Journal of Experimental Botany, Vol. 53, No. 376, pp. 1949-1957,
September 1, 2002
© 2002 Oxford University Press
Developing fruit direct post-floral morphogenesis in Helleborus niger L.
Received 3 January 2002; Accepted 31 May 2002
2
1 Ruðer Bo
kovi
Institute, Bijenika c. 54, PO Box 180, HR-10002 Zagreb, Croatia
2 National Institute of Biology, Vena pot 111, SI-1000 Ljubljana, Slovenia
Abbreviations: BA, N6-benzyladenine; 4-Cl-IAA, 4-chloroindole-3-acetic acid; DHZ, dihydrozeatin; DHZ-9-G, dihydrozeatin-9-glucoside; DHZR, dihydrozeatin riboside; GA, gibberellic acid; HPLC, high-performance liquid chromatography; IAA, indole-3-acetic acid; iP, N6-(2-isopentenyl)adenine; iPA, N6-(2-isopentenyl)adenosine; iP-9-G, N6-(2-isopentenyl)adenine-9-glucoside; PB, paclobutrazol; Z, trans-zeatin; Z-9-G, trans-zeatin-9-glucoside; ZR, trans-zeatin riboside.
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Abstract |
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In fertilized flowers of Helleborus niger L., the sepals (the showy elements of the perianth at anthesis) grow, spread, and turn green, and the peduncles elongate. These processes did not proceed to completion when the pistils were removed at the bud stage, but could be restored by the application of plant growth regulators. Cytokinins and gibberellins stimulated the formation of well-developed chloroplasts in, and spreading of, the sepals; the gibberellin, GA3, and the auxin, 4-chloroindole-3-acetic acid, promoted peduncle elongation. In fruit-bearing flowers, on the other hand, paclobutrazol, an inhibitor of gibberellin biosynthesis, reduced chlorophyll formation in the sepals, reversed sepal spreading, and inhibited peduncle elongation. Of the endogenous growth regulators in developing fruit, the following cytokinins were identified: zeatin, dihydrozeatin, N6-(2-isopentenyl)adenine and their ribosides and 9-glucosides. Zeatin riboside drastically increased in abundance (about 200 times), shortly after fertilization, when chlorophyll accumulation in the sepals occurred at the fastest rate, and remained the most prominent identified cytokinin until seed ripening.
Key words: Key words: Christmas rose, cytokinin analysis, Helleborus niger L., plant growth regulator, post-floral morphogenesis.
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Introduction |
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Pollination is a critical event in the life cycle of a flower, initiating, in most cases, processes such as changes in pigmentation, a decrease in the production of scent and nectar, followed by the abscission of stamens, nectaries and the showy elements of the perianth (O’Neill, 1997; van Doorn and Stead, 1997). In addition, the pedicel and peduncle may grow and carry out complex movements which eventually ascertain the optimal spreading of the ripe seeds (Kaldewey, 1957). Exceptionally, in a number of species, pollination and/or the presence of developing fruit increase the life span of the perianth which then tends to turn green (or darker green) and engage in photosynthesis. Examples studied in detail include some orchids (van Doorn, 1997), the dicots Chrysosplenium alternifolium L., C. oppositifolium L. (Sitte, 1974), and Nuphar luteum Sibth. et Sm. (Grönegress, 1974), as well as the araceans Spathiphyllum wallisii Regel (Palandri, 1967), Zantedeschia aethiopica Spreng. (Pais, 1972; Chaves das Neves and Pais, 1980a, b) and Z. elliotiana Engl. (Grönegress, 1974), in which the spathe has assumed the function of a collective perianth for the entire inflorescence. In all these species, unpollinated or depistillated flowers or inflorescences senesce significantly before simultaneously pollinated ones. In Helleborus niger L., in contrast, flowers depistillated at the bud stage have the same life span as intact fruit-bearing flowers and thus provide a control for studies on every single phase of the metamorphosis initiated by pollination. The formation of a functional photosynthetic system in the perianth of fertilized flowers was recently described in detail (Salopek-Sondi et al., 2000). Additional aspects of the metamorphosis of fruit-bearing flowers, and possible correlative signals which trigger and maintain these processes, are investigated here.
