A Novel Rice Xylosyltransferase Catalyzes the Addition of 2-O-Xylosyl Side Chains onto the Xylan Backbone

A Novel Rice Xylosyltransferase Catalyzes the Addition of 2-O-Xylosyl Side Chains onto the Xylan... Abstract Xylan is a major hemicellulose in both primary and secondary walls of grass species. It consists of a linear backbone of β-1,4-linked xylosyl residues that are often substituted with monosaccharides and disaccharides. Xylosyl substitutions directly on the xylan backbone have not been reported in grass species, and genes responsible for xylan substitutions in grass species have not been well elucidated. Here, we report functional characterization of a rice (Oryza sativa) GT61 glycosyltransferase, XYXT1 (xylan xylosyltransferase1), for its role in xylan substitutions. XYXT1 was found to be ubiquitously expressed in different rice organs and its encoded protein was targeted to the Golgi, the site for xylan biosynthesis. When expressed in the Arabidopsis gux1/2/3 triple mutant, in which xylan was completely devoid of sugar substitutions, XYXT1 was able to add xylosyl side chains onto xylan. Glycosyl linkage analysis and comprehensive structural characterization of xylooligomers generated by xylanase digestion of xylan from transgenic Arabidopsis plants expressing XYXT1 revealed that the side chain xylosyl residues were directly attached to the xylan backbone at O-2, a substituent not present in wild-type Arabidopsis xylan. XYXT1 was unable to add xylosyl residues onto the arabinosyl side chains of xylan when it was co-expressed with OsXAT2 (Oryza sativa xylan arabinosyltransferase2) in the gux1/2/3 triple mutant. Furthermore, we showed that recombinant XYXT1 possessed an activity transferring xylosyl side chains onto xylooligomer acceptors, whereas recombinant OsXAT2 catalyzed the addition of arabinosyl side chains onto xylooligomer acceptors. Our findings from both an in vivo gain-of-function study and an in vitro recombinant protein activity assay demonstrate that XYXT1 is a novel β-1,2-xylosyltransferase mediating the addition of xylosyl side chains onto xylan. Introduction Xylan is the second most abundant polysaccharide after cellulose in plant biomass targeted for biofuel production, and, therefore, elucidation of genes required for xylan biosynthesis has important implications in our attempts at genetic modification of plant biomass composition better tailored for biofuel production (Carroll and Somerville 2009). Xylan is the major hemicellulose in the secondary walls of dicots and it is essential for normal secondary wall deposition and structure (Zhong and Ye 2015). In grasses, xylan is present in both primary and secondary walls (Ebringerova and Heinze 2000). Xylan is made of a linear chain of β-1,4-linked xylosyl residues with a degree of polymerization of around 100. Xylans in dicots are often named glucuronoxylan as the backbone xylosyl residues are substituted with α-1,2-linked glucuronic acid (GlcA) and methylglucuronic acid (MeGlcA). Xylans in grasses are named arabinoxylan or glucuronoarabinoxylan as the backbone xylosyl residues are branched with α-1,3 and/or α-1,2-linked arabinofuranose (Araf) in addition to the GlcA/MeGlcA side chains. The α-1,3-linked Araf may be further substituted at O-2 with another Araf or xylose (Xyl) to form disaccharide substituents, Araf–Araf and Xyl–Araf. The reducing end of xylans in gymnosperms, dicots and some monocots contains a distinct tetrasaccharide sequence, β-d-Xylp-(1→3)-α-l-Rhap-(1→2)-α-d-GalpA-(1→4)-d-Xylp (Shimizu et al. 1967, Johansson and Samuelson 1977, Andersson et al. 1983, Pena et al. 2007, Lee et al. 2009a, Pena et al. 2016). Genetic and biochemical analyses of secondary wall-associated genes in Arabidopsis have uncovered important roles for a number of them in xylan backbone elongation and side chain addition. It has been shown that glycosyltransferases from two GT families, GT43 [irregular xylem 9 (IRX9), I9H/IRX9L, IRX14 and I14H/IRX14] and GT47 (IRX10 and IRX10L), are essential for xylan backbone elongation (Brown et al. 2007, Pena et al. 2007, Brown et al. 2009, Wu et al. 2009, Lee et al. 2010, Wu et al. 2010, Jensen et al. 2014, Urbanowicz et al. 2014), three GT8 glycosyltransferases (GUX1, GUX2 and GUX3) mediate GlcA transfer onto xylan (Mortimer et al. 2010, Lee et al. 2012a) and three DUF579-containing methyltransferases (GXM1, GXM2 and GXM3/GXMT1) catalyze methylation of GlcA side chains (Lee et al. 2012b, Urbanowicz et al. 2012, Yuan et al. 2014). In addition to sugar substitutions, xylans are often heavily acetylated at O-2 and O-3 (Teleman et al. 2000). In Arabidopsis, three groups of proteins, i.e four RWA (reduced wall acetylation) proteins (RWA1, RWA2, RWA3 and RWA4) (Lee et al. 2011), AXY9 (Schultink et al. 2015) and nine DUF231 proteins [eskimo 1 (ESK1), trichome birefringence-like 3 (TBL3), TBL28, TBL30, TBL31, TBL32, TBL33, TBL34 and TBL35] (Xiong et al. 2013, Yuan et al. 2013, Urbanowicz et al. 2014, Yuan et al. 2016a, Yuan et al. 2016b, Yuan et al. 2016c, Zhong et al, 2017), have been implicated in xylan acetylation. The biosynthesis of xylan reducing end sequence involves glycosyltransferases from families GT47 [fragile fiber 8 (FRA8) and F8H] and GT8 (IRX8 and PARVUS) (Zhong et al. 2005, Brown et al. 2007, Lee et al. 2007b, Pena et al. 2007, Lee et al. 2009b). In grasses, homologs of IRX9, IRX14 and IRX10 have also been shown to be involved in xylan backbone elongation (Zeng et al., 2010, Chiniquy et al., 2013, Lovegrove et al. 2013, Lee et al. 2014). Besides the genes shared with Arabidopsis, grasses apparently possess additional xylan biosynthetic genes for the addition of 3-linked Araf and the side chains of disaccharides Araf–Araf and Xyl–Araf. It has been demonstrated that the addition of 3-linked Araf is mediated by several grass-specific GT61 glycosyltransferases in the clade A of the GT61 family (Anders et al. 2012). RNA interference (RNAi) inhibition of a wheat GT61 gene, TaXAT1, led to a significant decrease in 3-linked Araf in xylan of wheat endosperm. Another wheat GT61 gene, TaXAT2, was able to add Araf residues onto xylan when expressed in Arabidopsis, and so were its close rice homologs, OsXAT2 and OsXAT3. It was concluded that TaXAT1, TaXAT2, OsXAT2 and OsXAT3 are α-1,3-arabinosyltransferases involved in arabinosyl transfer onto xylan (Anders et al. 2012). Another rice GT61 gene, XAX1, was suggested to mediate xylosyl transfer onto the Araf side chains that are attached at O-3 of xylan based on the observation that its mutation caused a reduction in the Xyl–Araf side chains in rice xylan (Chiniquy et al. 2012). In this report, we carried out functional characterization of another member of the rice GT61 clade A, XYXT1 (xylan xylosyltransferase1), and demonstrated it to be a novel β-1,2-xylosyltransferase catalyzing the addition of xylosyl side chains directly onto the xylan backbone. Our finding uncovers a new biochemical function of members of grass-specific GT61 glycosyltransferases, which enriches our understanding of genes involved in xylan biosynthesis. Results XYXT1, a member of rice GT61 family, is able to mediate xylosyl substitutions of xylan Among the glycosyltransferases in clade A of the GT61 family, only five members from wheat and rice have previously been shown to be involved in xylan biosynthesis, namely TaXAT1, TaXAT2, OsXAT2, OsXAT3 and XAX1 (Fig. 1A). Other members in clade A of the GT61 family have not been characterized for their functions. To decipher their possible roles in xylan biosynthesis, we performed gain-of-function studies on one of them, Os06g49300 (GenBank accession number MG763173), by investigating its ability to substitute xylan in the Arabidopsis gux1/2/3 triple mutant. Since xylan in the Arabidopsis gux1/2/3 triple mutant is completely devoid of GlcA/MeGlcA side chains (Lee et al. 2012a), any new substitutions in xylan could be easily identified. Os06g49300 is named xylan xylosyltransferase1 (XYXT1) because it was able to mediate the addition of xylosyl side chains onto xylan when expressed in the gux1/2/3 mutant (see below). Quantitative PCR analysis showed that XYXT1 was expressed ubiquitously in different rice organs (Fig. 1B), which was consistent with the XYXT1 expression data from microarray analyses in the Rice eFP Browser (Jain et al. 2007) and the RiceXPro Rice Expression Profile database (Jung et al. 2011) (Supplementary Figs. S1, S2). Subcellular localization using yellow fluorescent protein (YFP)-tagged XYXT1 in Arabidopsis protoplasts revealed a punctate distribution pattern, which overlapped with that of cyan fluorescent protein (CFP)-tagged FRA8 (Fig. 1C; see enlarged images in Supplementary Fig. S3A), a known Golgi-localized glycosyltransferase involved in xylan biosynthesis (Zhong et al. 2005). A similar co-localization pattern was observed when green fluorescent protein (GFP)-tagged XYXT1 was co-expressed with mCherry-tagged FRA8 in tobacco leaf cells (Supplementary Fig. S3B). The observed Golgi localization of XYXT1 was congruent with the prediction of XYXT1 as a Golgi-localized type II transmembrane protein (Supplementary Fig. S4) by the TMHMM2.0 program (http://www.cbs.dtu.dk/services/TMHMM/) and the Golgi Predictor (http://ccb.imb.uq.edu.au/golgi/). Fig. 1 View largeDownload slide Phylogenetic and expression analyses of XYXT1. (A) Phylogenetic relationship of XYXT1 to other rice and Arabidopsis GT61 members in the GT61 family. The GT61 family is grouped into three clades, A, B and C (Anders et al. 2012). Two wheat GT61 proteins, TaXAT1 and TaXAT2, were also included in the tree. Arabidopsis GT61 members are marked with a green triangle. The phylogenetic tree was constructed using the Neighbor–Joining algorithm, and the 0.1 scale denotes 10% change. The numbers at each node represent the percentage bootstrap values from 1,000 replicates. (B) Quantitative PCR analysis of XYXT1 expression in different rice organs. Error bars denote the SD of three biological samples. (C) Subcellular localization of YFP-tagged XYXT1 (XYXT1–YFP) together with CFP-tagged FRA8 (FRA8–CFP) in Arabidopsis protoplasts. Differential interference contrast (DIC) image of a protoplast and its fluorescence images of YFP, CFP and their merged signals. Scale bars = 11 μm. Fig. 1 View largeDownload slide Phylogenetic and expression analyses of XYXT1. (A) Phylogenetic relationship of XYXT1 to other rice and Arabidopsis GT61 members in the GT61 family. The GT61 family is grouped into three clades, A, B and C (Anders et al. 2012). Two wheat GT61 proteins, TaXAT1 and TaXAT2, were also included in the tree. Arabidopsis GT61 members are marked with a green triangle. The phylogenetic tree was constructed using the Neighbor–Joining algorithm, and the 0.1 scale denotes 10% change. The numbers at each node represent the percentage bootstrap values from 1,000 replicates. (B) Quantitative PCR analysis of XYXT1 expression in different rice organs. Error bars denote the SD of three biological samples. (C) Subcellular localization of YFP-tagged XYXT1 (XYXT1–YFP) together with CFP-tagged FRA8 (FRA8–CFP) in Arabidopsis protoplasts. Differential interference contrast (DIC) image of a protoplast and its fluorescence images of YFP, CFP and their merged signals. Scale bars = 11 μm. To investigate whether XYXT1 is involved in xylan substitutions, we expressed the XYXT1 gene under the promoter of the secondary wall-specific cellulose synthase A catalytic subunit 7 (CesA7) gene in the Arabidopsis gux1/2/3 triple mutant (Supplementary Fig. S5A). The gux1/2/3 mutant had a mild reduction in plant growth and a mild deformation in some of the xylem vessels (Lee et al. 2012a). No apparent alterations in the mutant phenotypes were observed in gux1/2/3 expressing XYXT1. Cell walls from the stems of gux1/2/3, gux1/2/3 expressing XYXT1, and the wild type were digested with xylanase to release xylooligomers for subsequent chemical analyses. Matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF-MS) analysis showed that the xylooligomers from xylanase-digested wild-type xylan had two major ion peaks at m/z 745 and 759, which correspond to GlcA-substituted Xyl4 and MeGlcA-substituted Xyl4, respectively, and a minor ion peak at m/z 761 that is attributed to the pentasaccharide reducing end sequence, β-d-Xylp-(1→4)-β-d-Xylp-(1→3)-α-l-Rhap-(1→2)-α-d-GalpA-(1→4)-d-Xylp (X-X-R-GA-X; Fig. 2A). Because the Xyl residue at the reducing end of this pentasaccharide was converted into xylitol during alkaline extraction in the presence of sodium borohydride, the mass of the pentasaccharide shown in the MALDI-TOF-MS spectra is 761 Da instead of 759 Da. The xylooligomers from xylanase-digested gux1/2/3 mutant xylan exhibited only a single ion peak at m/z 761 corresponding to the reducing end sequence (Fig. 2A) because the xylan backbone was digested into xylose monomers/dimers (Lee et al. 2007a) due to the lack of GlcA/MeGlcA side chains. Analysis of xylooligomers from xylanase-digested xylan of XYXT1-expressing gux1/2/3 plants, XYXT1-OE, revealed a new prominent ion peak at m/z 701 corresponding to an oligomer with five pentosyl residues in addition to the ion peak for the reducing end sequence at m/z 761 (Fig. 2A), indicating that XYXT1 expression resulted in an alteration in xylan structure. Fig. 2 View largeDownload slide Chemical analysis of xylan from the gux1/2/3 mutant expressing XYXT1 or MUCI21. (A) MALDI-TOF-MS spectra of xylooligomers generated from