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Identification and biochemical characterization of four wood-associated glucuronoxylan methyltransferases in Populus.

Yuan Y, Teng Q, Zhong R, Ye ZH - PLoS ONE (2014)

Bottom Line: The PtrGXM genes were found to be highly expressed in wood-forming cells and their encoded proteins were shown to be localized in the Golgi.Kinetic analysis showed that PtrGXMs exhibited differential affinities toward the GlcA-substituted xylooligomer acceptor with PtrGXM3 and PtrGXM4 having 10 times higher K m values than PtrGXM1 and PtrGXM2.Together, these findings indicate that PtrGXMs are methyltransferases mediating GlcA methylation in Populus xylan during wood formation.

View Article: PubMed Central - PubMed

Affiliation: Department of Plant Biology, University of Georgia, Athens, Georgia, United States of America.

ABSTRACT
Wood is one of the promising bioenergy feedstocks for lignocellulosic biofuel production. Understanding how wood components are synthesized will help us design strategies for better utilization of wood for biofuel production. One of the major wood components is xylan, in which about 10% of xylosyl residues are substituted with glucuronic acid (GlcA) side chains. All the GlcA side chains of xylan in wood of Populus trichocarpa are methylated, which is different from Arabidopsis xylan in which about 60% of GlcA side chains are methylated. Genes responsible for methylation of GlcA side chains in Populus xylan have not been identified. Here, we report genetic and biochemical analyses of four DUF579 domain-containing proteins, PtrGXM1, PtrGXM2, PtrGXM3 and PtrGXM4, from Populus trichocarpa and their roles in GlcA methylation in xylan. The PtrGXM genes were found to be highly expressed in wood-forming cells and their encoded proteins were shown to be localized in the Golgi. When overexpressed in the Arabidopsis gxm1/2/3 triple mutant, PtrGXMs were able to partially complement the mutant phenotypes including defects in glucuronoxylan methyltransferase activity and GlcA methylation in xylan, indicating that PtrGXMs most likely function as glucuronoxylan methyltransferases. Direct evidence was provided by enzymatic analysis of recombinant PtrGXM proteins showing that they possessed a methyltransferase activity capable of transferring the methyl group onto GlcA-substituted xylooligomers. Kinetic analysis showed that PtrGXMs exhibited differential affinities toward the GlcA-substituted xylooligomer acceptor with PtrGXM3 and PtrGXM4 having 10 times higher K m values than PtrGXM1 and PtrGXM2. Together, these findings indicate that PtrGXMs are methyltransferases mediating GlcA methylation in Populus xylan during wood formation.

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MALDI-TOF mass spectra of xylooligosaccharides generated by xylanase digestion of xylan from the gxm1/2/3 mutant overexpressing PtrGXMs.The ions at m/z 745 and 759 are attributed to xylotetrasaccharides bearing a GlcA residue [(GlcA)Xyl4] and a methylated GlcA residue [(MeGlcA)Xyl4], respectively. The ion at m/z 761 corresponds to the xylan reducing end pentasaccharide, β-d-Xyl-(1→4)-β-d-Xyl-(1→3)-α-l-Rha-(1→2)-α-d-GalA-(1→4)-d-Xyl (X-X-R-GA-X). Note the partial restoration of the ion signal at m/z 759 corresponding to (MeGlcA)Xyl4 (red arrows) in the gxm1/2/3 mutant overexpressing PtrGXMs.
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pone-0087370-g006: MALDI-TOF mass spectra of xylooligosaccharides generated by xylanase digestion of xylan from the gxm1/2/3 mutant overexpressing PtrGXMs.The ions at m/z 745 and 759 are attributed to xylotetrasaccharides bearing a GlcA residue [(GlcA)Xyl4] and a methylated GlcA residue [(MeGlcA)Xyl4], respectively. The ion at m/z 761 corresponds to the xylan reducing end pentasaccharide, β-d-Xyl-(1→4)-β-d-Xyl-(1→3)-α-l-Rha-(1→2)-α-d-GalA-(1→4)-d-Xyl (X-X-R-GA-X). Note the partial restoration of the ion signal at m/z 759 corresponding to (MeGlcA)Xyl4 (red arrows) in the gxm1/2/3 mutant overexpressing PtrGXMs.

