Planta (1996)200:369 372 P l a n t ~

(c) Springer-Verlag 1996

A xylogalacturonan whose level is dependent on the size of cell clusters is present in the pectin from cultured carrot cells

Akira Kikuchi 1, Yusuke Edashige 2, Tadashi Ishii 2, Shinobu Satoh ~

1 Institute of Biological Sciences, University of Tsukuba, Tsukuba, Ibaraki 305, Japan z Forestry and Forest Products Research Institute, Tsukuba, Ibaraki 305, Japan

Received: 28 March 1996/Accepted: 23 August 1996

Abstract. A substantial level of xylose was detected in the pectic polysaccharides that had been extracted from car- rot (Daucus carota L.) calli and purified by gel-permeation and ion-exchange chromatography. The results of the removal of neutral sugar chains and 13-elimination in- dicated that the xylose was not included in the neutral sugar chains but was directly bound to a polygalac- turonic-acid backbone. Methylation analysis confirmed that the xylose was directly linked to galacturonic acid at position 2 or 3, as a terminal residue. The amount of xylose was positively correlated with the size of cell clus- ters in several lines of cultured carrot cells.

Key words: Callus - Daucus (cell walls) - Pectin - Xylo- galacturonan

Introduction

Intercellular at tachment is critical to the morphogenesis of higher plants, and pectin is known to be an important material in the adhesion of cell walls (Bonnet 1950). In an effort to enhance our understanding of the physiological and biochemical events related to the morphogenesis of higher plants, we have been studying the relationship between morphogenesis and the structure of pectin using cultured carrot cells as a model system.

Carrot embryogenic callus (EC) loses its embryogenic competence and becomes non-embryogenic callus (NC) during long-term culture (Reinert et al. 1970). Concomi- tant with the loss of competence, cell clusters become smaller, with a reduction in the extent of intercellular

Abbreviations: EC = embryogenic callus; 4-GalA = 4-1inked galac- turonic acid; NC =non-embryogenic callus; T-Xyl = terminal xylose; 3,4-GalA = 3,4-1inked galacturonic acid; 2,4-GalA = 2,4- linked galacturonic acid

Correspondence to: S. Satoh; FAX: 81 (298) 534579

attachment (Satoh et al. 1986). The ratio of the amount of arabinose to that of galactose in the pectin fraction is strongly and positively correlated with the size of cell clusters. We postulated that the neutral sugar chains of pectins should be important in the interactions of cell wall components and intercellular at tachment (Kikuchi et al. 1995). Moreover, a large number of xylosyl residues have been detected in the pectin and the amount of such resi- dues in EC was found to be twice that in NC (Kikuchi et al. 1996).

The level of xylose has been reported to be low in pectic polysaccharides (McNeil et al. 1980; Nistitani and Masuda 1980; Jarvis et al. 1981; Talbot t and Ray 1992; Lerouge et al. 1993; Mufloz et al. 1993). However, in our cultured carrot cells, xylose is present at a substantial level and is almost all terminal xylose (T-Xyl), even when the pectin is purified by gel-permeation and ion-exchange column chromatography (Kikuchi et al. 1996).

In this report, the structure of xylose-linked pectic polysaccharides is identified and a correlation between the size of cell clusters and the xylose content is demonstrated.

Materials and methods

Plant materials. Embryogenic callus of Daucus carota L. cv. US- Harumakigosun was induced from hypocotyl segments of seedlings on Murashige and Skoog's (Murashige and Skoog 1962) agar medium that contained 2,4-dichlorophenoxyacetic acid (1 mg" 1-1). Non-embryogenic callus was derived spontaneously from EC and entirely lacked embryogenic competence (Satoh et al. 1986. The average sizes of cell clusters in cultures were estimated as described by Kikuchi et al. (1996).

Purification of pectic polysaccharides. Cell walls were isolated and pectic polysaccharides were extracted as described by Kikuchi et al. (1996). For the elimination of protein, starch and nucleic acids, a mixture of phenol and chloroform (3:1, v/v), ct-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) plus ribonuclease I (from bovine pancreas; Nip- pon Gene; 3.1.27.5) were used, respectively (Kikuchi et al. 1995).

Pectic polysaccharides were purified by column chromato- graphy on Sephacryl S-500 HR (Pharmacia, Uppsala, Sweden),

370 A. Kikuchi et al.: Cell-cluster-size-related xylogalacturonan in carrot calli

diethylaminoethyl (DEAE)-Sepharose CL-6B (Pharmacia) and Mono Q HR5/5 (Pharmacia), in that order, as described by Kikuchi et al. (1996).

Quantitation o['su.qars and analysis of sugar composition. 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.

Sugar composition was analyzed by methanolysis (York et al. 1985) and conversion to alditol acetates (Keegstra et al. 1973), with subsequent gas-liquid chromatography (GC).

