Characterisation of extracellular polysaccharides from ... - Springer Link

Planta (2000) 210: 261±268

Characterisation of extracellular polysaccharides from suspension cultures of members of the Poaceae Ian M. Sims1*, Kay Middleton2, Alan G. Lane2, Andrew J. Cairns3, Antony Bacic1 1

Cooperative Research Centre for Industrial Plant Biopolymers and Plant Cell Biology Research Centre, School of Botany, University of Melbourne, Parkville, Victoria 3052, Australia 2 Cooperative Research Centre for Industrial Plant Biopolymers and Food Science Australia, PO Box 52, North Ryde, New South Wales 2113, Australia 3 Environmental Biology Department, Institute of Grassland and Environmental Research, Plas Gogerddan, Aberystwyth, Ceredigion, SY23 3EB, United Kingdom Received: 27 April 1999 / Accepted: 5 June 1999

Abstract. Microscopic examination of suspensioncultured cells of Phleum pratense L., Panicum miliaceum L., Phalaris aquatica L. and Oryza sativa L. showed that they were comprised of numerous root primordia. Polysaccharides secreted by these suspension cultures contained glycosyl linkages consistent with the presence of high proportions of root mucilage-like polysaccharides. In contrast, suspension-cultured cells of Hordeum vulgare L. contained mostly undi€erentiated cells more typical of plant cells in suspension culture. The polysaccharides secreted by H. vulgare cultures contained mostly linkages consistent with the presence of glucuronoarabinoxylan. The soluble polymers secreted by cell-suspension cultures of Phleum pratense contained 70% carbohydrate, 14% protein and 6% inorganic material. The extracellular polysaccharides were separated into four fractions by anion-exchange chromatography using a gradient of imidazole-HCl at pH 7.0. From glycosyl-linkage analyses, ®ve polysaccharides were identi®ed: an arabinosylated xyloglucan (comprising 20% of the total polysaccharide), a glucomannan (6%), a type-II arabinogalactan (an arabinogalactanprotein; 7%), an acidic xylan (3%), and a root-slime-like polysaccharide, which contained features of type-II arabinogalactans and glucuronomannans (65%). Key words: Cell-suspension culture ± Compositional analysis ± Di€erentiation ± Extracellular polysaccharide ± Poaceae ± Root mucilage

*Present address: Industrial Research Limited, Grace®eld Research Centre, PO Box 31-310, Lower Hutt, New Zealand Abbreviations: AG = arabinogalactan; AGP = arabinogalactanprotein; Araf = L-arabinofuranose; Arap = L-arabinopyranose; ECP = extracellular polysaccharide; Fucp = L-fucopyranose; Galp = D-galactopyranose; GalAp = D-galacturonic acid; Glcp = D-glucopyranose; GlcAp = glucuronic acid; Manp = Dmannopyranose; Rhap = L-rhamnopyranose; XG = xyloglucan; Xylp = D-xylopyranose Correspondence to: A. Bacic; E-mail: [email protected]; Fax: 61(3) 9347 1071

Introduction Plant cells in suspension culture are generally considered as being in a relatively homogeneous and undi€erentiated state. However, plant cells have a tendency to grow as aggregates and large clumps of cells are often di€erentiated to some extent (Allan 1991). Suspensioncultured plant cells, particularly those of dicotyledons, have been used widely to study the structure of cell wall polysaccharides (McNeil et al. 1984; York et al. 1985). The structures of polysaccharides extracted from the cell walls have been shown to be representative of those found in intact plants, and polysaccharides secreted into the medium of suspension cultures have been shown to be similar to those in the walls (McNeil et al. 1984; York et al. 1985 and references therein). There have, however, been relatively few investigations of the structure of extracellular polysaccharides (ECPs) from suspensioncultured monocotyledons. Primary cell walls of graminaceous monocots are composed of 2±40% cellulose, together with a high proportion of highly substituted heteroxylans (glucuronoxylans) and variable amounts of (1 ® 3, 1 ® 4)-b-D-glucans (Bacic et al. 1988; Carpita and Gibeaut 1993). The walls of suspension-cultured monocot cells derived from endosperm tissue contain substantially larger amounts of (1 ® 3, 1 ® 4)-b-D-glucans (Anderson and Stone 1978) than those derived from somatic cells (Burke et al. 1974; Carpita et al. 1985). For monocot plant cells in suspension-culture, in some instances there is a strong correlation between the composition of the ECPs from suspension cultures and the cell walls. Burke et al. (1974) showed that ECPs from sugarcane and wheat contained mostly arabinoxylans, and contained essentially the same glycosyl linkages as arabinoxylans in the walls. However, ECPs from oats contained a high proportion of type-II arabinogalactan (AG), whereas the walls contained a high proportion of arabinoxylan linkages with type-II AGs present in only minor amounts (Burke et al. 1974). Arabinogalactanproteins (AGPs) have been isolated from the culture medium of suspension cultures of Lolium multi¯orum,

