Characterization of xylan in the early stages of secondary cell wall formation in tobacco bright yellow-2 cells

Carbohydrate Polymers 176 (2017) 381–391

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Carbohydrate Polymers journal homepage: www.elsevier.com/locate/carbpol

Characterization of xylan in the early stages of secondary cell wall formation in tobacco bright yellow-2 cells

MARK

⁎

Tadashi Ishiia, , Keita Matsuokaa,1, Hiroshi Onob, Mayumi Ohnishi-Kameyamac, Katsuro Yaoid, Yoshimi Nakanoe,2, Misato Ohtanie,f, Taku Demurae,f, Hiroaki Iwaia, Shinobu Satoha a

Faculty of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Ibaraki 305-8572, Japan Advanced Analysis Center, National Agriculture and Food Research Organization (NARO), Tsukuba, Ibaraki 305-8642, Japan c Food Research Institute, National Agriculture and Food Research Organization (NARO), Tsukuba, Ibaraki 305-8642, Japan d Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki 305-8566, Japan e Graduate School of Biological Sciences, Nara Institute of Science and Technology, Ikoma, Nara 630-0192, Japan f RIKEN Center for Sustainable Resource Sciences, Yokohama, Kanagawa 230-0045, Japan b

A R T I C L E I N F O

A B S T R A C T

Keyword: Cell wall polysaccharides Glucuronic acid Inducible VND7 system Tobacco BY-2 cell Xylan

The major polysaccharides present in the primary and secondary walls surrounding plant cells have been well characterized. However, our knowledge of the early stages of secondary wall formation is limited. To address this, cell walls were isolated from differentiating xylem vessel elements of tobacco bright yellow-2 (BY-2) cells induced by VASCULAR-RELATED NAC-DOMAIN7 (VND7). The walls of induced VND7-VP16-GR BY-2 cells consisted of cellulose, pectic polysaccharides, hemicelluloses, and lignin, and contained more xylan and cellulose compared with non-transformed BY-2 and uninduced VND7-VP16-GR BY-2 cells. A reducing end sequence of xylan containing rhamnose and galaturonic acid- residues is present in the walls of induced, uninduced, and nontransformed BY-2 cells. Glucuronic acid residues in xylan from walls of induced cells are O-methylated, while those of xylan in non-transformed BY-2 and uninduced cells are not. Our results show that xylan changes in chemical structure and amounts during the early stages of xylem differentiation.

1. Introduction Most of the studies on the chemical structure of plant cell walls have concentrated on the primary cell walls of growing plant tissues (Carpita & Gibeaut, 1993; McNeil, Darvill, Fry, & Albersheim, 1984; O’Neill & York 2003) and the lignified secondary cell walls of woody plants (Shimizu, 1991) and grasses (Åman, 1993). However, our knowledge of cell wall polymers associated with the early stages of secondary cell wall formation is limited. There have been some reports on cell wall polysaccharides from cambial tissues and differentiating zones of woody plants. Thornber and Northcote (1961a,b) reported changes in the chemical composition of a cambial cell during its differentiation into xylem and phloem tissue in trees. Simson and Timell (1978a) isolated cell walls from cambial tissues of aspen (Populus tremuloides) and basswood (Tilia americana) and characterized xyloglucan

Simson and Timell (1978b), arabinogalactan Simson and Timell (1978c), 4-O-methylglucuronoxylan and pectin Simson and Timell (1978d), and cellulose Simson and Timell (1978e). Edashige, Ishii, Hiroi, and Fujii (1995) isolated cell walls from xylem-differentiating zones of sugi (Cryptomeria japonica) and characterized pectic polysaccharides (Edashige & Ishii, 1996) and xyloglucan (Kakegawa, Edashige, & Ishii, 1997), rhamnogalacturonan I (RG-I) (Edashige & Ishii, 1997) and rhamnogalacturonan II (RG-II) (Edashige & Ishii, 1998). Ermel et al. (2000) reported on differential localization of arabinan and galactan of RG-I in cambial derivatives. Gene and metabolite profiles of cotton fiber during cell elongation and secondary cell wall formation were characterized by Gou, Wang, Chen, Hu, & Chen (2007). As far as we are aware there have been few reports on cell wall polysaccharides and lignin from the early stages of secondary cell wall formation. An in vitro differentiating system in which non-xylem cells of zinnia

Abbreviations: 2-AB, 2-aminobenzamide; BY-2, Bright Yellow-2; DEX, dexamethasone; EG, endo-(1 → 4)-glucanase; EPG, endo-polygalacturonase; ESI-MS, electrospray ionization mass spectrometry; LC, liquid chromatography; LC-ESI-MS, liquid chromatograph-electrospray ionization mass spectrometry; MALDI-TOF MS, matrix assisted laser desorption/ionization time of flight mass spectrometry; NAC, NAM/ATAF/CUC; RG-I, rhamnogalacturonan I; RG-II, rhamnogalacturonan II; TMS, trimethylsilyl; XG, xyloglucan; VND7, vascular-related NAC domain7 ⁎ Corresponding author. E-mail address: [email protected] (T. Ishii). 1 Department of Bioscience, Graduate School of Science and Engineering, Teikyo University, Utsunomiya, Tochigi 320-8551, Japan. 2 Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology, Tsukuba, Ibaraki 305-8566, Japan. http://dx.doi.org/10.1016/j.carbpol.2017.08.108 Received 16 May 2017; Received in revised form 4 August 2017; Accepted 22 August 2017 Available online 26 August 2017 0144-8617/ © 2017 Elsevier Ltd. All rights reserved.

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amounts of water to remove the phenol.

