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Chilling-injury in cucumbers. V. Polysaccharide changes in cell walls
Scientia Horticulturae, 8 (1978) 219--227 Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands
219
C H I L L I N G - I N J U R Y I N C U C U M B E R S . V. P O L Y S A C C H A R I D E C H A N G E S IN CELL WALLS
T. FUKUSHIMA and M. YAMAZAKII Faculty of Agriculture, Yamagata University, Tsuruoka, Yamagata (Japan) 1Present address: Mayeda Industrial Co., Ltd. Inc., Shibukawa-cho, Yao-shi, Osaka 581 (Japan) (Received 22 September 1977)
ABSTRACT Fukushima, T. and Yamazaki, M., 1978. Chilling-injury in cucumbers. V. Polysaccharide changes in cell walls. Scientia Hortic., 8: 219--227. A decline in the hot-water-soluble pectin and an increase in the hot-water-insoluble pectin were observed when cucumber fruits were subjected to chilling temperatures. Infrared absorption spectra revealed the presence of highly esterified carboxyl groups in the soluble pectin, and of free carboxyl groups in the insoluble pectin. An increase of the insoluble pectin during chilling was also found in other chilling-sensitive plants. From these results it is suggested that a de-esterification of pectin and the concomitant increase of polymeric pectin takes place during chilling, making cell walls firmer, and that these pectic changes may be a characteristic'common to a number of chilling-sensitive plants.
INTRODUCTION I n a p r e v i o u s r e p o r t ( F u k u s h i m a et al., 1 9 7 7 c ) , it was c o n c l u d e d t h a t chilling-injury in c u c u m b e r f r u i t is a t y p e o f water-stress injury, and t h a t water-stress i n j u r y o c c u r s o n l y in fruits w i t h rigid cell walls. F u r t h e r , it was ascertained t h a t t h e cell walls in chilled fruits b e c o m e rigid at t h e o n s e t o f chilling-injury. T h u s , cell wall rigidity appears t o p l a y an integral p a r t in t h e d e v e l o p m e n t o f chilling-injury, T h e p u r p o s e o f t h e p r e s e n t p a p e r is t o clarify t h e m e c h a n i s m b y w h i c h cell wall rigidity increases w i t h t i m e o f chilling. As a first step in this d i r e c t i o n , we u n d e r t o o k a q u a n t i t a t i v e s t u d y o f t h e l o w - t e m p e r a t u r e - i n d u c e d c h e m i c a l c h a n g e s in p e c t i n a n d o t h e r c o m p o n e n t s o f t h e cell walls o f c u c u m b e r fruits.
MATERIALS AND METHODS A cultivar o f c u c u m b e r , ' N a t s u s a i r a k u N o 3', harvested o n 17 J u n e 1 9 7 4 was used. T h e material, o f a b o u t 1 0 0 g fresh w e i g h t , was s t o r e d in t h e same
220 w a y as described in a previous report (Fukushima et al., 1977a). Groups of 3 cucumbers were taken o u t at 3-day intervals up to 15 days of chilling. A 50-gram piece of flesh tissue per fruit was cut, placed in a small polyethylene bag, and frozen a t - 2 0 ° C until required. Preparation o f cell walls. - - The frozen sample was plunged into 200 ml of
boiling ethanol and boiled for 1 hour under reflux. The mixture was then homogenized in a h o m o b l e n d e r and filtered with suction through a sinteredglass funnel. The residue on the filter was washed with 80%, subsequently 90%, finally 100%, ethanol and then allowed to dry in air. F r a c t i o n a t i o n o f the wall polysaccharides. - - Using a modified version of the
m e t h o d of Maki and Sato (1967), the dried preparation was first extracted with 100 ml of distilled water for 2 h at 100°C (hot-water-soluble pectin fraction) and then for an.additional hour at 100°C with 100 ml of 0.5% EDTA disodium salt solution buffered at pH 6.8 with 0.05 M sodium phosphates and NaOH (hot-water-insoluble pectin fraction). The residue from the EDTA extraction was extracted first with 100 ml of 5% NaOH for 2 h at 100°C (hemicellulose fraction) and then with 100 ml of 20% NaOH for 24 h at r o o m temperature (/3 and 7 cellulose). The final residue in a filter was washed exhaustively with distilled water and dried in air. The dry residue was immersed in 7.5 ml of 70% H2SO4, overnight. The m i x t u r e w a s diluted with distilled water sufficient to give a :final concentration of 5% H2SO4, heated at 100°C for 2 h, and then filtered (a cellulose fraction). Each extraction procedure was repeated twice, and combined extracts were brought to 250 ml b y adding distilled water. Each filtration was carried o u t b y using a sintered-glass funnel with suction. E s t i m a t i o n o f the wall polysaccharides. -- Polyuronide in b o t h the water-soluble
and insoluble pectin fractions was estimated b y the carbazole m e t h o d (McComb and McCready, 1952). Carbohydrates in all the fractions w e r e estimated b y the phenol--sulfuric acid m e t h o d (Dubois et al., 1956). Neutral sugar in the 2 pectin fractions was calculated for each sample b y subtracting the absorbance corresponding to polyuronide present from the absorbance of total carbohydrates. Infrared s p e c t r o s c o p y . - - T w o 0°C pectin extracts, the hot-water-soluble pectin
fraction from the fruit chilled f o r 3 days, and the insoluble-fraction from that chilled for 12 days, were separately adjusted to 80% ethanol solutions. After standing overnight, the gelatinous precipitates were collected b y centrifugation and dried. Infrared spectra for these dried samples were determined with a KBr disk using Jasco-IRA-2 infrared spectrophotometer. RESULTS Changes in p e c t i n during storage. ~ Figure I shows the results of analyses of hot-water-soluble pectin, which was expressed ir~ terms of polyuronide content.
