
Expression analysis of PtrXOAT genes.
(A) Quantitative PCR analysis of PtrXOAT gene expression in leaves, petioles, and stems with primary growth (Stem-I) and stems with secondary growth (Stem-II). The data were the average of three biological replicates. (B) Induction of expression of PtrXOAT genes by the wood-associated secondary wall NAC master switch PtrWND2B. Total RNAs from the control (transformed with an empty vector) and PtrWND2B overexpressors (PtrWND2B-OE) were examined for expression of PtrXOAT genes by quantitative PCR analysis. The expression level in the control was set to 1. The data were the average of three biological replicates.
Fig 4.
Production and xylan acetyltransferase activity assay of recombinant PtrXOAT proteins.
His-tagged recombinant PtrXOAT proteins without the N-terminal transmembrane sequence were expressed in the secreted form in mammalian HEK293F cells and purified for activity assay. (A) SDS-polyacrylamide gel electrophoresis showing the purified recombinant PtrXOAT proteins. Proteins were detected by staining with Coomassie Blue. (B) Assay of recombinant PtrXOAT proteins for xylan acetyltransferase activities. PtrXOATs were incubated with 14C-labeled acetyl CoA and the Xyl6 acceptor for examination of their ability to transfer the radiolabeled acetyl group from acetyl CoA onto the Xyl6 acceptor. PtrXOATs incubated with 14C-acetyl CoA without Xyl6 were used as controls (No acceptor). (C) Acetyltransferase activities of PtrXOATs toward xylooligomers with different degree of polymerization (DP). The data were the average of three biological replicates.
Fig 5.
1H NMR spectra of PtrXOAT-catalyzed reaction products.
Recombinant PtrXOAT proteins were incubated with acetyl CoA and Xyl6 (left panel) or (GlcA)Xyl4 (right panel), and the reaction products were examined by 1H NMR spectroscopy for acetyl substitutions at different positions of Xyl residues. The control reaction contained acetyl CoA and the acceptor only without recombinant proteins. The resonance peaks attributed to Xyl-2Ac, Xyl-3Ac, Xyl-2,3-Ac, and Xyl-3Ac-2GlcA are marked. The additional resonance peaks seen in the spectra of the reactions with (GlcA)Xyl4 as the acceptor (right panel) were likely due to the fact that acetyl CoA was not removed from the reaction products. The data shown were representatives of three biological replicates.
Fig 6.
Kinetic properties of the acetyltransferase activities of PtrXOATs.
Recombinant PtrXOATs were assayed for their xylan acetyltransferase activities by incubation with 14C-acetyl CoA and various concentrations of Xyl6. Lineweaver-Burk plots were used to calculate Vmax and Km values.
Fig 7.
Complementation of the esk1 mutant by PtrXOATs.
Full-length cDNAs of PtrXOATs driven by the CesA7 promoter were introduced into esk1 mutant plants and first-generation transgenic plants were examined for their growth. Note the restoration of rosette size (top panel) and inflorescence height (bottom panel) in PtrXOAT-complemented esk1 plants.
Fig 8.
1H NMR analysis of acetyl xylooligomers from PtrXOAT-complemented esk1 plants.
DMSO-extracted acetyl xylans were digested with xylanase and the acetylation patterns of released xylooligomers were analyzed using 1H NMR spectroscopy. (A) 1H NMR spectra of acetyl xylooligomers from the wild type, esk1, and PtrXOAT-complemented esk1. Acetyl xylooligomers from ESK1-complemented esk1 were used for comparison. The spectra in the left panel show the resonances attributed to carbohydrate (3.0–5.5 ppm) and acetyl groups (2.0–2.25 ppm). The enlarged spectra in the right panel show the resonances corresponding to acetyl groups in Xyl-2,3Ac, Xyl-3Ac, Xyl-2Ac, and Xyl-3Ac-2GlcA. There was a slight shift in the position of the resonance signal for Xyl-3Ac-2GlcA in some samples. (B) Integration analysis of the degree of acetyl substitution in xylans from the wild type, esk1, and PtrXOAT-complemented esk1 based on the resonance signals for the carbohydrate and the acetyl groups in (A). The relative amount of acetyl groups (% of WT) was determined by the ratio of acetyl groups in the xylans of PtrXOAT-complemented esk1 over that of the wild type (taken as 100).