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Materials and methods |
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Plant material was collected, and experiments were performed, at the natural habitat of the Christmas rose (Helleborus niger L.) in the mountain forests of Gorski kotar, Croatia (altitude: 800 m, dominant tree species: Abies alba Mill., Fagus silvatica L., Picea abies L.). As the flowers are proterogynous (Damboldt and Zimmermann, 1965) anthesis was considered to comprise the period between bud opening and the abscission of stamens and nectaries. The morphogenetic effects of fructification were studied by comparing post-floral development (a) following fertilization and fruit formation, and (b) when the pistils were removed before bud opening. For normally developing flowers, the developmental stages monitored were defined as follows: stage I, white flower buds immediately before anthesis; stage II, shortly after anthesis; immature fruit about 3 cm long, sepals greenish-yellow; and stage III, fruit just before maturity, about 5 cm long; sepals green.
Depistillated flowers were considered to pass through anthesis, and stages II and III, when the majority (c. 80%) of their fruit-bearing counterparts at the same locality was in the respective developmental phases.
A third group of flowers was permitted to develop normally until the abscision of stamens and nectaries. The sepals were then removed and further development was monitored.
To investigate the role of plant hormones in post-floral development, white, depistillated flowers immediately after anthesis were treated with a selection of auxins, gibberellins (GA), and cytokinins applied in lanolin (Mitchell and Livingston, 1968) to the inner surface of three (out of five) adjacent sepals; the remaining two served as an internal negative control. Using the same methodology and time of application, a number of intact flowers was treated with paclobutrazol, an inhibitor of GA biosynthesis. The growth regulators were applied in the following concentrations: 0.1, 1 and 10 mM for indole-3-acetic acid (IAA), 4-chloroindole-3-acetic acid (4-Cl-IAA), GA3, N6-benzyladenine (BA), zeatin (Z), N6-(2-isopentenyl)adenine (iP), N6-(2-isopentenyl)adenosine (iPA), and paclobutrazol; 0.01, 0.1 and 1 mM for GA4, GA7, zeatin riboside (ZR), dihydrozeatin (DHZ), and dihydrozeatin riboside (DHZR). The effects were recorded after 45 d, which is the upper limit of the period normally required to complete post-floral metamorphosis in fertilized flowers.
Of the parameters monitored to characterize post-floral development, the angle of flower opening (the angle 
in degrees enclosed by opposite sepals) was calculated using the equation:
where a is the length (cm) of the sepals, and b is the distance (cm) between the tips of opposite sepals.
The method of cytokinin analysis in Helleborus fruit was adopted from Kova and 
el (1994). In brief, the frozen (–70 °C) fruit (5 g) were ground in liquid nitrogen and extracted with cold 80% aqueous methanol. The filtered (Whatman no. 1) and concentrated extract was purified with polyvinylpolypyrrolidone at pH 3.1, followed by a commercial (Olchemim Ltd., Czech Republic) immunoaffinity column containing a mixture of polyclonal antibodies to trans-zeatin riboside (ZR), dihydrozeatin-riboside (DHZR), and isopentenyl adenosine (iPA). The column is able to bind free bases, ribosides, 9- and 3-glucosides, and 5-nucleotides of isoprenoid cytokinins. The affinity-purified cytokinins were fractionated by high-performance liquid chromatography (HPLC) using a Nova Pack C18 (Waters) column (150x3.9 mm, 4 µm spherical particles) equilibrated with 1 mM aqueous triethylamine acetate (pH 7) containing 5% of a mixture of methanol:acetonitrile (1:1, v/v). Elution was performed by increasing the concentration of the same organic solvent mixture to 20% over 30 min, followed by 5 min of isocratic elution (unchanged level of the organic solvent mixture) and a further increase of the solvent concentration to 30%, within 5 min. The effluent was passed through a WatersTM 996 photodiode array spectrophotometer and 1 ml fractions were collected. Cytokinins were identified by comparing retention times and UV spectra (220–320 nm) with those of authentic standards. Quantification was based on the comparison of peak areas (absorbance at 265 nm) to those of known amounts of standards. The results were corrected for recoveries (70–90%). The latter were determined, separately for each individual cytokinin, by comparing the peak areas of (a) authentic standards (50 pmol per compound) directly injected into the HPLC and (b) equal amounts of the same standards that had gone through the whole process of prepurification before the HPLC step.
The cytokinin activity of the collected 1 ml fractions was monitored using the Amaranthus betacyanin bioassay (Biddington and Thomas, 1973).
Student’s t-test was used to assess the statistical significance of numerical data.