221
The pectin content of the 0°C fruit decreased rapidly during the first 6 days, while that of the 5 ° C fruit remained relatively constant up to 6 days of storage and then decreased abruptly.
150t
~ 100~ E
2 5o
~
11°C
(D I
0
3
I
I
I
I
6
9
12
15
Storage time (days)
Fig. 1. Changes in hot-water-soluble pectin content during storage.
Figure 2 shows the results of analyses of hot-water-insoluble pectin. There was a continuous rise in content for both the 0°C fruit and the 5°C fruit throughout the storage period, but the rise was more rapid in the 0°C fruit than in the 5°C fruit. On the other hand, the content of the 10°C fruit remained generally constant.
uz 250 0
o 200 I0°C
0
~= 150 ~J
0
I
3
I
I
I
/
6 9 12 15 Storage time (days)
Fig. 2. Changes in hot-water-insoluble pectin content during storage.
222
Changes in o t h e r cell-wall p o l y s a c c h a r i d e s during storage. - - Figures 3 and 4 show the results of analyses of neutral polysaccharide in the hot-water-insoluble pectin fraction and of hemicellulose, respectively. In both the 0°C fruit and the 5°C fruit the c o n t e n t of these polysaccharides did not show definite trends of increase or decrease during the storage period. However, in the 10°C fruit, there was a considerable fall in b o t h with an increase in the length of storage period.
u:
o
~400
0
~m3oc 8
~
200
E~ 0
P
O°C
o,.
40C
5°C 8
30C 8
~
O°C 5°C
10°C
~-~110oc
Q. L Q.
b c~
~ 2oo ~00
o E
Z
0
3
6 9 12 15 Storage time (days)
0
6 9 12 1 StorGge time (days)
Fig. 3. Changes in neutral sugar content of the hot-water-insoluble fraction during storage. Fig. 4. Changes in hemicellulose content during storage.
Figures 5 and 6 show the results of analyses of ~+~/cellulose, and of s-cellulose, respectively. There was no significant change in the content t h r o u g h o u t the storage period. I n f r a r e d s p e c t r o s c o p y . - - Figures 7 and 8 show the infrared absorption spectrum
of the hot-water-soluble pectin and the hot-water-insoluble pectin, respectively. Both were obtained through alcohol precipitation from the corresponding fraction. The 2 compounds have very distinct infrared absorption spectra. The absorption between the 1750--1730 cm -1 region, indicating the presence of a possible ester group, was stronger in the soluble pectin than in the insoluble pectin. In the insoluble pectin, a strong peak, representing an ionized carboxyl group, was observed in the 1610--1550 cm -1 region. In contrast, there was no similar peak in the soluble pectin. The strong absorption in the region of 1650 cm -1, which was observed in the soluble pectin, indicates contamination of protein in this sample.
223
t~ O
g
LK 0
#- IO°C • ~5oc
Q.
0 400
1500
~0 °C
E
I0°0
o_
~ 30C
1oo
Q.
0 500 -5
200
I
+
6
0
9
112 I'5
Storage time
0
3
(days~
6 9 12 15 Storage time (days)
Fig. 5. Changes in # + 7 - c e l l u l o s e c o n t e n t during storage. Fig. 6. Changes in a - c e l l u l o s e c o n t e n t during storage.
1oo
?