xylanase digestion of xylans of the wild type, gux1/2/3 and gux1/2/3 expressing XYXT1 or MUCI21. The major ion peaks are marked with their mass sizes and corresponding structural identities. [GlcA]Xyl4, GlcA-substituted Xyl4; [MeGlcA]Xyl4, MeGlcA-substituted Xyl4; X-X-R-GA-X, xylan reducing end pentasaccharide sequence. Note the appearance of a new ion peak at m/z 701 in gux1/2/3 expressing XYXT1. (B) Glycosyl linkage analysis of xylooligomers from xylanase-digested xylans of gux1/2/3 and gux1/2/3 expressing XYXT1. N.D., not detected. Note the presence of 2,4-linked xylosyl residues in gux1/2/3 expressing XYXT1. Fig. 2 View largeDownload slide Chemical analysis of xylan from the gux1/2/3 mutant expressing XYXT1 or MUCI21. (A) MALDI-TOF-MS spectra of xylooligomers generated from xylanase digestion of xylans of the wild type, gux1/2/3 and gux1/2/3 expressing XYXT1 or MUCI21. The major ion peaks are marked with their mass sizes and corresponding structural identities. [GlcA]Xyl4, GlcA-substituted Xyl4; [MeGlcA]Xyl4, MeGlcA-substituted Xyl4; X-X-R-GA-X, xylan reducing end pentasaccharide sequence. Note the appearance of a new ion peak at m/z 701 in gux1/2/3 expressing XYXT1. (B) Glycosyl linkage analysis of xylooligomers from xylanase-digested xylans of gux1/2/3 and gux1/2/3 expressing XYXT1. N.D., not detected. Note the presence of 2,4-linked xylosyl residues in gux1/2/3 expressing XYXT1. To decipher the chemical structure of the altered xylan in XYXT1-OE plants, we first resorted to sugar linkage analysis to determine what sugar linkages were present in XYXT1-OE xylan. It was found that in addition to sugar linkages expected for xylooligomers from gux1/2/3 xylan (including terminal Xyl, 4-linked Xyl, 3-linked Rha and 2-linked GalA), a new sugar linkage, 2,4-linked Xyl, appeared in xylooligomers from XYXT1-OE xylan (Fig. 2B; Supplementary Fig. S5B), indicating that XYXT1 expression led to the addition of a side chain at O-2 of Xyl in xylan. The presence of side chains on the xylan backbone prevents its digestion into xylose monomers/dimers by xylanase, therefore generating oligomers with five pentosyl residues. The side chain in XYXT1-OE xylan could be Xyl or Ara residue as both Xyl-substituted Xyl4 oligomers and Ara-substituted Xyl4 oligomers could give rise to the ion peak at m/z 701 as seen in the MALDI-TOF-MS spectrum (Fig. 2A). Since Ara residues were absent in the sugar linkage analysis of XYXT1-OE xylooligomers (Supplementary Fig. S5B), the ion peak at m/z 701 most probably corresponded to the Xyl-substituted Xyl4 oligomer. A previous study proposed that MUCI21 (mucilage-related 21), an Arabidopsis GT61 glycosyltransferase, was involved in addition of Xyl side chains onto xylan in seed mucilage (Voiniciuc et al. 2015). We also expressed the MUCI21 gene under the CesA7 promoter in the Arabidopsis gux1/2/3 mutant and examined the xylooligomers from xylanase-digested xylan of MUCI21-expressing plants. MALDI-TOF-MS analysis of the xylooligomers did not reveal the presence of the characteristic ion peak at m/z 701 corresponding to Xyl-substituted xylotetraose as seen in the XYXT1-OE xylooligomers (Fig. 2A). The xylosyl substitutions of xylan mediated by XYXT1 occur at O-2 of xylosyl residues The presence of Xyl side chains in the XYXT1-OE xylooligomers was further substantiated using electrospray ionization ion trap mass spectrometry (ESI-MSn; n = the number of times that the isolation–fragmentation cycle is carried out) (Mazumder and York 2010). The free hydroxyl groups of the XYXT1-OE xylooligomers were first methylated to aid in the identification of newly generated fragments as subsequent gas-phase fragmentation of the methylated oligosaccharides generates fragments with a free hydroxyl group that has a 14 Da mass difference relative to a methylated site. After methylation, the original ion peak at m/z 701 was shifted to m/z 869 due to the addition of a methyl group onto each of the 12 free hydroxyl groups (Supplementary Fig. S6). Upon fragmentation of the ion at m/z 869, an abundant Y ion at m/z 695 was recorded in the ESI-MS2 spectrum (Fig. 3A), which could be formed by the loss of a terminal Xyl residue. Further fragmentation of the two ions at m/z 695 would theoretically result in two Y ions at m/z 521 (three Xyl residues with two unmethylated sites), two Y ions at m/z 375 (two Xyl residues with one unmethylated site), two B ions at m/z 343 (two Xyl residues with two unmethylated sites) and two B ions at m/z 503 (three Xyl residues with two unmethylated sites), which were consistent with the ions seen in the ESI-MS3 spectrum (Fig. 3B). The presence of these ions indicates that the backbone of the xylooligomer is branched, which is consistent with the sugar linkage analysis showing the existence of Xyl residues branched at O-2. Fig. 3 View largeDownload slide Determination of the branching sites of xylooligomers from gux1/2/3 expressing XYXT1 by ESI-MSn. The xylooligomer at m/z 701 from gux1/2/3 expressing XYXT1 was methylated at the free hydroxyl groups to give rise to a new mass at m/z 869, which was then subjected to fragmentation by ESI-MS. (A) ESI-MS2 spectrum of the per-O-methylated xylooligomer at m/z 869. (B) ESI-MS3 spectrum of the ion mass at m/z 695 generated from fragmentation of the per-O-methylated xylooligomer at m/z 869 in (A). Fragment ion types that arise by cleavage within the sugar moieties of the xylooligomer are depicted above each spectrum. Y ions represent fragments containing the reducing sugar unit, and B ions designate fragments containing a terminal sugar unit. Fig. 3 View largeDownload slide Determination of the branching sites of xylooligomers from gux1/2/3 expressing XYXT1 by ESI-MSn. The xylooligomer at m/z 701 from gux1/2/3 expressing XYXT1 was methylated at the free hydroxyl groups to give rise to a new mass at m/z 869, which was then subjected to fragmentation by ESI-MS. (A) ESI-MS2 spectrum of the per-O-methylated xylooligomer at m/z 869. (B) ESI-MS3 spectrum of the ion mass at m/z 695 generated from fragmentation of the per-O-methylated xylooligomer at m/z 869 in (A). Fragment ion types that arise by cleavage within the sugar moieties of the xylooligomer are depicted above each spectrum. Y ions represent fragments containing the reducing sugar unit, and B ions designate fragments containing a terminal sugar unit. The structure of the XYXT1-OE xylooligomers was confirmed by one-dimensional (1D) and 2D nuclear magnetic resonance (NMR) spectroscopy (Figs. 4, 5). The 1H NMR spectrum of the xylooligomers showed well-resolved anomeric proton signals from 4.4 to 5.5 p.p.m. (Fig. 4B). Anomeric configurations were assigned from the magnitude of J1,2, with a value of 3.7 Hz indicative of the equatorial–axial coupling of α-Xyl at 5.185 p.p.m., and 7.2–7.8 Hz for the diaxial coupling associated with β-Xyl at 4.460, 4.584, 4.635 and 4.640 p.p.m. The xylooligomers from xylanase-digested gux1/2/3 xylan exhibited resonances characteristic of the pentasaccharide reducing end sequence β-d-Xylp-(1→4)-β-d-Xylp-(1→3)-α-l-Rhap-(1→2)-α-d-GalpA-(1→4)-d-Xylp (H1 of α-GalA at 5.23 p.p.m., H1 of α-Rha at 5.07 p.p.m., H1 of 3-linked β-Xyl at 4.66 p.p.m., H4 of α-GalA at 4.33 p.p.m., H2 of α-Rha at 4.23 p.p.m. and H1 of unbranched β-Xyl at 4.46 p.p.m.) (Fig. 4B). In contrast, the 1H NMR spectrum of the xylooligomers from xylanase-digested xylan of XYXT1-expressing gux1/2/3 displayed not only the resonances of the pentasaccharide reducing end sequence, but also the resonances attributed to xylosyl residues of the xylan backbone as seen in the wild-type xylan, including H1 of reducing α-Xyl at 5.18 p.p.m., H1 of reducing β-Xyl at 4.58 p.p.m. and elevated resonances of H1 of unbranched β-Xyl at 4.46 p.p.m. (Fig. 4B). In addition, the XYXT1-OE xylooligomers exhibited prominent resonances at 4.64 p.p.m. of unknown identity (Fig. 4B). It was noted that there existed minor resonance signals at 5.41 p.p.m. in the 1H NMR spectra of xylooligomers from both XYXT1-OE in gux1/2/3 and the wild type (Fig. 4B). Although their exact identity was unknown, these resonance signals are distinct from the characteristic resonance peak for terminal Araf at 5.39 p.p.m. (Mazumder and York 2010, Chiniquy et al. 2012), indicating that they are unlikely to be attributed to Araf. Fig. 4 View largeDownload slide Structural analysis of xylan from the gux1/2/3 mutant expressing XYXT1. (A) Diagram of the xylooligomer generated from xylanase digestion of xylan from gux1/2/3 expressing XYXT1. (B) 1H NMR spectra of xylooligomers from the gux1/2/3 mutant, gux1/2/3 expressing XYXT1 and wild-type Arabidopsis. Resonance peaks are marked with the proton positions and the corresponding residue identities. HDO, hydrogen deuterium oxide. (C) Assignment of 1H and 13C NMR chemical shifts for the xylooligomer from gux1/2/3 expressing XYXT1 based on 1D and 2D NMR spectra. The numbering of positions of xylosyl residues is the same as in (A). Fig. 4 View largeDownload slide Structural analysis of xylan from the gux1/2/3 mutant expressing XYXT1. (A) Diagram of the xylooligomer generated from xylanase digestion of xylan from gux1/2/3 expressing XYXT1. (B) 1H NMR spectra of xylooligomers from the gux1/2/3 mutant, gux1/2/3 expressing XYXT1 and wild-type Arabidopsis. Resonance peaks are marked with the proton positions and the corresponding residue identities. HDO, hydrogen deuterium oxide. (C) Assignment of 1H and 13C NMR chemical shifts for the xylooligomer from gux1/2/3 expressing XYXT1 based on 1D and 2D NMR spectra. The numbering of positions of xylosyl residues is the same as in (A). Fig. 5 View largeDownload slide Determination of the glycosidic linkages in the xylooligomers from gux1/2/3 expressing XYXT1 based on the HSQC, H2BC, HMBC and ROESY spectra. The numbering of positions of xylosyl residues is the same as in Fig. 4A. (A) HSQC spectra showing the one-bond H1–C1 correlations between 1H and 13C of each Xyl. Note the H1–C1 signals for the side chain Xyl5 at 4.64 p.p.m. (1H) and 103.6 p.p.m. (13C). (B) H2BC spectra showing the two-bond H1–C2 correlations between 1H and 13C of each Xyl. (C) HMBC spectra showing interglycosidic cross-peaks between 1H and 13C. Note the cross-peak signals between H1 of Xyl5 at 4.64 p.p.m. and C2 of Xyl3 at 80.3 p.p.m., and those between H2 of Xyl3 at 3.520 p.p.m. and C1 of Xyl5 at 103.6 p.p.m. (D) ROESY spectrum showing interglycosidic cross-peaks between 1H and 1H. Note the cross-peak signals between H1 of Xyl5 at 4.64 p.p.m. and H2 of Xyl3 at 3.52 p.p.m. Fig. 5 View largeDownload slide Determination of the glycosidic linkages in the xylooligomers from gux1/2/3 expressing XYXT1 based on the HSQC, H2BC, HMBC and ROESY spectra. The numbering of positions of xylosyl residues is the same as in Fig. 4A. (A) HSQC spectra showing the one-bond H1–C1 correlations between 1H and 13C of each Xyl. Note the H1–C1 signals for the side chain Xyl5 at 4.64 p.p.m. (1H) and 103.6 p.p.m. (13C). (B) H2BC spectra showing the two-bond H1–C2 correlations between 1H and 13C of each Xyl. (C) HMBC spectra showing interglycosidic cross-peaks between 1H and 13C. Note the cross-peak signals between H1 of Xyl5 at 4.64 p.p.m. and C2 of Xyl3 at 80.3 p.p.m., and those between H2 of Xyl3 at 3.520 p.p.m. and C1 of Xyl5 at 103.6 p.p.m. (D) ROESY spectrum showing interglycosidic cross-peaks between 1H and 1H. Note the cross-peak signals between H1 of Xyl5 at 4.64 p.p.m. and H2 of Xyl3 at 3.52 p.p.m. Because of limited dispersion of 1H chemical shifts in 3.2–4.2 p.p.m., 2D NMR experiments were performed to identify and assign resonances for each Xyl residue in the XYXT1-OE xylooligomers. Heteronuclear single quantum coherence (HSQC) provides the correlation between each carbon and its attached protons. Fig. 5A shows the spectral region of the one-bond H1–C1 anomeric correlation of the XYXT1-OE xylooligomers. All the other proton–carbon one-bond correlations are shown in Supplementary Fig. S7A. H5eq, H5ax and H5–C5 correlations were easily identified from the edited version of HSQC, which shows -CH2 peaks in opposite phases (Supplementary Fig. S7B). Heteronuclear two-bond correlation (H2BC) (Nyberg et al. 2005) gives valuable help in spectra assignments as it contains only two-bond correlations. All C2 carbons in the H2BC spectrum of the XYXT1-OE xylooligomers could be assigned unequivocally. The spectral region of the two-bond H1–C2 correlation of the XYXT1-OE xylooligomers is shown in Fig. 5B and that of the two-bond H2–C1 correlation is shown in Supplementary Fig. S8. Both 1H-1H COSY (correlation spectroscopy) and HSQC-TOSCY (total correlation spectroscopy) were used to identify protons and carbons in each spin system. The H1 region of HSQC-TOCSY revealed H1 and all relayed carbons (C2, C3, C4 and C5) in each Xyl (Supplementary Fig. S9). Heteronuclear multiple bond correlation (HMBC) provides correlations between carbons and protons that are separated by two or more bonds. Although some of the H1–C2 correlations were missing due to vanishing 2J(H1–C2) coupling constants, the spectral data from HMBC (Fig. 5C) were quite complementary to those from H2BC and HSQC-TOCSY. The combination of the above 2D spectra led us to accomplish the assignment of the spectra of XYXT1-OE xylooligomers (Fig. 4C). All the assignments for Xyl residues in the XYXT1-OE xylooligomers are consistent with those for similar xylooligomers (branched with Araf or Araf-Xyl) previously published (Mazumder and York 2010, Chiniquy