For histological and ultrastructural studies, small pieces of the freshly collected sepals were fixed in 1% glutaraldehyde in cacodylate buffer (pH 7.2), post-fixed in 1% OsO4 in the same buffer and, after dehydration, embedded in Araldite. Semi-thin sections of the fixed tissue were stained with a mixture of 2% toluidine blue and 2% borax, (1:1), and examined under an optical microscope (Zeiss Axiovert 35). For electron microscopy (Zeiss EM 10A), thin sections of the same material were stained with uranyl acetate and lead citrate.
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Results |
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Morphology and post-floral changes in intact and depistillated flowers
The flowers of the Christmas rose (Helleborus niger L.) are characterized by a showy, white or rose, perianth whose, usually five, elements are mostly classified as sepals (Damboldt and Zimmermann, 1965). They enclose a whorl of c. 10 nectaries, a multitude of stamens and five to eight carpels (pistils). The plant populations studied started blooming in late December or early January. Following fertilization by February or March, the fruit developed and matured by May or June (depending on ambient temperatures); the accompanying changes in length and fresh weight are shown in Fig. 1. Simultaneously, the sepals spread, turned green, thus initiating photosynthesis (Salopek-Sondi et al., 2000), and eventually survived until seed ripening. When the pistils were removed before bud opening, the flowers unfolded more slowly and stopped at an early stage, even though survival was not affected. The resulting drastic differences in the overall appearance of stage III fertilized and depistillated flowers are shown in Fig. 2.
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The changes in selected morphological parameters are presented in Fig. 3. In fertilized flowers, the length of the peduncle increased by about a factor of four, as development proceeded from stage I to stage III; in depistillated flowers the corresponding elongation was only about 2-fold. The length and the width of the sepals increased markedly during anthesis and post-floral development. These changes were consistently, but not conspicuously, larger in fertilized than in depistillated flowers. Interestingly, the increase in sepal size was only in fertilized flowers matched by an increase in fresh weight. In depistillated flowers, the sepals lost weight while proceeding from stage II to stage III. The overall shape of Christmas rose flowers was not directly correlated to sepal size. While bell-shaped at anthesis, they became saucer-shaped after fertilization, as reflected by the distance between the tips of opposite sepals and the angle enclosed by these sepals (140° in stage III). Depistillated flowers, in contrast, remained bell-shaped (respective angle about 60°).
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The increase in sepal dimensions and fresh weight is based on cell enlargement and the expansion of intercellular spaces in the mesophyll (Fig. 4). These changes are practically complete in stage II and occur in both fertilized (Fig. 4B) and depistillated (Fig. 4D) flowers (with minor quantitative differences).
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Effect of exogenous hormones on the morphology of depistillated flowers
To assess the response of depistillated flowers to exogenous growth regulators, their morphogenetic effects were compared with two controls. The negative control comprises (a) non-treated depistillated flowers and (b) depistillated flowers treated with plain lanolin; the resulting developmental patterns were identical. The morphological parameters determined for fruit-bearing flowers were used as the positive control. Sepal histology in depistillated flowers was not markedly affected by the growth regulators tested, as illustrated in Fig. 4C for GA3-treated material.
The effect of exogenous hormones on the greening of the perianth was particularly obvious, as only the three treated sepals responded while the remaining two served as an internal negative control. As judged by visual screening, all cytokinins tested stimulated sepal greening in depistillated flowers, at concentrations in the range of 0.01 to 10 mM. Sepals treated with 1 mM BA, zeatin and ZR were practically indistinguishable from the positive controls. The gibberellins, GA3, GA4 and GA7 were also effective. If gibberellins induce greening in depistillated flowers then paclobutrazol should inhibit greening in intact flowers to the extent the inhibitor reaches the centres of gibberellin biosynthesis. This was indeed observed, but paclobutrazol concentrations around 10 mM were required to produce a clearly visible effect. The auxins, IAA and 4-Cl-IAA, did not induce any distinct sepal greening in depistillated flowers.
At the cellular level, depistillation and hormone treatment affected both the abundance of chloroplasts and their ultrastructure. In semi-thin cross-sections of untreated and auxin-treated stage III sepals of depistillated flowers, only c. 10% of the mesophyll cells screened showed, maximally two, chloroplasts per cross-section. By contrast, cytokinin- and gibberellin-treated sepals at the same developmental stage contained four to eight chloroplasts per cross-section in every mesophyll cell, which was within the range observed for intact fruit-bearing flowers.