262

I.M. Sims et al.: Polysaccharides from grass cell suspension cultures

although these cultures were also rich in arabinoxylans (Anderson et al. 1977). The ECPs from some maize suspension cultures, which grew as clumps of aberrant roots, also contained AGP-like molecules, with high levels of fucose, which indicated the presence of polysaccharides similar to those of seedling root-slime (Bacic et al. 1987). In this paper we report the morphological examination of the cells and the chemical characterisation of ECPs and cell walls from cell-suspension cultures of members of the Poaceae (Phleum pratense, Panicum miliaceum, Phalaris aquatica, Oryza sativa and Hordeum vulgare). Materials and methods Initiation and maintenance of cultures. Seeds of Phleum

pratense L., Panicum miliaceum L., Phalaris aquatica L., Oryza sativa L. and Hordeum vulgare L. were surface-sterilised with 4% NaOCl (10 min) and washed extensively with sterile H2O. The seeds were germinated on Murashige and Skoog (MS) basal medium (Murashige and Skoog 1962), supplemented with 2,4dichlorphenoxyacetic acid (2,4-D) and mixed cytokinins (Table 1) and solidi®ed with agar (5.0 g á l)1). Following germination, the growing shoots were cut o€ and the seedlings pushed together to induce formation of callus. The resulting calli were maintained at 27 °C in the dark and subcultured at 2- to 4-week intervals, depending on the species. Calli were transferred to liquid medium (10 ml in a 50-ml Erlenmeyer ¯ask) using the media formulation described for establishing callus cultures (Table 1) except that the agar was omitted. The cultures were repeatedly transferred (every 3 weeks) to larger volumes of fresh liquid medium to give the ®nal suspension cultures (50 ml in a 250-ml ¯ask). Suspension cultures of Phleum pratense, Panicum miliaceum, Phalaris aquatica and H.vulgare were maintained by sub-culturing every 2±3 weeks depending on the species. The suspension culture of O. sativa was maintained on basal MS liquid medium (Murashige and Skoog 1962) supplemented with 2,4-D (4 mg á l)1) and mixed cytokinins (2.15 mg á l)1) and sub-cultured every 3 weeks.

Microscopy. Clumps of cells from suspension cultures of Phleum

pratense, Panicum miliaceum, Phalaris aquatica, O. sativa and H. vulgare were stained with I2/KI, blotted to remove excess stain and photographed under a dissecting microscope. Nodules from the cell clumps of Phleum pratense were ®xed in 4% (w/v) paraformaldehyde, 2% (w/v) glutaraldehyde in 0.03 M Pipes bu€er (pH 7.0) for 1 h at room temperature. The tissue was dehydrated in a graded series of ethanol (30%±100%, v/v) and embedded in Spurrs resin (Spurr 1969). Longitudinal sections (2 lm

Table 1. Media used for establishing callus cultures Species

Phleum pratense Panicum miliaceum Phalaris aquatica Oryza sativa Hordeum vulgare a

MS basal medium supplemented with: 2,4-D (mg á L)1)

Cytokinins (mg á L)1)a

2 4 4 2 2

0 0 2.15 1.075 1.075

Mixed cytokinins consisting of 333.3 lM each of benzyladenine (75.0 mg á L)1), kinetin (71.7 mg á L)1) and isopentenyladenine (67.6 mg á L)1)

thick) were cut using an ultramicrotome, stained with toluidine blue O and transferred to microscope slides. Bright-®eld microscopy was carried out with an Olympus BH microscope ®tted with a tungsten lamp, and photographed using LBT-N and ND-6 ®lters.

