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First published online April 24, 2003; 10.1104/pp.102.016642 Plant Physiology 132:768-778 (2003) © 2003 American Society of Plant Biologists Analysis of Xyloglucan Fucosylation in Arabidopsis1Michigan State University-Department of Energy Plant Research Laboratory, East Lansing, Michigan (R.M.P., T.A.W., R.S., N.V.R., K.K.); and Complex Carbohydrate Research Center, 220 Riverbend Road, University of Georgia, Athens, Georgia (Z.J., M.A.O., W.S.Y.)
Xyloglucan (XyG) is a load-bearing primary wall component in dicotyledonous and non-graminaceous monocotyledonous plants. XyG fucosyltransferase (FUTase), encoded by the Arabidopsis gene AtFUT1, directs addition of fucose (Fuc) residues to terminal galactose residues on XyG side chains. Reverse transcription-polymerase chain reaction and analysis of promoter-  -glucuronidase transgenic plants indicated highest expression of AtFUT1 in the upper portion of elongating inflorescence stems of Arabidopsis. XyG FUTase activity was highest in Golgi vesicles prepared from growing Arabidopsis tissues and low in those isolated from mature tissues. There was no discernible correlation between the Fuc contents of XyG oligosaccharides derived from different Arabidopsis organs and the level of AtFUT1 expression in the organs. Thus, organ-specific variations in AtFUT1 expression and enzyme activity probably reflect differential rates of cell wall biosynthesis, rather than differences in levels of XyG fucosylation. The effects of manipulating AtFUT1 expression were examined using an Arabidopsis mutant (atfut1) containing a T-DNA insertion in the AtFUT1 locus and transgenic plants with strong constitutive expression of AtFUT1. No Fuc was detected in XyG derived from leaves or roots of atfut1. Plants overexpressing AtFUT1 had higher XyG FUTase activity than wild-type plants, but the XyG oligosaccharides derived from the transgenic and wild-type plants contained comparable amounts of Fuc, indicating that suitable acceptor substrates are limiting. Galactosyl residues had slightly higher levels of O-acetylation in XyG from plants that overexpressed AtFUT1 than in XyG from wild-type plants. O-Acetylation of galactose residues was considerably reduced in Fuc-deficient mutants (atfut1, mur1, and mur2) that synthesize XyG containing little or no Fuc. These results suggest that fucosylated XyG is a suitable substrate for at least one O-acetyltransferase in Arabidopsis.
Plant growth is largely delimited by cell wall biosynthesis. In the absence of cell wall biosynthesis, growing plant cells would eventually rupture because their walls would become so thin that they could no longer resist their internal turgor pressure (Cosgrove, 2000  ). Several genes encoding enzymes that synthesize cell wall polysaccharides have been identified (Arioli et al., 1998; Edwards et al., 1999; Perrin et al., 1999; Richmond and Somerville, 2000; Faik et al., 2002), but few of these genes have been characterized at the levels of gene expression, the activity of the enzyme they encode, or the quantity and structure of the synthesized glycan. Genes encoding cell wall biosynthetic enzymes are difficult to identify, because the proteins they encode do not have high sequence similarity to polysaccharide-synthesizing enzymes characterized in non-plant organisms (Perrin et al., 2001). Developing suitable enzymatic assays is challenging, because many plant glycosyltransferases and glycan synthases are unstable and often require polysaccharide acceptors that are not readily available.
Xyloglucan (XyG) fucosylation is one process in which analyses of gene expression, enzyme activity, and product structure are all achievable tasks. XyG is a quantitatively major hemicellulosic polysaccharide present in the primary walls of dicots and nongraminaceous monocots. The 1,4-linked
Expression of AtFUT1 in Arabidopsis Tissues The steady-state levels of AtFUT1 transcript abundance were determined using reverse transcription (RT)-PCR and RNA prepared from various tissues of mature, hydroponically grown Arabidopsis (Columbia-0 [Col-0]) plants. These tissues were also used to determine XyG FUTase activity and the extent of XyG fucosylation. Primers specific to AtFUT1 or the "housekeeping" gene cytochrome c were used to determine gene expression levels (Fig. 1). Amplification was halted at a cycle number (22–24 cycles for AtFUT1; 28 cycles for cytochrome c) within the linear range of product accumulation (data not shown). The highest level of AtFUT1 expression occurred in the apical, youngest region of the inflorescence stem, in siliques, and in roots. Lower levels of transcript were detected in the basal, oldest region of the stem and in rosette and cauline leaves.