Selected examples illustrating the differences in chloroplast ultrastructure are presented in Figs 5 and 6. The chloroplasts in stage III sepals of intact fruit-bearing flowers (Fig. 5A) contained a dense stroma with numerous ribosomes and just a few small, electron-dense plastoglobules. The thylakoid system was normally developed with grana composed of, on average, three to four stacked thylakoids. In the sepals of depistillated flowers a similar ultrastructural pattern could be induced by treatment with cytokinins and gibberellins. The effects of 1 mM BA (Fig. 5B) and 1 mM GA3 (Fig. 5C) are shown as representative examples documenting an even higher degree of grana stacking than in stage III sepals of green, fruit-bearing, flowers. By contrast, the chloroplast stroma of stage III sepals of untreated depistillated flowers (Fig. 6A), was relatively transparent, with sparse ribosomes and many large plastoglobules. Thylakoids were few in number and, in part, assembled into atypical grana. Essentially the same pattern was observed in most chloroplasts from auxin-treated sepals of depistillated flowers (Fig. 6B), but the thylakoid system was not always quite as degenerated.
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The influence of external growth regulators on sepal spreading is summarized in Fig. 7. In the negative controls, this angle increased by 6±1°, during the assay period. In depistillated flowers treated with growth regulators, all sepals responded equally (i.e. not just the three sepals to which the lanolin paste was applied). Cytokinins and the gibberellins, GA3 and GA7, consistently stimulated sepal spreading, in most cases in an obviously dose-dependent fashion. Vice versa, paclobutrazol, an inhibitor of gibberellin biosynthesis, not only inhibited sepal spreading in intact fruit-bearing flowers, but actually caused them to close by up to 35°. Depistillated flowers closed by 13° or, respectively, 21°, when treated with 10 mM IAA and 4-Cl-IAA.
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Peduncle elongation during the period following anthesis was more variable in depistillated than in normal, fertilized flowers, suggesting interference by unrecognized external and internal factors. On hormone treatment, only the elongation caused by 10 mM 4-Cl-IAA (3.8±0.7 cm) and 10 mM GA3 (3.3±0.2 cm) was significantly (P >95%) larger than in the negative control (2.3±0.3 cm). More clearly, for the peduncles of intact flowers treated with 0.1, 1 and 10 mM paclobutrazol, the elongation (0.9±0.2 cm, 1.6±0.4 cm, 0.5±0.2 cm) was markedly (P >>99%) less than that of the positive control (7.6±0.2 cm).
Cytokinin analyses
The cytokinins extracted from Helleborus fruit and sepals were, after purification by immunoaffinity chromatography identified and quantified by HPLC. A representative chromatogram for stage II fruit is presented in Fig. 8, illustrating the overall purification accomplished and the separation of the individual cytokinin peaks. Biological activity in the Amaranthus betacyanin induction bioassay (tested in consecutive 1 ml fractions of the effluent from the HPLC column) is also shown and corroborates the identification of the cytokinin peaks. The changing cytokinin pattern in developing Helleborus fruit is summarized in Table 1. Unpollinated pistils (stage I) contained only traces of cytokinins (in total c. 3.7 pmol per set of pistils in an individual flower), with an approximate ratio of free bases:ribosides:9-glucosides of 24:57:19. Following fertilization, as development proceeded to stage II, total cytokinins increased about 36 times, and the ratio (free bases:ribosides:9-glucosides = 13:78:9) changed markedly in favour of the ribosides. In particular, the concentration of ZR, which predominated, was about 230 times that in stage I. As fruit growth and maturation proceeded, free bases and 9-glucosides continued to accumulate, while the level of total ribosides decreased. The ratio of active cytokinins (free bases and ribosides): inactive glucosides (9-glucosides) increased from 4.6 in stage I to 9.7 in stage II, to return to 5.0 during maturation. The most abundant group of active cytokinins in developmental stages I and II were the ribosides which comprised 71% and 87% of the total active cytokinins (free bases+ribosides). Stage III fruit then contained free bases and ribosides in a ratio of 56:44, i.e. in almost equimolar amounts.
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Effect of sepal removal
When the sepals were removed after fertilization, but the carpels left in place, the peduncle reached about the same final length as in intact fruit-bearing flowers. However, in 29 out of 30 flowers deprived of their sepals, the fruit aborted before maturity. In the single recorded case in which the cluster of fruit remained viable, their length immediately before seed ripening reached only one-half, and their overall fresh weight one-third, of the values observed for intact fertilized flowers (shown in Fig. 1).
