Plant Cell Reports (1995) 14:279-284 Plant Cell Reports

Differences in pectic polysaccharides between carrot embryogenic and non-embryogenic calli

Akira Kikuchi 5 Shinobu Satoh l , Norio Nakamura ', and Tadashi Fujii

Institute of Biological Sciences, University of Tsukuba, Tsukuba, Ibaraki 305, Japan Department of Biology, Yokohama City University, 22-2 Seto, Kanazawa-ku, Yokohama, Kanagawa, 236 Japan

Received 23 March 1994lRevised version received 24 September 1994 - Communicated by A. Komamine

Summary. Cell walls and media were obtained from three kinds of carrot cell culture, namely, embryogenic callus (EC), non- embryogenic callus (NC) and somatic embryos (SE), and analyzed for their sugar content and sugar composition by electrophoresis and gas chromatography. EC formed large cell clusters while NC formed small clusters. Observations under the light microscope revealed that the intercellular contacts in NC were much more limited than those in EC. The analysis of pectic polysaccharides revealed that the level of neutral sugars was higher than that of acidic sugars in EC, while the opposite was true in NC. Gas- chromatographic analysis of neutral sugars in pectic fractions revealed that EC and SE were rich in arabinose, while NC was rich in galactose. On the basis of these results, we discuss the possible involvement of neutral sugars, and of arabinose and galactose in particular, in pectic polysaccharides in intercellular contacts.

Abbreviations EC, embryogenic callus; NC, non-embryogenic callus; SE, somatic embryo; MS, Murashige and Skoog; PAS, periodic acid-Schiff's reagent

Key Words: Carrot / Pectin / cell-to-cell attachment

Introduction

Carrot cells in culture are the most suitable available system for studies of the biochemical

Correspondence to: S. Satoh

and morphological events in the embryogenesis of higher plants because somatic embryos are induced with a high degree of synchrony. When embryogenic callus, induced from carrot hypocotyl in the presence of 2,4- dichlorophenoxyacetic acid (2,443) and capable of forming somatic embryos in the absence of auxins is maintained in culture for more than 6 months, it is gradually converted to non- embryogenic callus and loses embryogenic competence (Reinert et al., 1970).

We have demonstrated some differences between NC and EC in terms of gene expression (Kiyosue et al,, 1992a, Satoh e t al., 1992a), level of an endogenous hormone (Kiyosue et al., 1992b), and the morphological features of cell clusters (Satoh et al., 1986). NC forms much smaller cell clusters than EC. This result suggests that cell-to-cell attachment is important for morphogenesis, In fact, tight intercellular contacts have been observed in somatic embryos, as well as in zygotic embryos (Halperin 1966).

The strength of intercellular contacts should depend on cell wall structures, which include polysaccharide components, and in particular on pectin, which is found in the middle lamella of plant cells. Pectin is known to be related to processes involved in the breakdown and the loosening of cell wall (Goldberg et al., 1989). Moreover, there have been many reports about qualitative and quantitative changes in sugars in the pectic fraction during growth (Nevins et al., 1968, Nishitani and Masuda 1982, Sakurai et al., 1987, Tanimoto 1988).

Pectic substances are heterogeneous acidic polysaccharides. They are composed of a linear polymer, polygalacturonic acid, that

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forms the 'backbone' and at tached side chains, m a d e o f neutral sugars and k n o w n as 'neutral suga r b locks ' (S t todar t et al., 1967). The cha rac te r i s t i c s o f pec t ic subs tances depend main ly on the composi t ion o f the neutral sugar blocks and on the extent of methylat ion of the

c a r b o x y l g r o u p s o f p o l y g a l a c t u r o n i c ac id (Bonner 1950).

In th is r e p o r t , we d e s c r i b e the d i f fe rences in morphologica l features and cell wal l p o l y s a c c h a r i d e s b e t w e e n the cel ls o f

e m b r y o g e n i c callus, n o n - e m b r y o g e n i c callus and somatic embryos , and we discuss possible invo lvement o f side chains of pectic substances in the a t tachment o f cul tured carrot cells to one another.

Materials and Methods

Plant materials and cell culture. Embryogenic callus (EC) and Non-embryogenic callus (NC), which had lost embryogenic competence and failed to form large clusters of cells such as EC, were obtained from hypocotyl segments of Daucus carota L. cv. US- Harumakigosun as described in Kiyosue et al.,(1991) and Satoh et al., (1986), and cultured in Murashige and Skoog's (MS) (Murashige and Skoog 1962) liquid medium containing 2,4-dichlorophenoxyacetic acid (2,4- D ; 1 mg/1). Somatic embryos (SE) were produced by transferring the embryogenic callus to auxin free medium as also described in Satoh et al., (1986). After 2 weeks in culture, cells were separated from the culture medium by filtration through a 36 lam mesh, and used for further experiments after lyophilization.