~ 8C Q)
0
g60 E m 40 ~ 20 ' 2 0 0' 0
'
' ' 1800
' ' 1600
' ' 1~' O0 ' 1o' O0 ' 1400 Wove n u m b e r ( c m - O
8S 0
100
"~8o N 60 E ~ 40 F--
20 I
I
2000
I
I
1800
i
I
I
I
I
I
I
I
1600 1400 1200 1000 Wave n u m b e r (cm-O
I
I
800
Fig. 7. Infrared a b s o r p t i o n s p e c t r u m o f h o t - w a t e r - s o l u b l e pectin. Fig. 8. Infrared a b s o r p t i o n s p e c t r u m o f h o t - w a t e r - i n s o l u b l e pectin.
224
H o t - w a t e r - i n s o l u b l e p e c t i n in o t h e r crops. - - Pectic analyses were extended to
turnip roots, green peas, banana fruits and sweet p o t a t o roots, which were purchased from local markets. Figure 9 shows the insoluble-pectin content of these materials stored for 10 days at either 0°C or 15°C. The content of turnip roots and green peas (chilling-resistant crops) showed higher value in the samples stored at 15°C than at 0°C, while that of banana fruits and sweet p o t a t o roots (chilling-sensitive crops) was greater in the 0°C samples than in the 15°C samples. Green
peas L~ D)
O,
o.
C3~
E
.u c
o
(9
0
15
0 15 0 15 0 15 Storage temperature (°C)
Fig. 9. Hot-water-insoluble pectin content of turnip roots, green peas, banana fruits and sweet potato roots, stored for 10 days at 0°C or 15°C.
DISCUSSION
A decline in the hot-water-soluble pectin and an increase in the hot-waterinsoluble pectin were observed when cucumber fruit were subjected to chillingtemperatures. However, it should be noted that the present extraction scheme of pectin differs from the general method, in which fruit pectin has been fractioned b y successive extraction with cold water and some degrading agents. In the original m e t h o d of Maki and Sato (1967), which we modified to some extent, pectin has been extracted from 80% ethanol-insoluble dry sample first with 50% h o t methanol, followed b y cold water, h o t water, and 0.5% a m m o n i u m oxalate, in that order. Thus, they collected short chain pectin in 50% methanol and/or cold water. On the other hand, our procedure started with a direct hot-water extraction from the preparation, omitting the 50% h o t methanol and cold-water extraction. Our modified m e t h o d had the advantage of inducing artificial alterations in the compositions which occur
225 during extraction, and of fractionating pectin to highly esterified pectin and other. That is, if there is an excess of polyvalent cations in the preparation, all the pectin with free carboxyl groups, regardless of the length of its chain, would turn into polymeric pectin in combination with polyvalent cations liberated on heating. Thus, the pectin with free carboxyl groups which is found in short-chain pectin could be driven into the hot-water-insoluble pectin with other insoluble pectin. As a result, only simple chains of esterified pectin could become soluble in hot water. The infrared absorption spectra also support the above assumption, in addition, it can be inferred from the report of Jansen et al. (1960) that hot,water-soluble pectin, which is obtained from the residue after extraction of the cold-water-soluble fraction, is almost completely esterified with methyl groups. Thus, the present method seems to be o f great advantage in distinguishing nearly completely methyl-esterified pectin chains from other pectin chains with anhydride or metallic linkages. Several papers o n the mechanism and control o f plant growth stressed the significance of pectic substances. Tagawa and Bonner (1957) and Ordin et al. (1956) showed that indole acetic acid increases the rate of cell elongation by causing the cell wall to become more easily stretched. Ordin et aL (1957) attributed the ceU-wall loosening to the increase of methyl esterified pectin. This possibility, however, has been eliminated b y t h e finding that auxininduced cell elongation can occur even when auxin-induced pectin methylation is completely blocked by ethionine (Cleland, 1963). Although pectin methylation cannot account for auxin-induced cell-wall loosening and subsequent cell elongation, this should not discount the role of the methylation itself in the wall plasticity. Highly methyl-esterified pectin chains may form more dispersed systems of the filamentous macromolecules of pectin t h a n other pectin chains with free carboxyl groups, which can strongly associate either with neighboring pectin chains or with other cell-wall Constituents. This will make it possible to loosen the binding between cells. Tagawa and Bonnet (1957) presumed that polyvalent cations are associated primarily with pectin, and that cell walls are given rigidity by the formation of cross-linkages between neighboring pectin chains. Ordin et al. (1957) have stressed the possibility that pectin may be cross-linked, not only by calcium bridges but also by anhydride bridges, between adjacent carboxyl groups, the bonds of which contribute to cell-wall rigidity. Gould et al, (1965)have reported the presence in pectic preparations of a neutral araban fraction and an acidic polysaccharide fraction, composed mainly of galacturonosyl radicals, and suggested that the increased capacity for intermolecular bonding of the acidic fraction may result i n a decrease in the deformation of cell walls: This provides evidence for the idea that anhydride or polyvalent ion bridges between pectin chains may contribute to the rigidity of cell walls. Stoddart et al. (1967) have also found 2 similar components and have shown that the acidic fraction is the characteristic of young, actively dividing cells, a finding which may support the fact that young cucumber fruits are more rigid than adult ones (Fukushima et al., 1977b).