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Discussion |
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Removal of the pistils at the bud stage (stage I) prevents completion of the following processes which normally occur in fruit-bearing Christmas rose flowers: the development of functional chloroplasts in the perianth, sepal spreading, and peduncle elongation. These morphological changes must thus be triggered and/or maintained by signals originating in the developing fruit. The latter, and the seeds in particular, have in a number of other species been shown to be a rich source of auxins, cytokinins, and gibberellins (Bearder, 1980; van Staden, 1983). If the set of signals released by developing Christmas rose fruit includes these hormones they should, when applied to depistillated flowers, initiate and support at least part of the metamorphosis which normally occurs after fertilization. This was indeed observed, but the individual morphogenetic events responded to different plant growth regulators.
The effect of hormone application on the greening response in the sepals of depistillated flowers was particularly clear-cut. All cytokinins and gibberellins tested induced chlorophyll formation, while the auxins, IAA and 4-Cl-IAA, had no such effect. This was confirmed by monitoring chloroplast numbers per cell and chloroplast ultrastructure (Figs 5, 6) both of which, under cytokinin and gibberellin treatment, approached the pattern generally observed in photosynthetically active tissues, while grana stacking was even more pronounced. The sepals of depistillated flowers treated with auxin contained the same small number of chloroplasts as in untreated flowers; in both cases, low photosynthetic activity was also indicated by chloroplast ultrastructure.
In senescing leaves, cytokinins have long been known to prevent the degradation of chlorophyll, or to increase its abundance (Richmond and Lang, 1957; Mothes et al., 1959), intensely studied complex processes which include: reduction of chlorophyllase (EC 3.1.1.14) levels (Genkov et al., 1997; Trebitsh et al., 1993), induction of chlorophyll-synthesizing enzymes and light-harvesting chlorophyll-binding proteins (Kusnetsov et al., 1994, 1998, 1999; Zavaleta-Mancera et al., 1999a, b), and are accompanied by an increase in the activity of photosynthetic enzymes; Wingler et al., 1998). Such cytokinin effects on chloroplast physiology are not necessarily linked to senescence. This was, for instance, illustrated by a tobacco tissue culture line which could grow without external cytokinins, but, nevertheless, required them for chloroplast maturation and replication (Stetler and Laetsch, 1965; Boasson and Laetsch, 1969). When BA was applied to leaves still attached to their mother plants, grana stacking in the chloroplasts increased markedly (Naito et al., 1981; Wilhelmová and Kutík, 1995). Also, when the white spathe of Zantedeschia aethiopica regreens after anthesis, this is at least in part induced by aromatic cytokinins exported by the developing fruit (Pais, 1972; Chaves das Neves and Pais, 1980a, b).
While less extensively investigated, the role of gibberellins in plastid function and development appears to overlap with that of cytokinins. Thus, hormones of either type reduced chlorophyll loss in detached, senescing, lettuce leaves (Aharoni and Richmond, 1978) and, with subtle differences, in ripening citrus fruits (Coggins et al., 1960; Goldschmidt et al., 1977; García-Luis et al., 1986; Trebitsh et al., 1993). Both benzyladenine and GA3 enabled light-induced greening in cotyledons isolated from dormant cocklebur (Xanthium pennsylvanicum Wallr.) seeds which otherwise do not perform chlorophyll synthesis (Esashi et al., 1977). On a mechanistic level, both hormones increased the expression of NADPH-protochlorophyllide oxidoreductase (EC 1.3.1.33), an enzyme of chlorophyll biosynthesis (Kuroda et al., 1996). Interestingly, this enzyme was found down-regulated in the slender mutant of barley (Ougham et al., 2001) which is unable to respond to gibberellins.
Sepal spreading in depistillated Helleborus flowers also responded in a different way to auxins (sepal tips moving closer together) than to gibberellins and cytokinins (sepal tips moving apart). Moreover, the effect of paclobutrazol in fertilized flowers was exactly opposite to that of gibberellins in depistillated flowers. The angle enclosed by the tips of opposite sepals was used here as a parameter which can be routinely checked in living plants at their natural habitat. As Helleborus sepals are slightly concave, their tips may move closer together by increasing the degree of curvature, by closing the angle at the point of attachment to the peduncle, or both.