et al. 2012). The HMBC spectral region shown in Fig. 5C revealed how Xyl units were linked. The correlations between H1 of β-Xyl2 (4.460 p.p.m.) and C4 of β-Xyl1 (76.3 p.p.m.) and that between H1 of β-Xyl4 (4.460 p.p.m.) and C4 of β-Xyl3 (76.3 p.p.m.) indicate that Xyl4 and Xyl3 were β-(1→4) linked, and so were Xyl2 and Xyl1 (Fig. 5C, left panel). Although they were overlapping, the correlation between H4 of β-Xyl1 (3.767 p.p.m.) and C1 of β-Xyl2 (101.6 p.p.m.) and that between H4 of α-Xyl1 (3.741 p.p.m.) and C1 of β-Xyl2 (101.6 p.p.m.) confirmed that Xyl2 and Xyl1 were β-(1→4) linked (Fig. 5C, right panel). The correlation between H1 of β-Xyl3 (4.635 p.p.m.) and C4 of β-Xyl2 (76.3 p.p.m.) indicates that Xyl3 and Xyl2 are also β-(1→4) linked. Both the correlation between H1 of β-Xyl5 (4.640 p.p.m.; side chain Xyl in Fig. 4A) and C2 of β-Xyl3 (80.3 p.p.m.; branched Xyl in Fig. 4A) (Fig. 5C, left panel) and that between H2 of β-Xyl3 (3.520 p.p.m.) and C1 of β-Xyl5 (103.6 p.p.m.) (Fig. 5C, right panel) indicate that Xyl5 is attached to Xyl3 at the O-2 position. Furthermore, the spectrum from rotating-frame Overhauser effect spectroscopy (ROESY) revealed the correlation between H1 of β-Xyl5 and H2 of β-Xyl3 (Fig. 5D), further confirming that Xyl5 is attached to Xyl3 at the O-2 position. Based on the 1D and 2D NMR analyses, it was concluded that the XYXT1-OE xylooligomers contain the β-1,4-linked xylotetraose backbone that is substituted at O-2 with β-Xyl side chains (Fig. 4A). Recombinant XYXT1 catalyzes xylosyl substitutions of xylan The demonstration that XYXT1 was able to mediate the addition of Xyl side chains onto xylan when expressed in the Arabidopsis gux1/2/3 mutant indicates that XYXT1 is most probably a xylan xylosyltransferase. To prove its activity biochemically, we first attempted to express His-tagged XYXT1 in a secreted form in human embryonic kidney (HEK) 293F cells but failed to obtain any recombinant protein from the medium. We next expressed His-tagged full-length XYXT1 in HEK293F cells and isolated microsomes, which contained His-tagged XYXT1 as evidenced by Western blot analysis using anti-His antibody (Fig. 6A; see Supplementary Fig. S10 for whole gel images), for assaying its xylan xylosyltransferase activity. To do so, microsomes from cells transfected with the empty vector (control) or cells expressing XYXT1 were incubated with the xylotetraose acceptor and the xylosyl donor UDP-Xyl, and the reaction products were analyzed by MALDI-TOF-MS and NMR spectroscopy. The MALDI-TOF-MS analysis showed that the XYXT1-catalyzed reaction products had one new ion species at m/z 701 with an increase in mass of 132 Da over the mass of the xylotetraose acceptor (m/z 569) (Supplementary Fig. S11), indicating the addition of one Xyl residue onto xylotetraose. Examination of the 1H NMR spectra revealed the presence of resonance signals at 4.64 p.p.m. (Fig. 6C;Supplementary Fig. S12), which correspond to the side chain β-Xyl (Fig. 4B), in the reaction products of XYXT1-expressing microsomes but not in those of the control microsomes. These results confirm that XYXT1 is a xylosyltransferase adding Xyl side chains onto xylan. Fig. 6 View largeDownload slide Detection of xylan xylosyltransferase and arabinosyltransferase activities of recombinant XYXT1 and OsXAT2 proteins. His-tagged full-length XYXT1 protein was expressed in HEK293F cells and their microsomes were isolated for activity assay. His-tagged OsXAT2 protein was expressed in a secreted form in HEK293F cells, and the purified protein was used for arabinosyltransferase activity assay. (A) Immunoblot detection of His-tagged XYXT1 protein in HEK293F microsomes. Microsomal proteins (20 μg) were separated by SDS–PAGE and immunodetected with anti-His monoclonal antibody. Control, microsomal proteins isolated from HEK293F cells transfected with the empty vector. (B) SDS–PAGE detection of recombinant OsXAT2 protein (5 μg) that was visualized by Coomassie Blue staining. (C) 1H NMR spectra of the reaction products of the control microsomes (control) and the XYXT1-expressing microsomes (XYXT1) after incubation with the xylotetraose acceptor and UDP-Xyl. The resonance signals are marked with the proton positions and the corresponding residue identities. Note the appearance of the resonance signals at 4.64 p.p.m. corresponding to the side chain Xyl in the reaction products of the XYXT1-expressing microsomes. (D) 1H NMR spectra of the reaction products of the control microsomes (control), the XYXT1-expressing microsomes (XYXT1) and the recombinant OsXAT2 protein (OsXAT2) after incubation with the xylotetraose acceptor and UDP-Araf. The 1H NMR spectrum of xylooligomers from xylanase-digested rice xylan was included to show the resonance signals corresponding to the terminal Araf at 5.39 p.p.m. Note the presence of the terminal Araf signal at 5.39 p.p.m. in the reaction product catalyzed by OsXAT2 but not XYXT1. Fig. 6 View largeDownload slide Detection of xylan xylosyltransferase and arabinosyltransferase activities of recombinant XYXT1 and OsXAT2 proteins. His-tagged full-length XYXT1 protein was expressed in HEK293F cells and their microsomes were isolated for activity assay. His-tagged OsXAT2 protein was expressed in a secreted form in HEK293F cells, and the purified protein was used for arabinosyltransferase activity assay. (A) Immunoblot detection of His-tagged XYXT1 protein in HEK293F microsomes. Microsomal proteins (20 μg) were separated by SDS–PAGE and immunodetected with anti-His monoclonal antibody. Control, microsomal proteins isolated from HEK293F cells transfected with the empty vector. (B) SDS–PAGE detection of recombinant OsXAT2 protein (5 μg) that was visualized by Coomassie Blue staining. (C) 1H NMR spectra of the reaction products of the control microsomes (control) and the XYXT1-expressing microsomes (XYXT1) after incubation with the xylotetraose acceptor and UDP-Xyl. The resonance signals are marked with the proton positions and the corresponding residue identities. Note the appearance of the resonance signals at 4.64 p.p.m. corresponding to the side chain Xyl in the reaction products of the XYXT1-expressing microsomes. (D) 1H NMR spectra of the reaction products of the control microsomes (control), the XYXT1-expressing microsomes (XYXT1) and the recombinant OsXAT2 protein (OsXAT2) after incubation with the xylotetraose acceptor and UDP-Araf. The 1H NMR spectrum of xylooligomers from xylanase-digested rice xylan was included to show the resonance signals corresponding to the terminal Araf at 5.39 p.p.m. Note the presence of the terminal Araf signal at 5.39 p.p.m. in the reaction product catalyzed by OsXAT2 but not XYXT1. To examine whether XYXT1 also exhibits arabinosyltransferase activity, the XYXT1-expressing microsomes were incubated with xylotetraose and UDP-Araf, and the reaction products were examined with 1H NMR spectroscopy for the resonance signal characteristic of H1 of the terminal α-Araf at 5.39 p.p.m. (Pena et al. 2016). No signals at 5.39 p.p.m. were observed in the XYXT1-calayzed reaction products compared with the control (Fig. 6D). In contrast, incubation of recombinant His-tagged OsXAT2 (Fig. 6B; see Supplementary Fig. S10C for the whole gel image) with xylotetraose and UDP-Araf resulted in reaction products with a resonance signal at 5.39 p.p.m. that matched with that of the terminal α-Araf in the xylooligomers generated from xylanase digestion of rice xylan (Fig. 6D), which provided biochemical evidence demonstrating that OsXAT2 is a xylan arabinosyltransferase. Furthermore, we simultaneously expressed XYXT1 and OsXAT2 in the gux1/2/3 mutant (Supplementary Fig. S5A) to investigate whether XYXT1 could transfer Xyl residues onto the Araf side chains of xylan. The H1 of Araf side chains substituted at O-2 with Xyl residues in xylan exhibits a characteristic resonance peak at 5.54 p.p.m. as previously revealed by 1H NMR spectroscopy (Chiniquy et al. 2012, Pena et al. 2016). 1H NMR analysis showed that while the xylooligomers of the gux1/2/3 mutant expressing OsXAT2 had a resonance peak at 5.39 p.p.m. corresponding to H1 of terminal Araf side chains, those of gux1/2/3 expressing both XYXT1 and OsXAT2 displayed resonance peaks at 4.64 p.p.m. attributed to H1 of Xyl side chains in addition to those for the terminal Araf side chains (Fig. 7). No resonance signals at 5.54 p.p.m. for the H1 of 2-O-Araf were observed, indicating that XYXT1 was unable to add Xyl onto the Araf side chains of xylan. These results demonstrate that XYXT1 is a xylosyltransferase specifically catalyzing the addition of Xyl side chains directly onto the xylan backbone, an activity different from the arabinosyltransferase activity of OsXAT2 and the xylosyltransferase activity transferring Xyl onto O-2 of the Araf side chains of xylan. Fig. 7 View largeDownload slide 1H NMR spectra of xylooligomers released from xylanase-digested xylans of the gux1/2/3 mutant, gux1/2/3 expressing OsXAT2 (OsXAT2-OE in gux1/2/3), gux1/2/3 expressing XYXT1 (XYXT1-OE in gux1/2/3) and gux1/2/3 simultaneously expressing both OsXAT2 and XYXT1 (OsXAT2-OE XYXT1-OE in gux1/2/3). Resonance peaks are marked with the proton positions and the corresponding residue identities. Note the appearance of the resonance peak at 5.39 p.p.m. that is characteristic of H1 of terminal α-Araf and the resonance peak at 4.64 p.p.m. that corresponds to Xyl side chains in gux1/2/3 expressing both OsXAT2 and XYXT1 but a lack of any resonance signals at 5.54 p.p.m. attributed to Xyl attached to O-2 of α-Araf side chains of xylan. Fig. 7 View largeDownload slide 1H NMR spectra of xylooligomers released from xylanase-digested xylans of the gux1/2/3 mutant, gux1/2/3 expressing OsXAT2 (OsXAT2-OE in gux1/2/3), gux1/2/3 expressing XYXT1 (XYXT1-OE in gux1/2/3) and gux1/2/3 simultaneously expressing both OsXAT2 and XYXT1 (OsXAT2-OE XYXT1-OE in gux1/2/3). Resonance peaks are marked with the proton positions and the corresponding residue identities. Note the appearance of the resonance peak at 5.39 p.p.m. that is characteristic of H1 of terminal α-Araf and the resonance peak at 4.64 p.p.m. that corresponds to Xyl side chains in gux1/2/3 expressing both OsXAT2 and XYXT1 but a lack of any resonance signals at 5.54 p.p.m. attributed to Xyl attached to O-2 of α-Araf side chains of xylan. Discussion Our comprehensive analyses using MALDI-TOF-MS, ESI-MS, and 1D and 2D NMR have firmly established that the xylan from XYXT1-expressing gux1/2/3 is branched at O-2 with Xyl, indicating that XYXT1 is capable of transferring Xyl side chains onto xylan. Because Arabidopsis xylan lacks Xyl as side chains and the Arabidopsis genome has no close homologs of XYXT1, the addition of Xyl side chains in XYXT1-OE xylan can most probably be directly attributed to the activity of XYXT1. This result is further supported by the finding that recombinant XYXT1 expressed in mammalian cells possesses a xylosyltransferase activity catalyzing the transfer of Xyl residues from UDP-Xyl onto the xylotetraose acceptors. While recombinant OsXAT2 was shown to catalyze the addition of Araf residues from UDP-Araf onto xylotetraose, recombinant XYXT1 was unable to do so. In addition, XYXT1 was unable to transfer Xyl residues onto the Araf side chains of xylan when XYXT1 and OsXAT2 were co-expressed in the gux1/2/3 mutant. To our knowledge, this is the first report of a glycosyltransferase with an activity catalyzing the transfer of Xyl side chains directly onto the xylan backbone. The discovery of XYXT1 as a β-1,2-xylosyltransferase mediating 2-O-Xyl substitutions of xylan enriches our understanding of genes involved in the biosynthesis of xylan, the second most abundant polysaccharide in plant biomass. The presence of Xyl side chains directly attached to the xylan backbone has not been reported previously in grass species. The common sugar substitutions found in xylans from grasses, including wheat, barley, rice, and switchgrass, are 2-O-linked GlcA/MeGlcA, 3-O-linked Araf, 2,3-di-O-linked Araf, and 3-O-linked Araf that is further substituted at O-2 with Araf or Xyl (Araf–Araf or Araf–Xyl) (Hoffmann et al. 1992, Hoije et al. 2006, Mazumder and York 2010, Chiniquy et al. 2012, Lee et al. 2014). Glycosyl linkage analysis of rice xylan revealed that it had a high proportion of Xyl that was substituted at O-2 (Supplementary Fig. S13). Due to its low abundance, we were unable to perform structural analysis to confirm the presence of 2-O-linked Xyl side chains in rice xylan. Nevertheless, our finding that XYXT1 is a β-1,2-xylosyltransferase mediating xylosyl transfer at O-2 of xylan indicates that rice xylan is likely to be substituted with Xyl at O-2 in addition to other substituents. Sequence analysis showed that the genomes of several other grass species, including Zea mays, Sorghum bicolor, Brachypodium distachyon, Panicum virgatum, Triticum aestivum, Hordeum vulgare, Oropetium thomaeum and Setaria italica, harbor close homologs of rice XYXT1 (Supplementary Fig. S14). It is possible that Xyl substitutions in xylan are common in the cell walls of grasses. The presence of Xyl side chains directly attached to the xylan backbone has been reported in the seed mucilage polysaccharides of psyllium (Plantago ovata) and Arabidopsis (Fischer et al. 2004, Voiniciuc et al. 2015). In psyllium, a number of GT61 genes have been shown to be expressed during seed development and were suggested to be involved in the addition