Alterations in cell wall polysaccharides during ripening in distinct anatomical tissue regions of the fig (Ficus carica L.) fruit

Alterations in cell wall polysaccharides during ripening in distinct anatomical tissue regions of the fig (Ficus carica L.) fruit

Postharvest Biology and Technology 32 (2004) 67–77 Alterations in cell wall polysaccharides during ripening in distinct anatomical tissue regions of ...

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Postharvest Biology and Technology 32 (2004) 67–77

Alterations in cell wall polysaccharides during ripening in distinct anatomical tissue regions of the fig (Ficus carica L.) fruit Willis Omondi Owino, Ryohei Nakano, Yasutaka Kubo, Akitsugu Inaba∗ Laboratory of Postharvest Agriculture, Faculty of Agriculture, Okayama University, Tsushima, Okayama 700-8530, Japan Received 24 June 2003; accepted 12 October 2003

Abstract During the last stage of fig (Ficus carica L.) fruit development, profound cell wall modification processes occur as indicated by increase in fruit size and tissue softening. In this study, we characterized the changes in cell wall polysaccharides taking place within the distinct and separate tissues of the receptacle and the pulpy drupelets during sequential ripening in fig fruit. The pectic extracts had high uronic acid contents in addition to high amounts of Ara, Gal and Rha. The gel filtration profile of the water-soluble polymers at the ripening onset in drupelets were different when compared to those of the receptacle, even though in both tissues, these polymers underwent increased solubilization and depolymerization during ripening. The molecular downshift of the CDTA-soluble polymers and the decrease in the amounts of both the uronic acid and total sugars were more pronounced in the drupelets than in the receptacle. Major difference in the neutral sugar composition between the two tissues was only observed in the Na2 CO3 -soluble fraction. The xyloglucan polymers in 4% KOH fraction exhibited a molecular size downshift accompanied by a decline in Xyl and increase in Glc. In the 24% KOH fractions of both tissues, the total sugar and xyloglucan components decreased in amount and also exhibited a molecular size downshift during ripening. These data suggest that even though quantitative and qualitative changes in cell wall polysaccharides occurred during ripening in both tissues, qualitative variations between tissues occurred only in the pectic polymers but not in the hemicellulosic polymers. © 2003 Elsevier B.V. All rights reserved. Keywords: Fruit ripening; Ficus carica; Alcohol-insoluble solids; CDTA; Total sugars; Uronic acid

1. Introduction Fruit ripening is a complex process involving ultra-structural modifications of the cell wall due to concerted activities of cell wall enzymes. Fruit cell walls generally consist of pectins, hemicelluloses and cellulose polysaccharide polymers. Additional minor cell wall constituents include a broad range of struc∗ Corresponding author. Tel.: +81-86-251-8337; fax: +81-86-251-8338. E-mail address: [email protected] (A. Inaba).

tural and enzymatic proteins, hydrophobic compounds and inorganic molecules. The content and structural features of the fruit cell wall polymers vary depending on the species, developmental stage and the tissue type (Brownleader et al., 1999). Analysis of cell wall polysaccharides has revealed that large changes occur in both pectins and hemicelluloses during ripening, though the precise roles of particular cell wall alterations and of the cell wall-modifying enzymes bringing about these changes are not known (Brummell and Harpster, 2001). Pectin polymers are the most abundant and the most complex class of cell wall

0925-5214/$ – see front matter © 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.postharvbio.2003.10.003

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macromolecules that are degraded during ripening, undergoing both solubilization and depolymerization. The modification of hemicellulosic polysaccharides is also a common characteristic during ripening in most of the fruit species studied, although the extent and rate of modification is species-specific (Gross and Sams, 1984; Rose and Bennett, 1999). Considered botanically, the fig (Ficus carica L.) is a syconium, a very remarkable form of fruit, which stands alone in its peculiar arrangement of flowers. It is actually neither fruit nor flower though partaking of both, being really a hollow, fleshy receptacle, enclosing a multitude of flowers, which never see the light, yet come to full perfection and develop into drupelets within the fleshy receptacle. During the last stage of fig fruit development, profound cell wall modification processes occur within the tissues as indicated by increase in fruit size and tissue softening (Chessa, 1997). The fig is a highly perishable climacteric fruit subject to rapid physiological breakdown. Indeed the postharvest life of the fruit is considered not to extend beyond 7–10 days even when low temperature storage is used (Chessa, 1997). Basic studies on the processes that occur during ripening are essential for investigating systems in which the biological and physiological processes linked to maturation are involved in postharvest deterioration. In mature and ripe fig fruit the receptacle tissue and the pulpy tissue of drupelets within it are clearly distinct and separate (see cross-sectional diagram in Fig. 1). Therefore, analysis of the both tissues as a single mixture may obscure some cell wall changes