Peduncle elongation in depistillated flowers was stimulated by 10 mM GA3 and 10 mM 4-Cl-IAA, but remained smaller than in fertilized flowers. Peduncle growth in the latter was, however, drastically inhibited by 0.1–10 mM paclobutrazol. In a number of other species, both gibberellins (Izhaki et al., 1996) and auxins (Kaldewey, 1957; Hanks and Rees, 1975, 1977) produced by the gynaecium have been implicated in peduncle elongation and the complex interplay of these hormones has, for instance, been studied in Gerbera (Sachs, 1968) and tulip (Rietveld et al., 2000). The cytokinins tested did not significantly affect peduncle elongation in Helleborus.
To what extent do the effects of external hormones in depistillated flowers reflect the signals normally released by the developing fruit?
The application of plant growth regulators in lanolin to the inner surface of the sepals of depistillated flowers was a method which worked under outdoor conditions and led to morphogenetic events normally initiated by fertilization. This analogy may not be perfect in every respect.
The high concentration of the external growth regulators may appear unphysiological, but experience suggests that lanolin pastes release phytohormones slowly, at levels substantially below those in the paste. This is, for instance, illustrated by experiments in Fritillaria meleagris L.(Kaldewey, 1957). Its buds and developing fruit release auxin at a rate of 4–13 pmol h–1. To mimic their effect on stem elongation in decapitated stems, lanolin pastes containing IAA concentrations up to 500 mmol kg–1 were required. Ross et al. (2000) applied 3000 mg (17 mmol) IAA kg–1 of lanolin to decapitated pea stems to establish a stationary IAA concentration of 550 µg (3 µmol) kg–1 fresh weight in the adjacent internode. The concentrations of plant regulators in lanolin (0.01, 0.1, 1, and 10 mmol kg–1) used with Helleborus, therefore, did not exceed the customary range.
As the application of cytokinins in lanolin to depistillated Helleborus flowers reproduced the most obvious morphogenetic processes observed in fertilized flowers, the cytokinins produced by the developing fruit were analysed by a combination of immunoaffinity chromatography, HPLC, and bioassays. Except for BA, all cytokinins which promoted greening and sepal spreading in depistillated flowers were found in pistils and developing fruit. Moreover, the concentration of active cytokinins (free bases and ribosides) in the fruit was highest in stage II, when sepal greening occurred at the fastest rate (Salopek-Sondi et al., 2000).
Application of GA3 or the auxin, 4-Cl-IAA, just weakly stimulated peduncle elongation in depistillated flowers, while the inhibitor of gibberellin biosynthesis, paclobutrazol, drastically inhibited peduncle growth in intact, fertilized flowers. Should in situ GA biosynthesis be required for peduncle elongation, as was recently shown for stem elongation in pea (Ross et al., 2000)? This will be revealed by complete analyses of hormone levels and metabolic patterns, which are in preparation.
Interaction of the developing fruit and the sepals includes cross-talk, as suggested by the fact that the fruit abort or degenerate when the sepals are removed shortly after fertilization. A similar phenomenon has recently been described in the orchid, Phalaenopsis (Ketsa and Rugkong, 1999). In the Christmas rose, photosynthesis in the green sepals should be an important source of assimilates for the developing fruit, which are ripening while last year’s generation of leaves is dying back and the new generation is just starting to unfold.
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Acknowledgements |
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This work was supported by the Joint Board for Scientific and Technological Cooperation between the Republic of Croatia and the Republic of Slovenia and the Ministries of Science and Technology of the two states. We thank Mrs Lidija Mati
i for excellent technical support and Dr Nikola Ljubei for practical advice and helpful discussions.
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References |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Aharoni N, Richmond AE. 1978. Endogenous gibberellin and abscisic acid content as related to senescence of detached lettuce leaves. Plant Physiology 62, 224–228.
Bearder JR. 1980. Plant hormones and other growth substances—their background, structures, and occurrence. In: MacMillan J, ed. Encyclopedia of plant physiology, New series, Vol. 9. Hormonal regulation of development I. Berlin, Heidelberg, New York: Springer-Verlag, 9–112.
Biddington NL, Thomas H. 1973. A modified Amaranthus betacyanin bioassay for the rapid determination of cytokinins in plant extracts. Planta 111, 183–186.
Boasson R, Laetsch WM. 1969. Chloroplast replication and growth in tobacco. Science 166, 749–751.