Methylation analysis. The dried pectic polysaccharides were methyl- ated by a modified version (Sandford and Conrad 1966) of Hakomori's procedure (Hakomori 1964). The methylated polysac- charides were reduced with 1 M lithium triethylborodeuteride in tetrahydrofuran (Aldrich, Milwaukee, Wis., USA). The carboxyl- reduced methylated polysaccharides were hydrolyzed and converted to partially methylated alditol acetates (York et al. 1985). These products were analyzed by GC and gas-liquid chromatography- mass spectrometry (GC-MS; QP2000, Shimadzu, Kyoto, Japan).

[3-Elimination of pectin. 13-Elimination was performed by the method described by McNeil et al. (1980). Fully methylated non-uronosyl- reduced pectin from NC was treated with dimethyl sulfoxide ion. The reaction products were reduced, and subsequent steps were performed as described above for methylation analysis.

Removal of neutral su~lar chains. A solution of pectic polysacchar- ides (5 gg/gl) prepared from NC was mixed with an equal volume of 0.2 M oxalic acid and incubated at 99 ~ for 3 h (Matsuura 1984). The solution was then mixed with four volumes of ethanol and centrifuged at 8000.g for 15 min at 4 ~C. The pellet was suspended in 80% ethanol and centrifuged again under the same conditions. The final pellet was air-dried and then dissolved in 10 mM imidazole buffer (pH 7.0), and the solution was applied to a column of DEAE- Sepharose CL-6B. The eluate in 1.0 MNaC1 was collected and subjected to analyses of sugar composition and glycosyl linkages.

Results

Removal of neutral sugar chains from pectin. Almost all the xylose in the purif ied 1300-kDa pect in was detected as te rminal xylose (T-Xyl; K ikuch i et al. 1996). Because the neut ra l sugar -neu t ra l sugar l inkage is known to be cut by mi ld acid hydro lys i s ( M a t s u u r a 1984; Selvendran and O 'Ne i l l 1987), pectic po lysacchar ide p repared from N C were t rea ted with 0.1 M oxal ic acid and the sugars in the residue were examined (Table 1). Levels of xylose and ga lac tu ron ic acid increased but those of o ther sugars decreased upon such t rea tment , ind ica t ing tha t the xylose was not a c o m p o n e n t of the neut ra l sugar chains.

f3-Elimination of pectin. Table 2 shows the results of the analysis of sugar compos i t i on before and after 13-elimina- t ion (degrada t ion of u ronic acid under a lkal ine condi t ion) of pect in tha t had been p repa red fi 'om NC. The levels of r hamnosy l and xylosyl residues were decreased by [3-elim- ina t ion but those of a rab inosy l and galactosyl were not. This result indicates that r hamnose and xylose were l inked to po lyga l ac tu ron i c acid chains. The rhamnosy l residues might have been der ived from rhamnoga lac - t u ronan - I which is an integral pa r t of the pectic poly- saccharides .

Methylation analysis of the pectin after removal of neutral sugar chains. The results of me thy la t ion analysis of pect in that had been p repared from NC, after the remova l of neutra l sugar chains, are shown in Table 3. The xylose in the pectin consis ted main ly of T-Xyl. G a l a c t u r o n a n is c o m p o s e d of ga lac turon ic acid residues l inked at pos i t ion 0-4 (McNei l et al. 1980). Since the amoun t of T-Xyl was equal to the a m o u n t of 2,4-1inked ga lac turonic acid (2,4- GalA) plus 3,4-GalA residues and the a m o u n t of 3 ,4-GalA was abou t 2.5 t imes of that of 2 ,4-GalA (tool/tool), it appea red possible that T-Xyl might have been direct ly l inked to pos i t ions 2 and 3 of ga lac turonic acid, with the

Table 1. Glycosyl residues in pectin from carrot NC, analyzed be- fore and after treatment with oxalic acid

Glycosyl residue Before (mol%) After (mol%)

Rhamnosyl 6.4 4.7 Fucosyl 2.2 0.0 Arabinosyl 25.4 0. l Xylosyl 13.0 22.7 Mannosyl 2.9 0.3 Galactosyl 37.7 18.9 Glucosyl 4.3 5.4 Galacturonosyl 7.2 47.2 Glucuronosyl 0.7 0.9

Table 2. Neutral glycosyl residues in pectin from carrot NC, ana- lyzed before and after 13-elimination

Glycosyl residue Before (tool%) After (tool%)

Rhamnosyl 10.2 3.9 Fucosyl 0.4 0.9 Arabinosyl 28.7 30.2 Xylosyl 12.9 0.0 Mannosyl 0.4 1.2 Galactosyl 45.1 62.4 Glucosyl 2.3 6.7

Table 3. Glycosyl linkages in pectin from carron NC after the elim- ination of neutral sugar chains

