The Poplar Glycosyltransferase GT47C is ... - Oxford Academic

Plant Cell Physiol. 47(9): 1229–1240 (2006) doi:10.1093/pcp/pcj093, available online at www.pcp.oxfordjournals.org ß The Author 2006. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: [email protected]

The Poplar Glycosyltransferase GT47C is Functionally Conserved with Arabidopsis Fragile Fiber8 Gong-Ke Zhou 1, Ruiqin Zhong 1, Elizabeth A. Richardson 1, W. Herbert Morrison III 2, C. Joseph Nairn 3, Alicia Wood-Jones 3 and Zheng-Hua Ye 1, * 1

Department of Plant Biology, University of Georgia, Athens, GA 30602, USA Richard B. Russell Agriculture Research Center, US Department of Agriculture, Agriculture Research Service, Athens, GA 30604, USA 3 Daniel B. Warnell School of Forestry and Natural Resources, University of Georgia, Athens, GA 30602, USA 2

Xylan is the major hemicellulose in dicot wood. Unraveling genes involved in the biosynthesis of xylan will be of importance in understanding the process of wood formation. In this report, we investigated the possible role of poplar GT47C, a glycosyltransferase belonging to family GT47, in the biosynthesis of xylan. PoGT47C from the hybrid poplar Populus alba tremula exhibits 84% sequence similarity to Fragile fiber8 (FRA8), which is involved in the biosynthesis of glucuronoxylan in Arabidopsis. Phylogenetic analysis of glycosyltransferase family GT47 in the Populus trichocarpa genome revealed that GT47C is the only close homolog of FRA8. In situ hybridization showed that the PoGT47C gene was expressed in developing primary xylem, secondary xylem and phloem fibers of stems, and in developing secondary xylem of roots. Sequence analysis suggests that PoGT47C is a type II membrane protein, and study of the subcellular localization demonstrated that fluorescent protein-tagged PoGT47C was located in the Golgi. Immunolocalization with a xylan monoclonal antibody LM10 revealed a nearly complete loss of xylan signals in the secondary walls of fibers and vessels in the Arabidopsis fra8 mutant. Expression of PoGT47C in the fra8 mutant restored the secondary wall thickness and xylan content to the wildtype level. Together, these results suggest that PoGT47C is functionally conserved with FRA8 and it is probably involved in xylan synthesis during wood formation. Keywords: Arabidopsis thaliana — Glycosyltransferase — Poplar — Secondary wall synthesis — Xylan. Abbreviations: CaMV, cauliflower mosaic virus; fra8, fragile fiber8; GFP, green fluorescent protein; ECFP, enhanced cyan fluorescent protein; EYFP, enhanced yellow fluorescent protein; GlcA, glucuronic acid; GT, glycosyltransferase; 4-O-Me-GlcA, 4-O-methyl-glucuronic acid; RT–PCR, reverse transcription–PCR. The nucleotide sequences described in Figs. 1 and 2 can be found in GenBank with the following accession numbers: PoGT47C (DQ899955), FRA8 (DQ182567), At5g22940 (BT011629), NpGUT1 (AB080676), AtGUT1 (BT022053), AtGUT2 (AY054180) and MUR3 (AY195743).

Introduction Wood formation is a complex developmental process, including the differentiation of vascular cambial initials into various xylem cell types, cell elongation, secondary wall synthesis and programmed cell death. In dicot wood, fibers and vessels are the two cell types undergoing massive secondary wall thickening, which largely determines wood quality. The secondary walls in dicot wood are mainly composed of cellulose, xylan and lignin. For example, in poplar wood, cellulose, xylan and lignin constitute 43–48, 18–28 and 19–21%, respectively, of total dry weight. In addition to xylan, another hemicellulose, glucomannan, represents a small proportion (5%) of poplar wood (Mellerowicz et al. 2001). The biosynthetic pathway of lignin has been intensively studied, and a number of lignin pathway genes have been down-regulated in poplar to study their essential roles in lignin biosynthesis during wood formation (Boerjan et al. 2003). In contrast, genes involved in secondary wall polysaccharide synthesis during wood formation are less well understood. A number of cellulose synthase genes expressed in wood have been identified based on sequence analysis, and it is thought that similarly to secondary wall synthesis in Arabidopsis, at least three cellulose synthase genes are required for secondary wall synthesis during wood formation (Djerbi et al. 2004, Joshi et al. 2004, Nairn and Haselkorn 2005). Genes involved in the synthesis of xylan in dicot wood, which is the second most abundant wall polysaccharide, have not been investigated. Xylan is composed of b-(1,4)-linked D-xylosyl linear backbones to which single residues of a-D-glucuronic acid (GlcA) and/or 4-O-methyl-a-D-glucuronic acid (4-O-Me-GlcA) are attached at O-2 of one out of every 6–12 xylosyl residues. The xylosyl residues can also be substituted with arabinosyl and acetyl residues. In dicot wood, xylan contains single side chains of 2-linked 4-O-MeGlcA (Ebringerova´ and Heinze 2000). Immunolocalization showed that xylan is evenly distributed in secondary walls

