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JXB Advance Access originally published online on June 9, 2006
Journal of Experimental Botany 2006 57(10):2237-2247; doi:10.1093/jxb/erj190
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© The Author [2006]. Published by Oxford University Press [on behalf of the Society for Experimental Biology]. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

RESEARCH PAPER

Cytokinins in the perianth, carpels, and developing fruit of Helleborus niger L.

Petr Tarkowski1 *, Danuse Tarkowská2, Ondrej Novák2, Snjezana Mihaljevic3, Volker Magnus3, Miroslav Strnad2 and Branka Salopek-Sondi3,{dagger}

1Umeå Plant Science Centre, Department of Forest Genetics and Plant Physiology, Umeå, Sweden
2Laboratory of Growth Regulators, Palacky University and IEB ASCR, Olomouc, Czech Republic
3Rudjer Boskovic Institute, Zagreb, Croatia

{dagger}To whom correspondence should be addressed. E-mail: salopek{at}irb.hr

Received 12 September 2005; Accepted 9 March 2006


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results and discussion
 References
 
Reproductive development in the Christmas rose (Helleborus niger L.) differs from that in commonly investigated model plants in two important aspects: (i) the perianth develops a photosynthetic system, after fertilization, and persists until seed ripening; and (ii) the ripe seed contains an immature embryo which continues to mature off the mother plant. The possible roles of cytokinins in these processes are investigated here by analysing extracts of the perianth and the carpels/maturing fruit prepared during anthesis and four stages of post-floral development. trans-Zeatin, dihydrozeatin, N6-({Delta}2-isopentenyl)adenine, and their ribosides were identified by tandem mass spectrometry. Single ion monitoring in the presence of deuterated internal standards demonstrated the additional presence of the corresponding riboside-5'-monophosphates, O-glucosides, and 9-glucosides, and afforded quantitative data on the whole set of endogenous cytokinins. Fruit cytokinins were mostly localized in the seeds. Their overall concentrations increased dramatically during early seed development and remained high for 6–8 weeks, until shortly before seed ripening (the last time point covered in this work). Overall cytokinin levels in the perianth did not change markedly in the period covered, but the level of N6-({Delta}2-isopentenyl)adenine-type cytokinins appeared to increase slightly and transiently during the greening phase. The perianths of unpollinated or depistillated flowers, which survived, but did not pass through the complete greening process, contained significantly less cytokinins than observed in fruit-bearing flowers. This suggests that perianth greening requires defined cytokinin levels and supports the role of the developing fruit in their maintenance.

Key words: Christmas rose, cytokinin identification and quantification, fruit and seed development, Helleborus niger L., perianth greening


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results and discussion
 References
 
It is believed that the photosynthetic system adapts, both anatomically and functionally, to the rate of assimilate consumption by metabolic ‘sinks’, such as fruits and storage tubers (Brenner, 1987), but the complexity of most whole-plant systems imposes practical limits on detailed mechanistic studies. A simple model system can be found in the flowers of the Christmas rose (Helleborus niger L., Ranunculaceae), a herbaceous, winter-green, perennial native to south-eastern Europe, which is also widely grown as an ornamental. In mild winters, the flowers may indeed appear at Christmas time, resembling wild roses with respect to size and colour (white to pink). After pollination, the showy elements of the Christmas rose perianth (usually characterized as sepals) develop a functional photosynthetic system and persist until fruit ripening (Salopek-Sondi et al., 2000; Aschan and Pfanz, 2003). Comparable processes occur in fertilized flowers of a few other plants (Sitte, 1974; van Doorn, 1997), and in the spathes of some Araceans (Palandri, 1967; Pais, 1972; Grönegress, 1974; Chaves das Neves and Pais, 1980a, b) but, in these species, leaves are present at the same time to carry out the bulk of photosynthesis. In the Christmas rose, the overwintering leaves are often pressed to the ground by snow and covered with debris. They also senesce during fruit ripening, and the appearance of the new generation of leaves can be delayed by drought and low temperatures. Apart from the stores in the roots, the green perianth thus represents the most reliable, if not the only, source of assimilates for the developing seeds. Notably, flowers ignored by pollinators survive about as long as their fertilized neighbours, but only assume a faint greenish tinge (unpublished observations). The greening process is also arrested when the developing fruit are removed (Salopek-Sondi et al., 2000). It is resumed when the sepals are treated with cytokinins (Salopek-Sondi et al., 2002). Endogenous cytokinins could thus play a role in the greening process, and indeed preliminary analyses by high-performance liquid chromatography (HPLC) suggested dramatic quantitative changes during fruit development (Salopek-Sondi et al., 2002). This prompted the analysis of the endogenous cytokinins in Christmas rose fruit and sepals using a liquid chromatography–mass spectrometry (LC–MS) approach which provides more reliable identifications and more complete quantitative data. In fruits, cytokinins tend to be concentrated in the seeds, changing in kind and quantity as endosperm and embryo development proceed (Morris, 1997; Emery et al., 1998, 2000). In the species studied so far, these processes proceed to completion before seed ripening. In contrast, in Helleborus, embryo maturation is still in progress when the seeds are released by the mother plant, and this also appears to be the case in many perennial ornamentals. Cytokinin housekeeping during the development of this type of seeds is addressed here for the first time. This also appears to be the first detailed report on cytokinins in the perianth during its entire life cycle.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results and discussion
 References
 