of side chain substituents in xylan (Phan et al. 2016). In Arabidopsis, MUCI21, which is a member of clade B of the GT61 family (Fig. 1A), has been proposed to be involved in the addition of Xyl residues directly onto the xylan backbone in seed mucilage (Voiniciuc et al. 2015). This proposed function of MUCI21 was based on glycosyl linkage analysis showing a reduction of 2,4-linked Xyl in the seed mucilage of the muci21 mutant. However, structural analysis of the muci21 mutant xylan was not performed and the proposed xylosyltransferase activity has not been biochemically confirmed (Voiniciuc et al. 2015). Furthermore, overexpression of MUCI21 driven by the CesA7 promoter in gux1/2/3 did not result in an addition of Xyl side chains onto xylan (Fig. 2A). Therefore, it is currently unknown whether MUCI21 is a β-1,2-xylosyltransferase adding Xyl side chains directly onto the xylan backbone. Among the 19 GT61 glycosyltransferases in the clade A of rice GT61 family (Fig. 1A;Anders et al. 2012), three of them, OsXAT2, OsXAT3 and XAX1, have previously been shown to be involved in xylan biosynthesis. OsXAT2 and OsXAT3 were proposed to be arabinosyltransferases mediating the arabinosyl transfer at O-3 of xylan based on their ability to add Araf residues onto xylan when expressed in wild-type Arabidopsis plants (Anders et al. 2012). XAX1 was believed to be a xylosyltransferase involved in disaccharide substitutions in rice xylan, i.e. adding 1,2-linked Xyl onto Ara that is attached at O-3 of the xylan backbone. This hypothesis was based on the observation that the xylooligomers released from xylanase digestion of xylan from the xax1 mutant lacked an ion peak corresponding to xylooligomers branched with Xyl–Araf disaccharide compared with those from the wild type (Chiniquy et al. 2012). Activity assay of recombinant OsXAT2 in our study confirmed that it is an arabinosyltransferase catalyzing the transfer of Araf residues onto the xylan backbone. The enzymatic activities of OsXAT3 and XAX1 as an arabinosyltransferase and a xylosyltransferase, respectively, remain to be biochemically confirmed. Our finding that XYXT1 exhibits a β-1,2-xylosyltransferase activity catalyzing Xyl transfer directly onto the xylan backbone indicates that it functions distinctly from OsXAT2, OsXAT3 and XAX1. It is interesting to note that AtXYLT, which is a clade C member of the Arabidopsis GT61 family, has been shown to be a β-1,2-xylosyltransferase transferring Xyl residues onto N-linked glycans (Pagny et al. 2003). Our identification of XYXT1 as a novel xylan β-1,2-xylosyltransferase enriches our understanding of roles of GT61 glycosyltransferases in xylan biosynthesis. Since clade A of the GT61 family is dramatically expanded in grasses and many of its members have no known dicot orthologs (Anders et al. 2012), it is possible that in addition to OsXAT2, OsXAT3, XAX1 and XYXT1, some other clade A GT61 members are also involved in xylan substitutions. Further functional characterization of other GT61 members will probably help us understand the complex biochemical process of xylan biosynthesis in general. Materials and Methods Expression analysis Rice (Oryza sativa var. japonica) tissues, including 2-week-old seedlings, different organs (leaves, stems, inflorescence and roots) of 2-month-old plants, and developing seeds, were collected for isolation of total RNA using a Qiagen RNA isolation kit. First-strand cDNAs were reverse-transcribed from the RNA and subsequently used as templates for real-time quantitative PCR analyses. The PCR primers for XYXT1 were 5'-tcaccggaggaggtggagggcctt-3' and 5'-gacatgaacatgaggtacctgcag-3'. The XYXT1 expression level was calculated by normalizing its PCR threshold cycle number with that of the rice EF1α reference gene and quantitated based on the standard curve of a plasmid control. The data were the mean of three biological replicates. Subcellular localization XYXT1 fused with the N-terminus of YFP was generated by fusion of its full-length cDNA with that of YFP, which was subsequently cloned under the Cauliflower mosaic virus (CaMV) 35S promoter in a modified pBI221 vector. The YFP-tagged XYXT1 was co-expressed with a Golgi marker, CFP-tagged FRA8 (Zhong et al. 2005), in Arabidopsis leaf protoplasts. In addition, GFP-tagged XYXT1 together with mCherry-tagged FRA8, both of which were driven by the CaMV 35S promoter in a modified pBI121 vector, were transformed into leaves of Nicotiana benthamiana by Agrobacterium-mediated infiltration (Zhong et al. 2017). The fluorescent signals in the transfected Arabidopsis protoplasts and tobacco leaf cells were recorded using a Zeiss LSM 510 META confocal microscope. At least 10 Arabidopsis protoplasts and 10 infiltrated tobacco leaves were imaged, and representative images are shown. Generation of transgenic gux1/2/3 plants expressing XYXT1 and OsXAT2 The full-length cDNAs of XYXT1 and OsXAT2 driven by the 2 kb CesA7 promoter were cloned into a modified pGPTV binary vector to create the XYXT1 and OsXAT2 expression constructs. For simultaneous expression of XYXT1 and OsXAT2, the expression cassette composed of the 2 kb CesA7 promoter, the full-length OsXAT2 cDNA and the nopaline synthase terminator was inserted into the multiple cloning site of the XYXT1 expression construct to create the XYXT1–OsXAT2 expression construct. The XYXT1, OsXAT2 and XYXT1–OsXAT2 expression constructs were introduced into Arabidopsis gux1/2/3 mutant plants by Agrobacterium-mediated transformation. Transgenic plants were selected by sowing transformed seeds on agar plates containing hygromycin, and >100 independent transgenic plants were generated and used for subsequent gene expression and cell wall analyses. For gene expression analysis, RNA was isolated from stems of three separate pools of 10 independent transgenic plants (each of them represented a biological replicate) for each construct using a Qiagen RNA isolation kit. The isolated RNA was used for examination of expression of XYXT1 and OsXAT2 in the transgenic gux1/2/3 plants expressing XYXT1 and/or OsXAT2 by the reverse transcription–PCR approach. Generation of xylooligomers by xylanase digestion Mature inflorescence stems from the first-generation transgenic gux1/2/3 plants expressing XYXT1 and/or XAT2 were used for chemical and structural analyses of xylan. In order to obtain sufficient cell wall materials, stems were collected from three separate pools of transgenic plants, with each pool containing 30 independent first-generation transgenic plants. Thus, each pool represents a biological replicate. Alcohol-insoluble cell walls were isolated as previously described (Zhong et al. 2005). The cell walls were first extracted with ammonium oxalate to remove pectins and then extracted for xylan with 1 N KOH in the presence of sodium borohydride. The KOH-extracted xylan was then digested with endo-1,4-β-xylanase M6 from a rumen microorganism (Megazyme) to release xylooligomers, which were passed through a Sephadex G25 column to remove monomers and dimers (Zhong et al. 2005). Glycosyl linkage analysis The xylooligomers released from xylanase digestion of cell walls were first permethyalted with potassium dimsyl anion and methyl iodide, and then reduced using sodium borodeuteride according to Heiss et al. (2009). After re-permethylation with methyl iodide and sodium hydroxide (Ciucanu and Kerek 1984), the permethylated xylooligomers were hydrolyzed by trifluoroacetic acid and further derivatized to alditol acetates. The alditol acetates of sugars were analyzed on a PerkinElmer Clarus 500 gas–liquid chromatograph equipped with a silica capillary column (30 m×0.25 mm). Xylooligomers isolated from three separate pools of samples were analyzed, and representative data are shown. ESI-MS The xylooligomers were first permethylated with methyl iodide and sodium hydroxide according to the procedure described by Ciucanu and Kerek (1984) and then subjected to multiple-stage ESI-MS analysis using a Bruker Esquire 3000 Plus ion trap instrument. The MS/MS data were collected using manual mode with an isolation window width of 4 and fragmenation amplitude of 1. Xylooligomers isolated from three separate pools of samples were permethylated and analyzed. MALDI-TOF-MS The xylooligomers were subjected to MALDI-TOF-MS analysis using a Burker Autoflex TOF mass spectrometer in reflection mode (Zhong et al. 2005). The spectra were the averages of 250 laser shots. Xylooligomers isolated from three separate pools of samples were analyzed, and representative spectra are shown. NMR spectroscopy The xylooligomers were analyzed with a Varian Inova 500 MHz spectrometer. One-dimensional and two-dimensional (HSQC, H2BC, HMBC, ROESY and COSY) NMR spectra were recorded using standard Varian pulse sequences. The proton positions and residue identities in the NMR spectra were assigned based on our 1D and 2D NMR spectral data and the published NMR spectral data for xylans (Pena et al. 2007, Mazumder and York 2010, Chiniquy et al. 2012). Xylooligomers isolated from three separate pools of samples were examined. Recombinant protein expression We attempted to express two forms of recombinant XYXT1 protein heterologously in HEK293F cells; one was in a secreted form with the removal of the N-terminal transmembrane domain and the other was the full-length protein. For production of secreted XYXT1, the XYXT1 cDNA with deletion of the N-terminal transmembrane domain was cloned between the murine Igκ chain leader sequence (for protein secretion) and the c-myc epitope and a six tandem histidine tag in the pSecTag2 mammalian expression vector (Invitrogen), and the expression construct was tranfected into HEK293F cells using the Invitrogen FreeStyle 293 Expression System according to the manufacturer’s protocol. After 5 d of culture of the transfetced cells, the culture medium was passed through a nickel resin column for purification and detection of expressed recombinant proteins. For production of the full-length recombinant XYXT1, the full-length XYXT1 cDNA was cloned into the pcDNA3.1/myc-His mammalian expression vector (Invitrogen), and the expression construct was tranfected into HEK293F cells. After 4 d of culture, the transfected cells were collected for microsome isolation (Bozidis et al. 2007). The presence of XYXT1 in the isolated microsomes was examined by SDS–PAGE and immunodetection with the anti-HIS monoclonal antibody. Recombinant OsXAT2 was expressed in a secreted form by cloning the OsXAT2 cDNA with deletion of the N-terminal transmembrane domain between the murine Igκ chain leader sequence (for protein secretion) and the c-myc epitope and six tandem histidine tag in the pSecTag2 mammalian expression vector, and transfecting into HEK293F cells. The secreted recombinant His-tagged OsXAT2 protein was purified by passing the culture medium through a nickel resin column as previously described (Zhong et al. 2017) and examined by SDS–PAGE. Xylosyltransferase activity assay The isolated HEK293F microsomes containing XYXT1 were examined for xylosyltransferase activity by incubating 200 μg of microsomal proteins with the xylotetraose acceptor (200 μg), 1 mM UDP-Xyl (CarboSource), 50 mM HEPES buffer (pH 7.0), 1 mM MgCl2 and 0.5% Triton-X100 at 21°C for 16 h. After passing through Dowex 1X4 resin, the reaction products were analyzed using MALDI-TOF-MS as well as with NMR spectroscopy for the presence of the side chain Xyl. Arabinosyltransferase activity assay The purified recombinant OsXAT2 protein (100 μg) was incubated with xylotetraose (200 μg) and 0.3 mM UDP-Araf (Peptide Institute) in 50 mM HEPES buffer (pH 7.0) at 37°C for 16 h for detection of xylan arabinosyltransferase activity. After passing through the Dowex 1X4 resin, the reaction products were analyzed with NMR spectroscopy for the presence of the side chain Araf. Supplementary Data Supplementary data are available at PCP online. 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Google Scholar CrossRef Search ADS PubMed  Abbreviations Abbreviations Araf arabinofuranose AXY altered xyloglucan CaMV Cauliflower mosaic virus CFP cyan fluorescent protein COSY homonuclear correlation spectroscopy ESI-MS electrospray ionization-mass spectrometry ESK eskimo FRA fragile fiber GalA galacturonic acid GFP green fluorescent protein GlcA glucuronic acid GT glycosyltransferase GUX glucuronic acid substitution of xylan GXM glucuronoxylan methyltransferase H2BC heteronuclear two-bond correlation HEK human embryonic kidney HMBC heteronuclear multiple bond correlation HSQC heteronuclear single quantum correlation IRX irregular xylem MALDI-TOF-MS matrix-assisted laser desorption ionization-time-of-flight mass spectrometry MeGlcA 4-O-methyl-glucuronic acid MUCI mucilage-related NMR nuclear magnetic resonance Rha rhamnose ROESY rotating-frame Overhauser effect spectroscopy RWA reduced wall acetylation TBL trichome birefringence-like TOSCY total correlation spectroscopy XAT xylan arabinosyltransferase XAX xylosyl arabinosyl substitution of xylan Xyl xylose XYXT xylan xylosyltransferase YFP yellow fluorescent protein © The Author(s) 2018. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Plant and Cell Physiology Oxford University Press

A Novel Rice Xylosyltransferase Catalyzes the Addition of 2-O-Xylosyl Side Chains onto the Xylan Backbone

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Oxford University Press
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© The Author(s) 2018. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com