Glycosyl residue Deduced linkage mol%

Rhamnosyl 2,4-Linked 2.4 2,3,4- 5.2

Arabinosyl 2,3,5- 2.3 Xylosyl Terminal 14.3

2-Linked 0.5 4- 0.3

Galactosyl Terminal 12.1 4-Linked 2.5 6- 1.5 4,6- 0.8 2,4,6- 2.1 3,4,6- 1.9

Glycosyl 4-Linked 1.4 4,6- 4.8

Galacturonosyl Terminal 1.5 4-Linked 32.8 2,4- 3.8 3,4- 9.3

A. Kikuchi et al.: Cell-cluster-size-related xylogalacturonan in carrot calli

amount of the 3-1inked T-Xyl being about 2.5 times that of the 2-1inked T-Xyl. The fact that the ratio of linearly linked GalA (4-GalA) to branched GalA (3,4-Gala plus 2,4-GalA) is nearly 5/2 (Table 3), indicates that linkage of xylose occurred twice for every seven galacturonic acid residues. The galactosyl residues that we detected might have been due to residues that remained after the removal of neutral sugar chains.

Correlation between xylose content and the size of cell clusters. Pectic polysaccharides were prepared from cell clusters of various sizes (EC, NC and various calli that were intermediates between EC and NC) and the correla- tion between the average size of cell clusters and the xylose content was examined. Pectic polysaccharides from EC and NC contained xylose at 30 and 13% (mol/mol), re- spectively. The other calli contained intermediate levels of xylose. A positive correlation (r = 0.79) was found be- tween the average size of cell clusters and the xylose content of pectic polysaccharides (Fig. 1).

Discussion

Pectins of the cell walls of higher plants are composed of a main chain of galacturonic acid residues with some neutral sugar chains, which consist mainly of rhamnose, arabinose and galactose (McNeil et al. 1980). Xylose is known as a minor component of pectins (Nishitani and Masuda 1980; Jarvis et al. 1981; Quemener and Thibault 1990; Talbott and Ray 1992; Lerouge et al. 1993; Mufioz et al. 1993). However, the pectin from carrot calli was found to contain a large amount of xylose (Kikuchi et al. 1995). In order to eliminate any contamination by non-pectic polysaccharides such as xylan, xyloglucan and arabinoxylan, the pectic polysaccharide from carrot was further purified by gel-permeation and ion- exchange column chromatography. However, even after further purification, the same level of xylose was detec- ted in the purified pectic polysaccharides (Kikuchi et al. 1996).

371

The xylosyl residues were not from the neutral sugar chains composed of arabinose and galactose (Table 1). In this study we demonstrated that single-unit side chains of xylosyl residues were linked directly to positions 2 and 3 of the galacturonosyl residues of polygalacturonic acid (Tables 2, 3).

Aspinall et al. (1967a, b) found xylose linked to galac- turonic acid residues at position 3 in pectin prepared from apples. Furthermore, a pectic fraction from apples was found to contain a xylogalacturonan which had a (1,4)-Ct-D-galacturonan backbone with some inserted rhamnosyl residues, as well as single-unit side-chains of xylosyl residues linked to position 3 of the galacturonosyl residues (Voragen et al. 1993; Schols et al. 1995). Our xylogalacturonan had two types of linkage, with xylosyl residues linked to positions 2 and 3 of the galacturonosyl residues (Table 3). Therefore, 2-1inked xylose is novel in xylogalacturonans, The distribution of these xylogalac- turonans is of interest. The xylose could be attached to separate molecules at position 2 or position 3, or it could be attached to the same molecule at both positions 2 and 3.

We previously studied the relationship between inter- cellular at tachment and pectic polysaccharides, and we demonstrated that the neutral sugars (arabinose and ga- lactose), rather than the polygalacturonic acid appear to be related to intercellular at tachment (Kikuchi et al. 1995, 1996). Figure 1 indicates that the xylose content is also positively correlated (r = 0.79) to the size of cell clusters. These results indicate that the xylogalacturonan is related to the formation of cell clusters and to intercellular attach- ment. Because di-ferulic acid is known to link the polysac- charides in the cell wall (Fry 1983; Ishii and Hiroi 1990), it is possible that the terminal xylose of the xylogalacturonan is bound to the hemicellulosic polysaccharides (e.g. xylo- glucan) of the cell wall through phenolic compounds.

Part of this work was supported by a research grant from the Science and Technology Agency of Japan and a Grand-in-Aid from the Ministry of Education, Science and Culture, Japan. The authors are grateful to Dr. Koichi Kakegawa of the Forestry and Forest Prod- ucts Research Institute for his encouragement throughout this research.

40

35

30

25

2o 0

>~lS

�9 i �9 i i �9 i �9

1 0 ' i . I i t , i ,

100 200 300 400 500 600 Size of ceil clusters (~tm)

Fig. 1. Correlation between the xylose content and the average size of carrot cell clusters. The correlation between the xylose content and the average size of cell clusters was examined using calli that were intermediate between EC and NC, which had begun to form small cell clusters and had low embryogenic competence, r, coeffic- ient of the correlation between the xylose content and the average size of cell clusters

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