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Corresponding author: E-mail, [email protected]; Fax, þ1-706-542-1805. 1229

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PoGT47C is functionally conserved with FRA8

of wood (Awano et al. 1998). Previous studies have focused on the biochemical characterization of xylosyltransferases and glucuronyltransferases involved in xylan synthesis. Activities of xylosyltransferases and glucuronyltransferases were detected in developing wood of several tree species, including sycamore and poplars (Dalessandro and Northcote 1981), as well as in a number of herbaceous plants (Baydoun et al. 1983, Suzuki et al. 1991, Porchia and Scheller 2000, Kuroyama and Tsumuraya 2001, Gregory et al. 2002). Using the antibodies raised against the partially purified xylan synthase, it was demonstrated that xylan synthase is localized in vesicles on both the cis- and trans-Golgi sides (Gregory et al. 2002). In addition, arabinosyltransferase activities involved in arabinoxylan synthesis were characterized using Golgi membranes from wheat seedlings (Porchia et al. 2002). It has been proposed that cellulose synthase-like genes in the cellulose synthase superfamily might be involved in the synthesis of the xylan backbone (Richmond and Somerville 2000, Dhugga et al. 2004, Liepman et al. 2005), but no genetic or biochemical evidence is available to support this hypothesis. Recent genomic study of wood formation provides an unprecedented opportunity to tackle the long-standing question of how xylan is synthesized in wood. By isolation of developing secondary xylem cells at different developmental stages with microtome sectioning and subsequent microarray analysis of gene expression patterns, Aspeborg et al. (2005) identified 25 glycosyltransferases (GTs) that are highly expressed during secondary wall synthesis in poplar. Among these, four are cellulose synthases and one is a cellulose synthase-like protein, PttGT2a, which is a close homolog of the Arabidopsis AtCSLA9 shown to be involved in mannan synthesis (Liepman et al. 2005). The rest of the poplar xylem-associated GTs belong to different GT families and their roles in wood formation are unknown. Because the major hemicellulose in poplar wood is xylan, it is likely that some of these GTs might be involved in the biosynthesis of xylan. In Arabidopsis, mutation of three GT genes, which belong to the GT families GT47, GT8 and GT43, respectively, have been shown to cause a reduction in xylose content in inflorescence stems and a collapsed vessel phenotype (Brown et al. 2005, Persson et al. 2005, Zhong et al. 2005). Gene expression analysis revealed that the FRA8 (Fragile fiber8) gene is expressed in cells undergoing secondary wall thickening, including extraxylary fibers, primary xylem and secondary xylem (Zhong et al. 2005). This expression pattern is consistent with the fra8 mutant phenotypes showing that secondary walls of both fibers and vessels are drastically reduced in thickness. Chemical analysis of cell walls demonstrated that both xylan and cellulose are dramatically decreased in fra8, and xylan in fra8 is devoid of GlcA residues compared with the

wild-type xylan in which both GlcA and 4-O-Me-GlcA residues are present. In addition, FRA8 was found to be localized in Golgi. Based on these findings, it was concluded that FRA8, a putative GT, is involved in the biosynthesis of glucuronoxylan during secondary wall synthesis (Zhong et al. 2005). One of the poplar wood-associated GT genes, GT47C, shows high sequence similarity to FRA8. To ascertain whether the poplar GT47C is a functional ortholog of Arabidopsis FRA8, we investigated its expression pattern, subcellular localization and ability to complement the fra8 mutant phenotypes functionally. We show that the PoGT47C gene is expressed in primary xylem, secondary xylem and extraxylary fibers, and its encoded protein is localized in the Golgi. We demonstrate that expression of the PoGT47C gene in fra8 restores the stem strength and the thickness of secondary walls of fibers and vessels to the wild-type level. It also leads to restoration of the xylose content and xylan level in the stems. Our results provide strong evidence indicating that PoGT47C performs the same biochemical function as FRA8 and it most probably participates in xylan synthesis during wood formation.