that are crucial in understanding the cell wall modification processes during ripening. Presently there is no information on cell wall modification and composition during ripening in figs. In an effort to understand the essential features of the cell wall modification processes occurring in the receptacle and the drupelets tissues, we isolated and characterized both the pectic and hemicellulosic polysaccharides in the two anatomical tissue regions of the fig fruit.

2. Materials and methods 2.1. Plant material and tissue sampling The fig (F. carica L. cv Houraishi) fruit were obtained from a commercial fig fruit farmer in Nadasaki Cho, Okayama, Japan, and were harvested at sequential stages of ripening as assessed by almost uniform fruit diameter and coloration on the receptacle surface. The fruit diameter was measured at the point of greatest width by a vernier caliper and fruit of about 35–40 mm were selected. Based on the surface coloration, the selected fruit were classified as follows: ripening onset, the receptacle had a yellowish green coloration; ripe, the receptacle had three-fourths reddish-purple coloration; and over-ripe, receptacle had a deep purple coloration. The fruit firmness was determined using a penetrometer (Tensilon, Model STM-T-50P, Baldwin Co. Ltd., Tokyo) fitted with a plunger of 6 mm radius. The firmness of each fruit was measured at three points along the equatorial region of the fruit. The fruit was then cut into two and the receptacle was manually separated from the pulpy tissue of the drupelets. Tissues were immediately frozen in liquid nitrogen and stored at −80 ◦ C until subsequent analysis. 2.2. Cell wall isolation and the extraction of cell wall polysaccharides

Fig. 1. Cross-section diagram of a ripe Houraishi fig fruit.

The crude cell wall material was prepared from approximately 50 g of the frozen tissue using the method described by Rose et al. (1998) with slight modifications. The frozen tissue was boiled in 200 ml of ethanol for 45 min and then homogenized. The homogenate was filtered through Miracloth (Calbiochem), and washed sequentially with 500 ml of boiling ethanol,

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500 ml of chloroform:methanol (1:1, v/v) and 500 ml acetone; oven dried at 30 ◦ C and stored in a desiccator, constituting the alcohol-insoluble solids (AIS). One gram of the crude cell wall material (AIS) was extracted in 50 ml water containing 0.02% NaN3 for 4 h at room temperature, and the pellet was extracted twice in 25 ml water containing 0.02% NaN3 for 15 min. The water-soluble fractions were combined, lyophilized, resuspended in 50 ml of 90% (v/v) DMSO and stirred for 16 h at 20 ◦ C to solubilize starch. The suspension was centrifuged (20,000 × g for 20 min), and the pellet washed twice with 25 ml 90% (v/v) DMSO, three times with 25 ml 80% (v/v) ethanol and three times with 25 ml acetone, oven-dried at 30 ◦ C and stored in a dessicator at room temperature. The pellet was designated the water-soluble fraction. The water-insoluble pellets were then extracted twice for 12 h in 50 ml of 50 mM CDTA containing 50 mM sodium acetate, pH 6.5 and then the CDTA-insoluble pellets were extracted twice in 50 ml of 50 mM Na2 CO3 , containing 20 mM NaBH4 at 1 ◦ C. Both fractions were exhaustively dialyzed (molecular size cut off, 6–8 kDa) against distilled water for 48 h at 4 ◦ C, lyophilized and stored in a dessicator at room temperature. The Na2 CO3 -insoluble residue was extracted twice with 4% KOH (w/v) containing 0.1% NaBH4 (total volume, 50 ml) at room temperature for 48 h. The supernatants were combined, adjusted to pH 5 with acetic acid, chilled and clarified by centrifugation. The 4% KOH-insoluble materials were then extracted twice with 25 ml of 24% KOH (w/v) containing 0.1% NaBH4 and 0.5 M H3 BO3 for 48 h and centrifuged. The supernatants were similarly