Mentions: To find out whether expression of PtrGXMs restored the GlcA methylation of xylan in the gxm1/2/3 mutant, we isolated xylan from the pooled stems of the 8 transgenic lines as shown in Fig. 5 for each expression contruct of PtrGXMs. The isolated xylan was digested with xylanase to generate xylooligosaccharides, which were subsequently subjected to MALDI-TOF-MS (Fig. 6). Xylooligosaccharides from wild-type xylan exhibited a prominent ion peak at m/z 759, which corresponds to MeGlcA-substituted Xyl4, and a less prominent ion peak at m/z 745, which corresponds to GlcA-substituted Xyl4. Simultaneous mutations of the Arabidopsis GXM1, GXM2 and GXM3 genes in the gxm1/2/3 triple mutant resulted in a complete loss of the ion peak (m/z 759) attributed to MeGlcA-substituted Xyl4. As a result, the GlcA-substituted Xyl4 peak at m/z 745 was significantly elevated in the gxm1/2/3 mutant compared with that in the wild type. Expression of PtrGXMs in gxm1/2/3 led to a partial restoration of the ion peak at m/z 759 corresponding to MeGlcA-substituted Xyl4 (Fig. 6). The partial restoration of the GlcA methylation was further verified by 1H nuclear magnetic resonance (NMR) spectroscopy (Fig. 7). The wild-type xylan displayed resonances characteristic of H1 of GlcA and MeGlcA side chains at 5.31 and 5.29 ppm, respectively, and H5 of GlcA and MeGlcA side chains at 4.42 and 4.39 ppm, respectively. It also exhibited resonances at 4.63 and 4.46 ppm that are attributed to H1 of branched and unbranched backbone xylosyl residues, respectively. Xylan from the gxm1/2/3 mutant lacked all the resonances attributed to MeGlcA side chains, including H1 of MeGlcA, H5 of MeGlcA, and H1 of MeGlcA-branched β-Xyl. Analysis of xylan from gxm1/2/3 lines expressing PtrGXMs revealed a partial restoration of all the resonances attributed to MeGlcA side chains (Fig. 7). Integration analysis of the GlcA and MeGlcA signal peaks revealed that although the gxm1/2/3 mutant completely lacks GlcA methylation in xylan, 8% to 21% of the GlcA side chains in xylan were methylated in gxm1/2/3 expressing PtrGXMs (Table 1). The resonance intensities for the xylan reducing end tetrasaccharide sequence, β-d-Xyl-(1→3)-α-l-Rha-(1→2)-α-d-GalA-(1→4)-d-Xyl (H1 of 3-linked β-D-Xyl, H1 of α-L-Rha, H1 of α-D-GalA, H2 of α-L-Rha, and H4 of α-D-GalA), were unaltered in gxm1/2/3 expressing PtrGXMs compared with the gxm1/2/3 mutant and the wild type. Together, these results provide genetic evidence demonstrating that expression of each of the four PtrGXM genes in the gxm1/2/3 mutant is able to partially restore the glucuronoxylan methyltransferase activity and the GlcA methylation in xylan, indicating that PtrGXMs are functional orthologs of the Arabidopsis GXMs catalyzing the methylation of GlcA side chains in xylan.


Identification and biochemical characterization of four wood-associated glucuronoxylan methyltransferases in Populus.

Yuan Y, Teng Q, Zhong R, Ye ZH - PLoS ONE (2014)

MALDI-TOF mass spectra of xylooligosaccharides generated by xylanase digestion of xylan from the gxm1/2/3 mutant overexpressing PtrGXMs.The ions at m/z 745 and 759 are attributed to xylotetrasaccharides bearing a GlcA residue [(GlcA)Xyl4] and a methylated GlcA residue [(MeGlcA)Xyl4], respectively. The ion at m/z 761 corresponds to the xylan reducing end pentasaccharide, β-d-Xyl-(1→4)-β-d-Xyl-(1→3)-α-l-Rha-(1→2)-α-d-GalA-(1→4)-d-Xyl (X-X-R-GA-X). Note the partial restoration of the ion signal at m/z 759 corresponding to (MeGlcA)Xyl4 (red arrows) in the gxm1/2/3 mutant overexpressing PtrGXMs.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3921138&req=5