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0032-0781
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1471-9053
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10.1093/pcp/pcy003
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Abstract

Abstract Xylan is a major hemicellulose in both primary and secondary walls of grass species. It consists of a linear backbone of β-1,4-linked xylosyl residues that are often substituted with monosaccharides and disaccharides. Xylosyl substitutions directly on the xylan backbone have not been reported in grass species, and genes responsible for xylan substitutions in grass species have not been well elucidated. Here, we report functional characterization of a rice (Oryza sativa) GT61 glycosyltransferase, XYXT1 (xylan xylosyltransferase1), for its role in xylan substitutions. XYXT1 was found to be ubiquitously expressed in different rice organs and its encoded protein was targeted to the Golgi, the site for xylan biosynthesis. When expressed in the Arabidopsis gux1/2/3 triple mutant, in which xylan was completely devoid of sugar substitutions, XYXT1 was able to add xylosyl side chains onto xylan. Glycosyl linkage analysis and comprehensive structural characterization of xylooligomers generated by xylanase digestion of xylan from transgenic Arabidopsis plants expressing XYXT1 revealed that the side chain xylosyl residues were directly attached to the xylan backbone at O-2, a substituent not present in wild-type Arabidopsis xylan. XYXT1 was unable to add xylosyl residues onto the arabinosyl side chains of xylan when it was co-expressed with OsXAT2 (Oryza sativa xylan arabinosyltransferase2) in the gux1/2/3 triple mutant. Furthermore, we showed that recombinant XYXT1 possessed an activity transferring xylosyl side chains onto xylooligomer acceptors, whereas recombinant OsXAT2 catalyzed the addition of arabinosyl side chains onto xylooligomer acceptors. Our findings from both an in vivo gain-of-function study and an in vitro recombinant protein activity assay demonstrate that XYXT1 is a novel β-1,2-xylosyltransferase mediating the addition of xylosyl side chains onto xylan. Introduction Xylan is the second most abundant polysaccharide after cellulose in plant biomass targeted for biofuel production, and, therefore, elucidation of genes required for xylan biosynthesis has important implications in our attempts at genetic modification of plant biomass composition better tailored for biofuel production (Carroll and Somerville 2009). Xylan is the major hemicellulose in the secondary walls of dicots and it is essential for normal secondary wall deposition and structure (Zhong and Ye 2015). In grasses, xylan is present in both primary and secondary walls (Ebringerova and Heinze 2000). Xylan is made of a linear chain of β-1,4-linked xylosyl residues with a degree of polymerization of around 100. Xylans in dicots are often named glucuronoxylan as the backbone xylosyl residues are substituted with α-1,2-linked glucuronic acid (GlcA) and methylglucuronic acid (MeGlcA). Xylans in grasses are named arabinoxylan or glucuronoarabinoxylan as the backbone xylosyl residues are branched with α-1,3 and/or α-1,2-linked arabinofuranose (Araf) in addition to the GlcA/MeGlcA side chains. The α-1,3-linked Araf may be further substituted at O-2 with another Araf or xylose (Xyl) to form disaccharide substituents, Araf–Araf and Xyl–Araf. The reducing end of xylans in gymnosperms, dicots and some monocots contains a distinct tetrasaccharide sequence, β-d-Xylp-(1→3)-α-l-Rhap-(1→2)-α-d-GalpA-(1→4)-d-Xylp (Shimizu et al. 1967, Johansson and Samuelson 1977, Andersson et al. 1983, Pena et al. 2007, Lee et al. 2009a, Pena et al. 2016). Genetic and biochemical analyses of secondary wall-associated genes in Arabidopsis have uncovered important roles for a number of them in xylan backbone elongation and side chain addition. It has been shown that glycosyltransferases from two GT families, GT43 [irregular xylem 9 (IRX9), I9H/IRX9L, IRX14 and I14H/IRX14] and GT47 (IRX10 and IRX10L), are essential for xylan backbone elongation (Brown et al. 2007, Pena et al. 2007, Brown et al. 2009, Wu et al. 2009, Lee et al. 2010, Wu et al. 2010, Jensen et al. 2014, Urbanowicz et al. 2014), three GT8 glycosyltransferases (GUX1, GUX2 and GUX3) mediate GlcA transfer onto xylan (Mortimer et al. 2010, Lee et al. 2012a) and three DUF579-containing methyltransferases (GXM1, GXM2 and GXM3/GXMT1) catalyze methylation of GlcA side chains (Lee et al. 2012b, Urbanowicz et al. 2012, Yuan et al. 2014). In addition to sugar substitutions, xylans are often heavily acetylated at O-2 and O-3 (Teleman et al. 2000). In Arabidopsis, three groups of proteins, i.e four RWA (reduced wall acetylation) proteins (RWA1, RWA2, RWA3 and RWA4) (Lee et al. 2011), AXY9 (Schultink et al. 2015) and nine DUF231 proteins [eskimo 1 (ESK1), trichome birefringence-like 3 (TBL3), TBL28, TBL30, TBL31, TBL32, TBL33, TBL34 and TBL35] (Xiong et al. 2013, Yuan et al. 2013, Urbanowicz et al. 2014, Yuan et al. 2016a, Yuan et al. 2016b, Yuan et al. 2016c, Zhong et al, 2017), have been implicated in xylan acetylation. The biosynthesis of xylan reducing end sequence involves glycosyltransferases from families GT47 [fragile fiber 8 (FRA8) and F8H] and GT8 (IRX8 and PARVUS) (Zhong et al. 2005, Brown et al. 2007, Lee et al. 2007b, Pena et al. 2007, Lee et al. 2009b). In grasses, homologs of IRX9, IRX14 and IRX10 have also been shown to be involved in xylan backbone elongation (Zeng et al., 2010, Chiniquy et al., 2013, Lovegrove et al. 2013, Lee et al. 2014). Besides the genes shared with Arabidopsis, grasses apparently possess additional xylan biosynthetic genes for the addition of 3-linked Araf and the side chains of disaccharides Araf–Araf and Xyl–Araf. It has been demonstrated that the addition of 3-linked Araf is mediated by several grass-specific GT61 glycosyltransferases in the clade A of the GT61 family (Anders et al. 2012). RNA interference (RNAi) inhibition of a wheat GT61 gene, TaXAT1, led to a significant decrease in 3-linked Araf in xylan of wheat endosperm. Another wheat GT61 gene, TaXAT2, was able to add Araf residues onto xylan when expressed in Arabidopsis, and so were its close rice homologs, OsXAT2 and OsXAT3. It was concluded that TaXAT1, TaXAT2, OsXAT2 and OsXAT3 are α-1,3-arabinosyltransferases involved in arabinosyl transfer onto xylan (Anders et al. 2012). Another rice GT61 gene, XAX1, was suggested to mediate xylosyl transfer onto the Araf side chains that are attached at O-3 of xylan based on the observation that its mutation caused a reduction in the Xyl–Araf side chains in rice xylan (Chiniquy et al. 2012). In this report, we carried out functional characterization of another member of the rice GT61 clade A, XYXT1 (xylan xylosyltransferase1), and demonstrated it to be a novel β-1,2-xylosyltransferase catalyzing the addition of xylosyl side chains directly onto the xylan backbone. Our finding uncovers a new biochemical function of members of grass-specific GT61 glycosyltransferases, which enriches our understanding of genes involved in xylan biosynthesis. Results XYXT1, a member of rice GT61 family, is able to mediate xylosyl substitutions of xylan Among the glycosyltransferases in clade A of the GT61 family, only five members from wheat and rice have previously been shown to be involved in xylan biosynthesis, namely TaXAT1, TaXAT2, OsXAT2, OsXAT3 and XAX1 (Fig. 1A). Other members in clade A of the GT61 family have not been characterized for their functions. To decipher their possible roles in xylan biosynthesis, we performed gain-of-function studies on one of them, Os06g49300 (GenBank accession number MG763173), by investigating its ability to substitute xylan in the Arabidopsis gux1/2/3 triple mutant. Since xylan in the Arabidopsis gux1/2/3 triple mutant is completely devoid of GlcA/MeGlcA side chains (Lee et al. 2012a), any new substitutions in xylan could be easily identified. Os06g49300 is named xylan xylosyltransferase1 (XYXT1) because it was able to mediate the addition of xylosyl side chains onto xylan when expressed in the gux1/2/3 mutant (see below). Quantitative PCR analysis showed that XYXT1 was expressed ubiquitously in different rice organs (Fig. 1B), which was consistent with the XYXT1 expression data from microarray analyses in the Rice eFP Browser (Jain et al. 2007) and the RiceXPro Rice Expression Profile database (Jung et al. 2011) (Supplementary Figs. S1, S2). Subcellular localization using yellow fluorescent protein (YFP)-tagged XYXT1 in Arabidopsis protoplasts revealed a punctate distribution pattern, which overlapped with that of cyan fluorescent protein (CFP)-tagged FRA8 (Fig. 1C; see enlarged images in Supplementary Fig. S3A), a known Golgi-localized glycosyltransferase involved in xylan biosynthesis (Zhong et al. 2005). A similar co-localization pattern was observed when green fluorescent protein (GFP)-tagged XYXT1 was co-expressed with mCherry-tagged FRA8 in tobacco leaf cells (Supplementary Fig. S3B). The observed Golgi localization of XYXT1 was congruent with the prediction of XYXT1 as a Golgi-localized type II transmembrane protein (Supplementary Fig. S4) by the TMHMM2.0 program (http://www.cbs.dtu.dk/services/TMHMM/) and the Golgi Predictor (http://ccb.imb.uq.edu.au/golgi/). Fig. 1 View largeDownload slide Phylogenetic and expression analyses of XYXT1. (A) Phylogenetic relationship of XYXT1 to other rice and Arabidopsis GT61 members in the GT61 family. The GT61 family is grouped into three clades, A, B and C (Anders et al. 2012). Two wheat GT61 proteins, TaXAT1 and TaXAT2, were also included in the tree. Arabidopsis GT61 members are marked with a green triangle. The phylogenetic tree was constructed using the Neighbor–Joining algorithm, and the 0.1 scale denotes 10% change. The numbers at each node represent the percentage bootstrap values from 1,000 replicates. (B) Quantitative PCR analysis of XYXT1 expression in different rice organs. Error bars denote the SD of three biological samples. (C) Subcellular localization of YFP-tagged XYXT1 (XYXT1–YFP) together with CFP-tagged FRA8 (FRA8–CFP) in Arabidopsis protoplasts. Differential interference contrast (DIC) image of a protoplast and its fluorescence images of YFP, CFP and their merged signals. Scale bars = 11 μm. Fig. 1 View largeDownload slide Phylogenetic and expression analyses of XYXT1. (A) Phylogenetic relationship of XYXT1 to other rice and Arabidopsis GT61 members in the GT61 family. The GT61 family is grouped into three clades, A, B and C (Anders et al. 2012). Two wheat GT61 proteins, TaXAT1 and TaXAT2, were also included in the tree. Arabidopsis GT61 members are marked with a green triangle. The phylogenetic tree was constructed using the Neighbor–Joining algorithm, and the 0.1 scale denotes 10% change. The numbers at each node represent the percentage bootstrap values from 1,000 replicates. (B) Quantitative PCR analysis of XYXT1 expression in different rice organs. Error bars denote the SD of three biological samples. (C) Subcellular localization of YFP-tagged XYXT1 (XYXT1–YFP) together with CFP-tagged FRA8 (FRA8–CFP) in Arabidopsis protoplasts. Differential interference contrast (DIC) image of a protoplast and its fluorescence images of YFP, CFP and their merged signals. Scale bars = 11 μm. To investigate whether XYXT1 is involved in xylan substitutions, we expressed the XYXT1 gene under the promoter of the secondary wall-specific cellulose synthase A catalytic subunit 7 (CesA7) gene in the Arabidopsis gux1/2/3 triple mutant (Supplementary Fig. S5A). The gux1/2/3 mutant had a mild reduction in plant growth and a mild deformation in some of the xylem vessels (Lee et al. 2012a). No apparent alterations in the mutant phenotypes were observed in gux1/2/3 expressing XYXT1. Cell walls from the stems of gux1/2/3, gux1/2/3 expressing XYXT1, and the wild type were digested with xylanase to release xylooligomers for subsequent chemical analyses. Matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF-MS) analysis showed that the xylooligomers from xylanase-digested wild-type xylan had two major ion peaks at m/z 745 and 759, which correspond to GlcA-substituted Xyl4 and MeGlcA-substituted Xyl4, respectively, and a minor ion peak at m/z 761 that is attributed to the pentasaccharide reducing end sequence, β-d-Xylp-(1→4)-β-d-Xylp-(1→3)-α-l-Rhap-(1→2)-α-d-GalpA-(1→4)-d-Xylp (X-X-R-GA-X; Fig. 2A). Because the Xyl residue at the reducing end of this pentasaccharide was converted into xylitol during alkaline extraction in the presence of sodium borohydride, the mass of the pentasaccharide shown in the MALDI-TOF-MS spectra is 761 Da instead of 759 Da. The xylooligomers from xylanase-digested gux1/2/3 mutant xylan exhibited only a single ion peak at m/z 761 corresponding to the reducing end sequence (Fig. 2A) because the xylan backbone was digested into xylose monomers/dimers (Lee et al. 2007a) due to the lack of GlcA/MeGlcA side chains. Analysis of xylooligomers from xylanase-digested xylan of XYXT1-expressing gux1/2/3 plants, XYXT1-OE, revealed a new prominent ion peak at m/z 701 corresponding to an oligomer with five pentosyl residues in addition to the ion peak for the reducing end sequence at m/z 761 (Fig. 2A), indicating that XYXT1 expression resulted in an alteration in xylan structure. Fig. 2 View largeDownload slide Chemical analysis of