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combined, adjusted to pH 5 with acetic acid, chilled and clarified by centrifugation. Both the 4 and 24% KOH fractions were exhaustively dialyzed against deionized distilled at 4 ◦ C for 48 h, lyophilized and stored in a dessicator. 2.3. Total sugars (TS), uronic acid (UA) and GC analyses Five milligrams of samples were hydrolyzed by sulphuric acid (Ahmed and Labavitch, 1977) and uronic acid content assayed by the m-hydroxydiphenyl method (Blumenkrantz and Asboe-Hansen, 1973). For GC analysis of the AIS and extracted fractions, ca. 5 mg of the lyophilized samples were hydrolyzed in 2 M trifluoroacetic acid for 1 h at 121 ◦ C. The sugars released were reduced, acetylated to their respective alditols and suspended in 1 ml dichloromethane (Blakeney et al., 1983). One microliter was analyzed by GLC (Shimadzu model 14A) fitted with a flame-ionization detector and a capillary column (TC-17, GL Sciences, 15 m × 0.25 mm i.d.), using N2 as both the carrier and the auxiliary gas, and penta-erythritol as the internal standard. The initial oven temperature (160 ◦ C, 1 min) was programmed to 220 ◦ C at the rate of 2 ◦ C min−1 and the injection port and detector temperatures were 250 and 300 ◦ C, respectively. 2.4. Gel filtration chromatography Each pectic class (ca. 1–1.5 mg of uronic acid equivalents in 1 ml of buffer) was chromatographed

Table 1 Percentage yields, UA and the overall changes of neutral sugars (␮g mg−1 ) in the AIS of fig fruit during sequential ripening stages Stage and tissue

Fruit firmness

1A 2A 3A 1B 2B 3B

14.2 ± 0.7 4.7 ± 0.6 <1

Yield (%)

Total sugar

Uronic acid

Neutral sugar Rha

Fuc

Ara

Xyl

Man

Glc

Gal

3.7 3.4 2.8

387.5 352.4 309.5

236 221 201

8.3 11.8 12.3

4.5 5.3 3.7

25.9 14.6 12.4

28.7 21.9 13.5

3.7 4.2 3.9

19.8 29.2 33.8

60.6 45.2 29.2

4.0 3.2 2.5

360.8 349.8 303.7

228 234 191

6.6 13.8 8.7

4.4 6.9 5.2

32.4 13.9 12.0

12.2 16.4 22.8

4.6 5.1 5.7

27.5 33.3 36.0

45.1 26.4 22.3

Stages and tissue type are: 1A, ripening onset, drupelets; 2A, fully ripe, drupelets; 3A, over-ripe drupelets; 1B, ripening onset, receptacle; 2B, fully ripe, receptacle; 3B, over-ripe, receptacle. Percentage yield values are mean of n = 2. Neutral sugar values are means of n = 2, while UA values are means n = 3. The total sugar values are the sum of uronic acid content and the neutral sugar values.

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on a XK 16/70 column of Sephacryl S-400 HR gel (Amersham Pharmacia Biotech AB, Uppsala, Sweden) which was equilibrated with 80 mM sodium acetate buffer pH 5, containing 10 mM EDTA and 50 mM NaCl. The column was eluted with the same buffer, and fractions (4 ml) were collected at a flow rate of 18 ml h−1 and analyzed for the UA (Blumenkrantz and Asboe-Hansen, 1973) and total sugars (TS) contents (Dubois et al., 1956). The 4

and 24% KOH-soluble polymer samples (ca. 5 mg in 1 ml of NaOH buffer) were applied to the same column and 4 ml fractions were collected at a flow rate of 18 ml h−1 , neutralized with glacial acetic acid and assayed for TS content (Dubois et al., 1956) or xyloglucan content as described by Nishitani and Masuda (1981). The column elution profile was calibrated with three dextran standards (2000 kDa (blue dextran), 505 and 10 kDa) and glucose.

Table 2 Yields of the fractions after extraction of the AIS and the UA, TS and the neutral sugar levels (␮g mg−1 AIS) in the receptacle and drupelets of the fig fruit during sequential ripening stages Fraction

H2 O

CDTA

Na2 CO3

4% KOH

24% KOH

Stage

Yield (mg g−1 AIS)