Preparation of ECPs. Extracellular polysaccharides were

recovered from the culture medium 2 d after the sucrose was exhausted (14±28 d depending on species). Cells were removed by ®ltration under suction through four layers of gauze. The ®ltrate was transferred to a dialysis sac (molecular weight cut-o€ 10 000) and concentrated to approximately one-third the original volume by covering the sac with polyethylene glycol ¯akes (Polyethylene Glycol 6000; Ajax Chemicals, Australia) overnight at 1 °C. Na2 á EDTA . 2H2O was added to a ®nal concentration of 10 g á l)1 (EDTA was ®rst dissolved in 100±200 ml H2O by adjusting the pH to 8.0 using 3 M NH4OH). The EDTA solution was then added slowly to the concentrated ®ltrate while mixing and the pH readjusted to 8.0 with 0.5 M NH4OH. The concentrate was dialysed against H2O for 1±2 d at 1 °C and freeze-dried.

Preparation of cell walls. Cells from suspension cultures of

Phleum pratense were washed extensively with H2O to remove ECPs, frozen in liquid nitrogen and ground to a powder in a mortar and pestle. The powder was suspended in Hepes-KOH (25 mM, pH 7.0) containing 1 mM CaCl2 and sonicated on ice (2 ´ 1 min, setting 9, 20 Hz, Branson 250/450 Soni®er). Wall fragments were pelleted by centrifugation (100 g, 5 min), the supernatants were discarded, and the pellets were resuspended in bu€er and recentrifuged (100 g, 5 min). The wall suspensions were suspended in Hepes-KOH (20 mM, pH 7.2) containing 1 mM CaCl2, heated (80 °C, 5 min) to gelatinise the starch granules, cooled and incubated with porcine pancreatic a-amylase (Type 1-A; Sigma Chemical Company, St. Louis, Mo., USA) for 24 h at 20 °C to remove contaminating starch. The walls were recovered by centrifugation (1000 g, 5 min), washed once with Hepes-KOH bu€er, twice with water, and stored in 80% (v/v) ethanol at )20 °C.

Fractionation of ECPs. A solution of Phleum pratense poly-

mers (45 mg) in 20 mM imidazole-HCl bu€er (50 ml, pH 7.0) was applied to a column (10 cm long, 1.5 cm i.d.) of DEAE-Sepharose CL-6B equilibrated in the same bu€er and eluted at 4 ml á h)1 until no carbohydrate was detected in the eluate by the anthrone assay. Material which bound to the column was then eluted by a linear gradient (0.02±2 M) of imidazole-HCl (pH 7.0) over 100 ml, and fractions (2 ml) assayed for hexose, uronic acids and AGP as described below. Appropriate fractions were pooled to give four fractions (A, B, C and D), dialysed extensively against H2O and freeze-dried. Fraction A from anion-exchange chromatography (7 mg redissolved in 3.5 ml H2O) was treated by addition of ammonium sulphate to saturation (Akiyama and Kato 1982), left for 1 h at 20 °C and centrifuged (10 000 g, 10 min). The pellet was dissolved in H2O (5 ml), and the pellet (A-1) and supernatant (A-2) dialysed extensively against H2O, then freeze-dried.

Analytical methods. Total carbohydrate was determined by the

phenol-sulphuric acid method (Dubois et al. 1956) using galactose as a standard. Uronic acid was determined by the 3-phenylphenol method (Blumenkrantz and Asboe-Hansen 1973) using galacturonic acid as a standard. Hexoses were determined by the anthrone method (Dische 1962) using galactose as a standard. Arabinogalactan-protein was determined by radial di€usion against b-glucosyl Yariv reagent (Van Holst and Clarke 1985) using gum arabic (Sigma) as a standard. Total protein was determined by the Bio-Rad micro-assay method (Bradford 1976). Inorganic material was determined by ash content and was performed by National Analytical Laboratories (Melbourne, Australia).

Linkage analysis. Linkage analysis was performed following

reduction of uronic acids to their corresponding neutral sugars

I.M. Sims et al.: Polysaccharides from grass cell suspension cultures prior to methylation as described by Sims and Bacic (1995). Reduced samples (approx. 0.5 mg) were methylated using the NaOH/CH3I procedure of Ciucanu and Kerek (1984) as described by McConville et al. (1990). Methylated polysaccharides were hydrolysed with 2.5 M tri¯uoroacetic acid for 4 h at 100 °C, then dried in a stream of N2 at 40 °C and reduced with 1 M NaBD4 in 2 M NH4OH overnight at room temperature. Excess borodeuteride was destroyed by the addition of acetic acid and borate removed by repeated evaporation (5´) with 5% acetic acid in methanol. The partially methylated alditols were then acetylated with (CH3CO)2O for 2.5 h at 100 °C. Partially methylated alditol acetates were separated on a bonded phase BPX70 (SGE, Australia) column in a Finnigan MAT 1020B GC-MS (Lau and Bacic 1993).

