Characterisation of FUT4 and FUT6 α-(1 → 2)-fucosyltransferases reveals that absence of root arabinogalactan fucosylation increases Arabidopsis root growth salt sensitivity.

Plant type II arabinogalactan (AG) polysaccharides are attached to arabinogalactan proteins (AGPs) at hydroxyproline residues, and they are very diverse and heterogeneous structures. The AG consists of a β-(1 → 3)-linked galactan backbone with β-(1 → 6)-galactan side chains that are modified mainly with arabinose, but they may also contain glucuronic acid, rhamnose or other sugars. Here, we studied the positions of fucose substitutions in AGPs, and we investigated the functions of this fucosylation. Monosaccharide analysis of Arabidopsis leaf AGP extracts revealed a significant reduction in L-Fucose content in the fut4 mutant, but not in the fut6 mutant. In addition, Fucose was reduced in the fut4 mutant in root AGP extracts and was absent in the fut4/fut6 mutant. Curiously, in all cases reduction of fucose was accompanied with a reduction in xylose levels. The fucosylated AGP structures in leaves and roots in wild type and fut mutant plants were characterised by sequential digestion with AG specific enzymes, analysis by Polysaccharide Analysis using Carbohydrate gel Electrophoresis, and Matrix Assisted Laser Desorption/Ionisation (MALDI)-Time of Flight Mass spectrometry (MS). We found that FUT4 is solely responsible for the fucosylation of AGPs in leaves. The Arabidopsis thaliana FUT4 and FUT6 genes have been previously proposed to be non-redundant AG-specific fucosyltransferases. Unexpectedly, FUT4 and FUT6 enzymes both fucosylate the same AGP structures in roots, suggesting partial redundancy to each other. Detailed structural characterisation of root AGPs with high energy MALDI-Collision Induced Dissociation MS and NMR revealed an abundant unique AG oligosaccharide structure consisting of terminal xylose attached to fucose. The loss of this structure in fut4/fut6 mutants explains the reduction of both fucose and xylose in AGP extracts. Under salt-stress growth conditions the fut4/fut6 mutant lacking AGP fucosylation exhibited a shorter root phenotype than wild type plants, implicating fucosylation of AGPs in maintaining proper cell expansion under these conditions

EIC for the L-Fuc modified oligosaccharides from Arabidopsis root AGP extracts of wild-type (black line), <i>fut4</i> (red line), <i>fut6</i> (blue line) and <i>fut4/fut6</i> (magenta line) plants.

<p>Although all three fucosylated oligosaccharides were detected for wild-type, <i>fut4</i> and <i>fut6</i> samples, no FucAraGal₃ and FucAraGal₄ were detected from AGP root extracts from <i>fut4/fut6</i> plants and very small amount of FucPent₂Gal₃ oligosaccharide was detectable in root AGP extracts from <i>fut4/fut6</i> plants but its abundance relative to wild-type is significantly reduced.</p

HILIC purified oligosaccharide: HPAEC-PAD neutral monosaccharide analysis (% Mol).

<p>Values represent mean ± SD (n = 2).</p

RNA transcript levels of <i>FUT4</i> and <i>FUT6</i> genes by RT-PCR.

<p>RNA was isolated from roots 14 days after germination from homozygous <i>fut4</i>, <i>fut6</i>, <i>fut4/fut6</i> and wild-type seedlings and RT-PCR was performed using the primers listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0093291#pone.0093291.s002" target="_blank">Table S1</a>. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a control. In lane 1 the <i>fut4</i> left (LP) and right (RP) primers were used. In lanes 2 and 3, the <i>fut6</i> and <i>gapdh</i> primers (LP and RP) respectively, were used.</p

Characterisation of oligosaccharides released by the sequential digestion with the AG-specific enzymes α-arabinofuranosidase and endo-β-(1→6)-galactanase from Arabidopsis root AGP extracts.

<p>(a) Polysaccharide Analysis using Carbohydrate gel Electrophoresis (PACE). Oligosaccharide products from wild-type (Col-0), <i>fut4</i>, <i>fut6</i> and <i>fut4/fut6</i> were reductively aminated with 2-aminonaphthaline trisulfonic acid and separated by electrophoresis on acrylamide gels. An oligosaccharide ladder prepared from β-(1→6)-galactan was used as migration marker. The numbers indicate putatively fucosylated oligosaccharides with altered abundance in the wild-type and <i>fut</i> mutant samples. (b) MALDI-ToF-MS spectrum of <i>per</i>-methylated oligosaccharide products from wild-type plants. Peaks marked with an asterisk (*) were selected for high-energy MALDI-CID structural analysis. (c) Extracted ion chromatograms (EICs) for the fucosylated oligosaccharides originating from Arabidopsis leaf (blue lines) and root (red lines) AGP extracts hydrolysed sequentially by α-arabinofuranosidase, exo-β-(1→3)-galactanase, β-glucuronidase and endo-β-(1→6)-galactanase. Arabidopsis root AGP extracts contain the same three fucosylated oligosaccharides as leaves albeit in different relative abundances.</p