Results The PoGT47C gene is expressed in cells undergoing secondary wall thickening A full-length PoGT47C cDNA was isolated from a cDNA library prepared from stem RNAs of hybrid poplar (Populus alba tremula) using the FRA8 cDNA as a probe. The longest open reading frame in the PoGT47C cDNA is 1,329 bp long, and it encodes a protein of 442 amino acid residues with a predicted molecular mass of 50,704 Da and a predicted pI of 9.39. The deduced amino acid sequence of PoGT47C exhibits 66% identity and 84% similarity to Arabidopsis FRA8 (Fig. 1A). Within the GT signature motif, PoGT47C shares 84% identity and 92% similarity with FRA8. To determine whether PoGT47C has a close homolog in the poplar genome, we analyzed the phylogenetic relationship of poplar GT47 proteins. With the completion of sequencing of the Populus trichocarpa genome, a number of exostosin-like genes belonging to GT family 47 have been identified and their sequences are available from the DOE Joint Genome Institute website (http://genome.jgi-psf.org/ Poptr1/Poptr1.home.html). We retrieved the poplar GT47 protein sequences and analyzed their phylogenetic relationship (Fig. 2). The poplar genome contains a large family of GT47 genes, and the overall subgrouping of the phylogenetic tree is very similar to that in Arabidopsis (Zhong and Ye 2003). It was found that PoGT47C is the


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Fig. 1 Analysis of the PoGT47C protein sequence and its gene expression in various organs. (A) Amino acid sequence alignment of PoGT47C with FRA8. The numbers shown at the left of each sequence are the positions of amino acid residues in the corresponding proteins. Gaps (marked with dashes) were introduced to maximize the sequence alignment. Identical and similar amino acid residues are shaded with black and gray, respectively. The sequence corresponding to the GT signature motif is underlined. (B) RT–PCR analysis of the PoGT47C gene expression in poplar organs. The expression level of an actin gene was used as an internal control. Expanding leaves and petioles were from plants grown on MS medium. Stem I and stem II were from elongating and non-elongating parts of stems, respectively.

only close homolog of FRA8 in poplar and it resides in a subgroup different from the GUT1 subgroup (Fig. 2). Reverse transcription–PCR (RT–PCR) analysis showed that the PoGT47C gene was expressed in leaf blade, petiole and both elongating and non-elongating stems (Fig. 1B). To investigate further the expression pattern of PoGT47C, we used in situ hybridization to examine its expression in stems and roots. In elongating stems without secondary growth, the PoGT47C hybridization signal was predominantly present in primary xylem cells and phloem fibers (Fig. 3A, B), both of which were undergoing secondary wall thickening. In non-elongating stems in which secondary growth was evident, the signal was highly present in developing secondary xylem (Fig. 3D, E), which is consistent with previous microarray data (Aspeborg et al. 2005). Similarly, the PoGT47C signal was evident in developing secondary xylem of roots that were undergoing secondary growth (Fig. 3G, H). The controls hybridized with the sense PoGT47C probe did not show signals in developing xylem or fibers (Fig. 3C, F, I). These results demonstrated that the expression of PoGT47C

is closely associated with developing xylem and fibers in both stems and roots. PoGT47C is targeted to the Golgi Sequence analysis using the TMHMM2.0 program indicated that PoGT47C is a type II membrane protein that contains a short cytoplasmic N-terminus followed by a single transmembrane helix and a long non-cytoplasmic C-terminus (Fig. 4A). To study its subcellular location, PoGT47C was tagged with the green fluorescent protein (GFP) and expressed in transgenic Arabidopsis plants. Confocal microscopic examination of root epidermal cells showed that the PoGT47C–GFP signal exhibits a punctate pattern in the cytoplasm (Fig. 4D, E), indicating that the protein is localized in certain subcellular organelles. The control GFP alone had signals throughout the cytoplasm and nucleus (Fig. 4B, C). To determine its subcellular location further, enhanced yellow fluorescent protein (EYFP)-tagged PoGT47C was co-transfected into carrot protoplasts with enhanced cyan fluorescent protein (ECFP)-tagged MUR4, which