226
Returning to the present experiments, a decline in the soluble pectin and an increase in the insoluble pectin may show the progressive de-esterification and the concomitant increase of the pectin with free carboxyl groups during chilling, resulting in the increase of rigid cell walls. Thus, it is reasonably suggested that the increase of the more rigid cell walls in cucumber fruit during chilling (Fukushima et al., 1977b) m a y be due to the structural changes of pectin. Fukushima and Tsugiyama (1977) reported previously that the leakage of calcium ions and magnesium ions after 3 days of storage of cucumber fruits showed the highest value in the 10°C fruit, followed b y the 5°C fruit and the 0°C fruit, in that order. This lower value of leakage at chilling-temperatures may be attributed to the combination of these divalent cations with free pectic carboxyl groups formed b y de-esterification. Similar pectic changes have already been found b y Ben-Arie and Lavee (1971) in chilled peaches. Further, in the present study, an increase of the insoluble pectin was also found in chilled banana fruits and sweet p o t a t o roots, b o t h chilling-sensitive plants. Thus it is suggested that the de-esterification of pectin and the concomitant increase of polymeric pectin during chilling m a y be a characteristic c o m m o n to chilling-sensitive plants. A considerable loss in neutral sugar content of hot-water-insoluble fraction and in hemiceUulose content was observed in the 10°C fruit during storage. The significance of this loss is still unknown. Further studies are desirable on this point. ACKNOWLEDGEMENTS
The authors again wish to express their appreciation to Dr. T. Tomana, Dr. T. Iwata, Dr. S. Iwahori, W. Kelly and H. Miura, as in our former contribution, and newly to Dr. Sassa, Yamagata University, for his technical assistance on infrared spectroscopy.
REFERENCES Ben-Arie, R. and Lavee, S., 1971. Pectic changes occurring in Elberta peaches suffering from woolly breakdown. Phytochemistry, 10: 531--538. Cleland, R., 1963. Independence of effects of auxin on cell wall methylation and elongation. Plant Physiol., 38: 12--18. Dubois, M., Gilles, K.A., Hamilton, J.K., Rebers, P.A. and Smith, F., 1956. Colorimetric method for determination of sugars and related substances. Anal. Chem., 28: 350--356. Fukushima, T. and Tsugiyama, T., 1977. Chilling-injury in cucumber fruits. II. Chemical analyses of leakage substances and anatomical observation of symptoms. Scientia Hortic., 6: 199--206. Fukushima, T., Yamazaki, M. and Tsugiyama, T., 1977a. Chilling-injury in cucumber fruits. I. Effects of storage temperature on symptoms and physiological changes. Scientia Hortic., 6: 185--197. Fukushima, T., Yamazaki, M. and Odazima, T., 1977b~- Chilling-injury in cucumber fruits. III. Dynamic changes of osmotic quantities and the importance of cell wall rigidity. Scientia Hortic., 6: 311--321.
227
Fukushima, T., Yamazaki, M. and Odazima, T., 1977c. Chilling-injury in cucumber fruits: IV. The analogy between osmotic-stress injury and chilling-injury. Scientia Hortic., 6: 323--331. Gould, S.E.B., Rees, D.A., Richardson, N.G. and Steele, I.W., 1965. Pectic polysaccharides in the growth of plant cells: Molecular structural factors and their role in the germination of white mustard. Nature (London), 208: 876--878. Jansen, E.F., Jang, R., Albersheim, P. and Bonnet, J., 1960. Pectic metabolism of growing cell walls. Plant Physiol., 35: 87--97. Maki, M. and Sato, Y., 1967. Studies on the carbohydrates in vegetables (Part I). Fractionation of the carbohydrates in the leaves of Arternisica capiUaris. (in Japanese). J. Jpn. Soc. Food Nutr., 20: 373--377. McComb, E.A. and McCready, R.M., 1952. Colorimetric determination of pectic substances. Anal. Chem., 24: 1630--1632. Ordin, L., Applewhite, T.H. and Bonner, J., 1956. Auxin-induced water uptake by Avena sections. Plant Physiol., 31 : 44--53. Ordin, L., Cleland, R. and Bonner, J., 1957. Methyl esterification of cell wall constituents under the influence of auxin. Plant Physiol., 32 : 216--220. Stoddart, R.W., Barrett, A.J. and Northcote, D.H., 1967. Pectic polysaccharides of growing plant tissues. Biochem. J., 102: 194--204. Tagawa, T. and Bonnet, J., 1957. Mechanical properties of the Arena coleoptile as related to auxin and to ionic interactions. Plant Physiol., 32: 207--212.