Chaves das Neves HJ, Pais MSS. 1980a. Identification of a spathe regreening factor in Zantedeschia aethiopica. Biochemical and Biophysical Research Communications 95, 1387–1392.[Web of Science][Medline]
Chaves das Neves HJ, Pais MSS. 1980b. A new cytokinin from the fruits of Zantedeschia aethiopica. Tetrahedron Letters 21, 4387–4390.
Coggins Jr CW, Hield HZ, Garber MJ. 1960. The influence of potassium gibberellate on Valencia orange trees and fruit. Journal of the American Society for Horticultural Science 76, 193–198.
Damboldt J, Zimmermann W. 1965. 289. Helleborus. In: Hegi G, ed. Illustrierte Flora von Mittel-Europa, Vol. 3, Part 3. München: Carl Hanser Verlag, 91–107.
Esashi Y, Katoh H, Hata Y, Gotô N. 1977. Dormancy and impotency of cocklebur seeds. VII. Inability of dormant cotyledons to form chlorophyll. Plant Physiology 59, 122–125.
García-Luis A, Fornes F, Guardiola JL. 1986. Effects of gibberellin A3 and cytokinins on natural and post-harvest, ethylene-induced pigmentation of Satsuma mandarin peel. Physiologia Plantarum 68, 271–274.
Genkov T, Tsoneva P, Ivanova P. 1997. Effect of cytokinins on photosynthetic pigments and chlorophyllase activity in in vitro cultures of axillary buds of Dianthus caryophyllus L. Journal of Plant Growth Regulation 16, 169–172.
Goldschmidt EE, Aharoni Y, Eilati SK, Riov JW, Monselise SP. 1977. Differential counteraction of ethylene effects by gibberellin A3 and N6-benzyladenine in senescing citrus peel. Plant Physiology 59, 193–195.
Grönegress P. 1974. The structure of chromoplasts and their conversion to chloroplasts. Journal de Microscopie (Paris) 19, 183–192.
Hanks GR, Rees AR. 1975. Scape elongation in narcissus: the influence of floral organs. New Phytologist 75, 605–612.
Hanks GR, Rees AR. 1977. Stem elongation in tulip and narcissus: the influence of floral organs and growth regulators. New Phytologist 78, 579–591.
Izhaki A, Shoseyov O, Weiss D. 1996. Temporal, spatial and hormonal regulation of the S-adenosylmethionine synthetase gene in petunia. Physiologia Plantarum 97, 90–94.
Kaldewey H. 1957. Wuchsstoffbildung und Nutationsbewegungen von Fritillaria meleagris L. im Laufe der Vegetationsperiode. Planta 49, 300–344.
Ketsa S, Rugkong A. 1999. Senescence of Dendrobium ‘Pompadour’ flowers following pollination. Journal of Horticultural Science and Biotechnology 74, 608–613.
Kova M, el J. 1994. The effect of aluminium on the cytokinins in the mycelia of Lactarius piperatus. Plant Science 97, 137–142.
Kuroda H, Masuda T, Ohta H, Shioi Y, Takamiya K. 1996. Effects of light, developmental age and phytohormones on the expression of the gene encoding NADPH-protochlorophyllide oxidoreductase in Cucumis sativus. Plant Physiology and Biochemistry 34, 17–22.
Kusnetsov VV, Hermann RG, Kulaeva ON, Oelmüller R. 1998. Cytokinin stimulates and abscisic-acid inhibits greening of etiolated Lupinus luteus cotyledons by affecting the expression of light-sensitive protochlorophyllide oxidoreductase. Molecular and General Genetics 259, 21–28.
Kusnetsov VV, Landsberger M, Meurer J, Oelmüller R. 1999. The assembly of the CAA-box binding complex at a photosynthesis gene promotor is regulated by light, cytokinin, and the stage of the plastids. Journal of Biological Chemistry 274, 36009–36014.
Kusnetsov VV, Oelmüller R, Sarwat MI, Porfirova SA, Cherepneva GN, Herrmann RG, Kulaeva ON. 1994. Cytokinins, abscisic acid and light affect accumulation of chloroplast proteins in Lupinus luteus cotyledons without notable effect on steady-state mRNA levels. Planta 194, 318–327.[Web of Science]
Mitchell JW, Livingston GA. 1968. Methods of studying plant hormones and growth-regulating substances. Agriculture Handbook No. 336. Washington, DC, USA: United States Department of Agriculture, Agricultural Research Service.