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Fig. 2 Phylogenetic tree of the poplar glycosyltransferase family GT47. The phylogenetic relationship of Populus trichocarpa GTs was analyzed with ClustalW and TREEVIEW. Poplar GT sequences were retrieved from the DOE Joint Genome Institute website, and the numbers shown are the IDs of the proteins. Partial GT protein sequences that are shorter than 200 amino acid residues are not included in the phylogenetic tree. Arabidopsis GT47 members FRA8, At5g22940, AtGUT1, AtGUT2 and MUR3, and tobacco NpGUT1 are included in the tree to show their orthologs in poplar.

was previously shown to be located in the Golgi (Burget et al. 2003). It was found that the PoGT47C–EYFP signal was co-localized with that of the MUR4–ECFP signal (Fig. 4H–K), suggesting that PoGT47C is located in the Golgi. Transfection of carrot protoplasts with EYFP alone showed the presence of fluorescent signal throughout the cytoplasm and the nucleus (Fig. 4F, G). PoGT47C rescues the fra8 mutant phenotypes To test whether PoGT47C is a functional ortholog of FRA8, we investigated its ability to rescue the fra8

mutant phenotypes. The full-length PoGT47C cDNA driven by the cauliflower mosaic virus (CaMV) 35S promoter was expressed in the fra8 mutant plants. Ten transgenic lines were analyzed for the presence of the PoGT47C transgene in the homozygous fra8 mutant background (Fig. 5A). RT–PCR analysis demonstrated the expression of the PoGT47C transcript in these lines (Fig. 5B). The fra8 mutant has been shown to be smaller in size and its stems exhibit a fragile phenotype (Zhong et al. 2005). Expression of PoGT47C in fra8 was found to restore plant size and stem strength to the wild-type level

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Fig. 3 In situ localization of the PoGT47C mRNA in poplar stems and roots. Cross-sections of stems and roots were hybridized with the digoxigenin-labeled antisense (A, B, D, E, G and H) or sense (C, F and I) PoGT47C RNA probes; the hybridization signals were detected by alkaline phosphatase-conjugated antibodies and are shown in purple. (A) Cross-section of the elongating region of a stem showing the PoGT47C signal in primary xylem and phloem fibers. (B) A high magnification of (A) showing the PoGT47C signal in developing xylem cells and phloem fiber cells. (C) A control section of the elongating region hybridized with the sense probe showing the absence of hybridization signals. (D) Cross-section of the non-elongating region of a stem showing the PoGT47C signal in secondary xylem. (E) A high magnification of (D) showing the PoGT47C signal in developing secondary xylem cells including vessels (arrows) and xylary fibers. (F) A control section of the non-elongating region hybridized with the sense probe showing the absence of hybridization signals. (G) Crosssection of a root showing the PoGT47C signal in secondary xylem. (H) A high magnification of (G) showing the PoGT47C signal in developing xylem cells including vessels (arrows) and xylary fibers. (I) A control root section hybridized with the sense probe showing the absence of hybridization signals. co, cortex; hd, hypodermis; pf, phloem fiber; pi, pith; px, primary xylem; sx, secondary xylem. Bars ¼ 126 mm in (A), (D) and (G), and 55 mm in (B), (C), (E), (F), (H) and (I).

(Fig. 5C, D), indicating that PoGT47C can functionally complement the fra8 mutant. Examination of fibers and vessels in these PoGT47Ccomplemented fra8 plants showed that the thickness of interfascicular fiber walls and the shape of vessels were comparable with those of the wild type (Fig. 6A–F). Transmission electron microscopy confirmed that the wall thickness of fibers in these plants was nearly identical to that of the wild type (Fig. 6G–I, Table 1). The vessel wall thickness was also partially restored (Fig. 6J–L, Table 1). These results clearly demonstrated that the

restored stem strength in PoGT47C-complemented fra8 is a result of restored secondary wall thickness in fibers and vessels. The fra8 mutation has been shown to result in a nearly 60% reduction in xylose and a slight decrease in glucose in stem cell wall preparations (Zhong et al. 2005). In addition, several other monosaccharides, such as arabinose, mannose and galactose, were relatively elevated compared with the wild type. Cell wall composition analysis revealed that expression of PoGT47C in fra8 completely restored the level of xylose as well as other cell wall

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PoGT47C is functionally conserved with FRA8 1.2