xylan from the gux1/2/3 mutant expressing XYXT1 or MUCI21. (A) MALDI-TOF-MS spectra of xylooligomers generated from xylanase digestion of xylans of the wild type, gux1/2/3 and gux1/2/3 expressing XYXT1 or MUCI21. The major ion peaks are marked with their mass sizes and corresponding structural identities. [GlcA]Xyl4, GlcA-substituted Xyl4; [MeGlcA]Xyl4, MeGlcA-substituted Xyl4; X-X-R-GA-X, xylan reducing end pentasaccharide sequence. Note the appearance of a new ion peak at m/z 701 in gux1/2/3 expressing XYXT1. (B) Glycosyl linkage analysis of xylooligomers from xylanase-digested xylans of gux1/2/3 and gux1/2/3 expressing XYXT1. N.D., not detected. Note the presence of 2,4-linked xylosyl residues in gux1/2/3 expressing XYXT1. Fig. 2 View largeDownload slide Chemical analysis of xylan from the gux1/2/3 mutant expressing XYXT1 or MUCI21. (A) MALDI-TOF-MS spectra of xylooligomers generated from xylanase digestion of xylans of the wild type, gux1/2/3 and gux1/2/3 expressing XYXT1 or MUCI21. The major ion peaks are marked with their mass sizes and corresponding structural identities. [GlcA]Xyl4, GlcA-substituted Xyl4; [MeGlcA]Xyl4, MeGlcA-substituted Xyl4; X-X-R-GA-X, xylan reducing end pentasaccharide sequence. Note the appearance of a new ion peak at m/z 701 in gux1/2/3 expressing XYXT1. (B) Glycosyl linkage analysis of xylooligomers from xylanase-digested xylans of gux1/2/3 and gux1/2/3 expressing XYXT1. N.D., not detected. Note the presence of 2,4-linked xylosyl residues in gux1/2/3 expressing XYXT1. To decipher the chemical structure of the altered xylan in XYXT1-OE plants, we first resorted to sugar linkage analysis to determine what sugar linkages were present in XYXT1-OE xylan. It was found that in addition to sugar linkages expected for xylooligomers from gux1/2/3 xylan (including terminal Xyl, 4-linked Xyl, 3-linked Rha and 2-linked GalA), a new sugar linkage, 2,4-linked Xyl, appeared in xylooligomers from XYXT1-OE xylan (Fig. 2B; Supplementary Fig. S5B), indicating that XYXT1 expression led to the addition of a side chain at O-2 of Xyl in xylan. The presence of side chains on the xylan backbone prevents its digestion into xylose monomers/dimers by xylanase, therefore generating oligomers with five pentosyl residues. The side chain in XYXT1-OE xylan could be Xyl or Ara residue as both Xyl-substituted Xyl4 oligomers and Ara-substituted Xyl4 oligomers could give rise to the ion peak at m/z 701 as seen in the MALDI-TOF-MS spectrum (Fig. 2A). Since Ara residues were absent in the sugar linkage analysis of XYXT1-OE xylooligomers (Supplementary Fig. S5B), the ion peak at m/z 701 most probably corresponded to the Xyl-substituted Xyl4 oligomer. A previous study proposed that MUCI21 (mucilage-related 21), an Arabidopsis GT61 glycosyltransferase, was involved in addition of Xyl side chains onto xylan in seed mucilage (Voiniciuc et al. 2015). We also expressed the MUCI21 gene under the CesA7 promoter in the Arabidopsis gux1/2/3 mutant and examined the xylooligomers from xylanase-digested xylan of MUCI21-expressing plants. MALDI-TOF-MS analysis of the xylooligomers did not reveal the presence of the characteristic ion peak at m/z 701 corresponding to Xyl-substituted xylotetraose as seen in the XYXT1-OE xylooligomers (Fig. 2A). The xylosyl substitutions of xylan mediated by XYXT1 occur at O-2 of xylosyl residues The presence of Xyl side chains in the XYXT1-OE xylooligomers was further substantiated using electrospray ionization ion trap mass spectrometry (ESI-MSn; n = the number of times that the isolation–fragmentation cycle is carried out) (Mazumder and York 2010). The free hydroxyl groups of the XYXT1-OE xylooligomers were first methylated to aid in the identification of newly generated fragments as subsequent gas-phase fragmentation of the methylated oligosaccharides generates fragments with a free hydroxyl group that has a 14 Da mass difference relative to a methylated site. After methylation, the original ion peak at m/z 701 was shifted to m/z 869 due to the addition of a methyl group onto each of the 12 free hydroxyl groups (Supplementary Fig. S6). Upon fragmentation of the ion at m/z 869, an abundant Y ion at m/z 695 was recorded in the ESI-MS2 spectrum (Fig. 3A), which could be formed by the loss of a terminal Xyl residue. Further fragmentation of the two ions at m/z 695 would theoretically result in two Y ions at m/z 521 (three Xyl residues with two unmethylated sites), two Y ions at m/z 375 (two Xyl residues with one unmethylated site), two B ions at m/z 343 (two Xyl residues with two unmethylated sites) and two B ions at m/z 503 (three Xyl residues with two unmethylated sites), which were consistent with the ions seen in the ESI-MS3 spectrum (Fig. 3B). The presence of these ions indicates that the backbone of the xylooligomer is branched, which is consistent with the sugar linkage analysis showing the existence of Xyl residues branched at O-2. Fig. 3 View largeDownload slide Determination of the branching sites of xylooligomers from gux1/2/3 expressing XYXT1 by ESI-MSn. The xylooligomer at m/z 701 from gux1/2/3 expressing XYXT1 was methylated at the free hydroxyl groups to give rise to a new mass at m/z 869, which was then subjected to fragmentation by ESI-MS. (A) ESI-MS2 spectrum of the per-O-methylated xylooligomer at m/z 869. (B) ESI-MS3 spectrum of the ion mass at m/z 695 generated from fragmentation of the per-O-methylated xylooligomer at m/z 869 in (A). Fragment ion types that arise by cleavage within the sugar moieties of the xylooligomer are depicted above each spectrum. Y ions represent fragments containing the reducing sugar unit, and B ions designate fragments containing a terminal sugar unit. Fig. 3 View largeDownload slide Determination of the branching sites of xylooligomers from gux1/2/3 expressing XYXT1 by ESI-MSn. The xylooligomer at m/z 701 from gux1/2/3 expressing XYXT1 was methylated at the free hydroxyl groups to give rise to a new mass at m/z 869, which was then subjected to fragmentation by ESI-MS. (A) ESI-MS2 spectrum of the per-O-methylated xylooligomer at m/z 869. (B) ESI-MS3 spectrum of the ion mass at m/z 695 generated from fragmentation of the per-O-methylated xylooligomer at m/z 869 in (A). Fragment ion types that arise by cleavage within the sugar moieties of the xylooligomer are depicted above each spectrum. Y ions represent fragments containing the reducing sugar unit, and B ions designate fragments containing a terminal sugar unit. The structure of the XYXT1-OE xylooligomers was confirmed by one-dimensional (1D) and 2D nuclear magnetic resonance (NMR) spectroscopy (Figs. 4, 5). The 1H NMR spectrum of the xylooligomers showed well-resolved anomeric proton signals from 4.4 to 5.5 p.p.m. (Fig. 4B). Anomeric configurations were assigned from the magnitude of J1,2, with a value of 3.7 Hz indicative of the equatorial–axial coupling of α-Xyl at 5.185 p.p.m., and 7.2–7.8 Hz for the diaxial coupling associated with β-Xyl at 4.460, 4.584, 4.635 and 4.640 p.p.m. The xylooligomers from xylanase-digested gux1/2/3 xylan exhibited resonances characteristic of the pentasaccharide reducing end sequence β-d-Xylp-(1→4)-β-d-Xylp-(1→3)-α-l-Rhap-(1→2)-α-d-GalpA-(1→4)-d-Xylp (H1 of α-GalA at 5.23 p.p.m., H1 of α-Rha at 5.07 p.p.m., H1 of 3-linked β-Xyl at 4.66 p.p.m., H4 of α-GalA at 4.33 p.p.m., H2 of α-Rha at 4.23 p.p.m. and H1 of unbranched β-Xyl at 4.46 p.p.m.) (Fig. 4B). In contrast, the 1H NMR spectrum of the xylooligomers from xylanase-digested xylan of XYXT1-expressing gux1/2/3 displayed not only the resonances of the pentasaccharide reducing end sequence, but also the resonances attributed to xylosyl residues of the xylan backbone as seen in the wild-type xylan, including H1 of reducing α-Xyl at 5.18 p.p.m., H1 of reducing β-Xyl at 4.58 p.p.m. and elevated resonances of H1 of unbranched β-Xyl at 4.46 p.p.m. (Fig. 4B). In addition, the XYXT1-OE xylooligomers exhibited prominent resonances at 4.64 p.p.m. of unknown identity (Fig. 4B). It was noted that there existed minor resonance signals at 5.41 p.p.m. in the 1H NMR spectra of xylooligomers from both XYXT1-OE in gux1/2/3 and the wild type (Fig. 4B). Although their exact identity was unknown, these resonance signals are distinct from the characteristic resonance peak for terminal Araf at 5.39 p.p.m. (Mazumder and York 2010, Chiniquy et al. 2012), indicating that they are unlikely to be attributed to Araf. Fig. 4 View largeDownload slide Structural analysis of xylan from the gux1/2/3 mutant expressing XYXT1. (A) Diagram of the xylooligomer generated from xylanase digestion of xylan from gux1/2/3 expressing XYXT1. (B) 1H NMR spectra of xylooligomers from the gux1/2/3 mutant, gux1/2/3 expressing XYXT1 and wild-type Arabidopsis. Resonance peaks are marked with the proton positions and the corresponding residue identities. HDO, hydrogen deuterium oxide. (C) Assignment of 1H and 13C NMR chemical shifts for the xylooligomer from gux1/2/3 expressing XYXT1 based on 1D and 2D NMR spectra. The numbering of positions of xylosyl residues is the same as in (A). Fig. 4 View largeDownload slide Structural analysis of xylan from the gux1/2/3 mutant expressing XYXT1. (A) Diagram of the xylooligomer generated from xylanase digestion of xylan from gux1/2/3 expressing XYXT1. (B) 1H NMR spectra of xylooligomers from the gux1/2/3 mutant, gux1/2/3 expressing XYXT1 and wild-type Arabidopsis. Resonance peaks are marked with the proton positions and the corresponding residue identities. HDO, hydrogen deuterium oxide. (C) Assignment of 1H and 13C NMR chemical shifts for the xylooligomer from gux1/2/3 expressing XYXT1 based on 1D and 2D NMR spectra. The numbering of positions of xylosyl residues is the same as in (A). Fig. 5 View largeDownload slide Determination of the glycosidic linkages in the xylooligomers from gux1/2/3 expressing XYXT1 based on the HSQC, H2BC, HMBC and ROESY spectra. The numbering of positions of xylosyl residues is the same as in Fig. 4A. (A) HSQC spectra showing the one-bond H1–C1 correlations between 1H and 13C of each Xyl. Note the H1–C1 signals for the side chain Xyl5 at 4.64 p.p.m. (1H) and 103.6 p.p.m. (13C). (B) H2BC spectra showing the two-bond H1–C2 correlations between 1H and 13C of each Xyl. (C) HMBC spectra showing interglycosidic cross-peaks between 1H and 13C. Note the cross-peak signals between H1 of Xyl5 at 4.64 p.p.m. and C2 of Xyl3 at 80.3 p.p.m., and those between H2 of Xyl3 at 3.520 p.p.m. and C1 of Xyl5 at 103.6 p.p.m. (D) ROESY spectrum showing interglycosidic cross-peaks between 1H and 1H. Note the cross-peak signals between H1 of Xyl5 at 4.64 p.p.m. and H2 of Xyl3 at 3.52 p.p.m. Fig. 5 View largeDownload slide Determination of the glycosidic linkages in the xylooligomers from gux1/2/3 expressing XYXT1 based on the HSQC, H2BC, HMBC and ROESY spectra. The numbering of positions of xylosyl residues is the same as in Fig. 4A. (A) HSQC spectra showing the one-bond H1–C1 correlations between 1H and 13C of each Xyl. Note the H1–C1 signals for the side chain Xyl5 at 4.64 p.p.m. (1H) and 103.6 p.p.m. (13C). (B) H2BC spectra showing the two-bond H1–C2 correlations between 1H and 13C of each Xyl. (C) HMBC spectra showing interglycosidic cross-peaks between 1H and 13C. Note the cross-peak signals between H1 of Xyl5 at 4.64 p.p.m. and C2 of Xyl3 at 80.3 p.p.m., and those between H2 of Xyl3 at 3.520 p.p.m. and C1 of Xyl5 at 103.6 p.p.m. (D) ROESY spectrum showing interglycosidic cross-peaks between 1H and 1H. Note the cross-peak signals between H1 of Xyl5 at 4.64 p.p.m. and H2 of Xyl3 at 3.52 p.p.m. Because of limited dispersion of 1H chemical shifts in 3.2–4.2 p.p.m., 2D NMR experiments were performed to identify and assign resonances for each Xyl residue in the XYXT1-OE xylooligomers. Heteronuclear single quantum coherence (HSQC) provides the correlation between each carbon and its attached protons. Fig. 5A shows the spectral region of the one-bond H1–C1 anomeric correlation of the XYXT1-OE xylooligomers. All the other proton–carbon one-bond correlations are shown in Supplementary Fig. S7A. H5eq, H5ax and H5–C5 correlations were easily identified from the edited version of HSQC, which shows -CH2 peaks in opposite phases (Supplementary Fig. S7B). Heteronuclear two-bond correlation (H2BC) (Nyberg et al. 2005) gives valuable help in spectra assignments as it contains only two-bond correlations. All C2 carbons in the H2BC spectrum of the XYXT1-OE xylooligomers could be assigned unequivocally. The spectral region of the two-bond H1–C2 correlation of the XYXT1-OE xylooligomers is shown in Fig. 5B and that of the two-bond H2–C1 correlation is shown in Supplementary Fig. S8. Both 1H-1H COSY (correlation spectroscopy) and HSQC-TOSCY (total correlation spectroscopy) were used to identify protons and carbons in each spin system. The H1 region of HSQC-TOCSY revealed H1 and all relayed carbons (C2, C3, C4 and C5) in each Xyl (Supplementary Fig. S9). Heteronuclear multiple bond correlation (HMBC) provides correlations between carbons and protons that are separated by two or more bonds. Although some of the H1–C2 correlations were missing due to vanishing 2J(H1–C2) coupling constants, the spectral data from HMBC (Fig. 5C) were quite complementary to those from H2BC and HSQC-TOCSY. The combination of the above 2D spectra led us to accomplish the assignment of the spectra of XYXT1-OE xylooligomers (Fig. 4C). All the assignments for Xyl residues in the XYXT1-OE xylooligomers are consistent with