219
C H I L L I N G - I N J U R Y I N C U C U M B E R S . V. P O L Y S A C C H A R I D E C H A N G E S IN CELL WALLS
T. FUKUSHIMA and M. YAMAZAKII Faculty of Agriculture, Yamagata University, Tsuruoka, Yamagata (Japan) 1Present address: Mayeda Industrial Co., Ltd. Inc., Shibukawa-cho, Yao-shi, Osaka 581 (Japan) (Received 22 September 1977)
ABSTRACT Fukushima, T. and Yamazaki, M., 1978. Chilling-injury in cucumbers. V. Polysaccharide changes in cell walls. Scientia Hortic., 8: 219--227. A decline in the hot-water-soluble pectin and an increase in the hot-water-insoluble pectin were observed when cucumber fruits were subjected to chilling temperatures. Infrared absorption spectra revealed the presence of highly esterified carboxyl groups in the soluble pectin, and of free carboxyl groups in the insoluble pectin. An increase of the insoluble pectin during chilling was also found in other chilling-sensitive plants. From these results it is suggested that a de-esterification of pectin and the concomitant increase of polymeric pectin takes place during chilling, making cell walls firmer, and that these pectic changes may be a characteristic'common to a number of chilling-sensitive plants.
INTRODUCTION I n a p r e v i o u s r e p o r t ( F u k u s h i m a et al., 1 9 7 7 c ) , it was c o n c l u d e d t h a t chilling-injury in c u c u m b e r f r u i t is a t y p e o f water-stress injury, and t h a t water-stress i n j u r y o c c u r s o n l y in fruits w i t h rigid cell walls. F u r t h e r , it was ascertained t h a t t h e cell walls in chilled fruits b e c o m e rigid at t h e o n s e t o f chilling-injury. T h u s , cell wall rigidity appears t o p l a y an integral p a r t in t h e d e v e l o p m e n t o f chilling-injury, T h e p u r p o s e o f t h e p r e s e n t p a p e r is t o clarify t h e m e c h a n i s m b y w h i c h cell wall rigidity increases w i t h t i m e o f chilling. As a first step in this d i r e c t i o n , we u n d e r t o o k a q u a n t i t a t i v e s t u d y o f t h e l o w - t e m p e r a t u r e - i n d u c e d c h e m i c a l c h a n g e s in p e c t i n a n d o t h e r c o m p o n e n t s o f t h e cell walls o f c u c u m b e r fruits.
MATERIALS AND METHODS A cultivar o f c u c u m b e r , ' N a t s u s a i r a k u N o 3', harvested o n 17 J u n e 1 9 7 4 was used. T h e material, o f a b o u t 1 0 0 g fresh w e i g h t , was s t o r e d in t h e same
220 w a y as described in a previous report (Fukushima et al., 1977a). Groups of 3 cucumbers were taken o u t at 3-day intervals up to 15 days of chilling. A 50-gram piece of flesh tissue per fruit was cut, placed in a small polyethylene bag, and frozen a t - 2 0 ° C until required. Preparation o f cell walls. - - The frozen sample was plunged into 200 ml of
boiling ethanol and boiled for 1 hour under reflux. The mixture was then homogenized in a h o m o b l e n d e r and filtered with suction through a sinteredglass funnel. The residue on the filter was washed with 80%, subsequently 90%, finally 100%, ethanol and then allowed to dry in air. F r a c t i o n a t i o n o f the wall polysaccharides. - - Using a modified version of the
m e t h o d of Maki and Sato (1967), the dried preparation was first extracted with 100 ml of distilled water for 2 h at 100°C (hot-water-soluble pectin fraction) and then for an.additional hour at 100°C with 100 ml of 0.5% EDTA disodium salt solution buffered at pH 6.8 with 0.05 M sodium phosphates and NaOH (hot-water-insoluble pectin fraction). The residue from the EDTA extraction was extracted first with 100 ml of 5% NaOH for 2 h at 100°C (hemicellulose fraction) and then with 100 ml of 20% NaOH for 24 h at r o o m temperature (/3 and 7 cellulose). The final residue in a filter was washed exhaustively with distilled water and dried in air. The dry residue was immersed in 7.5 ml of 70% H2SO4, overnight. The m i x t u r e w a s diluted with distilled water sufficient to give a :final concentration of 5% H2SO4, heated at 100°C for 2 h, and then filtered (a cellulose fraction). Each extraction procedure was repeated twice, and combined extracts were brought to 250 ml b y adding distilled water. Each filtration was carried o u t b y using a sintered-glass funnel with suction. E s t i m a t i o n o f the wall polysaccharides. -- Polyuronide in b o t h the water-soluble
and insoluble pectin fractions was estimated b y the carbazole m e t h o d (McComb and McCready, 1952). Carbohydrates in all the fractions w e r e estimated b y the phenol--sulfuric acid m e t h o d (Dubois et al., 1956). Neutral sugar in the 2 pectin fractions was calculated for each sample b y subtracting the absorbance corresponding to polyuronide present from the absorbance of total carbohydrates. Infrared s p e c t r o s c o p y . - - T w o 0°C pectin extracts, the hot-water-soluble pectin
fraction from the fruit chilled f o r 3 days, and the insoluble-fraction from that chilled for 12 days, were separately adjusted to 80% ethanol solutions. After standing overnight, the gelatinous precipitates were collected b y centrifugation and dried. Infrared spectra for these dried samples were determined with a KBr disk using Jasco-IRA-2 infrared spectrophotometer. RESULTS Changes in p e c t i n during storage. ~ Figure I shows the results of analyses of hot-water-soluble pectin, which was expressed ir~ terms of polyuronide content.