Plant material
Flowers of the Christmas rose (H. niger L. ssp. niger sensu Damboldt and Zimmermann, 1965) were collected at their natural habitat in the mountain forests of Gorski kotar, Croatia (altitude, 800 m; dominant tree species, Abies alba L., Fagus silvatica L., and Picea abies L.). The six stages of the normal developmental cycle sampled for cytokinin analysis are shown in Table 1. Two stages of anthesis were recognized. The (‘proterogynous’) flowers first passed through their female phase during which the stigmata were receptive and the immature anthers were arranged in a ring at the base of the cluster of carpels. The male phase began with the elongation of the filaments, and ended with anther abortion. Fruit development and the accompanying metamorphosis of the perianth were monitored at four subsequent stages: ‘initial perianth greening’ (sepals with first greenish tinge, barely noticeable fruit growth); ‘advanced perianth greening’ (light green sepals, fruit weight twice that in late anthesis); ‘perianth greening complete’ (chlorophyll content in the sepals entering the stationary phase, fruit weight three times that in late anthesis); and ‘2–3 weeks before seed ripening’ (sepals green, fruit fully grown, weight ~12 times that in late anthesis).


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  		Table 1 Stages of the life cycle of Christmas rose flowers sampled for cytokinin analysis

 
Non-pollinated flowers (same carpel size as in late anthesis) and flowers depistillated at bud opening were collected when the seeds of fertilized flowers at the same location were 2–3 weeks before ripening.

For cytokinin analysis, the perianths and the clusters of carpels or developing fruit were separated, immediately frozen, and kept in sealed plastic bags, at –80 °C, until work-up. Stamens and nectaries were discarded.

Cytokinin identification
Cytokinins were extracted and separated, essentially as outlined by Åstot et al. (1998). Frozen plant material (~10 g fresh weight) was ground with a mortar and pestle in liquid nitrogen and extracted overnight in methanol–chloroform–formic acid–water (12:5:1:2, by vol.) (Bieleski, 1964). Passing the extract, in sequence, through a cation (SCX-cartridge) and an anion [DEAE-Sephadex combined with an SPE(C18)-cartridge] exchanger afforded fraction 1 containing the cytokinin free bases, ribosides, and glucosides, and fraction 2 containing the riboside-5'-monophosphates. Both fractions were purified further by immunoaffinity chromatography based on generic monoclonal anticytokinin antibodies, but fraction 2 was first treated with alkaline phosphatase. In fraction 1, the O-glucosides did not bind to the immunoaffinity column. The effluent was thus treated with ß-glucosidase and the hydrolysate was rechromatographed on the immunoaffinity column to yield the O-glucoside fraction (which actually contained just the aglycones of the original O-glucosides). The dried samples were propionylated using N,N-dimethylformamide–N-methylimidazole–propionic anhydride (5:3:1, by vol.) (Åstot et al., 1998), evaporated in vacuo and stored at –20 °C until further analysis.

For analysis, the samples were redissolved in 1% aqueous formic acid and subjected to LC–MS/MS. The chromatographic separation was performed on a capillary column (150 mmx0.3 mm; LC Packings, Amsterdam, The Netherlands) packed with Symmetry C18 packing material, particle size 3.5 µm (Waters). The eluent, applied at a flow rate of 4 µl min–1, was 1% aqueous formic acid (solvent A), mixed with 1% formic acid in acetonitrile (solvent B), as follows: 0–5.5 min, 10% B; 5.5–20 min, linear gradient to 70% B; followed by 3 min isocratic elution with 70% B. The effluent was introduced into a Micromass Quattro Ultima triple-stage quadrupole mass spectrometer via an electrospray ion source (capillary voltage, +2.9 kV; cone voltage, +70 V; source temperature, 90 °C; desolvation temperature, 120 °C; cone gas flow, 120 l h–1; desolvation gas flow, 520 l h–1; collision energy, 15 eV; dwell time, 0.35 s; scanning, 1 s per scan for mass range 0–600 amu).