A similar pattern of expression was observed in RNA samples analyzed by northern blot (Fig. 2). In this case, the expression of AtFUT1 was markedly higher in a floral bud sample, which was derived from clusters of unopened flowers, petioles, and uppermost top inflorescence stem, than a sample derived from individual fully opened flowers and associated petioles.
The spatial patterns of AtFUT1 expression in planta were investigated using an approximately 2-kb-long region upstream of the AtFUT1 coding region that was amplified by PCR and then used to direct expression of the GUS reporter gene. GUS activity was then determined in homozygous T3 plants carrying a single insertion of the transgene at various stages of plant development (Figs. 3 and 4) together with positive controls expressing GUS under control of the 35S reporter (data not shown). The cotyledon veins and emerging true leaves were stained in young seedlings (3–10 d old; Fig. 3). The venation-staining pattern observed in the cotyledons was not repeated in emerging true leaves, in which staining occurred first at the true leaf base and tip (Fig. 3), followed by staining only at the base of the midvein in mature leaves (Fig. 4). No staining was observed in roots, even though northern-blot and RT-PCR analyses had shown that AtFUT1 is expressed in roots. Staining patterns in cauline leaves, rosette leaves and old and young regions of the inflorescence stem generally corresponded to results obtained using RT-PCR and hybridization techniques (Figs. 1 and 2). In mature plants, a gradient of reporter activity was observed in the inflorescent stem, with most intense staining occurring in the youngest regions of the inflorescence (Fig. 4). No staining was observed in floral tissue itself, although petiole and inflorescence tissue directly below flowers did show reporter activity.
XyG FUTase is a Golgi-localized enzyme, and fucosylated XyG accumulates primarily in the trans-Golgi and trans-Golgi network (Brummell et al., 1990
A structural reporter approach was used to determine Fuc content of Arabidopsis XyG oligosaccharides. Alcohol insoluble residues (AIR) were prepared from the remaining Arabidopsis (Col-0) samples. The enzymatically depectinated AIR was treated with a XyG-specific endoglucanse (XEG) and then with 4 M KOH. XEG treatment of the AIR hydrolyzes the "enzyme-accessible domain" of the XyG, generating a mixture of O-acetylated XyG oligosaccharides. Subsequent treatment with 4 M KOH solubilizes much of the remaining XyG as a high Mr polysaccharide with no O-acetyl substituents, which are hydrolyzed under the alkaline extraction conditions. XEG treatment of the alkali-soluble XyG generates a mixture of oligosaccharides. Figure 6 illustrates the application of this structural reporter method, showing examples of 1H-NMR spectra of KOH- and XEG-extracted XyG oligosaccharides from 4.2 to 5.3 ppm. The amount of Fuc present per unit of oligosaccharide in the Arabidopsis organ AIR samples was determined by 1H-NMR spectroscopy (Table I). XyG oligosaccharide structures are abbreviated using the nomenclature described by Fry et al. (1993
Primers specific to AtFUT1 were used to screen T-DNA pools from the Arabidopsis Knockout Facility (Madison, WI), and plants homozygous for an insertion in the middle of the second exon (at Val-378) were identified. No full-length AtFUT1 mRNA transcripts were detected in the T-DNA mutant by RT-PCR, although transcripts of nonnative size were amplified from the atfut1 mutant using primers 5' to the insertion site. Arabidopsis plants containing the T-DNA insertion had no visible phenotype under laboratory growth conditions. Such a result is consistent with previously published data showing that the Arabidopsis loss-of-function mutant mur2-1, in which there is a point mutation in the AtFUT1 gene, also grows normally under laboratory conditions (Vanzin et al., 2002