Total sugar

Uronic acid

Neutral sugar Rha

Fuc

Ara

Xyl

Man

Glc

Gal

1A 2A 3A

121 ± 3.6 182 ± 4.8 220 ± 5.5

66.5 84.5 91.1

41 67 80

1.8 1.7 1.9

1.4 0.8 0.5

4.0 4.1 2.2

1.4 1.0 0.7

1.0 0.9 0.6

1.5 0.9 0.7

14.4 8.1 4.5

1B 2B 3B

144 ± 3.2 158 ± 2.4 190 ± 1.9

50.7 74.5 85.9

36 61 72

1.4 1.5 1.4

1.1 0.8 0.7

4.0 4.9 5.1

1.5 1.1 0.9

1.2 0.9 0.6

1.4 0.9 0.6

4.0 3.4 4.6

1A 2A 3A

248 ± 2.8 140 ± 3.4 144 ± 1.7

104.6 88.5 71.1

71 66 57

2.2 2.1 2.2

0.0 0.0 0.0

12.6 3.5 2.1

0.4 0.9 1.1

0.0 0.9 0.6

1.5 2.1 2.8

17.0 13.0 5.3

1B 2B 3B

221 ± 2.6 143 ± 3.2 151 ± 3.2

112.5 83.0 71.8

88 69 58

1.7 1.8 2.0

0.0 0.0 0.0

9.4 3.8 3.5

0.6 0.7 1.1

0.0 0.0 0.0

0.5 1.3 1.6

12.3 6.3 5.6

1A 2A 3A

74 ± 2.2 65 ± 3.1 54 ± 2.4

66.9 51.7 34.2

46 34 22

2.9 0.9 0.4

0.0 0.0 0.0

2.1 3.6 4.2

0.3 0.9 1.0

0.0 0.0 0.0

0.4 0.3 0.9

15.3 12.0 5.8

1B 2B 3B

69 ± 3.4 60 ± 4.0 44 ± 3.6

68.9 45.5 38.4

44 28 23

2.2 1.8 3.2

0.0 0.0 0.0

10.9 3.6 3.0

0.5 0.4 0.6

0.0 0.0 0.0

0.6 2.9 4.3

10.6 8.9 5.3

1A 2A 3A

86 ± 5.5 69 ± 3.9 42 ± 7.7

29.4 22.8 18.6

5 3 1

0.2 0.3 0.1

0.7 0.3 0.2

0.9 1.2 1.7

14.6 8.4 2.6

0.5 0.3 0.7

4.3 7.4 10.8

3.2 1.9 1.4

1B 2B 3B

93 ± 8.6 73 ± 5.5 41 ± 5.7

22.9 17.8 21.8

3 2 1

0.4 0.1 0.1

0.6 0.5 0.4

1.3 0.6 0.2

6.8 3.9 8.1

1.1 0.7 0.6

5.2 7.6 9.5

4.5 2.2 1.9

1A 2A 3A

77 ± 5.2 91 ± 6.7 73 ± 10.1

31.6 30.9 24.5

12 11 6

0.1 0.3 0.2

0.4 0.6 0.4

1.4 0.5 0.6

8.2 6.3 4.2

0.9 1.3 1.2

4.8 8.9 10.1

3.7 2.1 1.7

32.2 31.8 28.3

15 14 4

0.2 0.1 0.1

0.9 0.4 0.7

1.6 1.0 0.7

1.9 3.9 9.2

1.9 1.3 1.3

6.5 8.9 11.0

4.3 2.2 1.3

1B 2B 3B

84 ± 5.3 102 ± 3.0 88 ± 6.7

The values are means of n = 3. Stages and tissue type are: 1A, ripening onset, drupelets; 2A, fully ripe, drupelets; 3A, over-ripe, drupelets; 1B, ripening onset, receptacle; 2B, fully ripe, receptacle; 3B, over-ripe, receptacle.

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3. Results

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no consistent trends were observed for Rha and Man in the receptacle.

3.1. Overall composition of the AIS The figs showed a marked decrease in fruit firmness, which averaged 14.2, 4.7 and less than 1 N, respectively, at the ripening onset, ripe and over-ripe stages of ripening (Table 1). As ripening progressed there was a decrease in the UA contents of the drupelets while in the receptacle it increased at the ripe stage followed by a decrease at the over-ripe stage. The TS consistently declined throughout ripening in both tissues. The major neutral sugar components of the AIS were Ara, Xyl, Glc and Gal, with Gal being the most predominant sugar (Table 1). There was a general decline in the contents of Ara and Gal while Glc content increased in both tissues. The Xyl content decreased in the drupelets but increased in the receptacle. The Rha and Man contents increased in the drupelets but

3.2. Fractionation of the cell wall polysaccharide and analysis of their neutral sugars Individual polysaccharide fractions were obtained after successive extraction of the AIS with water, CDTA, Na2 CO3 , 4 and 24% KOH. The quantity of polysaccharides extracted with water increased during ripening (from about 121 to 220 and 144 to 190 mg g−1 of AIS in drupelets and receptacle, respectively), accompanied by a general decline in the CDTA-soluble fraction and in the Na2 CO3 -soluble fractions (Table 2). The total yields of the 4% KOHsoluble extracts decreased with ripening whereas in the 24% KOH-soluble extracts, higher yields were obtained at the ripe stage as compared to the ripening onset and the over-ripe stages (Table 2).