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Fig. 4 Subcellular localization of fluorescent protein-tagged PoGT47C. Fluorescent protein-tagged PoGT47C was expressed in Arabidopsis plants and carrot protoplasts, and their fluorescent signals were visualized under a laser confocal microscope. (A) PoGT47C is a type II membrane protein as predicted by the TMHMM2.0 program. PoGT47C is predicted to contain a short N-terminal region located on the cytoplasmic side of the membrane (inside), a transmembrane helix between amino acid residues 37 and 59, and a long stretch of C-terminal region located on the non-cytoplasmic side of the membrane (outside). (B) and (C) Differential interference contrast (DIC) image (B) and the corresponding fluorescent signal (C) of Arabidopsis root epidermal cells expressing GFP alone. Note the presence of the GFP signal throughout the cytoplasm and the nucleus. (D) and (E) DIC image (D) and the corresponding fluorescent signal (E) of Arabidopsis root epidermal cells expressing PoGT47C–GFP. Note the punctate pattern of the PoGT47C–GFP signal. (F) and (G) DIC image (F) and the corresponding fluorescent signal (G) of a carrot protoplast expressing EYFP alone. (H–K) DIC image (H) and the corresponding PoGT47C– EYFP signal (I), MUR4–ECYP signal (J) and a merged image (K) of a carrot cell expressing PoGT47C–EYFP and MUR4–ECFP. Note the superimposition of PoGT47C–EYFP and MUR4–ECFP signals. Bars ¼ 18 mm in (B–E) and 26 mm in (F–K).

monosaccharides to the wild-type level (Table 2). To examine whether expression of PoGT47C rescues the xylan deficiency in fra8, we performed immunolocalization of xylan using a xylan monoclonal antibody, LM10. The LM10 antibody was generated using a penta-1,4xylanoside-containing neoglycoprotein as an immunogen (McCartney et al. 2005). It has been shown that LM10 binds to birchwood xylan and 4-O-methylglucuronoxylan but not to arabinoxylan and glucuronoarabinoxylan, and it recognizes xylan epitopes in secondary walls of vascular and mechanical tissues in vascular plants (Carafa et al. 2005, McCartney et al. 2005). Immunostaining of

cross-sections of wild-type stems and roots revealed strong signals in the walls of interfascicular fibers and xylem cells (Fig. 7B, E), both of which have abundant xylan in secondary walls. No labeling was seen in parenchyma cells. Almost no xylan signal was detected in fra8 (Fig. 7A, D). In PoGT47C-complemented fra8, the xylan signals in the walls of both interfascicular fibers and xylem cells were restored to the wild-type level (Fig. 7C, F). Transmission electron microscopy further demonstrated that whereas the immunogold labeling density of xylan was drastically reduced in fra8, expression of PoGT47C in fra8 restored the immunogold labeling density of

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Fig. 5 Expression of the poplar PoGT47C gene in the Arabidopsis fra8 plants. Ten independent transgenic Arabidopsis lines were used for gene expression analysis and their stem strength measurement. (A) Presence of the PoGT47C transgene in the transgenic fra8 mutant plants (upper panel). All transgenic plants were confirmed to have a homozygous fra8 background by digestion of the PCR-amplified fra8 genomic DNA fragments with BanII, which shows loss of a BanII site in the fra8 mutant DNA compared with the wild type (lower panel). (B) RT–PCR detection of expression of the PoGT47C transgene in the transgenic fra8 plants. The expression of a ubiquitin gene was used as an internal control. (C) fra8 (left), PoGT47C-complemented fra8 (middle) and wild-type (right) plants. (D) Breaking force measurement showing that expression of the poplar PoGT47C gene restored the fra8 stem strength comparable with that of the wild type. Each bar represents the breaking force for the inflorescence stem of individual plants.

xylan to the wild-type level (Fig. 7G–I). These results indicate that PoGT47C performs a biochemical function identical to FRA8, thus leading to a restoration of normal xylan synthesis.

Discussion Although a number of GTs have previously been shown to be expressed during wood formation in poplar, their roles in cell wall synthesis are still elusive (Aspeborg et al. 2005). In this study, we demonstrated that one of these poplar GTs, PoGT47C, is a functional ortholog of Arabidopsis FRA8 that is known to be involved in xylan synthesis. This provides genetic evidence suggesting that PoGT47C is most probably involved in xylan synthesis during wood formation. Because none of the woodassociated GT genes have been shown to be involved in xylan synthesis, this study marks a first step toward molecular dissection of the process of xylan synthesis in trees.