221
The pectin content of the 0°C fruit decreased rapidly during the first 6 days, while that of the 5 ° C fruit remained relatively constant up to 6 days of storage and then decreased abruptly.
150t
~ 100~ E
2 5o
~
11°C
(D I
0
3
I
I
I
I
6
9
12
15
Storage time (days)
Fig. 1. Changes in hot-water-soluble pectin content during storage.
Figure 2 shows the results of analyses of hot-water-insoluble pectin. There was a continuous rise in content for both the 0°C fruit and the 5°C fruit throughout the storage period, but the rise was more rapid in the 0°C fruit than in the 5°C fruit. On the other hand, the content of the 10°C fruit remained generally constant.
uz 250 0
o 200 I0°C
0
~= 150 ~J
0
I
3
I
I
I
/
6 9 12 15 Storage time (days)
Fig. 2. Changes in hot-water-insoluble pectin content during storage.
222
Changes in o t h e r cell-wall p o l y s a c c h a r i d e s during storage. - - Figures 3 and 4 show the results of analyses of neutral polysaccharide in the hot-water-insoluble pectin fraction and of hemicellulose, respectively. In both the 0°C fruit and the 5°C fruit the c o n t e n t of these polysaccharides did not show definite trends of increase or decrease during the storage period. However, in the 10°C fruit, there was a considerable fall in b o t h with an increase in the length of storage period.
u:
o
~400
0
~m3oc 8
~
200
E~ 0
P
O°C
o,.
40C
5°C 8
30C 8
~
O°C 5°C
10°C
~-~110oc
Q. L Q.
b c~
~ 2oo ~00
o E
Z
0
3
6 9 12 15 Storage time (days)
0
6 9 12 1 StorGge time (days)
Fig. 3. Changes in neutral sugar content of the hot-water-insoluble fraction during storage. Fig. 4. Changes in hemicellulose content during storage.
Figures 5 and 6 show the results of analyses of ~+~/cellulose, and of s-cellulose, respectively. There was no significant change in the content t h r o u g h o u t the storage period. I n f r a r e d s p e c t r o s c o p y . - - Figures 7 and 8 show the infrared absorption spectrum
of the hot-water-soluble pectin and the hot-water-insoluble pectin, respectively. Both were obtained through alcohol precipitation from the corresponding fraction. The 2 compounds have very distinct infrared absorption spectra. The absorption between the 1750--1730 cm -1 region, indicating the presence of a possible ester group, was stronger in the soluble pectin than in the insoluble pectin. In the insoluble pectin, a strong peak, representing an ionized carboxyl group, was observed in the 1610--1550 cm -1 region. In contrast, there was no similar peak in the soluble pectin. The strong absorption in the region of 1650 cm -1, which was observed in the soluble pectin, indicates contamination of protein in this sample.
223
t~ O
g
LK 0
#- IO°C • ~5oc
Q.
0 400
1500
~0 °C
E
I0°0
o_
~ 30C
1oo
Q.
0 500 -5
200
I
+
6
0
9
112 I'5
Storage time
0
3
(days~
6 9 12 15 Storage time (days)
Fig. 5. Changes in # + 7 - c e l l u l o s e c o n t e n t during storage. Fig. 6. Changes in a - c e l l u l o s e c o n t e n t during storage.
1oo
?
~ 8C Q)
0
g60 E m 40 ~ 20 ' 2 0 0' 0
'
' ' 1800
' ' 1600
' ' 1~' O0 ' 1o' O0 ' 1400 Wove n u m b e r ( c m - O
8S 0
100
"~8o N 60 E ~ 40 F--
20 I
I
2000
I
I
1800
i
I
I
I
I
I
I
I
1600 1400 1200 1000 Wave n u m b e r (cm-O
I
I
800
Fig. 7. Infrared a b s o r p t i o n s p e c t r u m o f h o t - w a t e r - s o l u b l e pectin. Fig. 8. Infrared a b s o r p t i o n s p e c t r u m o f h o t - w a t e r - i n s o l u b l e pectin.