those for similar xylooligomers (branched with Araf or Araf-Xyl) previously published (Mazumder and York 2010, Chiniquy et al. 2012). The HMBC spectral region shown in Fig. 5C revealed how Xyl units were linked. The correlations between H1 of β-Xyl2 (4.460 p.p.m.) and C4 of β-Xyl1 (76.3 p.p.m.) and that between H1 of β-Xyl4 (4.460 p.p.m.) and C4 of β-Xyl3 (76.3 p.p.m.) indicate that Xyl4 and Xyl3 were β-(1→4) linked, and so were Xyl2 and Xyl1 (Fig. 5C, left panel). Although they were overlapping, the correlation between H4 of β-Xyl1 (3.767 p.p.m.) and C1 of β-Xyl2 (101.6 p.p.m.) and that between H4 of α-Xyl1 (3.741 p.p.m.) and C1 of β-Xyl2 (101.6 p.p.m.) confirmed that Xyl2 and Xyl1 were β-(1→4) linked (Fig. 5C, right panel). The correlation between H1 of β-Xyl3 (4.635 p.p.m.) and C4 of β-Xyl2 (76.3 p.p.m.) indicates that Xyl3 and Xyl2 are also β-(1→4) linked. Both the correlation between H1 of β-Xyl5 (4.640 p.p.m.; side chain Xyl in Fig. 4A) and C2 of β-Xyl3 (80.3 p.p.m.; branched Xyl in Fig. 4A) (Fig. 5C, left panel) and that between H2 of β-Xyl3 (3.520 p.p.m.) and C1 of β-Xyl5 (103.6 p.p.m.) (Fig. 5C, right panel) indicate that Xyl5 is attached to Xyl3 at the O-2 position. Furthermore, the spectrum from rotating-frame Overhauser effect spectroscopy (ROESY) revealed the correlation between H1 of β-Xyl5 and H2 of β-Xyl3 (Fig. 5D), further confirming that Xyl5 is attached to Xyl3 at the O-2 position. Based on the 1D and 2D NMR analyses, it was concluded that the XYXT1-OE xylooligomers contain the β-1,4-linked xylotetraose backbone that is substituted at O-2 with β-Xyl side chains (Fig. 4A). Recombinant XYXT1 catalyzes xylosyl substitutions of xylan The demonstration that XYXT1 was able to mediate the addition of Xyl side chains onto xylan when expressed in the Arabidopsis gux1/2/3 mutant indicates that XYXT1 is most probably a xylan xylosyltransferase. To prove its activity biochemically, we first attempted to express His-tagged XYXT1 in a secreted form in human embryonic kidney (HEK) 293F cells but failed to obtain any recombinant protein from the medium. We next expressed His-tagged full-length XYXT1 in HEK293F cells and isolated microsomes, which contained His-tagged XYXT1 as evidenced by Western blot analysis using anti-His antibody (Fig. 6A; see Supplementary Fig. S10 for whole gel images), for assaying its xylan xylosyltransferase activity. To do so, microsomes from cells transfected with the empty vector (control) or cells expressing XYXT1 were incubated with the xylotetraose acceptor and the xylosyl donor UDP-Xyl, and the reaction products were analyzed by MALDI-TOF-MS and NMR spectroscopy. The MALDI-TOF-MS analysis showed that the XYXT1-catalyzed reaction products had one new ion species at m/z 701 with an increase in mass of 132 Da over the mass of the xylotetraose acceptor (m/z 569) (Supplementary Fig. S11), indicating the addition of one Xyl residue onto xylotetraose. Examination of the 1H NMR spectra revealed the presence of resonance signals at 4.64 p.p.m. (Fig. 6C;Supplementary Fig. S12), which correspond to the side chain β-Xyl (Fig. 4B), in the reaction products of XYXT1-expressing microsomes but not in those of the control microsomes. These results confirm that XYXT1 is a xylosyltransferase adding Xyl side chains onto xylan. Fig. 6 View largeDownload slide Detection of xylan xylosyltransferase and arabinosyltransferase activities of recombinant XYXT1 and OsXAT2 proteins. His-tagged full-length XYXT1 protein was expressed in HEK293F cells and their microsomes were isolated for activity assay. His-tagged OsXAT2 protein was expressed in a secreted form in HEK293F cells, and the purified protein was used for arabinosyltransferase activity assay. (A) Immunoblot detection of His-tagged XYXT1 protein in HEK293F microsomes. Microsomal proteins (20 μg) were separated by SDS–PAGE and immunodetected with anti-His monoclonal antibody. Control, microsomal proteins isolated from HEK293F cells transfected with the empty vector. (B) SDS–PAGE detection of recombinant OsXAT2 protein (5 μg) that was visualized by Coomassie Blue staining. (C) 1H NMR spectra of the reaction products of the control microsomes (control) and the XYXT1-expressing microsomes (XYXT1) after incubation with the xylotetraose acceptor and UDP-Xyl. The resonance signals are marked with the proton positions and the corresponding residue identities. Note the appearance of the resonance signals at 4.64 p.p.m. corresponding to the side chain Xyl in the reaction products of the XYXT1-expressing microsomes. (D) 1H NMR spectra of the reaction products of the control microsomes (control), the XYXT1-expressing microsomes (XYXT1) and the recombinant OsXAT2 protein (OsXAT2) after incubation with the xylotetraose acceptor and UDP-Araf. The 1H NMR spectrum of xylooligomers from xylanase-digested rice xylan was included to show the resonance signals corresponding to the terminal Araf at 5.39 p.p.m. Note the presence of the terminal Araf signal at 5.39 p.p.m. in the reaction product catalyzed by OsXAT2 but not XYXT1. Fig. 6 View largeDownload slide Detection of xylan xylosyltransferase and arabinosyltransferase activities of recombinant XYXT1 and OsXAT2 proteins. His-tagged full-length XYXT1 protein was expressed in HEK293F cells and their microsomes were isolated for activity assay. His-tagged OsXAT2 protein was expressed in a secreted form in HEK293F cells, and the purified protein was used for arabinosyltransferase activity assay. (A) Immunoblot detection of His-tagged XYXT1 protein in HEK293F microsomes. Microsomal proteins (20 μg) were separated by SDS–PAGE and immunodetected with anti-His monoclonal antibody. Control, microsomal proteins isolated from HEK293F cells transfected with the empty vector. (B) SDS–PAGE detection of recombinant OsXAT2 protein (5 μg) that was visualized by Coomassie Blue staining. (C) 1H NMR spectra of the reaction products of the control microsomes (control) and the XYXT1-expressing microsomes (XYXT1) after incubation with the xylotetraose acceptor and UDP-Xyl. The resonance signals are marked with the proton positions and the corresponding residue identities. Note the appearance of the resonance signals at 4.64 p.p.m. corresponding to the side chain Xyl in the reaction products of the XYXT1-expressing microsomes. (D) 1H NMR spectra of the reaction products of the control microsomes (control), the XYXT1-expressing microsomes (XYXT1) and the recombinant OsXAT2 protein (OsXAT2) after incubation with the xylotetraose acceptor and UDP-Araf. The 1H NMR spectrum of xylooligomers from xylanase-digested rice xylan was included to show the resonance signals corresponding to the terminal Araf at 5.39 p.p.m. Note the presence of the terminal Araf signal at 5.39 p.p.m. in the reaction product catalyzed by OsXAT2 but not XYXT1. To examine whether XYXT1 also exhibits arabinosyltransferase activity, the XYXT1-expressing microsomes were incubated with xylotetraose and UDP-Araf, and the reaction products were examined with 1H NMR spectroscopy for the resonance signal characteristic of H1 of the terminal α-Araf at 5.39 p.p.m. (Pena et al. 2016). No signals at 5.39 p.p.m. were observed in the XYXT1-calayzed reaction products compared with the control (Fig. 6D). In contrast, incubation of recombinant His-tagged OsXAT2 (Fig. 6B; see Supplementary Fig. S10C for the whole gel image) with xylotetraose and UDP-Araf resulted in reaction products with a resonance signal at 5.39 p.p.m. that matched with that of the terminal α-Araf in the xylooligomers generated from xylanase digestion of rice xylan (Fig. 6D), which provided biochemical evidence demonstrating that OsXAT2 is a xylan arabinosyltransferase. Furthermore, we simultaneously expressed XYXT1 and OsXAT2 in the gux1/2/3 mutant (Supplementary Fig. S5A) to investigate whether XYXT1 could transfer Xyl residues onto the Araf side chains of xylan. The H1 of Araf side chains substituted at O-2 with Xyl residues in xylan exhibits a characteristic resonance peak at 5.54 p.p.m. as previously revealed by 1H NMR spectroscopy (Chiniquy et al. 2012, Pena et al. 2016). 1H NMR analysis showed that while the xylooligomers of the gux1/2/3 mutant expressing OsXAT2 had a resonance peak at 5.39 p.p.m. corresponding to H1 of terminal Araf side chains, those of gux1/2/3 expressing both XYXT1 and OsXAT2 displayed resonance peaks at 4.64 p.p.m. attributed to H1 of Xyl side chains in addition to those for the terminal Araf side chains (Fig. 7). No resonance signals at 5.54 p.p.m. for the H1 of 2-O-Araf were observed, indicating that XYXT1 was unable to add Xyl onto the Araf side chains of xylan. These results demonstrate that XYXT1 is a xylosyltransferase specifically catalyzing the addition of Xyl side chains directly onto the xylan backbone, an activity different from the arabinosyltransferase activity of OsXAT2 and the xylosyltransferase activity transferring Xyl onto O-2 of the Araf side chains of xylan. Fig. 7 View largeDownload slide 1H NMR spectra of xylooligomers released from xylanase-digested xylans of the gux1/2/3 mutant, gux1/2/3 expressing OsXAT2 (OsXAT2-OE in gux1/2/3), gux1/2/3 expressing XYXT1 (XYXT1-OE in gux1/2/3) and gux1/2/3 simultaneously expressing both OsXAT2 and XYXT1 (OsXAT2-OE XYXT1-OE in gux1/2/3). Resonance peaks are marked with the proton positions and the corresponding residue identities. Note the appearance of the resonance peak at 5.39 p.p.m. that is characteristic of H1 of terminal α-Araf and the resonance peak at 4.64 p.p.m. that corresponds to Xyl side chains in gux1/2/3 expressing both OsXAT2 and XYXT1 but a lack of any resonance signals at 5.54 p.p.m. attributed to Xyl attached to O-2 of α-Araf side chains of xylan. Fig. 7 View largeDownload slide 1H NMR spectra of xylooligomers released from xylanase-digested xylans of the gux1/2/3 mutant, gux1/2/3 expressing OsXAT2 (OsXAT2-OE in gux1/2/3), gux1/2/3 expressing XYXT1 (XYXT1-OE in gux1/2/3) and gux1/2/3 simultaneously expressing both OsXAT2 and XYXT1 (OsXAT2-OE XYXT1-OE in gux1/2/3). Resonance peaks are marked with the proton positions and the corresponding residue identities. Note the appearance of the resonance peak at 5.39 p.p.m. that is characteristic of H1 of terminal α-Araf and the resonance peak at 4.64 p.p.m. that corresponds to Xyl side chains in gux1/2/3 expressing both OsXAT2 and XYXT1 but a lack of any resonance signals at 5.54 p.p.m. attributed to Xyl attached to O-2 of α-Araf side chains of xylan. Discussion Our comprehensive analyses using MALDI-TOF-MS, ESI-MS, and 1D and 2D NMR have firmly established that the xylan from XYXT1-expressing gux1/2/3 is branched at O-2 with Xyl, indicating that XYXT1 is capable of transferring Xyl side chains onto xylan. Because Arabidopsis xylan lacks Xyl as side chains and the Arabidopsis genome has no close homologs of XYXT1, the addition of Xyl side chains in XYXT1-OE xylan can most probably be directly attributed to the activity of XYXT1. This result is further supported by the finding that recombinant XYXT1 expressed in mammalian cells possesses a xylosyltransferase activity catalyzing the transfer of Xyl residues from UDP-Xyl onto the xylotetraose acceptors. While recombinant OsXAT2 was shown to catalyze the addition of Araf residues from UDP-Araf onto xylotetraose, recombinant XYXT1 was unable to do so. In addition, XYXT1 was unable to transfer Xyl residues onto the Araf side chains of xylan when XYXT1 and OsXAT2 were co-expressed in the gux1/2/3 mutant. To our knowledge, this is the first report of a glycosyltransferase with an activity catalyzing the transfer of Xyl side chains directly onto the xylan backbone. The discovery of XYXT1 as a β-1,2-xylosyltransferase mediating 2-O-Xyl substitutions of xylan enriches our understanding of genes involved in the biosynthesis of xylan, the second most abundant polysaccharide in plant biomass. The presence of Xyl side chains directly attached to the xylan backbone has not been reported previously in grass species. The common sugar substitutions found in xylans from grasses, including wheat, barley, rice, and switchgrass, are 2-O-linked GlcA/MeGlcA, 3-O-linked Araf, 2,3-di-O-linked Araf, and 3-O-linked Araf that is further substituted at O-2 with Araf or Xyl (Araf–Araf or Araf–Xyl) (Hoffmann et al. 1992, Hoije et al. 2006, Mazumder and York 2010, Chiniquy et al. 2012, Lee et al. 2014). Glycosyl linkage analysis of rice xylan revealed that it had a high proportion of Xyl that was substituted at O-2 (Supplementary Fig. S13). Due to its low abundance, we were unable to perform structural analysis to confirm the presence of 2-O-linked Xyl side chains in rice xylan. Nevertheless, our finding that XYXT1 is a β-1,2-xylosyltransferase mediating xylosyl transfer at O-2 of xylan indicates that rice xylan is likely to be substituted with Xyl at O-2 in addition to other substituents. Sequence analysis showed that the genomes of several other grass species, including Zea mays, Sorghum bicolor, Brachypodium distachyon, Panicum virgatum, Triticum aestivum, Hordeum vulgare, Oropetium thomaeum and Setaria italica, harbor close homologs of rice XYXT1 (Supplementary Fig. S14). It is possible that Xyl substitutions in xylan are common in the cell walls of grasses. The presence of Xyl side chains directly attached to the xylan backbone has been reported in the seed mucilage polysaccharides of psyllium (Plantago ovata) and Arabidopsis (Fischer et al. 2004, Voiniciuc et al. 2015). In psyllium, a number of