The complementation study demonstrated that PoGT47C can rescue the fra8 phenotypes, indicating that PoGT47C performs the same biochemical function as FRA8. Previous chemical analysis showed that the fra8 mutation causes a marked reduction in xylan. This phenotype is further proven by immunolocalization revealing that little xylan is present in the secondary walls of fibers and vessels (Fig. 7). Expression of PoGT47C in fra8 restores not only the cell wall xylose level but also the xylan immunolocalization signal to the wild-type level. In addition, PoGT47C is shown to be located in Golgi, which is known to be the site of xylan synthesis (Gregory et al. 2002). These results indicate that PoGT47C is functionally conserved with FRA8. It will be extremely interesting to investigate the role of PoGT47C in wood formation further by down-regulating the expression of PoGT47C in poplar plants. A previous microarray study by Aspeborg et al. (2005) revealed that GT47C is highly induced during development of secondary xylem in poplar stems. We showed that PoGT47C is expressed not only in the secondary xylem of

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Fig. 6 Effect of expression of the poplar PoGT47C gene on secondary wall thickness of fibers and vessels in the transgenic Arabidopsis fra8 plants. The bottom internodes of 10-week-old plants were sectioned for examination of fibers and vessels. (A–C) Cross-sections of interfascicular regions of stems of fra8 (A), PoGT47C-complemented fra8 (B) and wild type (C) showing the restoration of fiber wall thickness by expression of the poplar PoGT47C gene (B). (D–F) Cross-sections of stems showing vascular bundles of fra8 (D), PoGT47Ccomplemented fra8 (E) and wild type (F). Expression of the poplar PoGT47C gene rescued the collapsed vessel phenotype (E). (G–I) Transmission electron micrographs of interfascicular fiber cells of fra8 (G), PoGT47C-complemented fra8 (H) and wild type (I) showing the restoration of thick fiber walls by expression of the poplar PoGT47C gene (H). (J–L) Transmission electron micrographs of vessel walls of fra8 (J), PoGT47C-complemented fra8 (K) and wild type (L) showing increased vessel wall thickness by expression of the poplar PoGT47C gene (K). co, cortex; if, interfascicular fiber; ph, phloem; ve, vessel; xf, xylary fiber. Bars ¼ 95 mm in (A–F), 5.8 mm in (G–I) and 1.8 mm in (J–L).

stems, but also in primary xylem and phloem fibers of stems and secondary xylem of roots. This expression pattern indicates that PoGT47C is most probably required for all cell types that undergo secondary wall thickening. The biosynthesis of xylan requires multiple enzymatic steps, including initiation of synthesis, elongation of the xylan backbone, addition of side chains onto the xylan backbone and modification of side chain residues. Consequently, a number of GTs, acetyltransferases and methyltransferases are involved in xylan synthesis. The exact biochemical function of FRA8 and PoGT47C in xylan synthesis is still elusive due to the lack of proper in vitro activity assay conditions (Zhong et al. 2005). FRA8 is proposed to be a glucuronyltransferase participating in glucuronoxylan synthesis based on the chemical analysis showing that xylan from fra8 is deficient in GlcA

Table 1 Wall thickness of fibers and vessels in stems of fra8, fra8 complemented with PoGT47C and wild type Sample

Fiber cells

Vessels

fra8 fra8 þ PoGT47C Wild type

0.85 0.09 1.73 0.16 2.07 0.12

0.35 0.17 0.55 0.08 0.89 0.18

Wall thickness was measured from transmission electron micrographs of fibers and vessels. Data are means (mm) SE from 10 cells.

residues compared with the wild type. However, the possibility that FRA8 is involved in the initiation of synthesis or elongation of xylan backbone could not be excluded (Zhong et al. 2005). Like other GTs participating

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Table 2 Monosaccharide composition of cell walls from the stems of fra8, fra8 complemented with PoGT47C and wild type Sample

Glucose

Xylose

Mannose

Galactose

Arabinose

Rhamnose

Fucose

fra8 fra8 þ PoGT47C Wild type

205 23 258 19 257 20

60.9 2.5 136 13 143 4.2

33 1.6 18.6 0.1 18.1 0.9

21.7 0.7 11.8 1.6 12.3 0.8

24 0.5 11.4 0.9 7.9 5.8

8.4 0.4 6.6 1.0 9.8 5.5

5.5 2.4 2.3 0.8 4.5 3.9

Cell walls were prepared from stems of 10-week-old plants. Data are means (mg g1 dry cell wall) SE of three independent assays.

