Open Access

Functional conservation and divergence of Miscanthus lutarioriparius GT43 gene family in xylan biosynthesis

BMC Plant BiologyBMC series – open, inclusive and trusted201616:102

https://doi.org/10.1186/s12870-016-0793-5

Received: 13 January 2016

Accepted: 21 April 2016

Published: 26 April 2016

Abstract

Background

Xylan is the most abundant un-cellulosic polysaccharides of plant cell walls. Much progress in xylan biosynthesis has been gained in the model plant species Arabidopsis. Two homologous pairs Irregular Xylem 9 (IRX9)/9L and IRX14/14L from glycosyltransferase (GT) family 43 have been proved to play crucial roles in xylan backbone biosynthesis. However, xylan biosynthesis in grass such as Miscanthus remains poorly understood.

Results

We characterized seven GT43 members in M. lutarioriparius, a promising bioenergy crop. Quantitative real-time RT-PCR (qRT-PCR) analysis revealed that the expression of MlGT43 genes was ubiquitously detected in the tissues examined. In-situ hybridization demonstrated that MlGT43A-B and MlGT43F-G were specifically expressed in sclerenchyma, while MlGT43C-E were expressed in both sclerenchyma and parenchyma. All seven MlGT43 proteins were localized to Golgi apparatus. Overexpression of MlGT43A-E but not MlGT43F and MlGT43G in Arabidopsis irx9 fully or partially rescued the mutant defects, including morphological changes, collapsed xylem and increased xylan contents, whereas overexpression of MlGT43F and MlGT43G but not MlGT43A-E complemented the defects of irx14, indicating that MlGT43A-E are functional orthologues of IRX9, while MlGT43F and MlGT43G are functional orthologues of IRX14. However, overexpression of all seven MlGT43 genes could not rescue the mucilage defects of irx14 seeds. Furthermore, transient transactivation analyses of MlGT43A-E reporters demonstrated that MlGT43A and MlGT43B but not MlGT43C-E were differentially activated by MlSND1, MlMYB46 or MlVND7.

Conclusion

The results demonstrated that all seven MlGT43s are functionally conserved in xylan biosynthesis during secondary cell wall formation but diversify in seed coat mucilage xylan biosynthesis. The results obtained provide deeper insight into xylan biosynthesis in grass, which lay the foundation for genetic modification of grass cell wall components and structure to better suit for next-generation biofuel production.

Keywords

Miscanthus lutarioriparius Glycosyltransferase family 43 Xylan biosynthesis Secondary cell wall Seed coat mucilage

Highlight

The functional roles of M. lutarioriparius GT43 family genes are conserved and diversified in xylan biosynthesis.

Background

Plant cell walls are complex and dynamic structures composed mainly of polysaccharides (cellulose, hemicellulose and pectin), phenolic compounds (lignin) and glycoproteins [1]. Xylans are the major hemicellulosic saccharides in the primary cell walls of grasses and the secondary cell walls of grasses and dicots, ranking as the second most abundant polysaccharides in nature [2]. Xylans are mainly composed of a linear backbone of β-(1,4)-linked D-xylosyl residues with various sidechains that vary among different plant species and tissue types [3]. Based on the sidechain substitutions, xylans can generally be classified as (methyl)glucuronoxylan (GX), arabinoxylan (AX), and glucuronoarabinoxylan (GAX) [3]. As the major xylan in dicot plants, GX is usually decorated with α-1,2-linked glucuronic acid (GlcA) or 4-O-methylglucuronic acid (MeGlcA), and acetylated at C-2 or C-3 [3, 4]. AX has α-1,3-linked arabinose (Ara) sidechains, and presents as typical hemicellulose components in starchy endosperm of cereal grains [3]. GAX is the predominant hemicellulose in grass cell walls, and has sidechains of α-1,2 or α-1,3-linked arabinose (Ara) and GlcA residues [3]. In addition, GX in angiosperm and GAX in several gymnosperm species contain a tetrasaccharide sequence [β-D-Xyl-(1,3)-α-L-Rha-(1,2) -α-D-GalA-(1,4)-D-Xyl] at the reducing end [57]. However, no such oligosaccharide has yet been identified for xylans in grasses [8, 9]. It is still in controversy whether this oligosaccharide functions as a primer or as a terminator in xylan backbone biosynthesis [10].

Several xylan-related mutants named as irregular xylem (irx) due to secondary cell wall deficiencies have been identified in Arabidopsis by reverse genetics approaches [11, 12]. Most of these identified genes encode putative glycosyltransferases (GT) that are involved in the biosynthesis of xylan. IRX9/IRX9L and IRX14/IRX14L from GT43 family as well as IRX10/IRX10L from GT47 family are responsible for the biosynthesis of xylan backbone [1319]. IRX9, IRX10 and IRX14 play dominant roles in xylan backbone biosynthesis, and mutations in each gene lead to reduced xylan content and growth defect. By contrast, IRX9L, IRX10L and IRX14L seem to perform partially redundant roles together with their close homologues, as loss-function of these genes have no observable phenotypes and they only partially complement the phenotypes of irx9, irx10 and irx14 mutants. In addition, double mutations in each gene pairs dramatically enhance the phenotypes of the single mutant [13, 14, 18, 19]. However, a recent study proposed that these gene pairs play equivalent roles in xylan biosynthesis [20]. Furthermore, two members of DUF579 domain-containing proteins, IRX15 and IRX15L, are essential for the normal elongation of xylan backbone [21, 22]. IRX7/IRX7L from GT47 family, IRX8 and PARVUS from GT8 family are required for the biosynthesis of the reducing end oligosaccharide [5, 2326]. Mutations in these genes lead to almost entirely loss of the tetrasaccharide accompanied with reduced xylan contents, while the xylan backbone elongation activity is not disturbed [5, 2326].

Recently biochemical and genetic studies have also led to the identification of several genes that are required for the sidechain modifications of xylan. For instance, GLUCURONIC ACID SUBSTITUTION OF XYLAN (GUX) 1, GUX2, GUX4 and GUX5 from GT8 family are proposed to catalyze the addition of GlcA and MeGlcA sidechains to GX backbone [20, 2729]. GLUCURONOXYLAN METHYLTRANSFERASE (GXMT) 1, a DUF579 domain protein, has been revealed to be responsible for the 4-O-methylation of GlcA residues in GX [30]. In addition, ESKIMO1/TRICHOME BIREFRINGENCE-LIKE (TBL) 29, a DUF231 domain protein, is required for the O-acetylation of xylan backbone [31, 32]. Moreover, several XYLAN ARABINOSYLTRANSFERASE (XAT), members of GT61 family proteins from rice and wheat, are responsible for transferring the Ara residues onto xylan backbone [33, 34]. XYLOSYL ARABINOSYL SUBSTITUTION OF XYLAN (XAX) 1, another member from GT61 family in rice, is involved in transferring the Xyl residues in β-Xylp-(1 → 2)-α-Araf -(1 → 3) sidechain [34].

Grass xylans have several unique features compared to those from dicots. GX is the most abundant hemicellulose in dicots, while grass xylans usually contain many Ara residue substitutions and thus are termed as GAX or AX [3]. Even though there are clear differences in xylan structure between grasses and dicots, accumulating evidence implicates that GT43 members are functionally conserved in xylan biosynthesis between dicots and monocots. For example, four rice IRX9 orthologues OsGT43A, OsGT43C, OsGT43E and OsGT43F can fully or partially rescue the xylan defect phenotype of irx9, while OsGT43J is able to complement the xylan defect phenotype of irx14 in Arabidopsis [35, 36]. Three poplar IRX9 orthologues PtrGT43A, PtrGT43B and PtrGT43E are capable of rescuing the defects of irx9, whereas the other two IRX14 orthologues PtrGT43C and PtrGT43D are able to complement the phenotypes of irx14 [37]. Furthermore, it has been demonstrated that rice and poplar GT43 family proteins are evolved to retain two functionally non-redundant groups involved in xylan backbone biosynthesis [3638]. Additionally, two GT43 members GhGT43A1 and GhGT43C1 from cotton have been revealed to be functional orthologues of Arabidopsis IRX9 and IRX14, respectively, and have been shown to participate in xylan backbone biosynthesis during fiber development [39].

Miscanthus is a perennial rhizomatous grass with superior characteristics as a bioenergy plant such as high photosynthetic efficiency, low fertilizer and water demand, wide adaptability and high biomass yield. It has attracted increasing attention and concern worldwide as an ideal lignocellulosic feedstock for next-generation bioenergy production [4042]. Hemicelluloses account for 29–42 % of the Miscanthus cell walls [43], and the most abundant hemicellulosic polysaccharide is AX [43, 44], which is also the typical xylan in grass cell walls [45]. It has been shown that hemicellulose exerts dominant and positive effects on biomass digestibility by affecting cellulose crystallinity after pre-treatment with alkali or acid [46]. Although much progress has been gained in the understanding of xylan biosynthesis in the model plant Arabidopsis thaliana, relatively less is known about xylan biosynthesis in grasses. To the best of our knowledge, none of GTs responsible for the biosynthesis of xylan has been isolated and characterized in Miscanthus as yet.