Fig. 2. Molecular size distribution profiles of water-soluble pectins from ripening fig fruit. Pectins were prepared from the AIS of receptacle and drupelets tissue and approximately 1–1.5 mg (galacturonic acid equivalents) were applied to the column as described in Section 2. Column fractions (4.0 ml) were assayed for UA content (upper panel) using the m-hydroxybiphenyl method (Blumenkrantz and Asboe-Hansen, 1973) or for TS content (lower panel) using the phenol–sulphuric acid method (Dubois et al., 1956). Dextran molecular mass markers in kilodaltons (kDa) used as a calibration scale are shown at the top; Vg, elution volume of glucose. Ripening stages are: (䊏) ripening onset; (䊊) fully ripe; () over-ripe.

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Quantitatively, the CDTA-soluble fraction was the major pectic fraction at the ripening onset, accounting for UA content of 71 and 85 ␮g mg−1 AIS in the drupelets and receptacle, respectively (Table 2). However, though the UA contents of both the CDTA and Na2 CO3 -soluble fractions decreased with ripening, that of water-soluble fractions increased. The TS of the three pectic fractions decreased or increased in a similar pattern to the UA contents. For the alkali extracts, the major proportion of the TS and UA in both tissues was extracted by the 24% KOH. The TS in the 24%. KOH-soluble and the 4% KOH-soluble fraction decreased with ripening (Table 2). The water-soluble fraction of both receptacle and drupelets was predominantly enriched in Ara and Gal, and these neutral sugars showed a decrease with ripening. In the CDTA-soluble fraction Ara and Gal decreased while Glu increased (Table 2). The Na2 CO3 -soluble fraction was enriched in Rha, Ara and Gal. The contents of Rha and Ara showed an inverse relationship in drupelets and receptacle during

ripening. Rha decreased in drupelets but increased in the receptacle, while Ara increased in drupelets but decreased in the receptacle (Table 2). The neutral sugar composition revealed that the 4 and 24% KOH fractions were particularly enriched in Ara, Xyl, Glc and Gal (Table 2). In the 4 and 24% KOH fraction of both tissues, the Xyl content decreased with ripening in the drupelets. However in the receptacle of both tissues there was a general decline in Xyl content. An increase in Glc content was observed in both the 4 and 24% KOH-soluble fractions during ripening (Table 2). The pectin associated sugars such as Rha and Ara were also detected in appreciable amounts in the 24% KOH fraction. 3.3. Gel filtration analysis The water-soluble polymers of the receptacle gave a single peak of UA and TS and with ripening the elution pattern shifted towards lower molecular weight regions (Fig. 2). In the drupelets, the UA and TS

Fig. 3. Molecular size distribution profiles of CDTA-soluble pectins from ripening fig fruit. CDTA-soluble pectins were extracted from the receptacle and the drupelets tissue from the AIS previously treated to remove water-soluble pectins. Other details are as described for Fig. 2.

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eluted as heterogeneous molecular size fractions of low and broad peaks. However at the over-ripe stage, the water-soluble polyuronides eluted as a relatively symmetrical peak. In the receptacle, at the ripening onset, the CDTA-soluble polymers were characterized by a broad population of polymers, which exhibited a molecular downshift with ripening (Fig. 3). In the drupelets, the CDTA-soluble polymers eluted as a high molecular size peak at the ripening onset but underwent a molecular downshift with ripening. The gel filtration profile of the Na2 CO3 -soluble fraction showed a bimodal molecular size distribution; high molecular size and low molecular size regions (Fig. 4). The elution pattern of this fraction exhibited only a slight downshift in the molecular weight size from the ripening onset to ripe stage. In general the TS and UA profiles of the water, CDTA and Na2 CO3 -soluble fractions showed similar patterns