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Fig. 7 Immunolocalization of xylan in fibers and xylem using the monoclonal antibody LM10 that was generated against plant cell wall (1,4)-b-D-xylan. Xylan signals were detected with fluorescein isothiocyanate-conjugated secondary antibodies and visualized under a laser confocal microscope (A–F), or detected with gold-conjugated secondary antibodies and visualized under a transmission electron microscope (G–I). (A–C) Fluorescent labeling of cross-sections of stems of fra8 (A), PoGT47C-complemented fra8 (B) and wild type (C) showing that expression of the PoGT47C gene in fra8 restored the LM10-labeled signal (white color) to the wild-type level. (D–F) Fluorescent labeling of cross-sections of roots of fra8 (D), PoGT47C-complemented fra8 (E) and wild type (F) showing that expression of the PoGT47C gene in fra8 restored the LM10-labeled signal to the wild-type level. (G–I) Immunogold labeling of secondary walls of fibers of fra8 (G), PoGT47C-complemented fra8 (H) and wild type (I). Expression of the PoGT47C gene in fra8(H) restored the immunogold labeling density (black dots) to the wild-type level. if, interfascicular fiber; sx, secondary xylem; xy, xylem. Bar in (A) ¼ 97 mm for (A–F), and bar in (G) ¼ 0.46 mm for (G–I).

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in the biosynthesis of cell wall polysaccharides, it will be challenging to detect the in vitro biochemical activity of PoGT47C and FRA8 although an activity assay is required to establish their exact role in xylan synthesis. Our finding that expression of PoGT47C in fra8 sufficiently rescues the xylan deficiency phenotype indicates that GTs involved in xylan synthesis are functionally conserved between the herbaceous plant Arabidopsis and the woody species poplar. This finding also suggests the feasibility of using Arabidopsis GT mutants as a complementary approach to ascertain functions of poplar GTs in wood formation. All of the poplar wood-associated GTs have close homologs in Arabidopsis (Aspeborg et al. 2005), and many of them are highly expressed in the Arabidopsis inflorescence stems (Ye et al. 2006). Several of these Arabidopsis GTs have been shown to be important for vessel morphology (Brown et al. 2005, Persson et al. 2005). It is conceivable that complementation of Arabidopsis GT mutants with corresponding poplar GT homologs could provide an alternative approach to reveal the functional roles of poplar wood-associated GTs in cell wall synthesis. It is expected that further investigation of PoGT47C and other poplar GTs will help understand the complex process of wood formation.

Materials and Methods Isolation of the PoGT47C cDNA and sequence analysis The full-length PoGT47C cDNA was isolated by screening a poplar stem cDNA library (Zhong et al. 2000), and sequenced using a dye-based cycle sequencing kit (Applied Biosystems, Foster City, CA, USA). The GT47 homologs from other plants were retrieved by searching public databases at the National Center for Biotechnology Information. The GT sequences were aligned using the ClustalW 1.8 program (http://www.ebi.ac.uk/ clustalw/), and the resulting alignment parameters were used to generate the phylogenetic tree using TREEVIEW (Page, 1996). Expression analysis Poplar (Populus tremula Populus alba; a gift from S.H. Strauss and C. Ma) plants were esthetically propagated on modified Murashige and Skoog medium (Murashige and Skoog 1962) as described by Leple et al. (1992). Total RNA was isolated using a Qiagen RNA isolation kit (Qiagen, Valencia, CA, USA). A 1 mg aliquot of purified RNA was treated with DNase I to remove any potential genomic DNA contamination and then used for first-strand cDNA synthesis. One-twentieth of the first-strand cDNA was used for PCR amplification. The PCR was performed for variable cycles to determine the logarithmic phase of amplifications. The RT–PCRs were repeated three times, and identical results were obtained. RNA without reverse transcription was used for PCR amplification, and no PCR product was detected. The expression of a ubiquitin gene was used as an internal control to determine the RT–PCR amplification efficiency among different samples. In situ hybridization The poplar stems and leaves were used for in situ mRNA localization according to McAbee et al. (2005). Tissues were fixed