Glycosyl composition analysis indicated that the cell walls prepared from mature rosettes, expanding leaves, and flowers of the atfut1 mutant contained 50% to 75% less Fuc than wild-type (Wassilewskija [WS] ecotype) walls (data not shown). Comparable amounts of Fuc were present in rhamnogalacturonans I and II isolated from the AIR of wild-type (WS2 ecotype) and atfut1 plants (data not shown). However, no Fuc was detected by 1H-NMR spectroscopic analyses of the oligosaccharides generated by XEG treatment of the 4 M KOH-soluble XyG from the shoots and roots of 7-d-old seedlings and from mature rosette leaves (Table II). These results confirm the observation that that AtFUT1 specifically fucosylates XyG (Faik et al., 2000
The effects of overexpression of AtFUT1 were determined by generating transgenic Arabidopsis (Col-0) plants containing a construct with cDNA encoding full-length AtFUT1 regulated by the cauliflower mosaic virus (CaMV) 35S strong constitutive promoter. The presence of the transgene in hygromycin-resistant plants was confirmed by PCR amplification using primers that span the intron of AtFUT1, allowing differentiation between the transgene and the endogenous genomic version of the gene (data not shown). Multiple lines were identified by RT-PCR with elevated expression of AtFUT1 in leaves as compared with wild-type plants, and six were chosen for further analysis. Golgi-enriched vesicle fractions were prepared from leaves of these plants, and their FUTase activities determined (Fig. 7). The transgenic lines all showed elevated levels of XyG FUTase activity compared with Golgi vesicles derived from wild-type leaves. Nevertheless, there were no visible phenotypic differences between 35S::AtFUT1 plants and wild-type plants grown under laboratory growth conditions.
AIR were prepared from 11 different AtFUT1-overexpressing plants, and the Fuc contents of their XyGs were then determined (Table III). No increase in fucosylation was detected by 1H-NMR spectroscopic analysis of the XyG oligosaccharides derived from any of the 35S::AtFUT1 plants. However, there was a consistent increase in the O-acetylation of Gal residues in the Fuc-Gal-Xyl side chains of the XyG from 35S::AtFUT1 plants (Table III).
Side chains terminated by a D-Gal residue are potential acceptor substrates for the AtFUT1 enzyme. Therefore, 1H-NMR spectroscopy was used to determine the number and position of terminal D-Gal residues in XyG from wild-type and AtFUT1-overexpressing plants. XyG oligosaccharides were treated with sodium borohydride, converting them to oligosaccharide alditols. 1H-NMR analysis of these oligoglycosyl alditols allows the galactosyl residues of "suitable" AtFUT1 acceptor substrates (e.g. XXLG) to be distinguished from galactosyl residues that are "unsuitable" AtFUT1 acceptor substrates (e.g. XLLG and XLXG; see Fig. 8). The observed distinctions between suitable and unsuitable acceptors correspond with observations by Faik et al. (2000
Our data (Table III) when taken together with the results of a previous study (Pauly et al., 2001a
Cell wall biosynthesis is believed to be correlated with plant growth, but few studies have examined the relationship between these two processes at the molecular level. This study demonstrates that fucosylation of XyG can be investigated at the levels of gene regulation, enzyme activity, and product composition.