Mothes K, Engelbrecht L, Kulajewa O. 1959. Über die Wirkung des Kinetins auf Stickstoffverteilung und Eiweißsynthese in isolierten Blättern. Flora (Jena) 147, 445–464.
Naito K, Ueda K, Tsuji H. 1981. Differential effects of benzyladenine on the ultrastructure of chloroplasts in intact bean leaves according to their age. Protoplasma 105, 293–306.
O’Neill SD. 1997. Pollination regulation of flower development. Annual Reviews of Plant Physiology and Plant Molecular Biology 48, 547–574.[Web of Science]
Ougham HJ, Thomas, AM, Thomas BJ, Frick GA, Armstrong GA. 2001. Both light-dependent protochlorophyllide oxidoreductase A and protochlorophyllide oxidoreductase B are down-regulated in the slender mutant of barley. Journal of Experimental Botany 52, 1447–1454.
Pais MSS. 1972. Sur le reverdissement de la spathe de Zantedeschia aethiopica au cours de la frutification. Portugaliae Acta Biologica, Serie A: Morfologia, Fisiologia, Genetica e Biologia Geral 12, 101–121.
Palandri M. 1967. Modificazioni ultrastrutturali presentate dai plastidi della spata nel corso dell’inverdimento in Spathiphyllum wallisii Regel. Caryologia 20, 273–285.
Richmond AE, Lang A. 1957. Effect of kinetin on protein content and survival of detached Xanthium leaves. Science 125, 650–651.
Rietveld PL, Wilkinson C, Franssen HM, Balk PA, van der Plas LHW, Weisbeek PJ, de Boer DA. 2000. Low temperature sensing in tulip (Tulipa gesneriana L.) is mediated through an increased response to auxin. Journal of Experimental Botany 51, 587–594.
Ross JJ, O’Neill DP, Smith JJ, Kerckhoffs HJ, Elliott RC. 2000. Evidence that auxin promotes gibberellin A1 biosynthesis in pea. The Plant Journal 21, 547–552.[Web of Science][Medline]
Sachs RM. 1968. Control of intercalary growth in the scape of gerbera by auxin and gibberellic acid. American Journal of Botany 55, 62–68.
Salopek-Sondi B, Kova M, Ljubei N, Magnus V. 2000. Fruit initiation in Helleborus niger L. triggers chloroplast formation and photosynthesis in the perianth. Journal of Plant Physiology 157, 357–364.[Web of Science]
Sitte P. 1974. Plastiden-Metamorphose und Chromoplasten bei Chrysosplenium. Zeitschrift für Pflanzenphysiologie 73, 243–265.
Stetler DA, Laetsch WM. 1965. Kinetin-induced chloroplast maturation in cultures of tobacco tissue. Science 149, 1387–1388.
Trebitsh T, Goldschmidt EE, Riov J. 1993. Ethylene induces de novo synthesis of chlorophyllase, a chlorophyll degrading enzyme, in Citrus fruit peel. Proceedings of the National Academy of Sciences, USA 90, 9441–9445.
van Doorn WG. 1997. Effects of pollination on floral attraction and longevity. Journal of Experimental Botany 48, 1615–1622.
van Doorn WG, Stead AD. 1997. Abscission of flowers and floral parts. Journal of Experimental Botany 48, 821–837.
van Staden J. 1983. Seeds and cytokinins. Physiologia Plantarum 58, 340–346.
Wilhelmová N, Kutík J. 1995. Influence of exogenously applied 6-benzylaminopurine on the structure of chloroplasts and arrangement of their membranes. Photosynthetica 31, 559–570.
Wingler A, von Schaewen A, Leegood RC, Lea PJ, Quick PW. 1998. Regulation of leaf senescence by cytokinin, sugar, and light. Plant Physiology 116, 329–335.
Zavaleta-Mancera H, Franklin K, Ougham H, Thomas H, Scott I. 1999a. Regreening of senescent Nicotiana leaves. I. Reappearance of NADPH-protochlorophyllide oxidoreductase and light harvesting chlorophyll a/b-binding protein. Journal of Experimental Botany 50, 1677–1682.
Zavaleta-Mancera H, Thomas B, Thomas H, Scott I. 1999b. Regreening of senescent Nicotiana leaves. II. Redifferentiation of plastids. Journal of Experimental Botany 50, 1683–1689.
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