Figure 6

<p>(a) Structural characterisation of the <i>per</i>-methylated FucPent₂Gal₃ oligosaccharide by high-energy MALDI-CID. Arabidopsis root AGP extracts were sequentially hydrolysed with α-arabinofuranosidase and endo-<i>β</i>-(1→6)-galactanase and the hydrolysis products were purified, <i>per</i>-methylated and analysed by MALDI-ToF-MS as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0093291#pone-0093291-g004" target="_blank">Figure 4b</a>. The oligosaccharide with <i>m/z</i> 1175.3 as selected for MALDI-CID analysis and was identified as Xyl-(1→3)-<i>α</i>-L-Fuc-(1→2)-<i>α</i>-L-Ara-(1→3)-<i>β</i>-Gal<i>p</i>-(1→6)-<i>β</i>-Gal<i>p</i>-(1→6)-Gal<i>p</i>. Glycosidic cross-ring fragments were identified according to the Domon and Costello nomenclature (1988). (b) HPAEC-PAD monosaccharide analysis of neutral sugars for the HILIC purified root oligosaccharide. Arabidopsis root AGP extracts were sequentially hydrolysed with α-arabinofuranosidase and endo-<i>β</i>-(1→6)-galactanase and the hydrolysis products were reductively aminated with 2-AA and separated with HILIC. The FucPent₂Gal₃ oligosaccharide was collected from the HILIC column and hydrolysed by TFA. The sugar content was identified by HPAEC-PAD monosaccharide analysis. (b) NMR analysis of the FucPent₂Gal₃ hexasaccharide. (i) H-1 strip plots from 2D ¹H-¹H TOCSY (blue) and ROESY (red) spectra showing the NOE connectivity arising from the Xyl-(1→3)-<i>α</i>-L-Fuc-(1→2)-<i>α</i>-L-Ara-(1→3)-<i>β</i>-Gal<i>p</i> glycosidic linkages. (ii) 2D ¹H-¹H ROESY spectrum of the <i>α</i>-H1 region showing the Fuc H-1/Ara H-1 NOE arising from their close proximity due to the <i>α</i>-L-Fuc-(1→2)-<i>α</i>-L-Ara glycosidic linkage. (iii) 2D ¹³C HSQC and H2BC spectra showing the assignment of ¹H,¹³C HSQC peaks using H2BC, which exclusively reveals sequential connections over two covalent bonds (Nyberg et al., 2005). The H-1 chemical shift of the non-reducing-end Xyl is consistent with an <i>α</i> configuration; the ¹³C resonance positions of Fuc C-3, Ara C-2 and Gal C-3 are downfield shifted consistent with their involvement in glycosidic linkages. (NB: Fuc C-6 was aliased in the spectra; the actual resonance frequency c. 16 ppm is shown for clarity.).</p

Capillary HILIC-MALDI-ToF-MS using stable isotope tagging of fucosylated oligosaccharides from Arabidopsis leaf AGP extracts of Columbia wild-type (Col-0; black line), <i>fut4</i> (red line), <i>fut6</i> (blue line) and <i>fut4/fut6</i> (magenta line) plants.

<p>Purified leaf AGP extracts were subjected to sequential digestion with α-arabinofuranosidase, exo-β-(1→3)-galactanase, β-glucuronidase and endo-β-(1→6)-galactanase. The oligosaccharide products were purified on a C₁₈ cartridge (elution with 5% acetic acid) and cation exchange resin (Dowex; elution with 5% acetic acid) and were reductively aminated with [¹²C₆]-aniline (wild-type oligosaccharides; black line) and [¹³C₆]-aniline (<i>fut4</i>, <i>fut6</i> and <i>fut4/fut6</i>; red, blue and magenta lines respectively). The labelled oligosaccharides were purified from the reductive amination reagents on a normal phase cartridge (Glyko clean S) and the purified glycans were separated by HILIC and analysed by MALDI-ToF-MS. Although all three fucosylated oligosaccharides were detected for wild-type and <i>fut6</i> samples, no corresponding glycans were detected from AGP leaf extracts from <i>fut4</i> and <i>fut4/fut6</i> plants. Background peaks are marked with an asterisk (*). On panel b the Col-0 trace (black line) partly coincides with, and therefore obscures, the <i>fut6</i> (blue line) trace.</p

Phenotypic analysis of Arabidopsis <i>fut</i> mutants compared to wild-type plants grown on solid medium.

<p>Wild-type, <i>fut4</i>, <i>fut6</i> and <i>fut4/fut6</i> seedlings were grown on MS medium for 7 days and then transferred to cellophane layered MS-agar medium plates without any supplements (a), with 100 mM NaCl (b) and with 100 mM mannitol (c) and grown for additional 7 days. (d) <i>fut4/fut6</i> double mutants are salt sensitive. The root growth response of <i>fut4</i>, <i>fut6</i> and <i>fut4/fut6</i> mutant plants to various NaCl concentrations was compared with those of wild-type. Data are presented as percentage relative to the growth of wild-type on MS medium. Bars represent mean ± SD (n = 3). A significant difference was identified between wild type and <i>fut4/fut6</i> mutant plants as indicated by <i>p</i>>0.05 in students <i>t-</i>test. (e) <i>fut4/fut6</i> double mutants are not sensitive to osmotic stress. The root growth response of <i>fut4</i>, <i>fut6</i> and <i>fut4/fut6</i> mutant plants to various mannitol concentrations was compared with those of wild-type. Data are presented as percentage relative to the growth of wild-type on MS medium. Bars represent mean ± SD (n = 3).</p