A mutant referred to as mur2-1 has a point mutation in AtFUT1 and synthesizes XyG that contains less than 2% of the amounts of Fuc than the XyG derived from wild-type plants in various tissues including leaves, stems, flowers, and roots (Vanzin et al., 2002 Distinct patterns of gene expression and XyG FUTase activity were observed in Arabidopsis stems, leaves, and flowers. Gene expression and enzyme activity levels were highest in rapidly growing tissues and lowest in the oldest tissues of the mature plant. Flowers showed high enzyme activity, but RT-PCR analysis of the same sample did not show a high level of gene expression. The variation in gene expression in floral tissue may be due to the amount of pedicel and inflorescence that is present, because promoter-reporter analysis indicated that expression of AtFUT1 is particularly high in those tissues. In that respect, it is interesting to note that northern analysis of gene expression indicated higher expression of AtFUT1 in floral bud clusters, which included a portion of top stem and pedicel tissue, than in isolated mature flowers and associated pedicels. AtFUT1::GUS transgenic plants did not exhibit strong reporter staining in reproductive tissue itself. No reporter activity was detected in roots of AtFUT1::GUS lines, although expression of AtFUT1 was observed in roots analyzed by RT-PCR and northern analysis. It is possible that regulatory regions (such as the single intron present in this gene or the 86-nucleotide-long 3'-untranslated region) directing expression of AtFUT1 in root tissues were not present in the construct used for the promoter-reporter analysis. The low reporter activity observed in mature rosette leaves and the oldest regions of the stem and high activity observed in the youngest inflorescence stem tissue paralleled AtFUT1 expression results obtained using RT-PCR and hybridization techniques. No correlations between the level of AtFUT1 expression and the Fuc content of XyG oligosaccharides were observed in the different organs of wild-type plants. Notably, the amounts of fucosylated XyG did not increase in the upper inflorescence stem even though this tissue had increased levels of AtFUT1 expression and XyG FUTase activity. Such a result suggests that differing levels of AtFUT1 gene expression and enzyme activity reflect the rate and amounts of cell wall deposition occurring in planta, rather than variations in XyG fucosylation patterns.
The results of XyG side-chain analysis indicate that side chains terminating in Gal are more abundant in leaf and silique XyG than in XyG from other tissues. This is consistent with a previous analysis demonstrating that the "central" side chain of the oligosaccharide subunits is more likely to be L (i.e. terminated with Ectopic expression of a gene is often used to study the effects of gene manipulation in planta. Transgenic plants expressing AtFUT1 under control of a strong constitutive promoter show higher gene expression and XyG FUTase activity than wild-type plants. Nevertheless, the overexpressing and wild-type plants synthesized XyG with comparable structures, which suggests that FUTase is not a limiting factor for XyG fucosylation. Our data show that the overexpression of AtFUT1 could not have resulted in a significant increase in XyG fucosylation, because the acceptor substrates (XLLG and XXLG) for the enzyme are almost fully fucosylated even in wild-type plants. The enzyme transfers a fucosyl to the galactosyl residue that terminates the side chain that is adjacent to the unbranched glucosyl residue, to produce XLFG and XXFG, respectively. Virtually no XFFG or XFLG is present in Arabidopsis XyG because side chains that are not adjacent to an unbranched glucosyl residue are not efficiently fucosylated by AtFUT1. In fact, Gal-Xyl side chains that are not in an appropriate position to act as AtFUT1 substrates are found in both wild-type and AtFUT1-overexpressing plants.
Previous studies of sycamore (Acer pseudoplatanus) XyG have shown that the major site of O-acetylation is O-6 of Gal residues. O-Acetylation also occurs at the O-4 and O-3 positions, and some Gal residues may be di-O-acetylated (York et al., 1988
The amounts of O-acetylated Gal residues in Arabidopsis XyG are positively correlated with the Fuc content of the XyG or AtFUT1 enzyme abundance. The amount of O-acetylation was consistently higher in plants that overexpress the AtFUT1 gene than in wild-type plants. Conversely, the levels of O-acetylation are considerably reduced in XyG isolated from atfut1 or mur2 plants, which have lesions in XyG FUTase, or in mur1-2 plants, which have a defect in the GDP-Fuc biosynthetic pathway. The XyG of mur1 plants does contain some O-acetyl substituents, presumably due to the presence of the J side chain [
Despite the XyG structural changes observed in AtFUT1-overexpressing plants, atfut1 and mur2, no visible changes in phenotype were apparent. This suggests that fucosylation and O-acetylation of XyG are not required for normal growth and development under laboratory conditions. However, fucosylation and O-acetylation of XyGs are widely observed in a taxonomically diverse range of dicotyledenous plants (Hayashi, 1989 In conclusion, AtFUT1 expression and XyG FUTase activity were positively correlated with growth in the aerial portions of Arabidopsis. Determination of the gene expression profile of AtFUT1 provides a baseline of comparison with other genes thought to encode enzymes involved in primary cell wall biosynthesis. Disruption of the AtFUT1 locus by insertion of a T-DNA in the second exon results in XyG containing no detectable Fuc and reduced O-acetylation of Gal residues. Increasing the expression of AtFUT1 using the strong constitutive CaMV 35S promoter results in increased XyG FUTase activity and increased XyG O-acetylation. This result, when taken together with the virtual absence of O-acetylation in atfut1, mur1-2, and mur2 XyGs, provides evidence that there is a positive correlation between XyG O-acetylation and fucosylation.