Results and discussion Morphology of cultures. Freshly initiated calli of Phleum pratense, Panicum miliaceum, Phalaris aquatica and H. vulgare were all visibly slimy, with those of Phleum pratense and Panicum miliaceum embedded in large amounts of clear, glistening mucilage. In contrast, the callus culture of O. sativa tended to be drier, more granular and free from any visible mucilage. Suspension cultures of each of the species investigated grew as clumps of cells ranging in size from approximately 2 to 5 mm across. The cell clumps from suspension cultures of Phleum pratense (Fig. 1A), Phalaris aquatica (Fig. 1B), O. sativa (Fig. 1C) and Panicum miliaceum (data not shown) were lumpy and covered with numerous nodules which stained with iodine. The pattern of iodine-staining of the clumps of cells in these species showed that starch was present in the nodules, and suggested that the nodules consisted of amyloplastcontaining root-cap cells. These morphological features suggested that, similar to some suspension cultures of Z. mays (Bacic et al. 1987), suspension cultures of Phleum pratense, Panicum miliaceum, Phalaris aquatica and O. sativa were composed of clumps of aberrant roots. In contrast, cell clumps of H. vulgare (Fig. 1D) were rather amorphous and did not stain with iodine and appeared to be composed of undi€erentiated cells. Longitudinal sections through nodules of cell clumps of Phleum pratense stained with toluidene blue O are shown in Fig. 1E and F. There is a region of more intense staining which has more disorganised ®les of cells extending from right to left, and which appears similar to the apical meristem of roots (Fig. 1E). The surrounding cells are loosely packed and not organised. These features resemble those of root primordia and developing lateral roots (McCully 1975; Salisbury and Ross 1992). The darker-staining region is composed of relatively small cells which have large nuclei (Fig. 1F) and are similar to those of lateral-root meristems. In Z. mays, cells of the epidermis of developing lateral roots secrete a polysaccharide-containing mucilage, and root-cap initials have amyloplasts containing starch (McCully 1975). The clumps of cells in suspension cultures of Phleum pratense became progressively smaller with successive subculture and the areas of iodine-staining less widely distributed (data not shown), until very little staining was observed. The decrease in the size of the cell clumps

263

and reduction in iodine-staining with successive subcultures of Phleum pratense cells indicated that the cultures were dedi€erentiating. These subcultured cells were not investigated further. Linkage compositions of ECPs. The linkage compositions of ECPs from Phleum pratense, Panicum miliaceum, Phalaris aquatica and O. sativa showed similar structural features (Table 2). There were high proportions of Araf and Galp linkages present, with various branched galactose (2,3-, 2,6-, 3,4-, 3,6- and 3,4,6-Galp) residues detected, and smaller amounts of Fucp linkages. This glycosyl linkage composition was similar to that of ECPs from suspension cultures of Z. mays (Bacic et al. 1987) and to the mucilage secreted by Z. mays seedling roots (Bacic et al. 1986). Morphologically, the Z. mays suspension cultures which secreted root-mucilage-like polysaccharides grew as clumps of aberrant roots rather than being undi€erentiated cell lines. Thus, it appears that ECPs from Phleum pratense, Panicum miliaceum, Phalaris aquatica and O. sativa contained varying amounts of root-mucilage-like polysaccharides. Extracellular polysaccharides from H. vulgare contained only low proportions of the arabinose and galactose linkages present in ECPs from Phleum pratense, Panicum miliaceum, Phalaris aquatica and O. sativa, but contained high proportions of linkages consistent with the presence of approximately 46% glucuronoarabinoxylan. Similar di€erences in the composition of ECPs from suspension cultures of monocotyledons have been reported previously (Burke et al. 1974). These results suggested that, except for H. vulgare, the ECPs from all the grasses were similar. We selected Phleum pratense ECP as a representative of this latter group for further analyses. Composition of ECPs from suspension cultures of Phleum pratense. The polymers secreted by Phleum pratense cells were composed of approximately 70% (w/w) carbohydrate and 14% (w/w) protein and 6% (w/w) inorganic material, accounting for 90% (w/w) of the total dry matter. The monosaccharide composition was similar to that reported by Hale et al. (1987), although in the present study the ECPs contained more glucose and less xylose. Comparison of the linkage composition of Phleum pratense ECPs (Table 2) to those of puri®ed polysaccharides, and quantitation of individual polysaccharides by summing the mol% of individual residues (Shea et al. 1989) initially indicated that they contained approximately 18% type-II AG, 12% arabinoxylan, 10% xyloglucan (XG) and 4% glucomannan. However, the AGP-speci®c b-glucosyl Yariv gel di€usion test showed that only small (1%) amounts of AGPs were present, and the unusual linkage types detected (3-Arap, 2,6-Galp, 3-Rhap) made it dicult to identify unequivocally the types and proportions of the various polysaccharides prior to fractionation. Fractionation of Phleum pratense ECPs by anionexchange chromatography gave an unbound neutral fraction (A) and three bound acidic fractions (B, C and D; Fig. 2). The overall yield of total carbohydrate from