224
H o t - w a t e r - i n s o l u b l e p e c t i n in o t h e r crops. - - Pectic analyses were extended to
turnip roots, green peas, banana fruits and sweet p o t a t o roots, which were purchased from local markets. Figure 9 shows the insoluble-pectin content of these materials stored for 10 days at either 0°C or 15°C. The content of turnip roots and green peas (chilling-resistant crops) showed higher value in the samples stored at 15°C than at 0°C, while that of banana fruits and sweet p o t a t o roots (chilling-sensitive crops) was greater in the 0°C samples than in the 15°C samples. Green
peas L~ D)
O,
o.
C3~
E
.u c
o
(9
0
15
0 15 0 15 0 15 Storage temperature (°C)
Fig. 9. Hot-water-insoluble pectin content of turnip roots, green peas, banana fruits and sweet potato roots, stored for 10 days at 0°C or 15°C.
DISCUSSION
A decline in the hot-water-soluble pectin and an increase in the hot-waterinsoluble pectin were observed when cucumber fruit were subjected to chillingtemperatures. However, it should be noted that the present extraction scheme of pectin differs from the general method, in which fruit pectin has been fractioned b y successive extraction with cold water and some degrading agents. In the original m e t h o d of Maki and Sato (1967), which we modified to some extent, pectin has been extracted from 80% ethanol-insoluble dry sample first with 50% h o t methanol, followed b y cold water, h o t water, and 0.5% a m m o n i u m oxalate, in that order. Thus, they collected short chain pectin in 50% methanol and/or cold water. On the other hand, our procedure started with a direct hot-water extraction from the preparation, omitting the 50% h o t methanol and cold-water extraction. Our modified m e t h o d had the advantage of inducing artificial alterations in the compositions which occur
225 during extraction, and of fractionating pectin to highly esterified pectin and other. That is, if there is an excess of polyvalent cations in the preparation, all the pectin with free carboxyl groups, regardless of the length of its chain, would turn into polymeric pectin in combination with polyvalent cations liberated on heating. Thus, the pectin with free carboxyl groups which is found in short-chain pectin could be driven into the hot-water-insoluble pectin with other insoluble pectin. As a result, only simple chains of esterified pectin could become soluble in hot water. The infrared absorption spectra also support the above assumption, in addition, it can be inferred from the report of Jansen et al. (1960) that hot,water-soluble pectin, which is obtained from the residue after extraction of the cold-water-soluble fraction, is almost completely esterified with methyl groups. Thus, the present method seems to be o f great advantage in distinguishing nearly completely methyl-esterified pectin chains from other pectin chains with anhydride or metallic linkages. Several papers o n the mechanism and control o f plant growth stressed the significance of pectic substances. Tagawa and Bonner (1957) and Ordin et al. (1956) showed that indole acetic acid increases the rate of cell elongation by causing the cell wall to become more easily stretched. Ordin et aL (1957) attributed the ceU-wall loosening to the increase of methyl esterified pectin. This possibility, however, has been eliminated b y t h e finding that auxininduced cell elongation can occur even when auxin-induced pectin methylation is completely blocked by ethionine (Cleland, 1963). Although pectin methylation cannot account for auxin-induced cell-wall loosening and subsequent cell elongation, this should not discount the role of the methylation itself in the wall plasticity. Highly methyl-esterified pectin chains may form more dispersed systems of the filamentous macromolecules of pectin t h a n other pectin chains with free carboxyl groups, which can strongly associate either with neighboring pectin chains or with other cell-wall Constituents. This will make it possible to loosen the binding between cells. Tagawa and Bonnet (1957) presumed that polyvalent cations are associated primarily with pectin, and that cell walls are given rigidity by the formation of cross-linkages between neighboring pectin chains. Ordin et al. (1957) have stressed the possibility that pectin may be cross-linked, not only by calcium bridges but also by anhydride bridges, between adjacent carboxyl groups, the bonds of which contribute to cell-wall rigidity. Gould et al, (1965)have reported the presence in pectic preparations of a neutral araban fraction and an acidic polysaccharide fraction, composed mainly of galacturonosyl radicals, and suggested that the increased capacity for intermolecular bonding of the acidic fraction may result i n a decrease in the deformation of cell walls: This provides evidence for the idea that anhydride or polyvalent ion bridges between pectin chains may contribute to the rigidity of cell walls. Stoddart et al. (1967) have also found 2 similar components and have shown that the acidic fraction is the characteristic of young, actively dividing cells, a finding which may support the fact that young cucumber fruits are more rigid than adult ones (Fukushima et al., 1977b).