To provide insight into xylan biosynthesis in Miscanthus, we identified seven GT43 genes in M. lutarioriparius and characterized their functional roles in xylan biosynthesis. Complementation assay including plant height, irregular xylem cells in stem cross sections and xylose content measurements revealed that MlGT43 genes have evolved into two distinct functional groups, in which MlGT43A-E are orthologous to IRX9, while MlGT43F and MlGT43G are orthologous to IRX14. Furthermore, our results indicated that substantial divergence has occurred in the functional roles of MlGT43s during xylan biosynthesis especially in seed coat mucilage. The results presented deepened our understanding of xylan biosynthesis in grasses and may lay the foundation for future genetic manipulation of Miscanthus cell wall structure and components.

Results

Isolation of GT43 genes in M. lutarioriparius

To identify the GT43 family in M. lutarioriparius, the amino acid sequences of four Arabidopsis GT43 members were used as query baits to BLAST against the draft genome sequences of M. lutarioriparius, and seven GT43 orthologous genes were identified. Specific primers were designed and seven candidate genes encoding putative GT43 proteins designated as MlGT43A to MlGT43G were obtained by PCR in M. lutarioriparius. As indicated in Fig. 1a, all seven proteins had a conserved structure and ranged in size from 358 to 451 amino acids. Pairwise comparison of the amino acid sequences showed that MlGT43C and MlGT43D shared the highest sequence similarity (75.3 %), while MlGT43D and MlGT43G shared the lowest sequence similarity (43.3 %) (Fig. 1b).
Fig. 1

Sequence alignment, identities and gene structure of MlGT43. a Sequence alignment of seven MlGT43 proteins. b Sequence identities and similarities among MlGT43 proteins. The highest and lowest in sequence identity and similarity are outlined. c Gene structure of MlGT43 genes. Exons and introns are represented by filled boxes and lines, respectively. The sizes of exons and introns are proportional to the scale at bottom

Deduced MlGT43A and MlGT43B amino acid sequences shared the highest sequence identities with Arabidopsis IRX9 (37 and 41 %), and MlGT43C-E shared relatively higher sequence identities with IRX9L (42, 48 and 53 %) than with IRX14 or IRX14L. By contrast, MlGT43F and MlGT43G proteins had the highest sequence identities with IRX14 and IRX14L (59 and 37 %) than with IRX9 (Additional file 1: Table S1).

Furthermore, the gene structure of each MlGT43 was obtained through the alignment of their coding sequences and genomic sequences (Fig. 1c). All MlGT43 genes shared very similar gene structure in terms of intron number and exon length. They all contained three exons and two introns. In addition, the intron phases with respect to codons were well conserved among different MlGT43 genes.

Phylogenetic analysis of GT43 members from M. lutarioriparius and other plant species

To gain insight into the origin and evolutionary history of the GT43 family, we further identified GT43 proteins from nine other currently sequenced genomes that cover a wide spectrum of plant taxonomic groups including moss (Physcomitrella patens), spikemoss (Selaginella moellendorffii), the monocot angiosperms (Oryza sativa, Brachypodium distachyon and Sorghum bicolor), and the dicot angiosperms (Arabidopsis thaliana, Populus trichocarpa, Medicago truncatula and Vitis vinifera). Totally 57 GT43 proteins were identified from these nine plant species (Additional file 2) and a phylogenetic tree was constructed with these GT43 proteins (Fig. 2a). The phylogenetic tree separated all GT43 proteins into three distinct subfamilies designated as IRX9, IRX9L and IRX14/IRX14L, which was similar to the previous studies [13, 38]. The seven GT43 proteins from Miscanthus were classified into the three subfamilies. MlGT43A and MlGT43B were clustered into the IRX9 subfamily, MlGT43C-E were classified into the IRX9L subfamily, while MlGT43F and MlGT43G were distributed into the IRX14/IRX14L subfamily.
Fig. 2

Phylogenetic analysis of GT43 family from Miscanthus and nine other plant species. a Phylogenetic tree of 64 GT43 proteins from ten plant species. The sequences of 64 GT43 proteins were aligned using ClustalW and their phylogenetic relationship was analyzed using the Neighbor-Joining method in MEGA 6.0. Numbers at nodes indicate the percentage bootstrap scores and only bootstrap values higher than 50% from 1,000 replicates are shown. MlGT43 proteins are marked with asterisks. b Distribution of the GT43 proteins from selected plant lineages. Pp, Physcomitrella patens; Sm, Selaginella moellendorffii; At, Arabidopsis thaliana; Pt, Populus trichocarpa; Mt, Medicago truncatula; Vv, Vitis vinifera; Os, Oryza sativa; Bd, Brachypodium distachyon; Sb, Sorghum bicolor; Ml, Miscanthus lutarioriparius

The distribution of the three subgroups among the ten plant species varied within each subfamily (Fig. 2b). It is noteworthy that the number of GT43 proteins in the monocot species seems to be higher than that of the dicot species, at least it is the case for the selected plant species. For example, there were 10, 10, 10 and 7 members in the monocot species O. sativa, B. distachyon, S. bicolor and M. lutarioriparius, whereas the number of GT43 in the dicot species A. thaliana, P. trichocarpa, M. truncatula and V. vinifera were 4, 7, 4 and 4, respectively. In addition, the members of IRX9 and IRX9L subfamilies in the monocot angiosperms were generally higher than those of the dicot species. For instance, the IRX9 subfamily accounted for 40, 40, 40 and 28 % in the monocot species O. sativa, B. distachyon, S. bicolor and M. lutarioriparius, respectively, whereas the percentages of the IRX9 subfamily in the dicot species A. thaliana, P. trichocarpa, M. truncatula and V. vinifera were 25, 25, 28 and 25 %, respectively. Noticeably, no IRX9 subfamily members were present in P. patens and S. moellendorffii.

MlGT43 genes are ubiquitously expressed and have specific expressions in stem cells

To investigate the expression patterns of MlGT43 genes, we first used the quantitative real-time RT-PCR (qRT-PCR) to examine their expressions across seven different tissues. As shown in Fig. 3a, all seven MlGT43 genes were ubiquitously expressed in seven different tissues examined, but their relative expression levels differed significantly. For example, MlGT43A, MlGT43D and MlGT43E genes shared similar expression patterns with predominant expressions in leaf, whereas the expressions of MlGT43B and MlGT43G genes were relatively lower. MlGT43C and MlGT43F genes were broadly expressed in the majority of the tissues, while especially higher expressions were detected in the basal stem. Furthermore, all MlGT43 genes except MlGT43B exhibited higher expressions in the basal stem than in the upper stem.
Fig. 3

Expression patterns of MlGT43 genes. a Expression analysis of MlGT43 genes by qRT-PCR. Relative expression levels in seven tissues were normalized using MlACT11 as the reference gene. For each gene, the tissues with the lowest expression level are set to 1. Data are the means ± SE of three biological replicates. b In situ localization of MlGT43 genes in Miscanthus stem. Cross-sections of stems were hybridized with digoxigenin-labeled antisense MlGT43A (b), MlGT43B (c), MlGT43C (d), MlGT43D (e), MlGT43E (f), MlGT43F (g), MlGT43G (h), or sense (i) RNA probes, and the hybridization signals were detected with alkaline phosphatase-conjugated antibody and were shown as purple color. pv, pitted vessel; x, xylem; ph, phloem; pa, parenchyma; sc, sclerenchyma. Bar = 100 μm

To obtain more detailed expression patterns of MlGT43 genes in specific cell types, we further performed the in situ hybridization analysis to examine their expressions in the 11th internode of the stem. For all seven genes, intense hybridization signals were observed in sclerenchyma cells and vascular bundle fiber cells, the cell types undergoing secondary wall thickening (Fig. 3b-h). Moreover, relatively weak hybridization signals were also observed for MlGT43C-E in parenchyma cells. By contrast, the control hybridized with sense probes did not show any signals in vascular bundle or sclerenchyma cells (Fig. 3g). These results suggest that MlGT43 genes may participate in diverse plant development processes especially in the secondary cell wall formation.