(Zinnia elegans) are induced to form xylem vessel elements has been used to study the mechanism of tracheary element differentiation (Fukuda, 1997; Roberts & McCann, 2000). Subsequently key regulators of xylem vessel differentiation including vascular-related NAC domain (VND) protein transcription factors in xylem vessel differentiation have been identified (Demura & Fukuda, 2007; Yamaguchi & Demura, 2010). A posttranslational induction system has been established, in which VND6 or VND7 was fused to the activation domain of herpes virus VP16 protein and the glucocorticoid receptor (GR) domain in their C-terminal region (VND6-VP16-GR or VND7-VP16-GR) under the control of the 35S promoter of the Cauliflower mosaic virus (CaMV), and this chimeric gene was activated upon treatment with the dexamethasone (DEX), a strong synthetic glucocorticoid. The transformed plants, for example, tobacco BY-2 cells expressing VND7-VP16-GR was transdifferentiated into xylem vessel elements after glucocorticoid treatment in a high yield. Yamaguchi et al. (2010), Goué et al. (2013), Li et al. (2016), and Ohtani et al. (2016) have been studied genes and proteins involved in the synthesis, modification and assembly of cell wall polymers. This induction system is also versatile tool for studying the types and structure of polysaccharides and lignin associated with the initial stages of secondary cell wall formation. In our previous works we reported that xylose and glucose residues in cell walls increased as the secondary cell wall formed, suggesting that xylan and cellulose increased (Yamaguchi et al., 2010; Goué et al., 2013). Here we isolated cell walls from tobacco VND7-VP16-GR BY-2 cells to differentiate and form xylem vessel elements and compared the amounts and structures of these polysaccharides with their counterparts in non-transformed BY-2 and uninduced cell walls. Our results provide that a reducing end sequence of xylan is present in non-transformed BY-2, uninduced, and induced cells and that glucuronic acid residues of xylan are 4-O-methylated as the xylem vessel elements are induced.

2.3. Extraction of Cell wall polysaccharides 2.3.1. Pectic polysaccharide Starch was removed by treating suspensions of the cell walls (200 mg 50 ml−1) in 50 mM potassium phosphate, pH 7, with α-amylase (Type I-A from porcine pancreas (Sigma-Aldrich Japan, Tokyo, Japan) under a drop of toluene. The reaction was carried out in polypropylene bottles placed in a shaking water bath for 18 h at 35 °C. After 18 h the suspension was centrifuged at 8,400 × g for 10 min, the supernatant was removed, and then the pellet washed once with buffer and once with water. The supernatant and the washes were combined, boiled for 5 min to inactive α-amylase, dialyzed against water at 4 °C with a 2,000 Da cut-off dialysis tubing. The retentate was concentrated using a rotary evaporator, and then lyophilized. The de-starched pellet was then suspended in 100 mM NaOH, and stirred for 4 h at 4 °C. After 4 h, 1 M acetic acid-sodium acetate buffer, pH 5.0 was added to adjust pH 5.0 and the walls treated for 18 h at 35 °C with endo-polygalacturonase (EPG) (Megazyme, Ireland). The soluble material was recovered as described above. Na2CO3 extraction of the EPG treated wall was carried out as described by Ishii, Thomas, Darvill, and Albersheim, (1989). The wall was suspended in 50 mM sodium carbonate for 2 h at room temperature. The wall suspension was centrifuged, the supernatant was removed, and the pellet washed with 50 mM sodium carbonate. The supernatant and the wash combined, dialyzed and lyophilized as described above. After Na2CO3 extraction, the wall was treated sequentially with EG from Trichoderma reesei (Megazyme). The residual wall remaining after Na2CO3 extraction was treated for 24 h at 35 °C with endo-(1 → 4)glucanase (EG) (Megazyme) in 50 mM sodium acetate, pH 5.0. The suspension was then centrifuged, the supernatant was removed, the pellet was washed once with water, the supernatant and the wash were combined, boiled for 5 min, and dialyzed against water in a 1,000 Da cut-off dialysis tubing. The retentate was concentrated using a rotary evaporator, and lyophilized. The EG-treated wall was then treated with 1 M KOH and 4 M KOH to solubilize xylan and xyloglucan (XG).

2. Materials and methods 2.1. Plant material and dexamethasone treatment Tobacco (Nicotiana tabacum L. cv.) bright yellow-2 (BY-2) and VND7-VP16-GR BY-2 suspension cultured cells were grown as described (Goué et al., 2013; Ohtani et al., 2016). The growth medium was supplemented with kanamycin (100 μg ml−1). For the induced VND7-VP16-GR BY-2 cell, the induction was proceeded by the addition of dexamethasone (DEX, 10 μM) at the beginning of the exponential growth phase. The cells were grown for 48 h at 27 °C with agitation in the absence of light. As induction rate after 48 h incubation reached about 70% (Ohtani et al., 2016), the cells incubated for 48 h were collected by filtration through filter paper using a vacuum pump. The cells were rinsed with 50 mM sodium phosphate buffer, pH 7.0 twice, and then kept at −80 °C. The cells were also collected from nontransformed BY-2 cells and VND7-VP16-GR BY-2 cells collected immediately after addition of DEX (uninduced cells).

2.3.2. Hemicellulose The EG-treated wall was suspended in 1 M KOH (50 ml containing 0.1 mM NaBH4) and stirred for 2 h at room temperature. The wall suspension was centrifuged, the supernatant was removed, then the pellet was washed once with 1 M KOH. The supernatant and the wash were combined, neutralized with acetic acid, dialyzed, and lyophilized. The wall was re-suspended in 4 M KOH (50 ml containing 0.1% NaBH4) and stirred at room temperature for 2 h. The 4 M KOH soluble material was treated as above and the wall residue was neutralized with acetic acid and dialyzed, and lyophilized. XG oligosaccharides were generated from the 1 M and 4 M KOH extracts as follows. The 1 M and 4 M KOH soluble extracts (2 mg) were treated for 16 h at 35 °C in 50 mM HCOONH4, pH 5.0 with a XG specific endo-(1 → 4)–β-glucanase (1 unit) from Geotricham sp. M128 (Yaoi & Mitsuishi, 2004). The enzyme reaction mixture was boiled for 1 min, centrifuged at 12,000 × g for 5 min, and the supernatant was passed through a cation exchange resin (Dowex 50W-X12), and lyophilized. Acidic xylan oligosaccharides were generated from the 1 M and 4 M KOH extracts as follows. The 1 M and 4 M KOH extracts (20 mg) in 1.9 ml of water were boiled for 5 min, cooled and then 0.1 ml of 1 M sodium acetate, pH 5.0 and 10 units of M1 xylanase from Trichoderma reesei (Megazyme) and a drop of toluene added. The mixture was kept for 16 h at 35 °C. The mixture was boiled for 5 min, centrifuged at 12,000 × g for 5 min, and the supernatant was passed through a cation exchange resin (Dowex 50W-X12). The eluent was applied on an anion exchange resin (Bio-Rad AG-1 × 8, acetate form). The neutral sugars were eluted with water (10 column volumes) and the acidic sugars were