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indicating that the TS and UA components were part of the same polysaccharide polymers. The TS gel filtration analysis of the 4% KOH-soluble polymers in the drupelets showed a molecular size profile with two peaks at the ripening onset stage (Fig. 5). During ripening, the amount of the high molecular size component declined accompanied by a concomitant increase in the amount of the low molecular size component. The xyloglucan components in the 4% KOH fraction eluted as a peak mainly coincident with the high molecular size polymer peak observed in the TS assay, but underwent a slight molecular downshift during ripening accompanied by a reduction in the amount of its components. In the receptacle, the TS profile of the 4% KOH fraction showed a bimodal molecular size distribution of high molecular size and low molecular size regions in all the stages of ripening, though the low molecular size

Fig. 4. Molecular size distribution profiles of Na2 CO3 -soluble pectins from ripening fig fruit. The Na2 CO3 -soluble pectins were extracted from the receptacle and the drupelets tissue from the AIS previously treated to remove water and CDTA-soluble pectins. Other details are as described for Fig. 3.

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Fig. 5. Molecular size distribution profiles of 4% KOH-soluble fraction from receptacle and drupelets of fig fruit derived from three ripening stages. Aliquots of the lyophilized samples were applied to the column as described in Section 2. Column fractions (4 ml) were assayed for TS (upper panel) using the phenol–sulphuric method (Dubois et al., 1956) or xyloglucan content (lower panel) using the iodine-staining method as described by Nishitani and Masuda (1981). Other details are as described for Fig. 2.

peak showed a significant downshift and a decrease in the amount of the TS. The TS and xyloglucan components of the drupelets extracted with 24% KOH eluted as a single symmetrical peak (Fig. 6). Both the TS and xyloglucan components of the 24% KOH fractions exhibited a slight molecular downshift and decreases in the amount of both the TS and xyloglucan polymers in both tissues during ripening. The elution profiles of the xyloglucan polymers in the 24% KOH fraction were more or less similar to that of TS suggesting that xyloglucans are the major components of this fraction.

4. Discussion The present study shows that there were quantitative and qualitative changes in the cell wall polysac-

charides between the distinct structural tissues of the fig fruit during sequential ripening stages. The decrease in percentage yields of AIS in both tissues (Table 1), during ripening may have been due to a gross decrease in cell wall synthesis with ripening, or an increase in solute accumulation relative to wall deposition. The loss of Gal and Ara in the AIS during ripening is consistent with the general loss of cell wall neutral sugars that has been reported in most fruit, and the significance of a particular wall change to fruit softening has not been satisfactorily resolved (Gross and Sams, 1984; Redgwell et al., 1997). At the ripening onset, water-soluble polymers of the drupelets eluted as a heterogeneous and broad distributed peak, a profile that was distinctively different from that of the receptacle (Fig. 3). However, the water-soluble polymers of both tissues exhibited a progressive shift towards the low molecular size regions accompanied

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Fig. 6. Molecular size distribution profiles of 24% KOH-soluble fraction from receptacle and drupelets tissue of fig fruit derived from three ripening stages. Aliquots of the lyophilized samples were applied to the column as described in Section 2. Other details are as described for Figs. 2 and 5.

by an incremental increase in UA and TS contents, suggesting both depolymerization and solubilization of these pectic polymers (Fig. 2 and Table 2). Similar results have been reported during ripening in avocado, tomato, blackberry, persimmon, plum and strawberry (Redgwell et al., 1997; Wakabayashi, 2000). The CDTA-soluble fraction exhibited a molecular downshift in both tissues even though a significant reduction in the amount of UA and TS components was observed in the drupelets but not in the receptacle (Fig. 3 and Table 2). The CDTA-soluble polymers are among the largest pectic molecules and the decrease in their molecular size as observed in this study can either be due to re-allocation of the smaller polymers from the Na2 CO3 -soluble fraction or possibly due to the action of PG on the homogalacturonan-type structures found in this fraction (Redgwell et al., 1992). In nectarines and peach, UA of pectic fractions (CDTA and Na2 CO3 ) decreased and the Ara residues increased (Lurie et al., 1994; Dawson et al., 1992;