MlGT43 members are targeted to Golgi apparatus

To investigate the subcellular localization of MlGT43 proteins, we constructed fluorescently tagged fusion proteins by fusing Yellow Fluorescent Protein (YFP) to the C terminus of each MlGT43 protein. The recombinant constructs were transiently co-expressed in Nicotiana benthamiana leaf epidermal cells with the Golgi marker Man49-mCherry [47]. Examination of the fluorescent signals revealed that seven YFP-tagged MlGT43s all exhibited a punctate distribution, and the pattern perfectly matched with that of Man49-mCherry (Fig. 4), whereas the YFP control protein had signals throughout the cytoplasm and the nucleus (data not shown). The co-localization of MlGT43 proteins with the Golgi marker indicate that MlGT43s are Golgi-localized proteins.
Fig. 4

Subcellular localization of YFP-tagged MlGT43 proteins. YFP-tagged MlGT43 proteins were transiently expressed in leaf epidermal cells of Nicotiana benthamiana, and their subcellular locations were examined with a laser scanning confocal microscope. The single-plane confocal micrographs of MlGT43 proteins fused with C-terminal YFP, the Golgi marker Man49-mCherry, differential interference contrast (DIC) image, and merged YFP and mCherry channels are shown. Note the superimposition of YFP-MlGT43s and Man49-mCherry signals. Bar = 20 μm

MlGT43 genes rescue the morphological defects of irx9 or irx14

To reveal whether MlGT43 genes perform the same functions as IRX9 and IRX14 orthologues in Arabidopsis, we examined their abilities to rescue the morphological defects of irx9 and irx14. Due to the severely dwarfed plant stature and poor fertility of homozygous irx9 plants [5], we used the heterozygous line for the transformation with the 35S:MlGT43s constructs. Positive transgenic lines for each construct were tested for the presence of MlGT43 genes in homozygous irx9 and irx14 background by semi-quantitative RT-PCR (Fig. 5a). Homozygous T2 plants from at least two independent transformants with higher expressions were used for the phenotypic analyses.
Fig. 5

Expression of seven MlGT43 genes in Arabidopsis irx9 or irx14 mutants. a RT-PCR detection of the MlGT43 transcripts in the complemented irx9 or irx14 plants. The Arabidopsis UBQ10 gene was used as a reference. b, d, f Phenotype of four-, six- and eight-week-old soil-grown WT, irx9 and MlGT43s complemented irx9 plants. c, e, g Phenotype of four-, six- and eight-week-old soil-grown WT, irx14 and MlGT43s complemented irx14 plants. h Stem height of the WT, irx9 and MlGT43s complemented irx9 plants through 40, 47, 57 days of growth. i Stem height of the WT, irx14 and MlGT43s complemented irx14 plants through 40, 47, 57 days of growth. Data are means ± SD from at least twelve plants for each background. Two homozygous T3 lines of MlGT43s complemented irx9 or irx14 were used in the analysis

The growth of the irx9 plants was characterized by the dwarf stature, smaller rosette size and dark-green leaves under our growth conditions, which is similar to the previous reports [5, 12]. Overexpression of MlGT43A-E genes in irx9 displayed an intermediate growth phenotype between the mutant and the wild type (WT) in terms of rosette size and inflorescence height. The rosette diameters of the complemented plants increased by two- to three-fold, and the inflorescence stems were two- to four-fold taller compared to the irx9 plants after four weeks of growth (Fig. 4b, d), suggesting that the irx phenotype may be partially complemented in these transformants. By contrast, transformants of MlGT43F or MlGT43G overexpression in irx9 mutant exhibited a morphology resembled of the irx9 mutant, indicating that MlGT43F and MlGT43G were unable to complement the irx9 phenotypes (Fig. 4b, d, f).

The growth of irx14 mutant did not show any other obvious phenotypes except for a slight reduction in plant height compared to WT (Fig. 4c, e) as described previously [14]. The height of all MlGT43 complemented irx14 plants was indistinguishable from that of irx14 or WT plants, thus it is hard to evaluate the ability of seven MlGT43 genes to complement the irx14 mutant merely judged from their growth phenotypes. Subsequently, xylem morphology, xylan immunolocalization and cell wall monosaccharide compositions will be further examined in the transgenic plants to determine the abilities of MlGT43s to complement the irx14 phenotypes.

Microscopic analysis of the secondary cell wall

To demonstrate whether the morphological complementation by MlGT43 genes could be accompanied with the rescue of xylem morphology, the basal inflorescence stems of each complemented line were sectioned and observed by light and transmission electron microscopy. Toluidine blue O (TBO) staining was performed on stem sections of WT, irx9, irx14 and complemented plants to examine the morphology of secondary cell walls. As shown in Fig. 6, all MlGT43A-E complemented irx9 plants exhibited dramatically thickened cell walls in interfascicular fibers compared to irx9. The majority of xylem vessels in MlGT43A and MlGT43B complemented irx9 plants were characterized by large open round cells comparable to those in WT plants (Fig. 6C1, D1, L1, M1). In addition, the xylem vessels of MlGT43C, MlGT43D or MlGT43E complemented irx9 plants were usually smaller in size with occasionally irregular shapes, probably due to the not fully thickened cell walls compared to WT (Fig. 6 E1-G1, N1-P1). By contrast, overexpression of MlGT43F or MlGT43G in irx9 could not restore the collapsed vessels and the weakly thickened interfascicular fibers in irx9 (Fig. 6 H1, I1, Q1, R1), which is in consistency with their growth phenotypes (Fig. 5b, d).
Fig. 6

Morphology of xylem and interfascicular fibers of WT, irx9, irx14 and MlGT43 complemented plants. Stems of eight-week-old plants were sectioned (8 μm-thick) and stained with TBO for examination of the morphology of vessels, xylary fibers and interfascicular fibers. A1-I1, interfasicular fibers for WT, irx9 and MlGT43 complemented irx9 plants. A2-I2, interfasicular fibers for WT, irx14 and MlGT43 complemented irx14 plants. J1-R1, xylary fibers and vessels for WT, irx9 and MlGT43 complemented irx9 plants. J2-R2, xylary fibers and vessels for WT, irx14 and MlGT43 complemented irx14 plants. At least two homozygous T3 lines of MlGT43s complemented irx9 or irx14 were used in the analysis. Images for each tissue are set as the same magnification. Bar = 50 μm

The homozygous irx14 plants also showed collapsed xylem vessels and thinner secondary cell walls, which is consistent with the previous study [15]. Overexpression of either MlGT43F or MlGT43G could almost fully rescue the irx phenotype of irx14 as witnessed by a relatively less irregular vessel cells compared to irx14. However, the complemented lines still retained relatively thinner cell walls in both interfascicular fibers and xylem vessels compared to WT (Fig. 6 H2, I2, Q2, R2). By contrast, overexpression of MlGT43A-E in irx14 displayed a collapsed xylem vessel and thinner fiber cell wall phenotype that was indistinguishable from the irx14 mutant (Fig. 6 C2-G2, L2-P2), indicating that MlGT43A-E genes could not rescue the defects of irx14.

Transmission electron microscopy confirmed that the thickness of interfascicular fiber cell walls of the MlGT43A-E complemented irx9 plants was intermediate between irx9 and WT (Fig. 7a and Table 1). Meanwhile, the wall thickness of xylary fibers and vessels in MlGT43A-E complemented irx9 lines was also significantly increased but not restored to the WT level. By contrast, the wall thickness of interfascicular fibers, xylary fibers and vessels of MlGT43F or MlGT43G complemented irx9 plants was similar to that of the irx9 mutant (Fig. 7a and Table 1). The wall thickness of interfascicular fibers, xylary fibers and vessels for MlGT43F or MlGT43G complemented irx14 plants was intermediate between irx14 and WT, while the wall thickness for MlGT43A-E complemented irx14 lines was similar to that of irx14 (Fig. 7b and Table 1). Together, these results indicate that MlGT43A-E can fully or partially rescue the irx9 but not the irx14 phenotypes, while MlGT43F and MlGT43G can complement the irx14 but not the irx9 defects.
Fig. 7

Transmission electron micrographs of stem sections of WT, irx9, irx14 and MlGT43 complemented plants. Stems of eight-week-old plants were cut into 70 nm-thick sections and observed with transmission electron microscope, indicating increased fiber and vessel wall thickness by expression of MlGT43 genes. a, Transmission electron micrographs of stem sections of MlGT43 complemented irx9 lines. b, Transmission electron micrographs of stem sections of MlGT43 complemented irx14 lines. At least two homozygous lines of MlGT43 complemented irx9 or irx14 were used in the analysis. ve, vessels, xf, xylary fibers. Bar = 5 μm

Table 1

Cell wall thickness of fiber and vessel cells in the stems of WT, irx9, irx14, and MlGT43s complemented plants

 

Interfascicular fiber (μm)

Vessel (μm)

Xylary fiber (μm)

WT

1.98 ± 0.11

1.35 ± 0.26

1.46 ± 0.28

irx9

1.15 ± 0.23

0.47 ± 0.10

0.59 ± 0.18

irx9 + MlGT43A

1.66 ± 0.19

1.21 ± 0.10

1.23 ± 0.10

irx9 + MlGT43B

1.68 ± 0.33

1.19 ± 0.14

1.23 ± 0.20

irx9 + MlGT43C

1.62 ± 0.25

0.97 ± 0.05

1.07 ± 0.15

irx9 + MlGT43D

1.36 ± 0.29

0.90 ± 0.08

0.97 ± 0.20

irx9 + MlGT43E

1.40 ± 0.18

0.95 ± 0.19

0.93 ± 0.14

irx9 + MlGT43F

1.26 ± 0.18

0.62 ± 0.14

0.63 ± 0.17

irx9 + MlGT43G

1.23 ± 0.26

0.59 ± 0.12

0.65 ± 0.11

irx14

1.49 ± 0.25

0.98 ± 0.08

1.01 ± 0.22

irx14 + MlGT43A

1.46 ± 0.30

0.97 ± 0.07

1.00 ± 0.19

irx14 + MlGT43B

1.47 ± 0.19

0.93 ± 0.30

0.95 ± 0.10

irx14 + MlGT43C

1.50 ± 0.13

0.96 ± 0.11

0.96 ± 0.15

irx14 + MlGT43D

1.46 ± 0.24

0.95 ± 0.13

0.97 ± 0.17

irx14 + MlGT43E

1.48 ± 0.21

0.97 ± 0.14

0.99 ± 0.13

irx14 + MlGT43F

1.53 ± 0.13

1.04 ± 0.16

1.12 ± 0.12

irx14 + MlGT43G

1.58 ± 0.11

1.10 ± 0.17

1.20 ± 0.11

At least two independent transgenic lines for each construct were used for measurement. WT, irx9, and irx14 were included for comparison. Eight-week-old plants for each background were used for analysis. Wall thickness was measured from transmission electron micrographs of fibers and vessels. Data are means (μm) ± SE from 20 cells