226
Returning to the present experiments, a decline in the soluble pectin and an increase in the insoluble pectin may show the progressive de-esterification and the concomitant increase of the pectin with free carboxyl groups during chilling, resulting in the increase of rigid cell walls. Thus, it is reasonably suggested that the increase of the more rigid cell walls in cucumber fruit during chilling (Fukushima et al., 1977b) m a y be due to the structural changes of pectin. Fukushima and Tsugiyama (1977) reported previously that the leakage of calcium ions and magnesium ions after 3 days of storage of cucumber fruits showed the highest value in the 10°C fruit, followed b y the 5°C fruit and the 0°C fruit, in that order. This lower value of leakage at chilling-temperatures may be attributed to the combination of these divalent cations with free pectic carboxyl groups formed b y de-esterification. Similar pectic changes have already been found b y Ben-Arie and Lavee (1971) in chilled peaches. Further, in the present study, an increase of the insoluble pectin was also found in chilled banana fruits and sweet p o t a t o roots, b o t h chilling-sensitive plants. Thus it is suggested that the de-esterification of pectin and the concomitant increase of polymeric pectin during chilling m a y be a characteristic c o m m o n to chilling-sensitive plants. A considerable loss in neutral sugar content of hot-water-insoluble fraction and in hemiceUulose content was observed in the 10°C fruit during storage. The significance of this loss is still unknown. Further studies are desirable on this point. ACKNOWLEDGEMENTS
The authors again wish to express their appreciation to Dr. T. Tomana, Dr. T. Iwata, Dr. S. Iwahori, W. Kelly and H. Miura, as in our former contribution, and newly to Dr. Sassa, Yamagata University, for his technical assistance on infrared spectroscopy.
REFERENCES Ben-Arie, R. and Lavee, S., 1971. Pectic changes occurring in Elberta peaches suffering from woolly breakdown. Phytochemistry, 10: 531--538. Cleland, R., 1963. Independence of effects of auxin on cell wall methylation and elongation. Plant Physiol., 38: 12--18. Dubois, M., Gilles, K.A., Hamilton, J.K., Rebers, P.A. and Smith, F., 1956. Colorimetric method for determination of sugars and related substances. Anal. Chem., 28: 350--356. Fukushima, T. and Tsugiyama, T., 1977. Chilling-injury in cucumber fruits. II. Chemical analyses of leakage substances and anatomical observation of symptoms. Scientia Hortic., 6: 199--206. Fukushima, T., Yamazaki, M. and Tsugiyama, T., 1977a. Chilling-injury in cucumber fruits. I. Effects of storage temperature on symptoms and physiological changes. Scientia Hortic., 6: 185--197. Fukushima, T., Yamazaki, M. and Odazima, T., 1977b~- Chilling-injury in cucumber fruits. III. Dynamic changes of osmotic quantities and the importance of cell wall rigidity. Scientia Hortic., 6: 311--321.
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Fukushima, T., Yamazaki, M. and Odazima, T., 1977c. Chilling-injury in cucumber fruits: IV. The analogy between osmotic-stress injury and chilling-injury. Scientia Hortic., 6: 323--331. Gould, S.E.B., Rees, D.A., Richardson, N.G. and Steele, I.W., 1965. Pectic polysaccharides in the growth of plant cells: Molecular structural factors and their role in the germination of white mustard. Nature (London), 208: 876--878. Jansen, E.F., Jang, R., Albersheim, P. and Bonnet, J., 1960. Pectic metabolism of growing cell walls. Plant Physiol., 35: 87--97. Maki, M. and Sato, Y., 1967. Studies on the carbohydrates in vegetables (Part I). Fractionation of the carbohydrates in the leaves of Arternisica capiUaris. (in Japanese). J. Jpn. Soc. Food Nutr., 20: 373--377. McComb, E.A. and McCready, R.M., 1952. Colorimetric determination of pectic substances. Anal. Chem., 24: 1630--1632. Ordin, L., Applewhite, T.H. and Bonner, J., 1956. Auxin-induced water uptake by Avena sections. Plant Physiol., 31 : 44--53. Ordin, L., Cleland, R. and Bonner, J., 1957. Methyl esterification of cell wall constituents under the influence of auxin. Plant Physiol., 32 : 216--220. Stoddart, R.W., Barrett, A.J. and Northcote, D.H., 1967. Pectic polysaccharides of growing plant tissues. Biochem. J., 102: 194--204. Tagawa, T. and Bonnet, J., 1957. Mechanical properties of the Arena coleoptile as related to auxin and to ionic interactions. Plant Physiol., 32: 207--212.