Journal of Experimental Botany, Vol. 51, No. 348, pp. 1261-1266,
July 2000
© 2000 Oxford University Press
Original papers |
The role of fructan in flowering of Campanula rapunculoides
K.U. Leuven, Laboratory for Developmental Biology, Botany Institute, Kardinaal Mercierlaan 92, B-3001 Heverlee, Belgium
Received 5 January 2000; Accepted 10 March 2000
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Abstract |
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Inulin type fructan was detected in all vegetative organs of Campanula rapunculoides L. plants. All flower parts contained fructan at some developmental stage. A steady decrease was found in sepals during development. Petals, however, stored fructan in the bud stage. A rapid breakdown during opening of the flower resulted in high concentrations of glucose and especially fructose that may contribute to the osmotic driving force involved in petal expansion. Before complete shrivelling, the hexoses were apparently exported from flower parts. Fructans were hydrolysed and exported from the stamen and style tissue upon flower opening. Similarly, the major fructan reserves in the ovary were broken down almost simultaneously with those in other flower parts. Hexoses did not reach high levels in the ovary, probably because they were rapidly metabolized and/or incorporated by developing seeds.
Key words: Campanula, inulin, fructan, flowering.
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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According to Hendry, fructan has been detected in 10 families of angiosperm plants (Hendry, 1987
). Five families of monocotyledonous plants contain a relatively complex mixture of fructan based on 6-kestose, neokestose or both and including branched molecules (Livingston et al., 1993). Five dicotyledonous families belonging to three orders contain the structurally much simpler inulin series harbouring one terminal glucose and several exclusively ß-(2–1)-linked fructose moieties. Under conditions when inulin is broken down and fructose accumulates, a second homologous series of reducing inulo-n-oses without the terminal glucose can appear (Van den Ende et al., 1996a). Apparently this is the result of 1-fructan:fructan fructosyltransferase activity with free fructose as fructosyl acceptor (Van den Ende et al., 1996b).
Suggested roles for fructan include long and short-term carbohydrate reserve, stress protectant (both cold and drought) and osmoregulation (reviewed by Hendry, 1987). Hendry concludes that ‘the natural function of fructan in angiosperms has less to do with water-stress tolerance, but much more with water uptake, water retention and growth by water driven cell inflation’ (Hendry, 1993). Examples of the latter can be found in the rapid hydrolysis of fructan during sprouting of Lycoris radiata bulbs (Nagamatsu et al., 1991), flowering of Haemerocallis (Bieleski, 1993) and inflorescence development in Phippsia algida (Solhaug and Aares, 1994).
Fructan metabolism in dicotyledonous plants has almost exclusively been investigated in the Asteraceae. Helianthus tuberosus (Edelman and Jefford, 1968; Koops and Jonker, 1996) and Cichorium intybus (Van den Ende and Van Laere, 1996) have been model plants in this respect. It is clear that in these plants the inulin in the tubers or tap root is a long-term overwintering reserve. In an attempt to improve current understanding of inulin functions in dicotyledonous plants, the distribution of fructans and their fluctuations during development of flowers on the inflorescences of Campanula rapunculoides L. of the Campanulaceae family was investigated. Moreover, flowers of this species are much larger and easier to dissect into individual flower parts than their Asteracean counterparts.
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Materials and methods |
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Seeds were collected from a specimen in the botanical garden of the Botany Institute in Heverlee. In the spring of 1998 they were sown in a greenhouse and later transplanted to a local garden. Vegetative parts were occasionally collected and analysed to compare with flower parts. Flower parts were collected at different developmental stages (Table 1
) on two consecutive days in June 1999 from several clonally related inflorescences. C. rapunculoides has large racemous inflorescences. The middle flowers or flower buds within each inflorescence open first. After dissection of the different flower parts, their fresh weights were recorded and rapidly frozen in liquid nitrogen until further analysis. After grinding in a mortar, extraction was performed in water and the homogenate was immediately boiled in a water bath. A neutral fraction was obtained by ion exchange chromatography as described previously (Van den Ende et al., 1996a). Carbohydrates were separated by anion exchange chromatography and quantified by pulsed amperometric detection (Dionex, Sunnyvale Ca, USA). Mannitol was used as an internal standard. Factors for Glc, Fru, Suc, 1-Kes, and 1,1-nystose were obtained by injecting the pure compounds. Since the higher oligofructans (DP>4) were not available, their response coefficients were estimated using the work of Chatterton et al. (Chatterton et al., 1993). Only the peaks exceeding the baseline noise by a factor of 10 were considered. A CarbopacTM PA-100 guard and CarbopacTM PA-100 (4x250) in series were equilibrated with 90 mM NaOH for 24 min. A Na-acetate gradient was applied as follows: 0–6 min, 10 mM; 6–16 min, 10–100 mM; 16–26 min, 100–175 mM; 26–36 min, 175–230 mM; 36–61 min, 230–315 mM; 61–86 min, 315–360 mM; 86–125 min, 360–400 mM. Regeneration was 5 min with 500 mM Na-acetate and 10 min with 500 mM NaOH.