Immunolocalization of xylan in MlGT43s complemented lines

To investigate whether the phenotypes of the complemented plants are correlated with xylan deposition in secondary cell walls, we performed immunolocalization of xylan using the xylan-directed monoclonal antibody LM10, which recognizes unsubstituted or low-substituted xylan [48], to examine the distribution of xylan in the cell walls. As indicated in Fig. 8, strong fluorescence signals were present in the cell walls of interfascicular fibers and xylem cells in the WT stem, however, relatively weaker signals were detected in the corresponding tissues of the irx9 plants, although the overall pattern of labeling was unchanged compared with the WT plants (Fig. 8 A1, B1). In MlGT43A and MlGT43B complemented irx9 lines, the intensity of fluorescence signals was almost restored to the WT level, and the overall pattern of labeling was almost identical to that of WT, indicating that the GX content in interfascicular fibers and xylem cells was nearly restored to the WT level (Fig. 8 C1, D1). The LM10 signals in the MlGT43C-E complemented irx9 plants were intermediate between irx9 and WT plants (Fig. 8 E1-G1). By contrast, the LM10 signals for MlGT43F and MlGT43G complemented irx9 lines were relatively weaker compared with the others, and the intensity was comparable to that of the irx9 mutant (Fig. 8 H1, I1). As for the irx14 background, the intensity of fluorescence signals of MlGT43F and MlGT43G complemented lines was comparable to that of WT in xylem cells and interfascicular fibers (Fig. 8 H2, I2). By contrast, MlGT43A-E complemented irx14 lines exhibited nearly equal signal intensity to the irx14 mutant (Fig. 8 C2-G2). These results indicate that MlGT43A-E perform a similar biochemical function as IRX9, whereas MlGT43F and MlGT43G share a conserved biochemical function with IRX14, thus leading to a restoration of normal xylan synthesis in their complemented plants.
Fig. 8

Immunolocalization of xylan using the monoclonal antibody LM10. Labelling was carried out on 8 μm-thick transverse sections from stem tissues of eight-week-old plants. A1-I1: xylan immumolocalization in WT, irx9 and MlGT43 complemented irx9 lines. A2-T2: xylan immunolocalization in WT, irx14 and MlGT43 complemented irx14 lines. Signals were detected with Alexa Fluor488-conjugated secondary antibody and observed with a BX51 fluorescence microscope (OLYMPUS). Bar = 50 μm

Analysis of cell wall composition

To determine whether the complementation of xylem morphology and xylan deposition is correlated with the restoration of chemical composition, we measured the monosaccharide composition, cellulose and lignin contents of the transgenic lines. Monosaccharide composition analysis was performed on cell wall preparations from eight-week-old inflorescence stems of WT, irx9, irx14 and MlGT43 complemented lines (Fig. 9). The xyl content in irx14 was decreased by 40 % compared to WT, whereas it was decreased more dramatically in irx9, with only 21 % of the WT. The transgenic plants overexpressing MlGT43A and MlGT43B in irx9 significantly increased the content of xyl to 73 and 82 % of the WT level, respectively. A modest increase was also observed in the MlGT43C-E complemented irx9 lines. However, no significant increases in xyl content were observed in MlGT43F or MlGT43G complemented irx9 lines compared to irx9. Overexpression of MlGT43F and MlGT43G in irx14 restored the xyl content to 92 and 83 % of the WT, respectively. The xyl content of MlGT43A-E complemented irx14 plants was individually increased by approximately 5 to 10 % compared to irx14.
Fig. 9

Monosaccharide composition of cell walls isolated from the stems of WT, irx9, irx14 and MlGT43 complemented plants. Cell walls were prepared from inflorescence stems of eight-week-old plants and their glycosyl compositions were determined by HPLC. Data are means ± SD of three independent analyses

In addition, mutations of irx9 and irx14 caused significant reductions in cellulose and lignin contents compared to WT. Not unexpectedly, overexpression of MlGT43A-E but not MlGT43F and MlGT43G in irx9 restored the contents of cellulose and lignin almost to the WT level. Similarly, overexpression of MlGT43F and MlGT43G but not MlGT43A-E in irx14 recovered the levels of cellulose and lignin nearly to the WT level (Additional file 3: Figure S1). These results further indicate that MlGT43A-E but not MlGT43F-G can partially restore the xylan biosynthesis in irx9, while MlGT43F-G but not MlGT43A-E are able to rescue the xylan biosynthesis defect in irx14, suggesting that MlGT43A-E are orthologous to IRX9, while MlGT43F and MlGT43G are orthologous to IRX14.

Transactivation assay for MlGT43 genes

SND1 (SECONDARY WALL-ASSOCIATED NAC DOMAIN PROTEIN 1), VND7 (VASCULAR-RELATED NAC-DOMAIN 7) and MYB46 have been shown to act as the master switches in the regulatory network of secondary cell wall biosynthesis [49]. To better understand the underlying regulatory mechanism of MlGT43 genes, we isolated the orthologues of SND1, VND7 and MYB46 in M. lutarioriparius and analyzed their transactivation abilities on proMlGT43A-E:GUS reporters using a transient transactivation assay (Fig. 10). The results showed that MlGT43A was transactivated by MlSND1, MlMYB46a, MlMYB46b and MlVND7. MlGT43B was also transactivated by MlSND1, MlMYB46a, but not by MlMYB46b and MlVND7. By contrast, MlGT43C-E were not transactivated by any effectors examined. These results indicate that MlGT43A and MlGT43B genes are differentially regulated by SND1, MYB46 and VND7 orthologues and there probably exist other transcriptional factors regulating the expression of MlGT43C-E genes besides the above effectors examined.
Fig. 10

Transactivation assay of the MlGT43A-E promoters by MlSND1, MlMYB46a/b or MlVND7. Diagrams indicate the effector and reporter constructs used for transactivation analysis. The effector constructs contain the MlSND1, MlMYB46a, MlMYB46b or MlVND7 cDNA driven by the 35S promoter. The reporter constructs consist of the GUS reporter gene driven by the MlGT43A-E promoters. Transactivation ability was represented by the relative GUS activities. The expression level of the GUS reporter gene in Arabidopsis leaf protoplasts transfected with no effector was used as a control and was set to 1

None of MlGT43 genes could rescue the mucilage defects of irx14 seeds

Since IRX14 has been shown to be responsible for the synthesis of xylan in seed coat mucilage and mutations in IRX14 lead to a defect in mucilage cohesiveness property [50, 51], we sought to examine whether MlGT43 genes could rescue the mucilage defect of irx14. The seeds of MlGT43 complemented lines in irx14 background were examined by ruthenium red staining (Additional file 4: Figure S2). When seeds were imbibed in water and subjected to gentle shaking, the seeds of seven MlGT43 complemented irx14 lines all exhibited a thin layer of mucilage phenotype similar to that of the irx14 seeds. By contrast, the WT seeds have a much thicker mucilage layer tightly attached to the seed. This result indicated that none of MlGT43 genes could rescue the mucilage defect of irx14.

We further determined the monosaccharide composition of seed mucilage for each complemented line. The xyl content was dramatically reduced in irx14 mucilage as previously reported [50, 51]. Not surprisingly, the xyl content in seven complemented lines was comparable to that of irx14 and not restored to the WT level (Additional file 5: Figure S3), suggesting that none of MlGT43s could synthesize the xylan in the seed coat mucilage.

Discussion

Much progress has been gained in xylan biosynthesis mainly in the model species Arabidopsis. Several GT43 family proteins have been revealed to participate in xylan backbone biosynthesis in secondary cell walls [13, 19, 3538]. By contrast, less knowledge regarding the biosynthesis of xylan is known in grass, despite that xylan especially arabinoxylan is the major hemicellulosic components in grass cell walls. In this study, we identified seven GT43 genes from M. lutarioriparius and revealed that they are functional orthologues of Arabidopsis IRX9 and IRX14. Phylogenetic analysis of GT43 proteins from nine representative plant species and Miscanthus revealed that these proteins were classified into three major clades, namely IRX9, IRX9L and IRX14/IRX14L (Fig. 2). Noteworthy, our results indicated that no IRX9 orthologues were present in the lower plant species moss (P. patens) and spikemoss (S. mellysellia). Moss has been demonstrated to be capable of synthesizing glucuronoxylans that are structurally similar to those present in the secondary cell walls of higher plants [52]. The glucuronoxylans are mainly located in primary cell walls in moss as no mechanical supporting tissues composed mainly of secondary cell walls have been evolved. As a basal vascular plant, spikemoss has evolved tissues containing secondary cell walls. Xylans have been shown to be one of the most abundant cell wall components in spikemoss [53]. Since IRX9 has been shown to be mainly responsible for the biosynthesis of xylans in secondary cell walls [13, 19, 20, 35, 38, 54], the absence of xylans in secondary cell walls in moss may partially explain why no IRX9 orthologues are present in moss genome. Thus, it seems likely that vascular plants have evolved a specialized isoform of IRX9, which is responsible for xylan biosynthesis in secondary cell walls. However, this hypothesis seems somewhat implausible because IRX9 orthologues are also lacking in spikemoss. Together, these results indicate that the specialization of IRX9 for xylan biosynthesis in primary and secondary cell walls is not necessary for the evolution of vascular tissue.