Hedge and Maness, 1996). Presuming that the Ara residues were present as a covalently linked pectin side chain, Gal loss, as would occur through combined endo and exo PG would result in the increase in Ara residue in Na2 CO3 -soluble polymers of the drupelets. In the receptacle however, UA contents, the contents of Ara and Gal declined suggesting that both sugars are components of the same polysaccharide and that this Gal–Ara rich pectin polymer is being solubilized or converted to a less cohesive bound form during ripening. Similar decreases in neutral sugar in the Na2 CO3 -soluble fraction has been reported in tomato pericarp and persimmon but not kiwifruit (Carrington et al., 1993; Cutillas-Iturralde et al., 1993; Redgwell et al., 1992). The different patterns among the neutral sugars Rha, Ara, Gal observed between the drupelets and receptacle in the Na2 CO3 -soluble fraction further suggests that either the pectic polysaccharide polymers present in these tissues are different and/or they undergo specific modifications during ripening of fig fruit.

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The molecular weight distributions of the hemicellulose fractions have been investigated in a number of fruits. For instance the 1 M KOH-soluble hemicelluloses of peach (Hedge and Maness, 1998) eluted as three peaks and those of melon as two peaks (Rose et al., 1998). Polysaccharides extracted by 4 M KOH from peach fruit exhibited four peaks, but the neutral fraction, mostly composed of xyloglucan, eluted as two peaks (Hedge and Maness, 1998). In melon the 4 M KOH-soluble polymers eluted as a single peak (Rose et al., 1998). In fig fruit, the total sugar component of the 4% KOH-soluble fraction eluted as two peaks while the xyloglucan component eluted as a single peak. These polymers underwent depolymerization as shown by the apparent molecular size downshift of the xyloglucan polymer profiles in the 4% KOH fraction (Fig. 5). High levels of Xyl and Glc were observed in the 4% KOH fraction at the ripening onset stage and as ripening progressed the contents of Xyl decreased and Glc increased suggesting a decline in Xyl-rich polymers and increased presence of Glc-rich polymers (Table 2). The 24% KOH-soluble fractions eluted as single peaks (Fig. 6). The contents of Xyl and Glc in both tissues increased in the 24% KOH fraction while the relative content of Gal and Ara decreased suggesting modification of Xyl-rich polymers (Table 2). The TS profile of the 24% KOH-soluble polymers of both tissues, was similar to the xyloglucan profile and both the TS and xyloglucan components decreased in amount and also exhibited a molecular size downshift during ripening, suggesting that the tightly bound xyloglucan polymers undergo depolymerization during ripening (Fig. 6). In general the average molecular size of the fig fruit hemicelluloses was similar to melon (Rose et al., 1998), kiwifruit (Redgwell et al., 1997) and tomato (Huber, 1983) which showed a decrease in their molecular size during ripening. These results suggested that hemicelluloses in both tissues of the fig fruit also undergo structural modifications during ripening.

of sugar participating residues while the gel filtration analysis reveals the relationship between polymer size and the state of tissue softening. The present study has shown that both quantitative and qualitative changes take place during ripening in both the pectic and hemicellulosic polysaccharides in the receptacle and drupelets tissues of the fig fruit. The pectic extracts exhibited sugar compositions close to those of other fruits, i.e. high galacturonic acid content in addition to high amounts of Ara, Glu and Rha. Qualitative variations between the two tissues were observed only in the pectic polymers but not in the hemicellulosic polymers. For instance the molecular size profiles of each were distinctively different, with the drupelets exhibiting often high amounts of UA and TS components. The water-soluble polymers at the ripening onset were heterogeneous in drupelets, as compared to the receptacle, even though in both tissues, these polymers underwent increased solubilization and depolymerization during ripening. A major difference in the neutral sugar composition of the pectic polymers between the two tissues was only observed in the Na2 CO3 -soluble fraction. For the hemicellulosic polysaccharides, even though qualitative changes occurred in both tissues during ripening, there were no clear differences in the changes between the receptacle and the drupelets. Therefore, in fig fruit, the loss of firmness that accompanied softening was due to the cell wall modification processes that took place in both the pectic and hemicellulosic polymers in the receptacle and drupelet tissues. Acknowledgements This work was supported in part by Grants-in-Aid for Scientific Research, Grant no. 14360023 to A.I. from the Ministry of Education, Science, Sports and Culture of Japan and by Research Grant for Encouragement of Postgraduate Students (2002) awarded to W.O.O. from the Graduate School of Natural Science and Technology, Okayama University, Japan.

5. Conclusion Quantification of cell wall polysaccharides gives an overall indication of changes in the disparate group of polymers. Qualitative analyses such as neutral sugar determination show modifications in the proportion

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