264

I.M. Sims et al.: Polysaccharides from grass cell suspension cultures

Fig. 1A±F. Light microscopy of cell clumps from suspension-cultured cells of Phleum pratense (A,E,F), Phalaris aquatica (B), Oryza sativa (C) and Hordeum vulgare (D). A±D Low-magni®cation images of cell clumps stained with iodine for detection of starch. E,F Higher-

magni®cation images of longitudinal sections through nodules of cell clumps of Phleum pratense stained with Toluidine blue O. Bars = 1 mm (A±D), 250 lm (E), 100 lm (F)

the anion-exchange column was 66% (w/w), and the yield of uronic acid was 63% (w/w; calculated from the relative proportions of glucuronic acid in the three fractions). The yield of protein from the column was only 15% (w/w), indicating that most probably remained bound to the anion-exchange resin. Fraction A (8 mg) accounted for 27% (w/w) of the recovered

material, and contained only neutral carbohydrate. No protein was detected in this fraction. Fraction B (3 mg) accounted for 10% (w/w) of the recovered material and contained 1% uronic acid; the b-glucosyl Yariv di€usion test, detected AGP only in this fraction (Fig. 2). Fraction B contained approximately 2% (w/w) protein. Fractions C (14 mg) and D (5 mg) accounted for 47%

I.M. Sims et al.: Polysaccharides from grass cell suspension cultures

265

Table 2. Linkage composition of suspension-culture ECPs from Phleum pratense, Panicum miliaceum, Phalaris aquatica, O. sativa and H. vulgare and cell walls from Phleum pratense Sugara

Deduced glycosidic linkageb

Linkage composition (mol%)c Phleum pratense Cell walls

ECPs

Panicum miliaceum ECPs

Phalaris aquatica ECPs

Oryza sativa ECPs

Hordeum vulgare ECPs

±

Rhap Fucp

3terminal 3-

± ± ±

4 tr 1

tr 2 5

1 4 5

±

3

± ± ±

Araf

terminal terminalp 233-p

7 1 1 2 ±

15 9 1 5 4

20 1 3 tr ±

22 tr tr 2 8

20 tr 3 1 ±

29 1 1 4 ±

2

tr

tr

±

3

4

5Xylp

terminal 243,42,3,4-

6 1 2 5 ±

5 1 2 5 ±

3 1 3 3 ±

3 tr 3 7 5

10 2 4 4 ±

4 2 8 13 4

Manp

42,3-

6 ±

2 tr

7 7

3 1

4 tr

2 ±

Galp

terminal 23462,32,63,43,63,4,6-

6 1 3 tr 1 ± ± ± ± ±

8 1 4 1 5 ± 5 ± 4 tr

13 1 2 tr 3 2 ± 5 6 2

6 11 4 1 1 ± 1 ± 4 ±

10 ± 10 1 1 tr ± 1 9 2

Glcp

terminal 44,6-

1 52 4

1 9 5

tr 3 2

1 7 1

1 5 2

2 8 1

GlcApd

terminal 43,4-

± ± ±

± 2 1

± 1 1

± 1 1

± tr tr

2 ± ±

±

7

2 tr 1 ± ± ± 4 ±

a

See Abbreviations for de®nitions Terminal Araf is deduced from 1,4-di-O-acetyl-2,3,5-tri-O-methylpentitol etc. Average of duplicate determinations d Uronic acids and their esters were reduced to 6,6¢-dideuterio neutral sugars before methylation tr, Trace (