Although the qRT-PCR analysis revealed that MlGT43A to MlGT43E in M. lutarioriparius exhibited broad expression patterns across the tissues examined, the in situ hybridization analysis unambiguously indicated that Miscanthus IRX9 orthologues MlGT43A and MlGT43B were preferentially expressed in cells undergoing secondary wall thickening, while the IRX9L orthologues MlGT43C-E were expressed in both parenchymal cells and sclerenchyma cells (Fig. 3). In addition, IRX9 orthologues MlGT43A and MlGT43B were both transcriptionally regulated by MlSND1, MlMYB46a or MlVND7, three candidate transcriptional switches governing secondary cell wall biosynthesis. By contrast, the Miscanthus IRX9L orthologues (MlGT43C-E) were not significantly transactivated by these transcription factors (Fig. 10). Similar results were reported for IRX9 orthologues in Arabidopsis, rice (OsGT43A and OsGT43E) and poplar (PtrGT43A and PtrGT43B), which were shown to be highly expressed in tissues with abundant secondary cell walls [13, 35, 38]. In addition, poplar IRX9 orthologues (PtrGT43A and PtrGT43B) were transcriptionally regulated by PtxtMYB021 (MYB46 orthologue) and PNAC085 (SND1 orthologue), master transcriptional switches involved in secondary cell wall formation [38]. Together, these results indicated that IRX9 orthologues are mainly involved in secondary cell wall biosynthesis, and its roles are highly conserved in angiosperm species.

In addition, the number of GT43 proteins in monocot species seems to be higher than that of dicot species, which was mainly due to a significantly expansion of IRX9 and IRX9L members in monocot species (Fig. 2b). In dicots, such as Arabidopsis and poplar, xylan is predominantly deposited in the secondary cell walls, whereas there is very limited amounts of xylan in the primary cell walls. By contrast, the monocot species including rice and Miscanthus have abundant amounts of xylan in both primary and secondary cell walls. This could partially explain why the number of IRX9 and IRX9L orthologues are over-presented in monocots compared with dicots.

Phylogenetic analysis also indicated that ancestral IRX9 orthologues emerged after the specification of the higher plants (Fig. 2a). In addition, IRX9 may possibly evolve from its IRX9L homologue through the duplication events during the evolutionary process as they share very high sequence identities [13, 38]. The functional diversification of IRX9 orthologues may be due to their expression specificities and their abilities to respond to the key transcriptional factors involved in secondary wall formation (Fig. 10). The different cis-regulatory elements present in the promoter of Miscanthus IRX9 and IRX9L orthologues may explain their functional divergences to some extent (Additional file 6: Table S2). In other words, Miscanthus IRX9 orthologues may have evolved to gain some key cis-regulatory elements, which confers their specific functions in xylan biosynthesis during secondary cell wall formation.

In Arabidopsis, IRX9 and IRX14 play independent roles in xylan biosynthesis, since the phenotypes of irx9 mutant cannot be rescued by the overexpression of IRX14 or IRX14L and vice versa [13, 19]. In addition, IRX9 and IRX14 are proposed to play dominant roles, whereas their homologues IRX9L and IRX14L are indicated to play partially redundant or minor roles in xylan backbone biosynthesis [13, 14, 19]. Contrary to this assumption, a recent study proposed that IRX9L and IRX14L play equally important roles with IRX9 and IRX14 in xylan biosynthesis [20]. The seven GT43 orthologues in Miscanthus were classified into three major subclades namely IRX9, IRX9L and IRX14/IRX14L. All five Miscanthus IRX9 and IRX9L orthologues (MlGT43A-E) could nearly fully or partially complement the phenotypes of irx9, while none of these genes could rescue the phenotypes of irx14. Similarly, two Miscanthus IRX14 and IRX14L orthologues (MlGT43F and MlGT43G) were able to rescue the phenotypes of irx14 but not irx9. These results indicated that GT43 genes have been evolved into two functional groups in Miscanthus, and the functions between the members in IRX9/IRX9L and IRX14/IRX14L groups have been diversified substantially. Likewise, the involvement of two distinctly functional groups of GT43 genes in xylan biosynthesis seems to be highly conserved in different plant species. For example, the rice orthologues of IRX9 (OsGT43A and OsGT43E) were able to rescue the phenotypes of irx9 but were not able to complement those of irx14. By contrast, the IRX14 orthologue OsGT43J was able to complement the irx14 phenotypes but unable to rescue those of irx9. Similarly, the poplar IRX9 orthologues (PtrGT43A, PtrGT43B and PtrGT43E) were able to rescue the xylan defects of irx9 but could not complement those of irx14, whereas the IRX14 orthologues (PtrGT43C and PtrGT43D) were capable of rescuing the defects of irx14 but not those of irx9.

Xylans are typically substituted with α-l-Araf residues at C2- and/or C3-position in arabinoxylans (AX) and less frequently with GlcpA and/or 4-O-Me-GlcpA sidechains at C2- position in glucuronoarabinoxylans (GAX) in grasses [3, 4]. AX is the major xylan in Miscanthus and the degree of Araf substitution positively affects the lignocellulose saccharification under various pretreatments [44, 45]. AX is also the major xylan of the seed mucilage in psyllium (Plantago ovata) [55]. During Arabidopsis seed differentiation, the seed coat epidermal cells synthesize and secrete large amounts of mucilage, which encapsulated the seed upon imbibition. Although the Arabidopsis seed coat mucilage are primarily composed of pectic RG I, minor amounts of xylan are also present in the mucilage and play an important role in maintaining the structure of seed coat mucilage [50, 51]. Unlike the typical xylan in dicot secondary cell walls, mucilage xylan has a unique structure with frequent substitutions with Xyl rather than with GlcA or Ara residues [50, 51]. IRX14 has been revealed to be responsible for the biosynthesis of xylan in Arabidopsis mucilage and loss function lead to a mucilage cohesiveness defect [50, 51]. It is noteworthy that none of the MlGT43 genes could be able to complement the irx14 mucilage defect (Additional file 4: Figure S2), suggesting that MlGT43s could not synthesize the mucilage xylan, which is involved in maintaining the structure of seed coat mucilage (Additional file 5: Figure S3). The reason might due to the fact that mucilage xylan is structurally different from that of the stem secondary walls, and the functions of Miscanthus GT43 proteins have diversified from those of Arabidopsis orthologues during the evolutionary process. Similarly, there is also lines of evidence highlighting that mucilage xylan biosynthesis is diversified in different plant species. For example, IRX10 but not IRX9 or IRX14 might be responsible for the synthesis of the xylan backbone in psyllium mucilage because IRX10 orthologues were highly presented in psyllium mucilage, while relatively very lower transcripts of IRX9 and IRX14 were detected in a transcriptome analysis [55].

Conclusion

In this study, we functionally identified seven GT43 genes from M. lutarioriparius. Our results provided the first line of genetic evidence demonstrating that Miscanthus has evolved to retain two functionally non-redundant groups of GT43 genes involved in xylan biosynthesis. MlGT43A-E are functional orthologues of IRX9, while MlGT43F and MlGT43G are functional orthologues of IRX14. Nevertheless, functional divergence of IRX14 orthologues in M. lutarioriparius has occurred as none of MlGT43 genes could rescue the mucilage defects of irx14 seeds. Furthermore, MlGT43A-E were differentially regulated by SND1, MYB46 or VND7 orthologues, the putative key regulators in secondary cell wall formation. The results obtained deepen our understanding of xylan biosynthesis in Miscanthus. Understanding how xylan polymers are synthesized may lay a foundation for the genetic modification of Miscanthus to be better suited for various economically important applications, including the more efficient utilization of xylan for biofuel production.

Methods

Plant materials and growth conditions

The M. lutarioriparius used in this study was provided by Shanghai Institute for Biological Sciences of the Chinese Academy of Sciences. The plants were clonally propagated by young rhizomes in greenhouse under 16 h light/8 h dark photoperiod at 25–28 °C.

T-DNA insertion mutants irx9 (SALK_058238) and irx14 (SALK_038212) were obtained from the Arabidopsis Biological Resource Center (ABRC). Seeds were surface sterilized and sowed on 1/2 MS plate. After stratified at 4 °C for 3 d, the plates were transferred to the growth chamber and germinated at 21 °C under 16 h light/8h dark photoperiod. Homozygous T-DNA insertions were identified by PCR of genomic DNA. The primers are listed in Additional file 7: Table S3.