Light microscopy. A suspension of carrot cells was mixed with fixative [2.5% glutaraldehyde in 0.2 M sodium-cacodylate buffer (pH 7.2)] for fixation, and then suspended in the same buffer supplemented with 1% osmium tetroxide for post-fixation. The fixed material was dehydrated in an ethanol series and then further dehydrated in absolute ethanol. The material was embedded in Spurr's resin as described in Inouye et al.,(1990).

Sections (1.5 l.tm thick) of resin-embedded material were stained with toluidine blue as described in Coetzee (1982). They were observed and photographed under a light microscope (Vanox-T; Olympus, Tokyo).

Preparation of cell walls. Lyophilized cells powder was incubated in boiling 85% ethanol for 10 min, and washed in 80% ethanol. Then the residuum was suspended in a mixture of chloroform and methanol (1:1,

v/v) and in acetone, collected on a glass-fiber filter (Whatman GF/F; Whatman International Ltd., Maidstone, U.K.), and used as the cell wall fraction after air-drying.

Fractionation of cell wall and extracellular polysaccharides. The cell wall fraction was suspended in boiling 40 mM ammonium oxalate (pH 4.0), incubated for 200 min at 99~ The supernatant was dialyzed overnight against water, lyophilized and used as the pectic fraction. The residuum was suspended in 24% KOH that contained 0.1% NaBH4, incubated for 24 hr at 25~ The residuum was used as the cellulose fraction. The pH of the supernatant was adjusted to 4.5 with acetic acid at 4~ The sediment was used as the hemicellulose A fraction. The supernatant was dialyzed overnight against water, lyophilized and used as the hemicellulose B fraction.

The culture medium was lyophilized and the residue was suspended in boiling 10 mM sodium acetate buffer (pH 4.0). After boiling for 10 min, the solution was dialyzed overnight against the same buffer, lyophilized and used as the medium fraction.

In order to eliminate proteins, the pectic, hemicellulose B and medium fractions were resuspended in water and combined with a mixture of phenol and chloroform (3:1, v/v). Then the aqueous fraction was collected. The hemicellulose A and cellulose fractions were resuspended in 10% sodium dodecyl sulfate boiled for 10 min and the resultant were collected. To eliminate starch and nucleic acids, the pectic and hemicellulose B fractions that had been treated with phenol and chloroform were incubated with a mixture of t~t-amylase (from porcine pancreas; Sigma, St. Louis, MO, USA; EC 3. 2. 1. 1) and deoxyribonuclease I (from bovine pancreas; Nippon Gene, Tokyo, Japan; EC 3. 1.21.1) and ribonuclease I (from bovine pancreas, Nippon Gene) (EC 3. 1.27.5) at 37~ for 12 hr and then they were dialyzed overnight against 10 mM sodium acetate buffer (pH 4.0).

Electrophoresis . The electrophoresis of pectic polysaccharides on cellulose acetate membranes and the detection of acidic and total sugars were performed by using pectin (Orange peel; Sigma) and polygalacturonic acid (Orange peel; Sigma) as standards as described by Kikuchi et al., (1992). In some cases, to allow recovery of polysaccharides after electrophoresis glass-fiber filter (GF/F; Whatman), previously baked for 4 hr at 450~ were used as the solid matrix instead of a cellulose acetate membrane.

De-esterification of pectic substances. The elimination of methyl residues from methyl esterified pectin was performed with pectin methylesterase (from orange; Sigma; EC 3.1.1.11) as described previously (Kikuchi et al., 1992).

Gas-chromatographic analysis of carbohydrates. After electrophoresis on glass-fiber filters, stained areas corresponding to sugars were cut out, and the polysaccharides on the filters were hydrolyzed. For the analysis of neutral sugars, the polysacchrides were hydrolyzed, converted into the corresponding alditol acetates and subjected to gas chromatography as described previously (Satoh et al., 1992b).

Amounts of total sugar and uronic acid were determined by the phenol-sulfuric acid method (Dubois et al., 1956) and the carbazole-sulfuric acid method (Galambos and McCain 1967), as glucose and galacturonic acid equivalents, respectively.