2.2. Cell wall preparation The cell walls were prepared according to a slight modified method described by Selvendran and O’Neill (1987). The tobacco cells were ground in liquid nitrogen with a pestle and mortar and then homogenized in aq. 0.5% SDS using a ball mill with ceramic balls (Fritsch Classic Line P-5/2, Fritsch, Idar-Oberstein, Germany). Milling was performed as follows; 400 rpm min−1, for 10 min, 10 min hold, this procedure was repeated 20 times. The homogenate was centrifuged (8400 × g for 10 min at 4 °C) and the pellet then washed twice with 0.5% SDS and then 5 times with water. To remove lipids, the cell wall material was extracted 3 times with chloroform/methanol (1:1, v/v). Protein was removed by three extractions with phenol/acetic acid/ water (2:1:1, v/v/v). After collection of cell wall material by centrifugation (8400 × g for 5 min), the wall was rinsed with sufficient 382

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eluted with 5 M acetic acid (3 column volumes). The eluents were concentrated with a rotary evaporator to a small volume and lyophilized.

3. Results and Discussion

2.4. 2-Aminobenzamide derivatization of XG specific endo-glucanasegenerated xyloglucan oligosaccharides and endo-xylanase-generated acidic xylan oligosaccharides

Cell walls were isolated from tobacco non-transformed BY-2 cells and from VND7-VP16-GR BY-2 cells grown for 2 days in the presence of DEX or VND7-VP16-GR BY-2 cells collected immediately after DEX addition. The induction of VND7-VP16-GR BY-2 cells to xylem vessel elements after 48 h induction was estimated to be about 70% by microscopy. No induction was observed in non-transformed BY-2 cells and VND7-VP16-GR BY-2 cells in the presence of DEX for 0 h induction. Gene expression responsible for secondary cell wall formation peaked within 2 days after induction (Goué et al., 2013). Enhanced gene expression relative to the uninduced cells is still observed 48 h after induction, but the extent is decreasing, probably due to programmed cell death (Yamaguchi et al., 2010; Goué et al., 2013). So we named cells isolated from VND7-VP16-GR BY-2 cells in the presence of DEX for 48 induction and VND7-VP16-GR BY-2 cells in the presence of DEX for 0 h induction as induced cells and uninduced cells, respectively. The walls from induced cells contained more cellulose (33%) and less uronic acid (4%) compared with non-transformed BY-2 (cellulose content 22% and uronic acid content 12%). The induced cell walls contained 8.5% lignin, whereas non-transformed BY-2 contained ∼0.5% lignin. Uninduced cell walls had cellulose (22%) and lignin (0.5%) content similar to nontransformed BY-2 cells. These results show that VND7-induced cells have characteristic of secondary cell walls as described previously.

3.1. Cellulose and lignin content

XG specific endo-glucanase-generated XG oligosaccharides and endo-xylanase-generated acidic xylan oligosaccharides were reacted for 2 h at 65 °C with 0.2 M 2-aminobenzamide (2-AB) in the presence of 1 M NaBH3CN at pH 5.5 to quantitatively convert the reducing ends to their fluorescent derivatives (Ishii, Ichida, Matsue, Ono, & Maeda, 2002). 2-AB-derivatized oligosaccharides were recovered by size-exclusion chromatography as described before (Ishii et al., 2002). 2.5. Analytical methods 2.5.1. Determination of cellulose and lignin Cellulose and lignin content were determined by the Updegraff method (Updegraff, 1969) and by the thioglycolic acid method (Suzuki, Suzuki, Yamamoto, & Umezawa, 2009), respectively. Neutral sugar and uronic acid contents were determined by phenol-sulfuric acid method using glucose as the standard (Hodge & Hofreiter, 1962), and m-hydroxybiphenyl method using galacturonic acid as the standard (Blumenkrantz & Asboe-Hansen, 1973), respectively.

3.2. Glycosyl residue composition analysis of VND7-VP16-GR BY-2 cells grown in the presence of dexamethasone

2.5.2. Glycosyl residue composition and glycosyl linkage analysis Cell walls and the extracted residues were hydrolyzed with 72% sulfuric acid method (Seaman et al., 1954). Solubilized cell wall polysaccharides were hydrolyzed with 2 M trifluoroacetic acid. Neutral glycosyl residue compositions were determined by gas-liquid chromatography (GC) analysis of the alditol acetate derivatives. Combined neutral and acidic glycosyl compositions were determined by GC of their trimethylsilyl (TMS) methyl-ester methyl glycoside derivatives (York, Darvill, McNeil, Stevenson, & Alersheim, 1985). Glycosyl linkage compositions were determined using a modification of the Hakomori procedure (Hakomori, 1964). Polysaccharides were per-O-methylated as described by York et al. (York et al., 1985) and the resulting products were isolated using a 2,000 mw cut-off dialysis tubing. Uronic acidcontaining polysaccharides were methylated and carboxyl residues were reduced with Super-Deuteride (1 M Li-triethylborodeuteride in tetrahydrofuran) (York et al., 1985). The glycosyl linkage compositions were then determined by gas-liquid chromatograph-mass spectrometry (GC/MS) of the partially methylated, partially acetylated alditol acetate derivatives (York et al., 1985).

