Limits...
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.

Show MeSH

Related in: MedlinePlus

Subcellular localization of PtrGXM1, PtrGXM2, PtrGXM3, and PtrGXM4.PtrGXM proteins tagged with yellow fluorescent protein (YFP) were expressed in Arabidopsis protoplasts, and the fluorescence signals were detected with a laser confocal microscope. (A) PtrGXMs are membrane proteins with one transmembrane helix near the N-terminus as predicted by the TMHMM2.0 program. Inside, the cytoplasmic side of the membrane; outside, the noncytoplasmic side of the membrane. (B) and (C) An Arabidopsis protoplast (B; differential interference contrast image) expressing YFP alone showing the fluorescence signals throughout the cytoplasm (C). (D) to (G) An Arabidopsis protoplast (D) co-expressing PtrGXM1-YFP (E) and the Golgi-localized FRA8-CFP (F). (H) to (K) An Arabidopsis protoplast (H) co-expressing PtrGXM2-YFP (I) and FRA8-CFP (J). (L) to (O) An Arabidopsis protoplast (L) co-expressing PtrGXM3-YFP (M) and FRA8-CFP (N). (P) to (S) An Arabidopsis protoplast (P) co-expressing PtrGXM4-YFP (Q) and FRA8-CFP (R). Note the superimposition of the fluorescence signals of PtrGXM1-YFP (G), PtrGXM2-YFP (K), PtrGXM3-YFP (O), and PtrGXM4 (S) with the Golgi marker FRA8-CFP.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3921138&req=5

pone-0087370-g004: Subcellular localization of PtrGXM1, PtrGXM2, PtrGXM3, and PtrGXM4.PtrGXM proteins tagged with yellow fluorescent protein (YFP) were expressed in Arabidopsis protoplasts, and the fluorescence signals were detected with a laser confocal microscope. (A) PtrGXMs are membrane proteins with one transmembrane helix near the N-terminus as predicted by the TMHMM2.0 program. Inside, the cytoplasmic side of the membrane; outside, the noncytoplasmic side of the membrane. (B) and (C) An Arabidopsis protoplast (B; differential interference contrast image) expressing YFP alone showing the fluorescence signals throughout the cytoplasm (C). (D) to (G) An Arabidopsis protoplast (D) co-expressing PtrGXM1-YFP (E) and the Golgi-localized FRA8-CFP (F). (H) to (K) An Arabidopsis protoplast (H) co-expressing PtrGXM2-YFP (I) and FRA8-CFP (J). (L) to (O) An Arabidopsis protoplast (L) co-expressing PtrGXM3-YFP (M) and FRA8-CFP (N). (P) to (S) An Arabidopsis protoplast (P) co-expressing PtrGXM4-YFP (Q) and FRA8-CFP (R). Note the superimposition of the fluorescence signals of PtrGXM1-YFP (G), PtrGXM2-YFP (K), PtrGXM3-YFP (O), and PtrGXM4 (S) with the Golgi marker FRA8-CFP.

Mentions: To further substantiate their involvement in xylan biosynthesis, we exmined whether PtrGXM proteins are targeted to the Golgi, in which xylan biosynthesis occurs. PtrGXMs are typical type II membrane proteins containing one transmembrane helix as predicted by the TMHMM2.0 program for prediction of transmembrane helices in proteins (http://www.cbs.dtu.dk/service/TMHMM-2.0) (Fig. 4A). To examine the subcellular localization of PtrGXMs, we coexpressed in Arabidopsis protoplasts yellow fluorescence protein-tagged PtrGXMs together with cyan fluorescence protein-tagged FRA8, a family GT47 glycosyltransferase known to be located in the Golgi [29]. While the control protoplasts expressing YFP alone showed fluorescent signals throughout the cytoplasm (Fig. 4B, C), the protoplasts expressing PtrGXM-YFP exhibited punctate signals, which were colocalized with the FRA8-CFP signals (Fig. 4D–S). These results demonstrate that PtrGXMs are localized in the Golgi, where xylan biosynthesis occurs.