Results

Morphological features of embryogenic and non-embryogenic calli

Figure 1 shows light micrographs of NC (A) and EC (B) cultured for two weeks after inoculation, after staining with toluidine blue. The cluster size of NC is uniformly small as shown in the figure and the intercellular contacts in NC are loose and the cells are large and vacuolated. There are open spaces between cells. By contrast, the intercellular contacts appear stronger in the EC and the cluster is composed of smaller cells. The same results were obtained from the larger clusters which are contained in the same EC culture.

Fig.1. Toluidine-blue staining of sections of suspension-cultured carrot cells grown for 2 weeks after inoculation. (A) Non-embryogenic callus. (B) Embryogenic callus. The bar indicates 10 ~tm.

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Electrophoretic analysis of polysaccharides in pectic fractions

Polysaccharides in pectic fractions of NC, EC and SE were analyzed by electrophoresis on cellulose acetate membrane and subsequent staining with toluidine blue (Fig. 3A), which allows the detection of acidic sugars, and with

5 = A [ ] NC

�9 EC _ i 4 I~SE

3

F 2 ..

i 1 : ili, ~ 2 - B

M P HA HB C

Fraction Fig.2. Sugar contents of cell wall fractions and media of suspension-cultured carrot cells. Culture medium (M), pectin (P), hemicellulose A (HA), hemicellulose B (HB) and cellulose (C) fractions were obtained from NC (hatched columns), EC (closed columns) and SE (dotted columns), and total sugar contents (A) and uronic acid contents (B) were determined by the phenol-sulfuric acid method and the carbazole-sulfuric acid method, respectively.

Contents of sugars in cell wall fractions and in the media

Figure 2 shows the level of total sugar and uronic acid in the cell wall fractions and media prepared from the cultures of EC, NC and SE. The most conspicuous feature is that the amount of total sugars in the pectic fraction of EC is more than twice that of NC and four times that of SE. No such differences are apparent in the level of uronic acid in the various pectic fractions.

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periodic acid-Schiffs reagent (PAS; Fig. 3B) which allows detect of the total sugars. In all samples, both type of staining gave a dark spot at a position of low mobility, which corresponded to that of standard pectin (open arrowhead). There was also a faint smear in a higher-mobility area, but there were no spots at a high-mobility position that corresponded to that of standard polygalacturonic acid (closed arrowhead). However, there was a large difference in the intensity of staining of the low-mobility main spots between NC and EC. In NC, the spot stained by toluidine blue was more intense than that stained by PAS. By contrast, in EC, staining with the toluidine blue was weaker than staining with PAS (see the positions indicated by open arrowheads). These results indicate that the acidic polysaccharides in the pectic fraction of EC contained much more neutral sugars than did those of NC. This difference might be reflected in the total sugar content of pectic fractions between NC and EC (Fig. 2). SE gave the faint stainings by both stains (Fig.2). When five times more sample was loaded on the membrane the main spots appeared in a low mobility area in both stains (Data not shown). SE appeared to be intermediate between NC and EC, namely, staining of the main spot by both stains gave a spot of almost the same intensity.

As shown in Figure 3, the main acidic polysaccharides in the pectic fraction migrated slowly during electrophoresis. This observation sugges t s the poss ib i l i ty that the polygalacturonic acid backbone is highly methylesterified and/or that the side chains, composed of neutral sugars, attached to the backbone are relatively large. In order to examine these possibilities, the extent of me thy l e s t e r i f i c a t i on of the acidic polysaccharides in the pectic fractions was evaluated by treatment with pectin- methylesterase.

On electrophoresis, the mobility of the highly methylesterified standard pectin changed, after treatment, from low to high, and the high mobility was identical to that of standard polygalacturonic acid (see Fig. 2 in Kikuchi et al., 1992). However, for all pectic

fractions extracted from carrot cells, there were no changes after the treatment with pectin methylesterase (data not shown). These results indicate that the polygalacturonic acid backbone of the pectic polysacchar ides was methylesterified to a small extent, if at all, but had large neutral side chains.

Fig.3. Analysis of pectic polysaccharides by electrophoresis on cellulose acetate membranes. Pectic fractions were obtained from the cell wall fraction (2.0 g of dry cell walls for staining with toluidine blue and 3.0 g of dry cell walls for staining with periodic acid-Schiffs reagent) of NC, EC and SE, and subject to electrophoresis on cellulose acetate membrane followed by stainings with toluidine blue (A) and periodic acid- Schiff's regent (B). Closed arrow, open arrowhead and closed arrowhead indicate the positions of the origin, standard pectin and standard polygalacturonic acid, respectively.