RNA isolation and Quantitative real-time RT-PCR (qRT-PCR) analysis

The total RNA was isolated from root, rhizome, stem, leaf and sheath of M. lutarioriparius using Trizol reagent (Invitrogen), then treated with RNase-free DNaseI (Promega) to remove genomic DNA contamination. First-strand cDNA was synthesized using M-MLV reverse transcriptase (TaKaRa, Japan) according to the manufacturer’s instructions. The cDNAs were used as templates for qRT-PCR with gene-specific primers (Additional file 7: Table S3). The qRT-PCR was carried out using LightCycler® 480 detection system (Roche) with SYBR® Premix Ex Taq II (TaKaRa). MlACT11 was used as an internal control.

Identification of MlGT43 genes

The Arabidopsis GT43 proteins (IRX9, IRX9L, IRX14 and IRX14L) were used as baits to search against the draft genome sequence of M. lutarioriparius (Lu et al., unpublished data). Specific primers were designed to isolate the full length MlGT43 cDNAs (Additional file 7: Table S3). The PCR products were purified, cloned into pMD19-T vector (TIANGEN) and sequenced. The exon/intron organization was illustrated with Gene Structure Display Server (GSDS) program (http://gsds.cbi.pku.edu.cn/) by alignment of the cDNAs with their corresponding genomic DNA sequences [56].

Phylogenetic analysis of GT43 family from other plant species

GT43 family protein sequences from nine other species including moss (P. patens), spikemoss (S. moellendorffii), monocot angiosperms (O. sativa, B. distachyon and S. bicolor), and dicot angiosperms (A. thaliana, P. trichocarpa, M. truncatula and V. vinifera) were obtained using BLASTP search against Phytozome10 database (https://phytozome.jgi.doe.gov/). Phylogenetic analysis was performed with MEGA6.0 by the Neighbor-Joining (NJ) method with 1000 bootstrap replicates with default parameters [57].

In situ mRNA hybridization

For the synthesis of antisense and sense probes, ~200 bp fragments of MlGT43A-G were amplified by PCR with their corresponding primers (Additional file 7: Table S3) and cloned into the pGM-T vector (TIANGEN). The RNA probes were synthesized with the DIG RNA labelling kit (Roche) according to the manufacturer’s instructions.

Miscanthus stem segments from the 11th internode were fixed in FAA solution (70 % ethanol, 5 % formaldehyde and 5 % acetic acid) at 4 °C overnight, followed by dehydration in gradient ethanol series (10 % increments). The samples were embedded in paraplast and cut into 8 μm-thick sections. The sections were mounted onto slides, and hybridized with DIG-labeled antisense or sense RNA probes. Images were captured with the OLYMPUS BX51 microscope.

Subcellular localization

The co-localization of fluorescent protein-tagged MlGT43A-G with the Golgi marker was examined using the tobacco leaf transient expression system [58]. The full-length MlGT43 genes without a terminator codon were amplified and fused with yellow fluorescent protein (YFP) in pEarleyGate101 vector [59] via LR recombination reactions (Invitrogen). The proteins generated thus encode fusion proteins of MlGT43s with YFP tagged at the C terminus. After 3 days post co-infiltration of YFP fusion proteins and the Golgi marker into tobacco leaves, leaf epidermal cells were examined for yellow fluorescence signal using a FluoView FV1000 Laser Scanning confocal microscope (OLYMPUS) equipped with 488 nm argon laser.

Overexpression vector construction and complementation

The full-length cDNA sequence of MlGT43s were amplified by PCR and ligated to the pGWC-T as described previously [60]. The products were sequenced and then transferred into the pEarleyGate 100 vector [59] via LR recombination reaction (Invitrogen) to produce the 35S CaMV overexpression constructs. The constructs were introduced into Agrobacterium tumefaciens strain EHA105 by electroporation.

For complementation analysis, the overexpression constructs were transformed into the Arabidopsis irx9 heterozygous or irx14 homozygous mutant via the floral dip method [61]. Positive T0 and T1 generation plants were screened by spraying BASTA solution (50 mg/L) onto one-week-old seedlings in soil. For irx9 complemented lines, transformed seedlings were further genotyped with PCR to verify the homozygous T-DNA insertions. Homozygous T3 transgenic lines were used for further analysis.

Microscopy and immunolocalization analysis

Arabidopsis inflorescence stems were taken 0.5 cm above the rosette of eight-week-old plants. Samples were fixed in FAA solution, dehydrated via a series of ethanol gradients, and embedded in paraplast. For light microscopy, 8 μm-thick sections were stained with 0.5 % (w/v) toluidine blue O (Sigma-Aldrich) for 2 min and rinsed with water. The sections were photographed with a BX51 light microscope (OLYMPUS).

For the immunolabelling, sections were incubated with the LM10 antibody (1/20 dilution) for 2 h, then washed three times with phosphate-buffered saline, followed by incubation with rabbit anti-rat Alexa Fluor488-conjugated secondary antibody (1/100 dilution) in the dark for 1 h. Images were captured using a BX51 light microscope (OLYMPUS) equipped with fluorescent light.

For transmission electron microscopy, samples were embedded in Spurr’s resin. Ultra-thin sections (70 nm) were viewed by a H-7650 electron microscope (HITACHI). Cell wall thickness was measured in metaxylem vessels and interfascicular fibres using the software SmileView (JEOL). For each construct, at least three transgenic lines with the most severe phenotypes were examined.

Cell wall monosaccharide composition analysis

To prepare cell-wall alcohol-insoluble residues (AIR), eight-week-old inflorescence stems from at least 20 independent plants were collected, frozen in liquid nitrogen, and freeze-dried overnight using a lyophilizer. For monosaccharide composition analysis, AIR was hydrolyzed in 2 M trifluoroacetic acid for 2 h at 120 °C. The released monosaccharides were derived by 1-phenyl-3-methyl-5-pyrazolone (PMP) and the derivatives were separated on a Thermo ODS-2 C18 column (4.6 × 250 mm) connected to a Waters HPLC system. The absorbance was monitored at 245 nm. Cellulose content was assayed with the anthrone reagent according to Updegraff [62]. Lignin composition was determined using the acetyl bromide spectrophotometric method as described [63].

Transcriptional activation analysis

The pBI221 vector was used to produce both effector and reporter constructs. The MlSND1, MlMYB46a/b and MlVND7 effector constructs were obtained by PCR using Miscanthus stem cDNA as the template (Additional file 7: Table S3). All effector constructs were individually ligated between the CaMV 35S promoter and the NOS terminator after removing GUS from the pBI221 vector. The MlGT43A-E promoters were cloned by hiTAIL-PCR [64] and ligated upstream of the GUS reporter gene after removing the 35S promoter region of pBI221 to create the reporter constructs.

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Availability of data and materials

The data supporting the results of this article are included as additional files. The MlGT43 gene and promoter sequences were deposited in the Genbank (https://www.ncbi.nlm.nih.gov/genbank) under accession numbers KX082754 to KX082765.

Abbreviations

IRX: 

irregular xylem

GT: 

glycosyltransferase

qRT-PCR: 

quantitative real-time RT-PCR

GX: 

(methyl)glucuronoxylan

AX: 

arabinoxylan

GAX: 

glucuronoarabinoxylan

GlcA: 

glucuronic acid

MeGlcA: 

methylglucuronic acid

Ara: 

arabinose

GUX: 

glucuronic acid substitution of xylan

GXMT: 

glucuronoxylan methyltransferase

TBL: 

trichome birefringence-like

XAT: 

xylan arabinosyltransferase

XAX: 

xylosyl arabinosyl substitution of xylan

Xyl: 

xylose

CDS: 

coding sequence

YFP: 

yellow fluorescent protein

WT: 

wild type

TBO: 

toluidine blue O

SND1: 

secondary wall-associated NAC domain protein 1

VND7: 

vascular-related NAC-domain 7

Declarations

Acknowledgments

This work was supported by the National Key Technology Support Program of China (2013BAD22B01), the National Natural Science Foundation of China (31370328 and 31470291), the Youth Innovation Promotion Association of CAS (2014187), the Taishan Scholar Program of Shandong (to G. Z.), and the Youth Talent Plan of Chinese Academy of Agricultural Sciences (to Y. K.).

Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Authors’ Affiliations

(1)
Qingdao Institute of Bioenergy and Bioprocess Technology, Key Laboratory of Biofuels, Qingdao Engineering Research Center of Biomass Resources and Environment, Chinese Academy of Sciences
(2)
University of Chinese Academy of Sciences
(3)
Shandong Institute of Agricultural Sustainable Development
(4)
State Key Laboratory for Conservation and Utilization of Subtropical Agrobioresources, South China Agricultural University
(5)
Tobacco Research Institute of Chinese Academy of Agricultural Sciences, Key laboratory of Tobacco Genetic Improvement and Biotechnology

References

  1. Carpita NC, McCann M. The Cell Wall. In: Buchanan BB, Wilhelm G, Jones RL, editors. Biochemistry and Molecular Biology of Plants. Rockville, MD: American Society of Plant Biologists; 2000. p. 52–108.Google Scholar
  2. Scheller HV, Ulvskov P. Hemicelluloses. Annu Rev Plant Biol. 2010;61:263–89.View ArticlePubMedGoogle Scholar
  3. Ebringerova’ A, Heinze T. Xylan and xylan derivatives – biopolymers with valuable properties, 1. Naturally occurring xylans structures, isolation procedures and properties. Macromol Rapid Commun. 2000;21:542–56.View ArticleGoogle Scholar
  4. Rennie EA, Scheller HV. Xylan biosynthesis. Curr Opin Biotechnol. 2014;26:100–7.View ArticlePubMedGoogle Scholar
  5. Pena MJ, Zhong R, Zhou GK, Richardson EA, O'Neill MA, Darvill AG, York WS, Ye ZH. Arabidopsis irregular xylem8 and irregular xylem9: implications for the complexity of glucuronoxylan biosynthesis. Plant Cell. 2007;19(2):549–63.Google Scholar
  6. Johansson MH, Samuelson O. Reducing end groups in birch xylan and their alkaline degradation. Wood Sci Technol. 1977;11:251–63.View ArticleGoogle Scholar
  7. Andersson SI, Samuelson O, Ishihara M, Shimizu K. Structure of the reducing end-groups in spruce xylan. Carbohydrate Res. 1983;111:283–8.View ArticleGoogle Scholar
  8. Ratnayake S, Beahan CT, Callahan DL, Bacic A. The reducing end sequence of wheat endosperm cell wall arabinoxylans. Carbohydr Res. 2014;386:23–32.View ArticlePubMedGoogle Scholar
  9. Doering A, Lathe R, Persson S. An update on xylan synthesis. Mol Plant. 2012;5(4):769–71.View ArticlePubMedGoogle Scholar
  10. York WS, O’Neill MA. Biochemical control of xylan biosynthesis - which end is up? Curr Opin Plant Biol. 2008;11(3):258–65.View ArticlePubMedGoogle Scholar
  11. Turner SR, Somerville CR. Collapsed xylem phenotype of Arabidopsis identifies mutants deficient in cellulose deposition in the secondary cell wall. Plant Cell. 1997;9(5):689–701.View ArticlePubMedPubMed CentralGoogle Scholar
  12. Brown DM, Zeef LAH, Ellis J, Goodacre R, Turner SR. Identification of novel genes in Arabidopsis involved in secondary cell wall formation using expression profiling and reverse genetics. Plant Cell. 2005;17:2281–95.View ArticlePubMedPubMed CentralGoogle Scholar
  13. Lee C, Teng Q, Huang W, Zhong R, Ye ZH. The Arabidopsis family GT43 glycosyltransferases form two functionally nonredundant groups essential for the elongation of glucuronoxylan backbone. Plant Physiol. 2010;153(2):526–41.View ArticlePubMedPubMed CentralGoogle Scholar
  14. Keppler BD, Showalter AM. IRX14 and IRX14-LIKE, two glycosyl transferases involved in glucuronoxylan biosynthesis and drought tolerance in Arabidopsis. Mol Plant. 2010;3(5):834–41.View ArticlePubMedGoogle Scholar
  15. Brown DM, Goubet F, Wong VW, Goodacre R, Stephens E, Dupree P, Turner SR. Comparison of five xylan synthesis mutants reveals new insight into the mechanisms of xylan synthesis. Plant J. 2007;52(6):1154–68.View ArticlePubMedGoogle Scholar
  16. Brown DM, Zhang Z, Stephens E, Dupree P, Turner SR. Characterization of IRX10 and IRX10-like reveals an essential role in glucuronoxylan biosynthesis in Arabidopsis. Plant J. 2009;57(4):732–46.View ArticlePubMedGoogle Scholar
  17. Lee C, O’Neill MA, Tsumuraya Y, Darvill AG, Ye ZH. The irregular xylem9 mutant is deficient in xylan xylosyltransferase activity. Plant Cell Physiol. 2007;48(11):1624–34.View ArticlePubMedGoogle Scholar
  18. Wu AM, Rihouey C, Seveno M, Hornblad E, Singh SK, Matsunaga T, Ishii T, Lerouge P, Marchant A. The Arabidopsis IRX10 and IRX10-LIKE glycosyltransferases are critical for glucuronoxylan biosynthesis during secondary cell wall formation. Plant J. 2009;57(4):718–31.View ArticlePubMedGoogle Scholar
  19. Wu AM, Hornblad E, Voxeur A, Gerber L, Rihouey C, Lerouge P, Marchant A. Analysis of the Arabidopsis IRX9/IRX9-L and IRX14/IRX14-L pairs of glycosyltransferase genes reveals critical contributions to biosynthesis of the hemicellulose glucuronoxylan. Plant Physiol. 2010;153(2):542–54.View ArticlePubMedPubMed CentralGoogle Scholar
  20. Mortimer JC, Faria-Blanc N, Yu X, Tryfona T, Sorieul M, Ng YZ, Zhang Z, Stott K, Anders N, Dupree P. An unusual xylan in Arabidopsis primary cell walls is synthesised by GUX3, IRX9L, IRX10L and IRX14. Plant J. 2015;83(3):413–26.View ArticlePubMedPubMed CentralGoogle Scholar
  21. Jensen JK, Kim H, Cocuron JC, Orler R, Ralph J, Wilkerson CG. The DUF579 domain containing proteins IRX15 and IRX15-L affect xylan synthesis in Arabidopsis. Plant J. 2011;66(3):387–400.View ArticlePubMedGoogle Scholar
  22. Brown D, Wightman R, Zhang ZN, Gomez LD, Atanassov I, Bukowski JP, Tryfona T, McQueen-Mason SJ, Dupree P, Turner S. Arabidopsis genes IRREGULAR XYLEM (IRX15) and IRX15L encode DUF579-containing proteins that are essential for normal xylan deposition in the secondary cell wall. Plant J. 2011;66(3):401–13.View ArticlePubMedGoogle Scholar
  23. Lee C, Teng Q, Huang W, Zhong R, Ye ZH. The F8H glycosyltransferase is a functional paralog of FRA8 involved in glucuronoxylan biosynthesis in Arabidopsis. Plant Cell Physiol. 2009;50(4):812–27.View ArticlePubMedGoogle Scholar
  24. Lee C, Zhong R, Richardson EA, Himmelsbach DS, McPhail BT, Ye ZH. The PARVUS gene is expressed in cells undergoing secondary wall thickening and is essential for glucuronoxylan biosynthesis. Plant Cell Physiol. 2007;48(12):1659–72.View ArticlePubMedGoogle Scholar
  25. Zhong R, Pena MJ, Zhou GK, Nairn CJ, Wood-Jones A, Richardson EA, Morrison WH, Darvill AG, York WS, Ye ZH. Arabidopsis fragile fiber8, which encodes a putative glucuronyltransferase, is essential for normal secondary wall synthesis. Plant Cell. 2005;17:3390–408.View ArticlePubMedPubMed CentralGoogle Scholar
  26. Persson S, Caffall KH, Freshour G, Hilley MT, Bauer S, Poindexter P, Hahn MG, Mohnen D, Somerville C. The Arabidopsis irregular xylem8 mutant is deficient in glucuronoxylan and homogalacturonan, which are essential for secondary cell wall integrity. Plant Cell. 2007;19:237–55.Google Scholar
  27. Lee C, Teng Q, Zhong R, Ye ZH. Arabidopsis GUX proteins are glucuronyltransferases responsible for the addition of glucuronic acid side chains onto xylan. Plant Cell Physiol. 2012;53(7):1204–16.View ArticlePubMedGoogle Scholar
  28. Rennie EA, Hansen SF, Baidoo EE, Hadi MZ, Keasling JD, Scheller HV. Three members of the Arabidopsis glycosyltransferase family 8 are xylan glucuronosyltransferases. Plant Physiol. 2012;159(4):1408–17.View ArticlePubMedPubMed CentralGoogle Scholar
  29. Bromley JR, Busse-Wicher M, Tryfona T, Mortimer JC, Zhang ZN, Brown DM, Dupree P. GUX1 and GUX2 glucuronyltransferases decorate distinct domains of glucuronoxylan with different substitution patterns. Plant J. 2013;74(3):423–34.Google Scholar
  30. Urbanowicz BR, Pena MJ, Moniz HA, Moremen KW, York WS. Two Arabidopsis proteins synthesize acetylated xylan in vitro. Plant J. 2014;80(2):197–206.View ArticlePubMedPubMed CentralGoogle Scholar
  31. Xiong G, Cheng K, Pauly M. Xylan O-acetylation impacts xylem development and enzymatic recalcitrance as indicated by the Arabidopsis mutant tbl29. Mol Plant. 2013;6(4):1373–5.View ArticlePubMedGoogle Scholar