Glycosyl residue composition analysis of cell walls following Saemen (Seaman, Moore, Mitchell, & Millet, 1954) hydrolysis showed that the xylose and glucose contents in the walls of the induced cells increased and the arabinose and galactose contents decreased (Supplemental Table S-1). These results indicated that inducing VND7 leads to walls that contain more xylan and cellulose and less pectin than the non-transformed BY-2 and uninduced cells. Galacturonic acid and glucuronic acid residues were not detected as TMS derivatives by GC, although uronic acid content determined by m-hydroxybiphenyl method was 4 11%. 3.3. Sequential extraction of cell wall polysaccharides To characterize the cell wall polysaccharides, the cell walls were sequentially treated with EPG, Na2CO3, EG, 1 M KOH, and 4 M KOH. The glycosyl residue composition (Supplemental Table S-2) and glycosyl linkage composition (Table 1) were then determined. The α-amylase, EPG, and Na2CO3-soluble fractions of induced, nontransformed BY-2, and uninduced cell walls showed that these fractions contained substantial amounts of arabinose, rhamnose, galactose, and galacturonic acid (Supplemental Table S-2). The α-amylase and EPGsoluble fractions also contained small amounts of 2-O-methyl fucose and 2-O-methyl xylose, which are diagnostic glycosyl residues of rhamnogalacturonan II (RG-II) (O’Neill, Ishii, Albersheim, & Darvill, 2004). Further separation of RG-II fractions was not performed because the available amounts of α-amylase and EPG-soluble fractions were limited. Glycosyl linkage analysis showed that the α-amylase and EPGsoluble fractions contained 2- and 2,4-linked rhamnosyl, 4-linked gacturonic acid, terminal- and 5-linked arabinofuranosyl residues, 3linked, 4-linked and 3,6-linked galactosyl residues (Table 1), indicating that these fractions are rich in rhamnogalacturonan I (RG-I)-like polysaccharides (Ishii et al., 1989). 4-Linked and 4,6-linked glucosyl residues were detected in α-amylase, EPG, and Na2CO3 soluble fractions (Table 1), which could be from residual starch. Glycosyl linkage analysis of the EG-soluble fractions showed the presence of 4-linked, 2,4-linked, 3,4-linked xylosyl residues (Table 1),

2.5.3. Mass spectrometry 2AB labeled XG oligosaccharides were analyzed using a 4800 plus matrix assisted laser desorption/ionization time of flight (MALDI-TOF/ TOF) analyzer (AB SCIEX, Framingham, MA, USA) operating in the positive-ion mode and with 2, 5-dihydroxybenzoic acid as the matrix (Lerouxel et al., 2002). 2-AB labeled endo-xylanase-generated oligosaccharides were analyzed by liquid chromatograph-electrospray ionization mass spectrometry (LC-ESI–MS) using a LTQ Oritrap Velos (Thermo Fisher Scientific, Waltham, MA, USA). Reversed phase LC was performed on an Ultimate 3000 RSLC system (Thermo Fisher Scientific) using an ACQUITY UPLC HSS C18 column (2.1 mm i.d × 100 mm, 1.8 μm, Waters). The gradient conditions were as follows; eluent A, 0.1% formic acid in water and eluent B, 0.1% formic acid in acetonitrile and a linear gradient of eluent B; hold at 1% for 2 min, and from 1% to 40% in 21 min, from 40% to 97% in 2 min, and at 97% for 5 min. The flow rate was 0.3 ml min−1 at 30 °C, and the eluent was monitored at 340 nm. Mass spectra were recorded in the positive- and negative-ion modes with a spray voltage of 3 kV and a capillary temperature of 230 °C in the range of m/z 100 to 1000. 383

384 BY-2

3.1 3.8 7.2 1.4 1.9

n.d. 13.8 ± 2.1 n.d. 4.3 ± 0.4

1.4 ± 0.1 12.1 ± 0.1

EG

BY-2

Gal T-Gal 3-Gal 4-Gal 6-Gal 3,6-Gal

Glc T-Glc 4-Glc 2,3-Glc 4,6-Glc

GalA T-GalA 4-GalA

Llinkage

Ara T-Araf T-Arap 3-Araf 5-Araf

1 M KOH

n.d. n.d. n.d.

Man T-Man 4-Man 4,6-Man

0.2 0.2 0.4 0.05 0.2

6.5 ± 0.2 T 1.1 ± 0.1 15.9 ± 0.9

± ± ± ± ±

0.1 0.1 0.1 0.2 0.1

± ± ± ± ±

0.1 0.2 0.1 0.1 0.2

6.3 ± 0.1 T 0.6 ± 0.3 13.1 ± 0.3

0h

1.9 ± 0.1 14.6 ± 0.2

n.d. 9.4 ± 0.1 n.d. 6.2 ± 2.0

4.0 6.4 7.5 1.9 3.1

n.d. n.d. n.d.

5.8 ± 0.1 1.0 ± 0.1 1.0 ± 0.1 n.d. n.d.

0.6 4.6 2.6 6.6 6.7

± ± ± ± ±

0.05 0.6 ± 0.1 0.6 ± 0.1 n.d. n.d.

0.2 0.2 0.5 0.7 0.9

± ± ± ± ±

0.2 0.9 0.5 0.1 0.2

4.1 ± 0.1 T 0.7 ± 0.1 13.6 ± 0.3

48 h

1.9 ± 0.1 9.4 ± 0.2

n.d. 5.2 ± 0.5 n.d. 14.3 ± 0.3

4.0 2.9 6.0 1.9 5.0

n.d. n.d. n.d.

6.4 ± 0.5 1.8 ± 0.1 1.8 ± 0.1 n.d. n.d.

0.7 ± 0.1 2.1 ± 0.1 0.9 ± 0.1 3.5 ± 0.1 14.9 ± 1.3

± ± ± ± ±

0.1 0.1 0.2 0.1 0.1

5.7 1.1 1.5 7.9

± ± ± ±

0.1 0.1 0.1 0.1

1.9 ± 0.1 13.4 ± 0.2

n.d. 6.2 ± 0.1 n.d. 2.9 ± 0.1

6.6 7.5 9.2 1.9 2.4

n.d. n.d. n.d.

2.5 ± 0.7 T 0.6 ± 0.1 n.d. n.d.

0.9 ± 0.1 3.7 ± 0.1 0.8 ± 0.2 12.4 ± 0.3 6.2 ± 0.1

0.6 ± 0.1

Xyl T-Xyl 2-Xyl 4-Xyl 2,4-Xyl 3,4-Xyl

± ± ± ± ±

T

2.4 3.7 2.0 4.5 9.9

n.d.

2.9 ± 0.3 T 0.9 ± 0.1 16.0 ± 0.8 n.d.

Rha T-Rha 2-Rha 3-Rha 2,4-Rha 2,3,4-Rha

0.4 0.2 0.2 0.6

n.d.

4.9 ± 1.2 ± 1.2 ± 9.8 ± n.d.