Plant Growth Conditions and Tissue Collection
Arabidopsis Col-0 plants were grown hydroponically as previously described (Gibeaut et al., 1997
Arabidopsis Col-0, mur1-2, and mur2 plants were grown in potting soil (Fafard 3B, Fafard, Inc., Anderson, SC) in a controlled environmental chamber with a 14-h-light and 10-h-dark cycle at 19°C and 15°C, respectively. Arabidopsis mur1-2 plants were also grown in the presence of 10 mM L-Fuc as previously described (O'Neill et al., 2001 Flowers, the uppermost 6 cm of the inflorescence stem, the lowest 6 cm of the inflorescence stem, siliques, cauline leaves, rosette leaves, and roots were harvested from mature hydroponically grown Arabidopsis plants. Tissues were collected on ice, weighed, and divided into portions to be used for Golgi vesicle, RNA, and cell wall preparations. Golgi vesicles were prepared using fresh tissue. RNA and cell wall samples were prepared using tissue that had been frozen in liquid nitrogen and kept at –80°C.
RNA was prepared by grinding tissue under liquid nitrogen to a fine powder and then suspending in 65°C extraction buffer (2% [w/v] hexadecyltrimethylammonium bromide, 2% [w/v] polyvinylpyrrolidone K 30, 100 mM Tris-HCl pH 8.0, 25 mM EDTA, 2.0 M NaCl, 0.5 g L–1 spermidine, and 2% [v/v]
RNA samples were treated with RNase-free DNase I using the DNA-free kit (Ambion, Austin, TX) according to the manufacturer's instructions. RNA (1 µg) was used in a RT reaction with 0.5 µg of oligo(dT) primer (Invitrogen, Carlsbad, CA), 1x first-strand buffer, 10 mM dithiothreitol (DTT), 5 mM dNTPs, and 200 units of Superscript II reverse transcriptase (Invitrogen). RNA, water, and the reverse primer were incubated at 70°C for 10 min, transferred to ice for 2 min, centrifuged, and mixed with first-strand buffer, DTT, and dNTPs. The samples were incubated at 42°C for 1 min and reverse transcriptase was added. The samples were kept at 42°C for 50 min and then at 70°C for 15 min. PCR was performed using equal volumes of the original RT reactions as template in 20-µL PCR reactions with 1x PCR buffer, 150 µM MgCl2, 125 µM each forward and reverse primer, and 5 units of Amplitaq (Roche Diagnostics, Mannheim, Germany). Primers used to amplify AtFUT1 were: forward primer, 5'-GAA GGG CTA CTT GCT TCT GGT TTT-3'; reverse primer, 5'-CCC GAT GAA TGT TTG GTC TCC TT-3'. These primers amplify a 578-bp fragment of AtFUT1 from nucleotides 571 to 1,149. Thermal cycling parameters for amplification of AtFUT1 were: 94°C for 1 min (hot start); 92°C for 30 s, 58°C for 1 min, 72°C for 1 min and 30 s for 22 to 24 cycles; and 72°C for 5 min. Primers used to amplify cytochrome c were: forward primer, 5'-TCG CTT ATT TGA AGG AAG TG-3'; reverse primer, 5'-CTC TTC ACA TCA ATA GCGT AAT-3'. These primers amplify a 212-bp fragment of cytochrome c (Arabidopsis thaliana Gene Index no. TC87099), located on chromosome 4. Thermal cycling parameters for the amplification of cytochrome c were: 94°C for 1 min (hot start); 94°C for 30 s, 55°C for 30 s, and 72°C for 1 min for 28 cycles.