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In an attempt to establish the various developmental stages of flower development on a physiological time scale, 38 individual flower buds from four inflorescences were marked and their developmental stage was scored daily. Development was quantified each day by dividing the number of occurrences of each stage by the number of buds. However, the exact time needed to pass through the successive developmental stages is a function of environmental conditions.
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Results |
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Flower development
A few racemous inflorescences (from the same plant) were sampled in order to cover the complete development of the flower. Fresh weights of flower parts in the different stages (Table 1
; Fig. 1) are given in Fig. 2. Separation of the petal tips was arbitrarily assigned day 0. Sepals showed a slow and regular increase throughout development. After a regular increase, petal weight dramatically increased in a very short time after splitting, but soon wilted and senescence occurred. Upon opening of the flower, ovaries gained weight rapidly thereafter. Peak contributions of stamen and styles to flower fresh weight were reached 4 d before and immediately after petal separation, respectively. It should be noted that beginning on day minus 4, pollen stuck to the style and was analysed with that organ. At the time the flower opened (day 0) pollen stuck almost completely to the style and anthers started shriveling. In stages 10 to 12 nectar-like material appeared on the filaments and the top of the ovary.
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) sepals, (
) petals, (
) stamens, (
) style, (
) ovary.
Fructans in vegetative parts
A regularly spaced single series pattern of fructan was observed in roots, stems, petioles, and even a minimal amount was found in leaves (Fig. 3). Similar fructan patterns were found in all flower parts up to senescence. Elution times of peaks were identical with authentic inulin-type chicory fructan and were converted to fructose (and some glucose) by mild acid hydrolysis (data not shown).
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A second series in young petals became prominent at stage eight. This series co-eluted with the inulo-n-ose type fructan from chicory (Van den Ende et al., 1996b
) and was destroyed by mild hydrolysis (data not shown).
Fructans in sepals
The concentration of mono- and disaccharides in the sepals remained relatively constant during early development but gradually decreased during petal expansion (Fig. 4A). Inulins with rather low DP were present but slowly disappeared after splitting of the petals (Fig. 4B). A small amount of inulo-n-oses was also detected (Fig. 4C).
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): intermediate DPs have intermediate patterns. (C) Inulobiose (
Fructans in petals
The most dramatic changes in carbohydrate content occurred in the petals. Concentrations of glucose, fructose, sucrose, and maltose remained relatively low up to shortly before petal opening (Fig. 5A). Inulin type fructan, including those of a high DP, were detected throughout the early stages of development. The concentrations remained roughly constant (Fig. 5B) although fresh weights were increasing, suggesting a continuing synthesis of fructan. Shortly before, during and after petal splitting, fructan was rapidly hydrolysed to form large amounts of glucose but mainly fructose with a peak in stage 10 at the time of maximal petal expansion. Later on hexose concentration (mainly glucose) also decreased. Before the petals were completely wilted, large amounts of sugar were apparently exported because both fresh weight and concentration decreased.
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Fructan breakdown is likely catalysed by fructan exohydrolase resulting in large amounts of fructose. Indeed, fructose became the most important single carbohydrate during petal expansion. Because the presence of fructose, inulins and the 1-FFT enzyme results in the production of inulo-n-ose type fructan (Van den Ende et al., 1996b
), it is not surprising that inulo-n-ose fructan (especially the disaccharide) peaked concomitantly with fructan breakdown (Fig. 5C).
Fructans in stamens
Fructans have a small DP and are less abundant in stamen tissue than in other plant parts. Moreover, fructan concentration gradually decreased and was near minimal when they reached their maximal weight at day minus 4. A subsequent slight increase was suddenly arrested by a complete breakdown of the fructan (Fig. 6B). Hexose concentrations were high in developing stamens. Fructose was especially high and reached peak values of 0.2 M (Fig. 6A) well after stamens had reached their maximum fresh weight. The loss of pollen makes an interpretation ambiguous, but the final upsurge in hexose, sucrose and 1-kestose concentration may be the result of variable amounts of nectar sticking to the filaments folded over the ovary.