Fuc T-Fuc

2.5 ± 0.1 1.0 ± 0.1 n.d. 12.7 ± 0.2 n.d.

4.4 ± 0.3 0.8 ± 0.1 2.2 ± 0.2 12.4 ± 0.63 n.d.

BY-2

48 ha

BY−2a

0 ha

EPG

α-amylase

Ara T-Araf T-Arap 3-Araf 5-Araf 3,5-Araf

Llinkage

5.4 1.4 1.2 4.0

0h

± ± ± ±

0.1 0.1 0.1 0.3

1.5 ± 0.2 12.8 ± 0.1

n.d. 7.5 ± 0.1 n.d. 3.3 ± 0.3

6.2 ± 0.1 6. 9 ± 0.2 9.1 ± 0.3 1.5 ± 0.1 3.6 ± 0.1

n.d. n.d. n.d.

3.0 ± 1.1 T 0.6 ± 0.1 n.d. n.d.

T 3.4 ± 0.1 1.2 ± 0.1 12.7 ± 0.2 4.3 ± 0.2

T

2.9 ± 0.4 T 0.8 ± 0.1 18.7 ± 0.4 n.d.

0h

± ± ± ± ±

0.1 0.1 0.1 0.2 0.1

3.2 0.7 0.6 4.8

± ± ± ±

48 h

0.3 0.1 0.1 0.1

2.0 ± 0.2 12.4 ± 0.6

n.d. 3.9 ± 0.1 n.d. 5.8 ± 0.1

5.9 6.9 6.5 2.0 4.4

n.d. n.d. n.d.

2.3 ± 0.7 1.1 ± 0.1 4.9 ± 0.2 n.d. n.d.

T 4.5 ± 0.1 1.7 ± 0.2 10.3 ± 0.2 5.6 ± 0.1

T

2.8 ± 0.2 T 1.0 ± 0.1 15.2 ± 1.1 n.d.

48 h

± ± ± ± ±

0.1 0.2 0.2 0.2 0.3

5.6 0.9 2.5 3.8

± ± ± ±

BY-2

0.1 0.1 0.1 0.2

４ M KOH

2.0 ± 0.2 13.5 ± 0.5

n.d. 6.5 ± 0.1 n.d. 3.5 ± 0.5

5.6 2.3 7.9 2.0 3.4

n.d. n.d. n.d.

1.3 ± 0.3 T 0.7 ± 0.1 n.d. n.d.

0.9 ± 0.1 6.8 ± 0.1 2.0 ± 0.1 10.5 ± 0.3 6.2 ± 0.2

0.8 ± 0.1

6. 3 ± 0.5 0.6 ± 0.1 1.4 ± 0.1 15.5 ± 0.6 T

BY-2

Na2CO3

± ± ± ± ±

0.1 0.5 1.7 0.3 0.1

5.1 1.0 1.4 2.3

0h

± ± ± ±

0.3 0.1 0.1 0.1

1.6 ± 0.2 13.1 ± 0.4

n.d. 5.4 ± 0.2 n.d. 3.3 ± 0.2

5.2 5.5 9.6 1.6 5.3

n.d. n.d. n.d.

1.2 ± 0.1 T 0.6 ± 0.1 n.d. n.d.

1.5 ± 0.1 6. 2 ± 0.1 2.1 ± 0.2 9.9 ± 0.1 5.7 ± 0.2

0.9 ± 0.2

5.4 ± 0.1 T 1.4 ± 0.1 13.7 ± 0.4 n.d.

0h

± ± ± ± ±

0.2 0.4 0.3 0.2 0.5

4.2 0.7 0.7 2.1

± ± ± ±

48 h

0.2 0.1 0.1 0.2

1.9 ± 0.2 12.8 ± 0.7

n.d. 3.5 ± 0.2 n.d. 6.5 ± 0.8

5.6 6.2 5.8 1.9 3.5

n.d. n.d. n.d.

2.1 ± 0.1 1.2 ± 0 3.9 ± 0.2 n.d. n.d.

1.1 ± 0.1 7.3 ± 0.3 2.7 ± 0.3 10.4 ± 0.4 9.5 ± 0.3

0.9 ± 0.1

4.0 ± 0.1 0.6 ± 0.1 0.8 ± 0.1 11.9 ± 0.5 n.d.

48 h

Table 1 Glycosyl linkage analysis of amylase, EPG, Na2CO3, EG, 1 M, and 4 M KOH-soluble fractions of cell walls isolated from non-transformed BY-2 cells, VND7-VP16-GR BY-2 cells with DEX for 0 h induction, and VND7-VP16-GR BY-2 cells with DEX for 48 h induction (mole%).

T. Ishii et al.

Carbohydrate Polymers 176 (2017) 381–391

0.8 ± 0.1 4.1 ± 0.2 n.d. 10.2 ± 0.3 8.6 ± 0.3

3.3 0.7 0.7 1.3 1.3

1.1 ± 0.4 T 1.4 ± 0.3

5.3 4.5 7.5 2.9 1.7

T 6.0 ± 0.2 n.d. 3.6 ± 0.5

1.1 ± 0.1 10.8 ± 0.7

Rha T-Rha 2-Rha 3-Rha 2,4-Rha 2,3,4-Rha

Xyl T-Xyl 2-Xyl 4-Xyl 2,4-Xyl 3,4-Xyl

Man T-Man 4-Man 4,6-Man

Gal T-Gal 3-Gal 4-Gal 6-Gal 3,6-Gal

Glc T-Glc 4-Glc 2,3-Glc 4,6-Glc

GalA T-GalA 4-GalA

385

0.2 0.2 0.2 0.1 0.4

± ± ± ± ±

0.1 0.3 0.3 0.5 0.4

0.1 0.1

0.1 0.1

± ± ± ± ±

0.1 0.1 0.1 0.2 0.1

1.4 ± 0.2 11.9 ± 0.3

1.1 ± 0.2 9.7 ± 0.1 n.d. 4.3 ± 0.3

6.5 4.6 7.8 1.7 1.4

1.8 ± 0.4 T T

3.0 0.7 1.3 2.1 1.0

0.6 ± 3.7 ± n.d. 9.8 ± 4.8 ±

0.6 ± 0.1

n.d.

