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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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Phylogenetic and expression analyses of four Populus GXM genes.(A) Phylogenetic relationship of DUF579-containing proteins from Populus and Arabidopsis. The amino acid sequences of DUF579-containing proteins from Populus and Arabidopsis were aligned using ClustalW and their phylogenetic relationship was analyzed using the neighbor-joining method in MEGA5.2 [40]. Bootstrap values resulted from 1,000 replicates are shown at the nodes. (B) Quantitative PCR analysis of the expression of PtrGXM genes in developing leaves, petioles, stems without secondary growth (stem I), and stems with secondary growth (stem II) of Populus. The expression level of each gene in leaves was taken as 1. (C) Quantitative PCR analysis of the induction of expression of PtrGXM genes in the leaves of transgenic Populus plants overexpressing PtrWND2B. The control is transgenic Populus plants transformed with an empty vector. Error bars in (B) and (C) denote the se of three biological replicates.
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pone-0087370-g002: Phylogenetic and expression analyses of four Populus GXM genes.(A) Phylogenetic relationship of DUF579-containing proteins from Populus and Arabidopsis. The amino acid sequences of DUF579-containing proteins from Populus and Arabidopsis were aligned using ClustalW and their phylogenetic relationship was analyzed using the neighbor-joining method in MEGA5.2 [40]. Bootstrap values resulted from 1,000 replicates are shown at the nodes. (B) Quantitative PCR analysis of the expression of PtrGXM genes in developing leaves, petioles, stems without secondary growth (stem I), and stems with secondary growth (stem II) of Populus. The expression level of each gene in leaves was taken as 1. (C) Quantitative PCR analysis of the induction of expression of PtrGXM genes in the leaves of transgenic Populus plants overexpressing PtrWND2B. The control is transgenic Populus plants transformed with an empty vector. Error bars in (B) and (C) denote the se of three biological replicates.

Mentions: To search for genes that are responsible for the above observed glucuronoxylan methyltransferase activity in Populus stems, we first analyzed the expression patterns of Populus DUF579 genes that are close homologs of Arabidopsis GXM genes. There are 11 DUF579 genes (Fig. 2A) in the Populus genome, of which four are closely grouped together with the Arabidopsis GXM1, GXM2 and GXM3 genes known to encode glucuronoxylan methyltransferases. These four Populus DUF579 genes (namely PtrGXM1/2/3/4) apparently fall into two pairs (PtrGXM1/PtrGXM2 and PtrGXM3/PtrGXM4), each of which was likely originated from genome duplication [28]. All four PtrGXM genes were found to be highly expressed in Populus stems undergoing secondary growth (Fig. 2B). In addition, the expression of PtrGXM1, PtrGXM2 and PtrGXM3 was shown to be significantly upregulated (Fig. 2C) in transgenic Populus plants overexpressing PtrWND2B, which is a transcriptional master switch activating secondary wall biosynthesis during wood formation [26], [27]. In situ hybridization further demonstrated that the PtrGXM genes were predominantly expressed in cells undergoing secondary wall thickening, including developing secondary xylem and phloem fibers (Fig. 3).


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

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

Phylogenetic and expression analyses of four Populus GXM genes.(A) Phylogenetic relationship of DUF579-containing proteins from Populus and Arabidopsis. The amino acid sequences of DUF579-containing proteins from Populus and Arabidopsis were aligned using ClustalW and their phylogenetic relationship was analyzed using the neighbor-joining method in MEGA5.2 [40]. Bootstrap values resulted from 1,000 replicates are shown at the nodes. (B) Quantitative PCR analysis of the expression of PtrGXM genes in developing leaves, petioles, stems without secondary growth (stem I), and stems with secondary growth (stem II) of Populus. The expression level of each gene in leaves was taken as 1. (C) Quantitative PCR analysis of the induction of expression of PtrGXM genes in the leaves of transgenic Populus plants overexpressing PtrWND2B. The control is transgenic Populus plants transformed with an empty vector. Error bars in (B) and (C) denote the se of three biological replicates.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0087370-g002: Phylogenetic and expression analyses of four Populus GXM genes.(A) Phylogenetic relationship of DUF579-containing proteins from Populus and Arabidopsis. The amino acid sequences of DUF579-containing proteins from Populus and Arabidopsis were aligned using ClustalW and their phylogenetic relationship was analyzed using the neighbor-joining method in MEGA5.2 [40]. Bootstrap values resulted from 1,000 replicates are shown at the nodes. (B) Quantitative PCR analysis of the expression of PtrGXM genes in developing leaves, petioles, stems without secondary growth (stem I), and stems with secondary growth (stem II) of Populus. The expression level of each gene in leaves was taken as 1. (C) Quantitative PCR analysis of the induction of expression of PtrGXM genes in the leaves of transgenic Populus plants overexpressing PtrWND2B. The control is transgenic Populus plants transformed with an empty vector. Error bars in (B) and (C) denote the se of three biological replicates.
Mentions: To search for genes that are responsible for the above observed glucuronoxylan methyltransferase activity in Populus stems, we first analyzed the expression patterns of Populus DUF579 genes that are close homologs of Arabidopsis GXM genes. There are 11 DUF579 genes (Fig. 2A) in the Populus genome, of which four are closely grouped together with the Arabidopsis GXM1, GXM2 and GXM3 genes known to encode glucuronoxylan methyltransferases. These four Populus DUF579 genes (namely PtrGXM1/2/3/4) apparently fall into two pairs (PtrGXM1/PtrGXM2 and PtrGXM3/PtrGXM4), each of which was likely originated from genome duplication [28]. All four PtrGXM genes were found to be highly expressed in Populus stems undergoing secondary growth (Fig. 2B). In addition, the expression of PtrGXM1, PtrGXM2 and PtrGXM3 was shown to be significantly upregulated (Fig. 2C) in transgenic Populus plants overexpressing PtrWND2B, which is a transcriptional master switch activating secondary wall biosynthesis during wood formation [26], [27]. In situ hybridization further demonstrated that the PtrGXM genes were predominantly expressed in cells undergoing secondary wall thickening, including developing secondary xylem and phloem fibers (Fig. 3).

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