pone-0087370-g006: MALDI-TOF mass spectra of xylooligosaccharides generated by xylanase digestion of xylan from the gxm1/2/3 mutant overexpressing PtrGXMs.The ions at m/z 745 and 759 are attributed to xylotetrasaccharides bearing a GlcA residue [(GlcA)Xyl4] and a methylated GlcA residue [(MeGlcA)Xyl4], respectively. The ion at m/z 761 corresponds to the xylan reducing end pentasaccharide, β-d-Xyl-(1→4)-β-d-Xyl-(1→3)-α-l-Rha-(1→2)-α-d-GalA-(1→4)-d-Xyl (X-X-R-GA-X). Note the partial restoration of the ion signal at m/z 759 corresponding to (MeGlcA)Xyl4 (red arrows) in the gxm1/2/3 mutant overexpressing PtrGXMs.
Mentions: To find out whether expression of PtrGXMs restored the GlcA methylation of xylan in the gxm1/2/3 mutant, we isolated xylan from the pooled stems of the 8 transgenic lines as shown in Fig. 5 for each expression contruct of PtrGXMs. The isolated xylan was digested with xylanase to generate xylooligosaccharides, which were subsequently subjected to MALDI-TOF-MS (Fig. 6). Xylooligosaccharides from wild-type xylan exhibited a prominent ion peak at m/z 759, which corresponds to MeGlcA-substituted Xyl4, and a less prominent ion peak at m/z 745, which corresponds to GlcA-substituted Xyl4. Simultaneous mutations of the Arabidopsis GXM1, GXM2 and GXM3 genes in the gxm1/2/3 triple mutant resulted in a complete loss of the ion peak (m/z 759) attributed to MeGlcA-substituted Xyl4. As a result, the GlcA-substituted Xyl4 peak at m/z 745 was significantly elevated in the gxm1/2/3 mutant compared with that in the wild type. Expression of PtrGXMs in gxm1/2/3 led to a partial restoration of the ion peak at m/z 759 corresponding to MeGlcA-substituted Xyl4 (Fig. 6). The partial restoration of the GlcA methylation was further verified by 1H nuclear magnetic resonance (NMR) spectroscopy (Fig. 7). The wild-type xylan displayed resonances characteristic of H1 of GlcA and MeGlcA side chains at 5.31 and 5.29 ppm, respectively, and H5 of GlcA and MeGlcA side chains at 4.42 and 4.39 ppm, respectively. It also exhibited resonances at 4.63 and 4.46 ppm that are attributed to H1 of branched and unbranched backbone xylosyl residues, respectively. Xylan from the gxm1/2/3 mutant lacked all the resonances attributed to MeGlcA side chains, including H1 of MeGlcA, H5 of MeGlcA, and H1 of MeGlcA-branched β-Xyl. Analysis of xylan from gxm1/2/3 lines expressing PtrGXMs revealed a partial restoration of all the resonances attributed to MeGlcA side chains (Fig. 7). Integration analysis of the GlcA and MeGlcA signal peaks revealed that although the gxm1/2/3 mutant completely lacks GlcA methylation in xylan, 8% to 21% of the GlcA side chains in xylan were methylated in gxm1/2/3 expressing PtrGXMs (Table 1). The resonance intensities for the xylan reducing end tetrasaccharide sequence, β-d-Xyl-(1→3)-α-l-Rha-(1→2)-α-d-GalA-(1→4)-d-Xyl (H1 of 3-linked β-D-Xyl, H1 of α-L-Rha, H1 of α-D-GalA, H2 of α-L-Rha, and H4 of α-D-GalA), were unaltered in gxm1/2/3 expressing PtrGXMs compared with the gxm1/2/3 mutant and the wild type. Together, these results provide genetic evidence demonstrating that expression of each of the four PtrGXM genes in the gxm1/2/3 mutant is able to partially restore the glucuronoxylan methyltransferase activity and the GlcA methylation in xylan, indicating that PtrGXMs are functional orthologs of the Arabidopsis GXMs catalyzing the methylation of GlcA side chains in xylan.

Bottom Line: The PtrGXM genes were found to be highly expressed in wood-forming cells and their encoded proteins were shown to be localized in the Golgi.Kinetic analysis showed that PtrGXMs exhibited differential affinities toward the GlcA-substituted xylooligomer acceptor with PtrGXM3 and PtrGXM4 having 10 times higher K m values than PtrGXM1 and PtrGXM2.Together, these findings indicate that PtrGXMs are methyltransferases mediating GlcA methylation in Populus xylan during wood formation.

View Article: PubMed Central - PubMed

Affiliation: Department of Plant Biology, University of Georgia, Athens, Georgia, United States of America.

ABSTRACT
Wood is one of the promising bioenergy feedstocks for lignocellulosic biofuel production. Understanding how wood components are synthesized will help us design strategies for better utilization of wood for biofuel production. One of the major wood components is xylan, in which about 10% of xylosyl residues are substituted with glucuronic acid (GlcA) side chains. All the GlcA side chains of xylan in wood of Populus trichocarpa are methylated, which is different from Arabidopsis xylan in which about 60% of GlcA side chains are methylated. Genes responsible for methylation of GlcA side chains in Populus xylan have not been identified. Here, we report genetic and biochemical analyses of four DUF579 domain-containing proteins, PtrGXM1, PtrGXM2, PtrGXM3 and PtrGXM4, from Populus trichocarpa and their roles in GlcA methylation in xylan. The PtrGXM genes were found to be highly expressed in wood-forming cells and their encoded proteins were shown to be localized in the Golgi. When overexpressed in the Arabidopsis gxm1/2/3 triple mutant, PtrGXMs were able to partially complement the mutant phenotypes including defects in glucuronoxylan methyltransferase activity and GlcA methylation in xylan, indicating that PtrGXMs most likely function as glucuronoxylan methyltransferases. Direct evidence was provided by enzymatic analysis of recombinant PtrGXM proteins showing that they possessed a methyltransferase activity capable of transferring the methyl group onto GlcA-substituted xylooligomers. Kinetic analysis showed that PtrGXMs exhibited differential affinities toward the GlcA-substituted xylooligomer acceptor with PtrGXM3 and PtrGXM4 having 10 times higher K m values than PtrGXM1 and PtrGXM2. Together, these findings indicate that PtrGXMs are methyltransferases mediating GlcA methylation in Populus xylan during wood formation.

Show MeSH
Related in: MedlinePlus