0.3 0.3

± ± ± ± ±

0.1 0.5 0.6 0.2 0.5

0.1 0.1

± ± ± ± ±

0.1 0.1 0.1 0.1 0.1

1.0 ± 0.2 11.2 ± 0.3

1.7 ± 0.3 4.9 ± 0.3 n.d. 4.6 ± 0.2

5.4 0.7 7.3 1.5 1.6

2. 5 ± 0.8 4.0 ± 0.2 n.d.

2.8 1.8 4.6 1.7 1.3

0.8 ± 4.9 ± n.d. 9.5 ± 7.0 ±

T

n.d.

± ± ± ± ±

0.3 0.2 0.1 0.1 0.1

n.d. n.d.

1.2 9.9 3.2 8.9

6.1 2.6 3.0 2.1 3.6 ± ± ± ±

± ± ± ± ± 0.1 0.1 0.1 0.6

0.1 0.1 0.1 0.1 0.5

1.1 ± 0.1 2.6 ± 0.1 4.6 ± 0.1

4.0 ± 0.1 4.9 ± 0.7 3. 9 ± 0.9 2.2 ± 0.1 1.5 ± 0.1

1.7 4.1 1.0 5.5 2.6

1.6 ± 0.2

1.8 ± 0.2

± ± ± ± ±

± ± ± ± ± 0.43 0.2 0.5 0.3 0.2

0.2 0.1 0.2 0.1 0.1

± ± ± ± ±

0.4 0.1 0.1 0.5 0.3

n.d. n.d.

0.7 ± 0.1 13.4 ± 0.5 2.4 ± 0.3 9.6 ± 0.4

5.9 2.4 1.9 2.8 3.4

1.7 ± 0.1 2.4 ± 0.1 5.8 ± 0.1

4.8 2.3 7.0 2.5 1.3

1.4 2.0 2.6 5.5 3.1

2.1 ± 0.2

1.1 ± 0.3

0h

± ± ± ± ±

0.1 0.5 0.1 0.1 0.1

n.d. n.d.

0.8 8.5 0.8 8.8

3.4 1.8 2.5 2.2 2.3 ± ± ± ±

± ± ± ± ±

0.1 0.4 0.1 0.4

0.1 0.4 0.5 0.3 0.3

0.7 ± 0.1 2.9 ± 0.3 3.6 ± 0.3

3.8 ± 0.1 2.9 ± 0.1 28.0 ± 0.5 5.3 ± 0.4 1.6 ± 0.1

0.7 2.2 0.8 3.5 1.3

1.0 ± 0.1

1.1 ± 0.3

48 h

± ± ± ± ±

0.1 0.2 0.4 0.1 0.1

0.1 0.1

0.1 0.1

± ± ± ± ±

0.2 0.1 0.1 0.1 0.1

n.d. n.d.

0.7 ± 0.3 17.7 ± 0.4 T 16.0 ± 0.5

6.7 0.9 1.2 1.5 0.7

0.9 ± 0.3 4.3 ± 0.1 8.1 ± 0.2

4.1 3.1 6.8 1.5 0.8

1.5 ± 5.0 ± n.d. 2.3 ± 2.3 ±

0.9 ± 0.1

T

BY-2

４ M KOH

± ± ± ± ±

0.4 0.2 0.5 0.3 0.1

0.4 0.1

0.1 0.2

± ± ± ± ±

0.6 0.2 0.5 0.1 0.5

n.d. n.d.

1.1 ± 0.1 19.5 ± 0.9 T 15.8 ± 0.4

8.9 0.9 1.1 1.4 0.8

0.8 ± 0 4.8 ± 0.3 11.4 ± 0.2

4.4 3.0 7.2 1.7 0.4

0.4 ± 2.9 ± n.d. 1.4 ± 1.6 ±

0.7 ± 0.2

T

0h

Induction rate after 48 h was estimated to be about 70%. No induction was observed in non-transformed BY-2 cells and VND7-VP16-GR BY-2 cells collected immidiately after DEX addition. Trace:0.5≦. n.d.:not determined. SE (n = 3) a BY-2, 0 h, and 48 h mean non-transformed BY-2, VND7-VP16-GR BY-2 cells in the presence of DEX for 0 h induction, and VND7-VP16-GR BY-2 cells in the presence of DEX for 48 h induction, respectively.

± ± ± ± ±

0.2 0.1 0.1 0.1 0.1

T

± ± ± ± ±

n.d.

Fuc T-Fuc

BY-2

48 h

BY-2

0h

1 M KOH

EG

3,5-Araf

Llinkage

Table 1 (continued)

0.1 0.2

0.1 0.1

± ± ± ± ±

0.85 0.1 0.1 0.1 0.1

n.d. n.d.

1.0 ± 0.1 15.0 ± 1.1 T 11.3 ± 0.5

6.9 1.7 0.7 0.9 0.5

1.5 ± 0.1 5.2 ± 0.2 8.3 ± 0.4

4.5 ± 0.3 2.5 ± 0.3 23.7 ± 1.9 2.0 ± 0.3 1.7 ± 0.5

0.6 ± 1.7 ± n.d. 0.8 ± 1.3 ±

0.5 ± 0.1

T

48 h

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Fig. 1. MALDITOF mass spectra of 2AB-labeled endo-xyloglucanase digests from the 4 M KOH fractions of non-transformed BY-2 (A), uninduced (B), and induced cell walls (C). The characters of H and P mean hexose and pentose, respectively.