GT61 genes have been shown to be expressed during seed development and were suggested to be involved in the addition of side chain substituents in xylan (Phan et al. 2016). In Arabidopsis, MUCI21, which is a member of clade B of the GT61 family (Fig. 1A), has been proposed to be involved in the addition of Xyl residues directly onto the xylan backbone in seed mucilage (Voiniciuc et al. 2015). This proposed function of MUCI21 was based on glycosyl linkage analysis showing a reduction of 2,4-linked Xyl in the seed mucilage of the muci21 mutant. However, structural analysis of the muci21 mutant xylan was not performed and the proposed xylosyltransferase activity has not been biochemically confirmed (Voiniciuc et al. 2015). Furthermore, overexpression of MUCI21 driven by the CesA7 promoter in gux1/2/3 did not result in an addition of Xyl side chains onto xylan (Fig. 2A). Therefore, it is currently unknown whether MUCI21 is a β-1,2-xylosyltransferase adding Xyl side chains directly onto the xylan backbone. Among the 19 GT61 glycosyltransferases in the clade A of rice GT61 family (Fig. 1A;Anders et al. 2012), three of them, OsXAT2, OsXAT3 and XAX1, have previously been shown to be involved in xylan biosynthesis. OsXAT2 and OsXAT3 were proposed to be arabinosyltransferases mediating the arabinosyl transfer at O-3 of xylan based on their ability to add Araf residues onto xylan when expressed in wild-type Arabidopsis plants (Anders et al. 2012). XAX1 was believed to be a xylosyltransferase involved in disaccharide substitutions in rice xylan, i.e. adding 1,2-linked Xyl onto Ara that is attached at O-3 of the xylan backbone. This hypothesis was based on the observation that the xylooligomers released from xylanase digestion of xylan from the xax1 mutant lacked an ion peak corresponding to xylooligomers branched with Xyl–Araf disaccharide compared with those from the wild type (Chiniquy et al. 2012). Activity assay of recombinant OsXAT2 in our study confirmed that it is an arabinosyltransferase catalyzing the transfer of Araf residues onto the xylan backbone. The enzymatic activities of OsXAT3 and XAX1 as an arabinosyltransferase and a xylosyltransferase, respectively, remain to be biochemically confirmed. Our finding that XYXT1 exhibits a β-1,2-xylosyltransferase activity catalyzing Xyl transfer directly onto the xylan backbone indicates that it functions distinctly from OsXAT2, OsXAT3 and XAX1. It is interesting to note that AtXYLT, which is a clade C member of the Arabidopsis GT61 family, has been shown to be a β-1,2-xylosyltransferase transferring Xyl residues onto N-linked glycans (Pagny et al. 2003). Our identification of XYXT1 as a novel xylan β-1,2-xylosyltransferase enriches our understanding of roles of GT61 glycosyltransferases in xylan biosynthesis. Since clade A of the GT61 family is dramatically expanded in grasses and many of its members have no known dicot orthologs (Anders et al. 2012), it is possible that in addition to OsXAT2, OsXAT3, XAX1 and XYXT1, some other clade A GT61 members are also involved in xylan substitutions. Further functional characterization of other GT61 members will probably help us understand the complex biochemical process of xylan biosynthesis in general. Materials and Methods Expression analysis Rice (Oryza sativa var. japonica) tissues, including 2-week-old seedlings, different organs (leaves, stems, inflorescence and roots) of 2-month-old plants, and developing seeds, were collected for isolation of total RNA using a Qiagen RNA isolation kit. First-strand cDNAs were reverse-transcribed from the RNA and subsequently used as templates for real-time quantitative PCR analyses. The PCR primers for XYXT1 were 5'-tcaccggaggaggtggagggcctt-3' and 5'-gacatgaacatgaggtacctgcag-3'. The XYXT1 expression level was calculated by normalizing its PCR threshold cycle number with that of the rice EF1α reference gene and quantitated based on the standard curve of a plasmid control. The data were the mean of three biological replicates. Subcellular localization XYXT1 fused with the N-terminus of YFP was generated by fusion of its full-length cDNA with that of YFP, which was subsequently cloned under the Cauliflower mosaic virus (CaMV) 35S promoter in a modified pBI221 vector. The YFP-tagged XYXT1 was co-expressed with a Golgi marker, CFP-tagged FRA8 (Zhong et al. 2005), in Arabidopsis leaf protoplasts. In addition, GFP-tagged XYXT1 together with mCherry-tagged FRA8, both of which were driven by the CaMV 35S promoter in a modified pBI121 vector, were transformed into leaves of Nicotiana benthamiana by Agrobacterium-mediated infiltration (Zhong et al. 2017). The fluorescent signals in the transfected Arabidopsis protoplasts and tobacco leaf cells were recorded using a Zeiss LSM 510 META confocal microscope. At least 10 Arabidopsis protoplasts and 10 infiltrated tobacco leaves were imaged, and representative images are shown. Generation of transgenic gux1/2/3 plants expressing XYXT1 and OsXAT2 The full-length cDNAs of XYXT1 and OsXAT2 driven by the 2 kb CesA7 promoter were cloned into a modified pGPTV binary vector to create the XYXT1 and OsXAT2 expression constructs. For simultaneous expression of XYXT1 and OsXAT2, the expression cassette composed of the 2 kb CesA7 promoter, the full-length OsXAT2 cDNA and the nopaline synthase terminator was inserted into the multiple cloning site of the XYXT1 expression construct to create the XYXT1–OsXAT2 expression construct. The XYXT1, OsXAT2 and XYXT1–OsXAT2 expression constructs were introduced into Arabidopsis gux1/2/3 mutant plants by Agrobacterium-mediated transformation. Transgenic plants were selected by sowing transformed seeds on agar plates containing hygromycin, and >100 independent transgenic plants were generated and used for subsequent gene expression and cell wall analyses. For gene expression analysis, RNA was isolated from stems of three separate pools of 10 independent transgenic plants (each of them represented a biological replicate) for each construct using a Qiagen RNA isolation kit. The isolated RNA was used for examination of expression of XYXT1 and OsXAT2 in the transgenic gux1/2/3 plants expressing XYXT1 and/or OsXAT2 by the reverse transcription–PCR approach. Generation of xylooligomers by xylanase digestion Mature inflorescence stems from the first-generation transgenic gux1/2/3 plants expressing XYXT1 and/or XAT2 were used for chemical and structural analyses of xylan. In order to obtain sufficient cell wall materials, stems were collected from three separate pools of transgenic plants, with each pool containing 30 independent first-generation transgenic plants. Thus, each pool represents a biological replicate. Alcohol-insoluble cell walls were isolated as previously described (Zhong et al. 2005). The cell walls were first extracted with ammonium oxalate to remove pectins and then extracted for xylan with 1 N KOH in the presence of sodium borohydride. The KOH-extracted xylan was then digested with endo-1,4-β-xylanase M6 from a rumen microorganism (Megazyme) to release xylooligomers, which were passed through a Sephadex G25 column to remove monomers and dimers (Zhong et al. 2005). Glycosyl linkage analysis The xylooligomers released from xylanase digestion of cell walls were first permethyalted with potassium dimsyl anion and methyl iodide, and then reduced using sodium borodeuteride according to Heiss et al. (2009). After re-permethylation with methyl iodide and sodium hydroxide (Ciucanu and Kerek 1984), the permethylated xylooligomers were hydrolyzed by trifluoroacetic acid and further derivatized to alditol acetates. The alditol acetates of sugars were analyzed on a PerkinElmer Clarus 500 gas–liquid chromatograph equipped with a silica capillary column (30 m×0.25 mm). Xylooligomers isolated from three separate pools of samples were analyzed, and representative data are shown. ESI-MS The xylooligomers were first permethylated with methyl iodide and sodium hydroxide according to the procedure described by Ciucanu and Kerek (1984) and then subjected to multiple-stage ESI-MS analysis using a Bruker Esquire 3000 Plus ion trap instrument. The MS/MS data were collected using manual mode with an isolation window width of 4 and fragmenation amplitude of 1. Xylooligomers isolated from three separate pools of samples were permethylated and analyzed. MALDI-TOF-MS The xylooligomers were subjected to MALDI-TOF-MS analysis using a Burker Autoflex TOF mass spectrometer in reflection mode (Zhong et al. 2005). The spectra were the averages of 250 laser shots. Xylooligomers isolated from three separate pools of samples were analyzed, and representative spectra are shown. NMR spectroscopy The xylooligomers were analyzed with a Varian Inova 500 MHz spectrometer. One-dimensional and two-dimensional (HSQC, H2BC, HMBC, ROESY and COSY) NMR spectra were recorded using standard Varian pulse sequences. The proton positions and residue identities in the NMR spectra were assigned based on our 1D and 2D NMR spectral data and the published NMR spectral data for xylans (Pena et al. 2007, Mazumder and York 2010, Chiniquy et al. 2012). Xylooligomers isolated from three separate pools of samples were examined. Recombinant protein expression We attempted to express two forms of recombinant XYXT1 protein heterologously in HEK293F cells; one was in a secreted form with the removal of the N-terminal transmembrane domain and the other was the full-length protein. For production of secreted XYXT1, the XYXT1 cDNA with deletion of the N-terminal transmembrane domain was cloned between the murine Igκ chain leader sequence (for protein secretion) and the c-myc epitope and a six tandem histidine tag in the pSecTag2 mammalian expression vector (Invitrogen), and the expression construct was tranfected into HEK293F cells using the Invitrogen FreeStyle 293 Expression System according to the manufacturer’s protocol. After 5 d of culture of the transfetced cells, the culture medium was passed through a nickel resin column for purification and detection of expressed recombinant proteins. For production of the full-length recombinant XYXT1, the full-length XYXT1 cDNA was cloned into the pcDNA3.1/myc-His mammalian expression vector (Invitrogen), and the expression construct was tranfected into HEK293F cells. After 4 d of culture, the transfected cells were collected for microsome isolation (Bozidis et al. 2007). The presence of XYXT1 in the isolated microsomes was examined by SDS–PAGE and immunodetection with the anti-HIS monoclonal antibody. Recombinant OsXAT2 was expressed in a secreted form by cloning the OsXAT2 cDNA with deletion of the N-terminal transmembrane domain between the murine Igκ chain leader sequence (for protein secretion) and the c-myc epitope and six tandem histidine tag in the pSecTag2 mammalian expression vector, and transfecting into HEK293F cells. The secreted recombinant His-tagged OsXAT2 protein was purified by passing the culture medium through a nickel resin column as previously described (Zhong et al. 2017) and examined by SDS–PAGE. Xylosyltransferase activity assay The isolated HEK293F microsomes containing XYXT1 were examined for xylosyltransferase activity by incubating 200 μg of microsomal proteins with the xylotetraose acceptor (200 μg), 1 mM UDP-Xyl (CarboSource), 50 mM HEPES buffer (pH 7.0), 1 mM MgCl2 and 0.5% Triton-X100 at 21°C for 16 h. After passing through Dowex 1X4 resin, the reaction products were analyzed using MALDI-TOF-MS as well as with NMR spectroscopy for the presence of the side chain Xyl. Arabinosyltransferase activity assay The purified recombinant OsXAT2 protein (100 μg) was incubated with xylotetraose (200 μg) and 0.3 mM UDP-Araf (Peptide Institute) in 50 mM HEPES buffer (pH 7.0) at 37°C for 16 h for detection of xylan arabinosyltransferase activity. After passing through the Dowex 1X4 resin, the reaction products were analyzed with NMR spectroscopy for the presence of the side chain Araf. Supplementary Data Supplementary data are available at PCP online. 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Google Scholar CrossRef Search ADS PubMed  Abbreviations Abbreviations Araf arabinofuranose AXY altered xyloglucan CaMV Cauliflower mosaic virus CFP cyan fluorescent protein COSY homonuclear correlation spectroscopy ESI-MS electrospray ionization-mass spectrometry ESK eskimo FRA fragile fiber GalA galacturonic acid GFP green fluorescent protein GlcA glucuronic acid GT glycosyltransferase GUX glucuronic acid substitution of xylan GXM glucuronoxylan methyltransferase H2BC heteronuclear two-bond correlation HEK human embryonic kidney HMBC heteronuclear multiple bond correlation HSQC heteronuclear single quantum correlation IRX irregular xylem MALDI-TOF-MS matrix-assisted laser desorption ionization-time-of-flight mass spectrometry MeGlcA 4-O-methyl-glucuronic acid MUCI mucilage-related NMR nuclear magnetic resonance Rha rhamnose ROESY rotating-frame Overhauser effect spectroscopy RWA reduced wall acetylation TBL trichome birefringence-like TOSCY total correlation spectroscopy XAT xylan arabinosyltransferase XAX xylosyl arabinosyl substitution of xylan Xyl xylose XYXT xylan xylosyltransferase YFP yellow fluorescent protein © The Author(s) 2018. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com

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Plant and Cell PhysiologyOxford University Press

Published: Mar 1, 2018

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