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Fructans in the style
No dramatic changes occurred in style tissue before day minus 4. Again, the contribution of adhering pollen makes interpretation difficult. Hexose, sucrose and DP3 concentrations increased with maturity. From day 1 forward however, all oligosaccharides were broken down. The resulting hexoses initially contributed to a maximal expansion, but were later completely exported before senescence of the style (Fig. 7).
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Fructans in the ovary
The largest concentration of high DP fructan was found in the developing ovary. Here too, fructan was rapidly broken down from day 0 onward and a concomitant (small) increase in inulo-n-ose (mainly inulobiose) occurred (Fig. 8). Hexose concentration increased but, probably due to its rapid utilization in seed growth, it never reached the levels present in other tissues.
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Asteraceae such as C. intybus (Van den Ende and Van Laere, 1996
), H. tuberosus (Koops and Jonker, 1996) or Cynara scolymus (Hellwege et al., 1998) are generally the model fructan-storing dicotyledonous plants. However, it is shown here that a similar pattern of fructan can be detected in all organs of C. rapunculoides. Since the flowers of C. rapunculoides are quite large, they can be easily dissected into individual parts. Moreover, vegetative propagation by stolons and the racemous inflorescences allows the collection of different developmental stages on the same day from the same (or a clonaly related) plant, excluding as far as possible the effect of environmental and genetic variables between different samples.
The largest fructan concentrations were found in petals and ovaries where the highest DPs are encountered (Figs 5, 8). Sepal development was very steady: initially the concentration of fructan and subsequently all carbohydrates decreased gradually during development and no sudden hydrolysis of fructan was observed. Carbohydrates essentially disappeared from the sepals soon after the petals wilted (Fig. 4).
Changes in fructan patterns were similar in most organs, but most spectacular in the petals (Fig. 5). Up to the stage of flower opening, fructan concentrations were high and hexose and disaccharide concentrations rather low. In a short period of 2–3 d, essentially all fructan was broken down and almost quantitatively converted to glucose and mainly fructose. This increase in sugar concentration (0.10 M for glucose and 0.15 M for fructose) slightly preceded the rapid expansion of the petals and, by lowering of the water potential, probably contributes to growth processes. Shortly after petal expansion and before the petals were completely dry, most of the sugar had been metabolized or exported to other organs. Some sugar was probably secreted as nectar and stuck to filaments and ovaries. The sudden increase in sugar concentration in senescing stamens was much larger than expected from endogenous fructan breakdown.
Large amounts of fructan were present in ovaries that were broken down simultaneously with the fructan in the other organs. Although ovaries are a major sink for other senescing flower parts, the increase in hexoses is rather moderate. It can be expected that imported and locally generated monosaccharides are rapidly metabolized in growth processes. It is likely that part of the fructan in senescing stems (unpublished results) is later mobilized and used in seed growth.
Comparable changes in carbohydrates have been observed during the opening of the ephemeral daylily flower (Bieleski, 1993), but sugar concentrations were higher in Campanula. During subsequent senescence of the petals the resulting carbohydrates were metabolized or exported (Bieleski, 1995).
A rapid breakdown of fructan has been reported in several monocotyledonous species, especially from the Poaceae, for example, tall fescue (Schnyder and Nelson, 1987), ryegrass (Prud'homme et al., 1992), barley (Bonnett and Incoll, 1993), and wheat (Schnyder, 1993). Although fructan mobilization in dicotyledonous plants is generally associated with a resumption of growth (Edelman and Jefford, 1968; Van den Ende et al., 1996a), a rapid remobilization of fructan in young Cichorium plants has been documented after defoliation (De Roover et al., 1999) or alleviation of nitrogen stress (Van den Ende et al., 1999). It is concluded that fructan is involved in both the rapid generation of osmotic potential and in the metabolism of sugars during flowering in Campanula.
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Acknowledgments |
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W Van den Ende was supported by grants from the FWO (Flanders).
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Notes |
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1 To whom correspondence should be addressed. Fax: +32 16 321967. E-mail: Andre.VanLaere{at}BIO.KULeuven.ac.be
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Abbreviations |
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DP, degree of polymerization; FFT, fructan : fructan 1-fructosyl transferase.
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