Neutral sugar compositions of low-mobility pectic polysaccharides

The low-mobility pectic polysaccharides (open arrowhead in Fig. 3) were subjected to an analysis of neutral sugar composition by gas chromatography. Figure 4 shows the neutral sugar compositions of NC, EC and SE. The major neutral sugars were arabinose and galactose. Galactose was predominant (50%) in NC but accounted for less than 30% in EC and SE. Other sugars were present at slightly higher level in EC and SE than in NC, with the exception of rhamnose which was present at the same relative level (about 10%). The ratio of arabinose to galactose (w/w) were 0.53, 1.30

and 1.72 in NC, EC and SE, respectively. Results showing the same tendency were obtained from cultures one and three weeks after inoculation, and also from the other two independent lines of EC and SE, and the other two different subcultures of NC.

. . . 6 0 ' . . . . . . ,

.~ m r~c 1 ' e 5 0 [] EC

~o 40 [] SE

30 ==

2O o~

o . . . . . Rha Fuc Ara Xyl Man Gal GIc

Fig.4. Relative levels of neutral sugars (as percentage of the total) in low-mobility pectic polysaccharides. The pectic polysaccharides in the low-mobility main spot (open arrowhead in Fig. 3) from NC (hatched column), EC (closed column) and SE (dotted column) were subjected to gas-chromatographic analysis.

D i s c u s s i o n

Pectins are thought to play important roles in intercellular attachment, since polygalacturonic acid is known to form a gel in the presence of calcium ions. The cementing activity of pectin is directly related to the level of polygalacturonic acid and decreases with increasing of methylesterification of carboxyl groups (Bonner 1950). By contrast, no positive role for the neutral polysaccharides of pectin has been demonstrated in intercellular attachment, although it seems likely that the large neutral polysaccharide side chains inhibit the formation of gels in the presence of calcium ions (Bonner 1950).

We found a positive relationship between levels of neutral sugars in the pectic fraction and intercellular contacts in calli cultured with 2,4-D (Fig. 1 and 3). Moreover, we found qualitative differences in the neutral polysaccharides in pectic substances among

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three type of material (Fig. 4). The ratio of arabinose to galactose (w/w) was directly related to visible intercellular contacts. The quantitative difference in neutral sugar may have been due to differences in the size of neutral sugar block and/or in their number. Since the relative level of rhamnose was constant (about 10%; Fig. 4), quantitative difference may be mainly due to the number of neutral sugar blocks. The same tendency was observed in the sugar composit ion of hemicellulose B fraction of EC and NC (Data not shown). This result indicates that the difference in neutral sugar composition of pectic fraction is not due to the difference in extractabilities of polysaccharides and the difference may be the character of whole matrix polysaccharides. These results indicate that an abundance of neutral sugar blocks containing arabinogalactan and/or the presence of arabinose-rich side chains may play a positive roles in cell-to-cell attachment.

It has been postulated from studies of the different cell l ines derived from C a t h a r a n t h u s roseus plants that arabinose is present at higher levels in pectic fractions of elongated cells than in those of spherical cells (Suzuki et al., 1990). However, such a system seems to be inappropriate for an analysis of the relationship between pectic polysaccharide components and intercellular attachment because cells of one strain contained large amounts of elongating cell walls which do not participate in intercellular attachment. By contrast, both NC and EC in our system were composed of similarly spherical cells. Though NC was vacuolated (Fig. 1A) it grew faster than EC. Therefore, the carrot caili in our system were suitable for analysis of the features of intercellular attachment.

The NC may arise as a result of spontaneous multiple mutations that are a consequence of somaclonal variations in EC. Because it is to be expected that strong intercellular attachment is essential for the morphogenesis such as embryogenesis, weak cell-to-cell attachment should be one of the causes for the loss of embryogenic competence in NC.

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W e now need to demons t ra te the structure of the neutral sugar blocks that appear to be responsible for the quantitative differences associated with arabinose and galactose if we are to conf i rm a n e w function for neutral polysaccharides in interceIIular attachment and the significance of such attachment in somatic embryogenes is .

Acknowledgments. We are grateful to Dr. I. Inouye, Dr. R. Amikura and Mr. D. Honda for their helpful on making section. We also would like to thank Mr. K. Higashi for his gift of embryogenic callus.

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