RNA was isolated from hydroponically grown wild-type (Col-0) Arabidopsis plants as described above. For the purposes of these experiments, "flower" samples refers to individual, fully opened flowers and associated petioles; "floral buds" refers to a cluster of unopened flowers, associated petioles, and approximately 2 mm of uppermost inflorescence stem; "top stem" refers to the uppermost 0.5 cm of inflorescence stem after removal of flowers; "low stem" refers to a segment of the inflorescence stem from 1.5 cm from the top of the stem (after removal of flowers) to the base of the stem; "leaves" refers to mature rosette leaves; and "roots" refers to the total root mass. Fifteen micrograms of total RNA was subjected to RNA gel-blot analysis using a hybridization probe specific for AtFUT1 (Sarria et al., 2001
Golgi vesicles were prepared using the method of Muñoz et al. (1996
XyG FUTase activity assays were conducted as previously described (Faik et al., 2000
Cell Wall Preparations
Partial Depectination of AIR
The partially depectinated AIR were suspended in 10 mL of 20 mM NaOAc, pH 5, containing 0.01% (w/v) thimerosal. XEG (10 units, supplied by Novozymes A/S) was added, and the suspensions were incubated at 24°C for 24 h in a shaking incubator and then filtered. The filtrate, containing XyG oligosaccharides, was applied to a C-18 cartridge (Supelcelan LC-18 SPE tube, Supelco, Bellefonte, PA), which was then washed with water (10 mL) to remove salts. The oligosaccharides were then eluted from the cartridge with 10 mL of aqueous 25% (v/v) methanol. The eluant was concentrated under vacuum and lyophilized. The oligosaccharides were further purified on a Superdex-75 HR10/30 column (Amersham Biosciences, Uppsala) eluted with 50 mM ammonium formate, pH 5.0, at a flow rate of 0.5 mL min–1. Carbohydrate in the eluant was monitored by refractive index. Fractions containing the oligosaccharides were pooled and lyophilized several times to remove volatile ammonium formate salts.
The insoluble residues remaining after XEG treatment were suspended in 10 mL of 1 N KOH containing 1% (w/v) NaBH4 and stirred at room temperature for 24 h. The suspensions were filtered, and the insoluble residues were then suspended in 10 mL of 4 N KOH containing 1% (w/v) NaBH4. The suspensions were stirred for 24 h at room temperature and then filtered. The filtrate was adjusted to pH 5 with glacial AcOH and dialyzed (3,500 Mr cutoff tubing, Spectrum Laboratories, Rancho Dominguez, CA) against six changes of deionized water over 2 d. The retentates were lyophilized to yield the 4 N KOH-solubilized XyG polysaccharides, which were digested with XEG (as described above) to generate XyG oligosaccharides, which were isolated from the reaction mixture as described above.