in 2.5% formaldehyde and 0.5% glutaraldehyde and embedded in paraffin. Sections (10 mm thick) were cut, mounted on slides and hybridized with digoxigenin-labeled PoGT47C antisense RNA probe synthesized using the DIG RNA labeling mix (Roche, Mannheim, Germany). The hybridization signals were detected by probing with alkaline phosphatase-conjugated antibodies against digoxigenin and subsequent color development with alkaline phosphatase substrates. Subcellular localization of fluorescent-tagged proteins The full-length PoGT47C cDNA was PCR amplified using a high-fidelity DNA polymerase and fused in-frame with the GFP cDNA (ABRC, Columbus, OH, USA). The PoGT47C–GFP fusion cDNA was cloned downstream of the CaMV 35S promoter in the binary vector pBI121. The PoGT47C–GFP construct was stably transformed into Arabidopsis plants (Bechtold and Bouchez 1994) and transgenic plants were selected on kanamycin. The GFP signal from roots of 3-day-old transgenic seedlings was observed using a Leica TCs SP2 spectral confocal microscope (Leica Microsystems, Heidelberg, Germany). Images were saved and processed with Adobe Photoshop Version 7.0 (Adobe Systems, San Jose, CA, USA). The co-localization of fluorescent-tagged PoGT47C with a Golgi marker was carried out in carrot protoplasts according to Liu et al. (1994). The PoGT47C cDNA was fused in-frame with EYFP cDNA and ligated between the CaMV 35S promoter and the nopaline synthase terminator in a high copy vector. The PoGT47C construct together with the MUR4–ECFP construct were co-transfected into carrot protoplasts. After 20 h incubation, the transfected protoplasts were examined for yellow and cyan fluorescent signals under a Leica TCs SP2 spectral confocal microscope. Generation of transgenic fra8 plants expressing PoGT47C The full-length PoGT47C cDNA was PCR amplified using high-fidelity DNA polymerase from cDNAs synthesized from poplar stems, and ligated downstream of the CaMV 35S promoter in the binary vector pBI121. The construct was introduced into the Arabidopsis fra8 plants by Agrobacterium-mediated transformation. Transgenic plants were selected on kanamycin, and the first generation of transgenic plants was used for analysis. Breaking force measurement Basal parts of the main inflorescence of 10-week-old Arabidopsis plants were measured for their breaking force using a digital force/length tester (Model DHT4-50; Larson System, Minneapolis, MN, USA). Ten individual wild-type plants, fra8 and fra8 complemented with PoGT47C were used for breaking force measurement. The breaking force was calculated as the force needed to break apart a stem segment (Zhong et al. 1997). Histology Basal internodes of 10-week-old inflorescence stems were fixed in 2% glutaraldehyde in phosphate-buffered saline (PBS; 33 mM Na2HPO4, 1.8 mM NaH2PO4 and 140 mM NaCl, pH 7.2) at 48C overnight. After fixation, tissues were post-fixed in 1% (v/v) OsO4 and then dehydrated through a gradient of ethanol and embedded in Spurr’s resin (Electron Microscopy Sciences, Fort Washington, PA, USA). Sections (1 mm thick) were cut with a microtome and stained with toluidine blue for light microscopy. For transmission electron microscopy, 85 nm thick sections were cut, post-stained with uranyl acetate and lead citrate, and

PoGT47C is functionally conserved with FRA8 visualized under a Zeiss EM 902A transmission electron microscope (Carl Zeiss, Jena, Germany). Cell wall composition analysis Inflorescence stems of 10-week-old plants were ground into a fine powder in liquid nitrogen and extracted in 70% ethanol at 708C. The resulting cell wall residues were dried and used for analysis of total monosaccharide composition. Cell wall monosaccharides (as alditol acetates) were determined following the procedure described by Hoebler et al. (1989). All samples were run in triplicate. Immunolocalization of xylan Basal internodes of 10-week-old inflorescence stems were fixed in 2% glutaraldehyde in PBS at 48C overnight. After fixation, tissues were dehydrated through a gradient of ethanol and embedded in LR White Resin (Electron Microscopy Sciences). Sections (1 mm thick) were cut with a microtome and incubated with the monoclonal antibody against xylan, LM10 (Plantprobes, Leeds, UK; McCartney et al., 2005) and fluorescein isothiocyanate-conjugated secondary antibodies. The fluorescencelabeled xylan signals were observed using a Leica TCs SP2 spectral confocal microscope. Images from single optical sections were collected and processed with Adobe Photoshop version 7.0. For transmission electron microscopy, 85 nm thick sections were cut and incubated with the xylan monoclonal antibody LM10 and gold (10 nm)-conjugated secondary antibodies. The immunogoldlabeled xylan signals were visualized under a Zeiss EM 902A transmission electron microscope.

Acknowledgments We thank the editor and the reviewers for their comments and suggestions. This work was supported by a grant from the US Department of Energy-Bioscience Division.

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(Received June 17, 2006; Accepted July 21, 2006)