[(hexose)4-(pentose)3-2AB + H]+, [(hexose)4-(pentose)4-2AB + H]+, and [(hexose)5-(pentose)3-2AB + H]+, respectively (Fig. 1). These oligosaccharides are XG subunits (Hoffman et al., 2005). Glycosyl residue composition analysis of the endo-xyloglucanase digests showed they contained arabinose, xylose, and glucose (∼1:1:1, molar ratio), indicating that BY-2 cell walls have an arabinose-containing XG subunit. Mori, Eda, and Katō (1986) identified arabinose-containing XG heptasaccharide Ara2Xyl2Glc3 from tobacco cell walls. MALDITOF-MS analysis of the endo-xyloglucanase-soluble fractions of induced, nontransformed BY-2, and uninduced cell walls gave almost the same mass spectra (Fig. 1), indicating that XG structure was similar between the walls of induced, non-transformed BY-2, and uninduced cells.

indicating the existence of xylan. EG-soluble fractions also contained glycosyl residues characteristic for RG-I-like polysaccharides and XG i.e. 2- and 2,4-linked rhamnosyl, 4-linked galacturonic acid, terminaland 5-linked arabinofuranosyl residues, and 4-linked and 4,6-linked glucosyl residues, respectively. This could be from residual pectin. 3.4. XG XG typically accounts for about 20% of dicot primary cell walls. The 1 M KOH and 4 M KOH fractions from the walls of induced cells, nontransformed BY-2, and uninduced cells contained 22 50% of xylosyl residues and 4–15% of glucosyl residues (Supplemental Table S-2). Glycosyl linkage analysis of the 1 M KOH and 4 M KOH fractions showed that these fractions contained 4-linked, and 4,6-linked glucosyl residues (Table 1), indicating that these fractions contained XG. To investigate XG, the 1 M and 4 M KOH fractions were treated with a XG specific endo-glucanase. The enzyme cleaves XG backbone after the non-substituted glucose residues and releases heptasaccharide (Xyl3Glc4) to decasaccharide (Xyl3GalFucGlc4) fragments, as well as the shorter pentasaccharide (Xyl2Glc3) and hexasaccharide (Xyl2Glc4) fragments. The products of the digests were derivatized with 2AB, and the 2AB-labeled oligosaccharides analyzed by MALDITOF MS. Peaks at m/z 1183, 1315, and 1345 were observed, which corresponded to

3.5. Xylan Xylan is the most abundant hemicellulose in dicot secondary cell walls. The 1 M KOH and 4 M KOH fractions from induced cell walls contained 45 50% of xylose residue (Supplemental Table S-2). These fractions contained 4-linked, 3-linked 2,4linked, and 3,4-linked xylosyl residues (Table 1), indicating the presence of xylan. For further characterization of xylan, the 1 M and 4 M KOH soluble fractions were treated with endo-xylanase, and the acidic xylan oligosaccharides isolated. The glycosyl residue composition analysis showed that they 386

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Fig. 2. ESI–MS analysis of acidic xylan oligosaccharides from endo-xylanase digests from the 1 M KOH fractions of induced cell walls. Selected ion chromatogram for m/z 739 ([M+H]+) (above) and m/z 761([M + Na]+) (bottom) (A). MS/MS mass spectrum of the ion at m/z 761([M + Na]+) of retention time at 1.28 min (peak 1) (B), MS/MS mass spectrum of ion at m/z 739 ([M+H]+) of retention time at 2.14 min (peak 2) (C), MS/MS mass spectrum of the ion at m/z 761([M + Na]+) of the standard reducing end oligosaccharide xylose-rhamnosegalacturonic acid-xylitol (D).

the digests were reductively aminated with 2-AB and the derivatized oligosaccharides analyzed by reversed-phase LC/MS. As induced cell walls contained high amounts of xylan, the induced cell wall xylan was analyzed at first. Two major peaks at retention time 1.28 min (peak 1) and 2.14 min (peak 2) (Fig. 2A) were detected. In the positive-ion mode, peak 1 gave ions at m/z 739 ([M+H]+) and m/z 761 ([M + Na]+). In the negative-ion mode it gave an ion at m/z 737 ([M−H]−). These results established that the component in peak 1 has a molecular weight of 738. MS/MS analysis of peak 1 at m/z 761 gave product ions at m/z 609 ([M+H−152]+), m/z 433 ([M +H−152−176]+), m/z 287 ([M+H−152−176−146]+) (Fig. 2B). Observed ions with mass difference of 152, 176, and 146 in the MS/MS spectra were corresponding to pentitol, hexuronic acid and

contained xylose, arabinose, glucuronic acid and 4-O-methyl glucuronic acid and small amounts of galacturonic acid and rhamnose, indicating the presence of the Rha-GalA-containing reducing end sequence (Peña et al., 2007; Shimizu, Ishihara, & Ishihara, 1976). The reducing end sequence of xylan, i.e. xylose-rhamnose-galacturonic acid-xylose, has been reduced to corresponding alditol, i.e. xylose-rhamnose-galacturonic acid-xylitol during 1 M KOH extraction in the presence of NaBH4. When 1 M or 4 M extracts were treated with xylanase, xylan oligosaccharides having reducing ends were generated. When the xylanase digests are reductively aminated, the xylan oligosaccharides are aminated, whereas the reducing end sequence is not. The aminated oligosaccharides and non-derivatized oligosaccharides can be separated by reversed-phase LC (Peña et al., 2007). A portion of 387

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Fig. 3. ESI–MS analysis of acidic xylan oligosaccharides from endo-xylanase digests from the 1 M KOH fractions of non-transformed BY-2 (A–C) and the uninduced cell walls (D–F). Selected ion chromatogram for m/z 737.2 ([M−H]−) of BY-2 (A) and uninduced cell walls (D). High resolution mass spectra in the negative-ion mode at 1.29 min (peak 1) of BY-2 (B) and of the uninduced cell walls (E). MS/MS mass spectrum of the ion at m/z 737.2 ([M−H]−) of retention time at 1.28 min (peak 1) of BY-2 (C) and uninduced cell walls (F) and the standard reducing oligosaccharide xylose-rhamnose-galacturonic acid-xylitol (G).