Solutions of XyG oligosaccharides in D2O (0.6 mL, 99.9%; Cambridge Isotope Laboratories, Andover, MA) were analyzed at 25°C using Inova NMR spectrometers (Varian Medical Systems, Palo Alto, CA) operating at 500 and 600 MHz. Five diagnostic regions in the anomeric region of the spectra (Fig. 6) were integrated: (a)
A 1,987-bp fragment upstream of the AtFUT1 open reading frame was amplified from Arabidopsis genomic DNA. The primers used for amplification were: 5'-GGG GGA TCC CTA TAG TGG CTG TCT GCT TGA GGA-3' and 5'-GGG CCA TGG ATT GCT CTT GAG GGA-3'. Primers included restriction sites for BamHI and NcoI digestion. Fragments were cloned in pGemT-Easy vector (Promega, Madison, WI) and then into BamHI and NcoI sites in the pCAMBIA 1303 vector. This caused a fusion of the first ATG codon originating from AtFUT1 with the gusA-mGFP5 double reporter present in the pCAMBIA 1303 vector. The constructs were introduced into Agrobacterium tumefaciens by standard transformation methods and transgenic Arabidopsis plants were generated by vacuum infiltration (Bechtold and Pelletier, 1998
Whole seedlings or plant tissues were harvested and suspended in cold aqueous 90% (v/v) acetone for 20 min on ice to permeabilize the tissues. Samples were washed three times for 15 min each in working solution (100 mM NaH2PO4 containing 10 mM EDTA, 0.5 mM ferrocyanide, 0.5 mM ferricyanide, and 0.1% [v/v] Triton X-100). Samples were then kept at 37°C for 16 to 36 h in staining solution (working solution containing 2 mM X-Gluc [Rose Scientific, Edmonton, Canada]). Samples were cleared of chlorophyll by washing with aqueous 70% (v/v) ethanol and photographed using a Coolpix 995 digital camera (Nikon, Tokyo) attached to a light microscope or stereoscope.
The T-DNA pools of the Wisconsin collection were screened as recommended by the Biotechnology Center-University of Wisconsin (http://www.biotech.wisc.edu/NewServicesAndResearch/Arabidopsis/Guidelines-Index.html). Forward and reverse primers used for the mutant screen were respectively, 5'-ACAATTAAACCATGTCGTGGAGACATGTT-3' and 5'-TCATACTAGCTTAAGTCCCCAGCTGATAT-3'. The reverse primer in combination with the T-DNA left border primer (JL202) amplified the insertion. RT-PCR of mutant and wild-type RNA was performed using 1 µg of total RNA and the One-Step RT-PCR kit (Qiagen, Germantown, MD) according to the manufacturer's directions. Primers for RT-PCR spanned the intron and are as follows: 5'-TTCTCGACGCCGGAGTTT-3' and 5'-CCCTCAGTATCAATCACCT-3'.
A cDNA fragment encoding the open reading frame of AtFUT1 was subcloned into the NcoI/XhoI sites of pET14-b. The AtFUT1 cDNA was then cloned into the SalI/XbaI sites of pCAMBIA 1300 MCS (a derivative of pCAMBIA with additional sites in the MCS generated by Dr. Anton Sanderfoot [Michigan State University]). This generated a construct in which the 35S CaMV strong constitutive promoter controls the expression of AtFUT1. The construct was introduced into A. tumefaciens by standard transformation methods and transformed into wild-type (Col-0) Arabidopsis plants using the vacuum infiltration method (Bechtold and Pelletier, 1998
We thank Dr. Carl Bergmann of the CCRC and Novozymes A/S for supplying enzymes used in the analysis of XyG. Received October 25, 2002; returned for revision November 17, 2002; accepted January 15, 2003.
Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.102.016642.
1 This work was supported by the Department of Energy Biosciences Program and the Plant Genome Program at the National Science Foundation, by the U.S. Department of Energy (grant no. DE–FG05–93ER20220 to Z.J., M.A.O., and W.S.Y.), and by the U.S. Department of Energy-funded Center for Plant and Microbial Complex Carbohydrates (grant no. DE–FG05–93ER20097 to Z.J., M.A.O., and W.S.Y.).
2 Present address: Genetics/Biotechnology Building, Room 3340, 445 Henry Mall, University of Wisconsin, Madison, WI 53706.
3 Present address: BASF Plant Science, 26 Davis Drive, Research Triangle Park, NC 27709.
4 Present address: Center for Plant Cell Biology and Department of Botany and Plant Sciences, Batchelor Hall 2109, University of California, Riverside, CA 92521. * Corresponding author; e-mail keegstra{at}msu.edu; fax 517–353–9168.
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TOP
ABSTRACT
RESULTS
-D-Xyl, 
2)-
4.617 to 