  32. Yuan Y, Teng Q, Zhong R, Ye ZH. The Arabidopsis DUF231 domain-containing protein ESK1 mediates 2-O- and 3-O-acetylation of xylosyl residues in xylan. Plant Cell Physiol. 2013;54(7):1186–99.View ArticlePubMedGoogle Scholar
  33. Anders N, Wilkinson MD, Lovegrove A, Freeman J, Tryfona T, Pellny TK, Weimar T, Mortimer JC, Stott K, Baker JM, et al. Glycosyl transferases in family 61 mediate arabinofuranosyl transfer onto xylan in grasses. Proc Natl Acad Sci U S A. 2012;109(3):989–93.View ArticlePubMedPubMed CentralGoogle Scholar
  34. Chiniquy D, Sharma V, Schultink A, Baidoo EE, Rautengarten C, Cheng K, Carroll A, Ulvskov P, Harholt J, Keasling JD, et al. XAX1 from glycosyltransferase family 61 mediates xylosyltransfer to rice xylan. Proc Natl Acad Sci U S A. 2012;109(42):17117–22.View ArticlePubMedPubMed CentralGoogle Scholar
  35. Chiniquy D, Varanasi P, Oh T, Harholt J, Katnelson J, Singh S, Auer M, Simmons B, Adams PD, Scheller HV, et al. Three Novel Rice Genes Closely Related to the Arabidopsis IRX9, IRX9L, and IRX14 Genes and Their Roles in Xylan Biosynthesis. Front Plant Sci. 2013;4:83.View ArticlePubMedPubMed CentralGoogle Scholar
  36. Lee C, Teng Q, Zhong R, Yuan Y, Ye ZH. Functional roles of rice glycosyltransferase family GT43 in xylan biosynthesis. Plant Signal Behav. 2014;9(1), e27809.View ArticlePubMedPubMed CentralGoogle Scholar
  37. Lee C, Teng Q, Zhong R, Ye ZH. Molecular dissection of xylan biosynthesis during wood formation in poplar. Mol Plant. 2011;4(4):730–47.View ArticlePubMedGoogle Scholar
  38. Ratke C, Pawar PM, Balasubramanian VK, Naumann M, Duncranz ML, Derba-Maceluch M, Gorzsas A, Endo S, Ezcurra I, Mellerowicz EJ. Populus GT43 family members group into distinct sets required for primary and secondary wall xylan biosynthesis and include useful promoters for wood modification. Plant Biotechnol J. 2015;13(1):26–37.View ArticlePubMedGoogle Scholar
  39. Li L, Huang J, Qin L, Huang Y, Zeng W, Rao Y, Li J, Li X, Xu W. Two cotton fiber-associated glycosyltransferases, GhGT43A1 and GhGT43C1, function in hemicellulose glucuronoxylan biosynthesis during plant development. Physiol Plant. 2014;152(2):367–79.View ArticlePubMedGoogle Scholar
  40. Brosse N, Dufour A, Meng XZ, Sun QN, Ragauskas A. Miscanthus: a fast-growing crop for biofuels and chemicals production. Biofuel Bioprod Bior. 2012;6:580–98.View ArticleGoogle Scholar
  41. Yan J, Chen W, Luo FAN, Ma H, Meng A, Li X, Zhu M, Li S, Zhou H, Zhu W, et al. Variability and adaptability of Miscanthus species evaluated for energy crop domestication. Glob Change Biol Bioenergy. 2012;4(1):49–60.View ArticleGoogle Scholar
  42. Lewandowski I, Clifton-Brown JC, Scurlock JMO, Huisman W. Miscanthus: European experience with a novel energy crop. Biomass Bioenergy. 2000;19(4):209–27.View ArticleGoogle Scholar
  43. Lygin AV, Upton J, Dohleman FG, Juvik J, Zabotina OA, Widholm JM, Lozovaya VV. Composition of cell wall phenolics and polysaccharides of the potential bioenergy crop - Miscanthus. Glob Change Biol Bioenergy. 2011;3:333–45.View ArticleGoogle Scholar
  44. Li F, Ren S, Zhang W, Xu Z, Xie G, Chen Y, Tu Y, Li Q, Zhou S, Li Y, et al. Arabinose substitution degree in xylan positively affects lignocellulose enzymatic digestibility after various NaOH/H2SO4 pretreatments in Miscanthus. Bioresour Technol. 2013;130:629–37.Google Scholar
  45. Kulkarni AR, Pattathil S, Hahn MG, York WS, O’ Neil MA. Comparison of Arabinoxylan Structure in Bioenergy and Model Grasses. Industrial Biotechnol. 2012;8(4):222–9.View ArticleGoogle Scholar
  46. Xu N, Zhang W, Ren S, Liu F, Zhao C, Liao H, Xu Z, Huang J, Li Q, Tu Y, et al. Hemicelluloses negatively affect lignocellulose crystallinity for high biomass digestibility under NaOH and H2SO4 pretreatments in Miscanthus. Biotechnol Biofuels. 2012;5(1):58.View ArticlePubMedPubMed CentralGoogle Scholar
  47. Nelson BK, Cai X, Nebenfuhr A. A multicolored set of in vivo organelle markers for co-localization studies in Arabidopsis and other plants. Plant J. 2007;51(6):1126–36.View ArticlePubMedGoogle Scholar
  48. McCartney L, Marcus SE, Knox JP. Monoclonal antibodies to plant cell wall xylans and arabinoxylans. J Histochem Cytochem. 2005;53(4):543–6.View ArticlePubMedGoogle Scholar
  49. Wang HZ, Dixon RA. On-off switches for secondary cell wall biosynthesis. Mol Plant. 2012;5(2):297–303.View ArticlePubMedGoogle Scholar
  50. Hu RB, Li JL, Wang XY, Zhao X, Wang Z, Yang XW, Tang Q, He G, Zhou GK, Kong YZ. Xylan synthesized by Irregular Xylem 14 (IRX14) maintains the structure of seed coat mucilage in Arabidopsis. J Exp Bot, accepted 2016Google Scholar
  51. Voiniciuc C, Gunl M, Schmidt MH, Usadel B. Highly Branched Xylan Made by IRREGULAR XYLEM14 and MUCILAGE-RELATED21 Links Mucilage to Arabidopsis Seeds. Plant Physiol. 2015;169:2481–95.View ArticlePubMedPubMed CentralGoogle Scholar
  52. Kulkarni AR, Pena MJ, Avci U, Mazumder K, Urbanowicz BR, Pattathil S, Yin Y, O'Neill MA, Roberts AW, Hahn MG, et al. The ability of land plants to synthesize glucuronoxylans predates the evolution of tracheophytes. Glycobiology. 2012;22(3):439–51.Google Scholar
  53. Harholt J, Sorensen I, Fangel J, Roberts A, Willats WG, Scheller HV, Petersen BL, Banks JA, Ulvskov P. The glycosyltransferase repertoire of the spikemoss Selaginella moellendorffii and a comparative study of its cell wall. PLoS One. 2012;7(5), e35846.View ArticlePubMedPubMed CentralGoogle Scholar
  54. Lee C, Zhong R, Ye ZH. Biochemical characterization of xylan xylosyltransferases involved in wood formation in poplar. Plant Signal Behav. 2012;7(3):332–7.View ArticlePubMedPubMed CentralGoogle Scholar
  55. Jensen JK, Johnson N, Wilkerson CG. Discovery of diversity in xylan biosynthetic genes by transcriptional profiling of a heteroxylan containing mucilaginous tissue. Front Plant Sci. 2013;4:183.View ArticlePubMedPubMed CentralGoogle Scholar
  56. Hu B, Jin J, Guo AY, Zhang H, Luo J, Gao G. GSDS 2.0: an upgraded gene feature visualization server. Bioinformatics. 2015;31(8):1296–7.View ArticlePubMedPubMed CentralGoogle Scholar
  57. Tamura K, Stecher G, Peterson D, Filipski A, Kumar S. MEGA6: Molecular Evolutionary Genetics Analysis version 6.0. Mol Biol Evol. 2013;30(12):2725–9.View ArticlePubMedPubMed CentralGoogle Scholar
  58. Sparkes IA, Runions J, Kearns A, Hawes C. Rapid, transient expression of fluorescent fusion proteins in tobacco plants and generation of stably transformed plants. Nat Protoc. 2006;1(4):2019–25.View ArticlePubMedGoogle Scholar
  59. Earley KW, Haag JR, Pontes O, Opper K, Juehne T, Song K, Pikaard CS. Gateway-compatible vectors for plant functional genomics and proteomics. Plant J. 2006;45:616–29.View ArticlePubMedGoogle Scholar
  60. Chen QJ, Zhou HM, Chen J, Wang XC. A Gateway-based platform for multigene plant transformation. Plant Mol Biol. 2006;62(6):927–36.View ArticlePubMedGoogle Scholar
  61. Clough SJ, Bent AF. Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J. 1998;16(6):735–43.View ArticlePubMedGoogle Scholar
  62. Updegraff DM. Semimicro determination of cellulose in biological materials. Anal Biochem. 1969;32:420–4.View ArticlePubMedGoogle Scholar
  63. Fukushima RS, Hatfield RD. Extraction and isolation of lignin for utilization as a standard to determine lignin concentration using the acetyl bromide spectrophotometric method. J Agri Food Chem. 2001;49:3133–9.View ArticleGoogle Scholar
  64. Liu YG, Chen Y. High-efficiency thermal asymmetric interlaced PCR for amplification of unknown flanking sequences. BioTechniques. 2007;43(5):649–54.View ArticlePubMedGoogle Scholar

Copyright

© Wang et al. 2016