388

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analyses of the ion at m/z 761 from non-transformed BY-2 cell walls gave the same product ions (data not shown) as those observed from the

deoxyhexose residues, respectively. By comparison with retention time and mass spectra of authentic xylose-rhamnose-galacturonic acid-xy-

Fig. 4. ESI–MS analysis of acidic xylan oligosaccharides from endo-xylanase digests of the 1 M KOH fractions from BY-2 (A and B) and induce cell walls (C and D). Total ion current chromatogram of non-transformed BY-2 cell walls (A). MS/MS spectrum of the ion at m/z 843([M+H]＋) of peak 1 from non-transformed BY-2cell walls (B). Total ion current chromatogram of induced cell walls (C). MS/MS spectrum of the ion at m/z 857([M+H]+) of peak 2 from induced cell walls (D). The characters of X, GlcA, and Me-GlcA mean xylose, glucuronic acid, and O-methyl glucuronic acid, respectively.

litol (Fig. 2D), peak 1 was identified as xylose-rhamnose-galacturonic acid-xylitol. These results showed that xylan from induced cells has the reducing end sequence present in xylan from hardwoods (Shimizu et al., 1976) and Arabidopsis stems (Peña et al., 2007). Non-transformed BY-2 and uninduced cell walls gave two small peaks at retention time 1.30 min (peak 1) and 2.20 min (peak 2) (Fig. 3A and D). In the negative-ion mode peak 1 gave an ion at m/z 737 ([M−H]−) (Fig. 3B and E). These results showed that the component in peak 1 has a molecular weight of 738. The positive-ion mode MS/MS

authentic oligosaccharide (Fig. 2D). The mass spectra from uninduced cell walls were too small to be interpreted. The MS/MS analyses of the ion at m/z 737 gave the same product ions (Fig. 3C and F) as those observed from the authentic oligosaccharide (Fig. 3G). These results showed that xylan from non-transformed BY-2 and uninduced cells has the reducing end sequence present in induced cells. The ion intensity at m/z 737 of non-transformed BY-2 and uninduced cells is about onetenth compared with that of induced ones, providing further evidence that walls from non-transformed BY-2 and uninduced cells have much 389

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and lignin, and less pectin than non-transformed BY-2 and uninduced cells. Induced, non-transformed, and uninduced BY-2cell walls contain at least five polysaccharides; these are homogalacturonan, RG-I, RG-II, XG, and xylan. Glucuronic acid residues are O-methylated in xylan from induced cells, but not in the xylan from non-transformed BY-2 and uninduced cells. A reducing end sequence of xylan is present in cell walls from induced, non-transformed BY-2 and uninduced cells. The induced cell walls contained higher amounts of the reducing end sequence than those of BY-2 and uninduced cell walls, which reflect xylan content in the cell walls.

less xylan than induced cells. Faik, Jiang, and Held (2014) suggested that a reducing end sequence is a secretion signal during glucuronoxylan synthesis in secondary cell wall or may be needed to attach glucuronoxylan at specific sites in the secondary walls. Present data suggested that the sequence is also involved in xylan biosynthesis in primary walls. Further experimental data are needed to understand the role of the reducing end sequence of xylan biosynthesis. The mass spectra at retention time at 2.19 min and 2.20 min (Fig. 3A and D) were too small to be interpreted. MS analysis of peak 2 (Fig. 2A) gave an ion peak at m/z 739 ([M +H]+). MS/MS analysis at m/z 739 gave product ions at m/z 607 ([M +H−132]+), m/z 475 ([M+H−2 × 132]+), m/z 417 ([M +H−190−132]+), m/z 285 ([M+H−190 − 2 × 132]+) (Fig. 2C). The observed ions with mass differences of 132 and 190 in the MS/MS spectra correspond to pentose and O-methyl hexuronic acid residues, respectively, confirming that the oligosaccharide was (O-methyl hexuronic acid)-(pentose)3-pentitol. This oligosaccharide likely originated from non-2-AB labeled O-methyl hexuronic acid-(pentose)4. The total current ion chromatogram of the 2AB labeled xylan oligosaccharides generated from non-transformed BY-2 and non-induced cell walls (Fig. 4A) showed an intense peak at a retention time of 8.21 min (peak 1). It gave an abundant ion at m/z 843 ([M+H]+). MS/ MS analysis of this ion gave product ions at m/z 711 ([M +H−132＋), m/z 667 ([M+H−176]+), m/z 579 ([M + +H−2 × 132] ), m/z 535 ([M+H−176−132]+), m/z 447 ([M +H−3 × 132]+), m/z 403 ([M+H−176−2 × 132]+), and m/z 271 ([M+H−176−3 × 132]＋) (Fig. 4B). Observed ions with mass difference of 132 and 176 in the MS/MS spectra were corresponding to pentose and hexuronic acid residues, respectively, suggesting that the oligosaccharide was hexuronic acid-(pentose)4-2AB. The induced cell walls gave an intense peak at retention time 8.84 min (peak 2) and a much small peak at 8.22 min (peak 1) (Fig. 4C). Peak 1 was identified as hexuronic acid-(pentose)4-2AB as described above. Peak 2 gave an intense ion at m/z 857 ([M+H]+). MS/MS analysis of the ion at m/z 857 gave product ions at m/z 725 ([M +H−132]＋), m/z 667 ([M+H−190]+), m/z 593 ([M + +H−2 × 132] ), m/z 535 ([M+H−190−132]+), m/z 461 ([M +H−3 × 132]+), m/z 403 ([M+H−190 − 2 × 132]+), and m/z 271 ([M+H−190−3 × 132]+) (Fig. 4D). The observed ions with mass difference of 132 and 190 in the MS/MS spectra correspond to pentose and O-methyl hexuronic acid residues, respectively, confirming that the oligosaccharide is O-methyl hexuronic acid-(pentose)4-2AB (OhnishiKameyama, 2016). O-methyl glucuronic acid-(xylose)4 was also identified by polysaccharide analysis using carbohydrate gel electrophoresis of the xylan oligosaccharides generated from induced VND7-VP16-GR BY-2 cells (Goué et el., 2013). The presence of the non-methylated fragment likely results from the fact that only 70% of the cells had undergone differentiation after 48 h and that some of the cells were under differentiated (Ohtani et al., 2016). Nevertheless, these results provide strong evidence that O-methylation of glucuronic acid residues occurs when xylem vessel elements with secondary walls are induced by expression of VND7 in tobacco BY-2 cells. Glucuronoxylan carrying a terminal 4-O-methly glucuronic acid residue and glucuronic acid residue were identified from lignified tobacco midribs (Eda, Watanabe, & Katō, 1977) and extracellular polysaccharides of suspension-cultured tobacco cell (Akiyama, Eda, & Katō, 1984), respectively. Extracellular polysaccharides are a good resource for primary cell walls (York et al., 1985). Mortimer et al. (2015) reported that xylan in Arabidopsis primary cell walls carries glucuronic acid and a pentose linked 1 → 2 to the α-1,2-D-glucuronic acid. Present data, together with the previous results indicate that O-methylation of glucuronic acid residues is associated with secondary wall formation.

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