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

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

Subcellular localization of PtrGXM1, PtrGXM2, PtrGXM3, and PtrGXM4.PtrGXM proteins tagged with yellow fluorescent protein (YFP) were expressed in Arabidopsis protoplasts, and the fluorescence signals were detected with a laser confocal microscope. (A) PtrGXMs are membrane proteins with one transmembrane helix near the N-terminus as predicted by the TMHMM2.0 program. Inside, the cytoplasmic side of the membrane; outside, the noncytoplasmic side of the membrane. (B) and (C) An Arabidopsis protoplast (B; differential interference contrast image) expressing YFP alone showing the fluorescence signals throughout the cytoplasm (C). (D) to (G) An Arabidopsis protoplast (D) co-expressing PtrGXM1-YFP (E) and the Golgi-localized FRA8-CFP (F). (H) to (K) An Arabidopsis protoplast (H) co-expressing PtrGXM2-YFP (I) and FRA8-CFP (J). (L) to (O) An Arabidopsis protoplast (L) co-expressing PtrGXM3-YFP (M) and FRA8-CFP (N). (P) to (S) An Arabidopsis protoplast (P) co-expressing PtrGXM4-YFP (Q) and FRA8-CFP (R). Note the superimposition of the fluorescence signals of PtrGXM1-YFP (G), PtrGXM2-YFP (K), PtrGXM3-YFP (O), and PtrGXM4 (S) with the Golgi marker FRA8-CFP.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0087370-g004: Subcellular localization of PtrGXM1, PtrGXM2, PtrGXM3, and PtrGXM4.PtrGXM proteins tagged with yellow fluorescent protein (YFP) were expressed in Arabidopsis protoplasts, and the fluorescence signals were detected with a laser confocal microscope. (A) PtrGXMs are membrane proteins with one transmembrane helix near the N-terminus as predicted by the TMHMM2.0 program. Inside, the cytoplasmic side of the membrane; outside, the noncytoplasmic side of the membrane. (B) and (C) An Arabidopsis protoplast (B; differential interference contrast image) expressing YFP alone showing the fluorescence signals throughout the cytoplasm (C). (D) to (G) An Arabidopsis protoplast (D) co-expressing PtrGXM1-YFP (E) and the Golgi-localized FRA8-CFP (F). (H) to (K) An Arabidopsis protoplast (H) co-expressing PtrGXM2-YFP (I) and FRA8-CFP (J). (L) to (O) An Arabidopsis protoplast (L) co-expressing PtrGXM3-YFP (M) and FRA8-CFP (N). (P) to (S) An Arabidopsis protoplast (P) co-expressing PtrGXM4-YFP (Q) and FRA8-CFP (R). Note the superimposition of the fluorescence signals of PtrGXM1-YFP (G), PtrGXM2-YFP (K), PtrGXM3-YFP (O), and PtrGXM4 (S) with the Golgi marker FRA8-CFP.
Mentions: To further substantiate their involvement in xylan biosynthesis, we exmined whether PtrGXM proteins are targeted to the Golgi, in which xylan biosynthesis occurs. PtrGXMs are typical type II membrane proteins containing one transmembrane helix as predicted by the TMHMM2.0 program for prediction of transmembrane helices in proteins (http://www.cbs.dtu.dk/service/TMHMM-2.0) (Fig. 4A). To examine the subcellular localization of PtrGXMs, we coexpressed in Arabidopsis protoplasts yellow fluorescence protein-tagged PtrGXMs together with cyan fluorescence protein-tagged FRA8, a family GT47 glycosyltransferase known to be located in the Golgi [29]. While the control protoplasts expressing YFP alone showed fluorescent signals throughout the cytoplasm (Fig. 4B, C), the protoplasts expressing PtrGXM-YFP exhibited punctate signals, which were colocalized with the FRA8-CFP signals (Fig. 4D–S). These results demonstrate that PtrGXMs are localized in the Golgi, where xylan biosynthesis occurs.

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