Cytokinin quantification
For quantitative analysis, the separation method was modified as follows (Novák et al., 2003). Aliquots of plant material (1 g) were processed by adding the following internal standards at the extraction stage: trans-[2H5]zeatin, trans-[2H5]zeatin riboside, trans-[2H5]zeatin 9-glucoside, [2H3]dihydrozeatin, [2H3]dihydrozeatin riboside, [2H3]dihydrozeatin 9-glucoside, [2H6]isopentenyladenine, [2H6]isopentenyladenine riboside, [2H6]isopentenyladenine 9-glucoside, trans-[2H5]zeatin O-glucoside, trans-[2H5]zeatin riboside O-glucoside, trans-[2H5]zeatin riboside-5'-monophosphate, [2H3]dihydrozeatin riboside-5'-monophosphate, and [2H6]isopentenyladenine riboside-5'-monophosphate (OlChemIm Ltd, Olomouc, Czech Republic). After fractionation and purification as for cytokinin identification (but no propionylation), the samples were subjected to HPLC on a reversed-phase column (150 mmx2.1 mm; particle size, 5 µm) (Symmetry C18, Waters) operated at 30 °C. The components of the eluent were (A) 15 mM ammonium formate, pH 4.0 and (B) methanol; they were mixed as follows (difference from 100% is A): 0 min, 10% B; 0–25 min, linear gradient to 50% B; 25–30 min, 50% B; 30–35 min, 100% B, all at a flow rate of 250 µl min–1. Using post-column splitting (1:1), the eluent was simultaneously introduced into a diode array detector (Waters PDA 996) and the electrospray source (source temperature, 100 °C; capillary voltage, +3.0 kV; cone voltage, +20 V; desolvation temperature, 250 °C) of a single-stage quadrupole mass spectrometer (ZMD 2000, Micromass, Manchester, UK). Nitrogen was used as both the desolvation gas (400 l h–1) and the cone gas (50 l h–1). Quantification was done by single ion monitoring of the quasi-molecular ([M+H]+) ions of the plant cytokinins and the corresponding deuterated internal standards.


   Results and discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
References
 
Perianth and fruit development
The metamorphosis of the sepals in pollinated Helleborus flowers has already been documented in detail (Salopek-Sondi et al., 2000, 2002). In brief, during anthesis, only the guard cells contain chloroplasts, resulting in bulk chlorophyll levels below 1 µg g–1 fresh weight (f. wt.). After fertilization, chloroplasts form in the entire perianth, eventually affording chlorophyll concentrations of ~350 µg g–1 f. wt. At the same time, the interior of the sepals assumes a sponge-like structure, due to the expansion of intercellular spaces. Cell divisions were not noticed; cell expansion was limited, and so was overall sepal growth. The small weight gain during the assimilatory phase shown in Table 1 was supported by further observations through several growth seasons (Salopek-Sondi et al., 2000, 2002, and unpublished data). The weight fluctuations observed in the present samples during anthesis and ‘initial sepal greening’ (Table 1) are not typical and appear to reflect intraspecific variability in flower size.

Simultaneously with perianth greening, the fruit clusters started to grow. Characteristic developmental stages of the follicles (Fig. 1A) and the seeds (Fig. 1B) are illustrated in Fig. 1. Interestingly, fruit growth was not restricted to the ovaries, but included the styles which developed into elongated beaks (Table 1). Most fruit weight was gained while the photosynthetic capacity of the perianth was fully established. The ratio between pericarp and seed weight depended on seed set (more or less successful pollination). In several samples analysed 3–4 weeks before ripening, up to two-thirds of the overall fruit weight was in the pericarp.


Figure 1
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  		Fig. 1 Fruit and seed growth in Helleborus niger L. (glutaraldehyde-fixed material). (A) Follicles split lengthwise at the following developmental stages (from left to right): white flower, male phase (i.e. shortly after fertilization), initial perianth greening, perianth greening complete, and 2–3 weeks before ripening. The upper half of the follicle wall was removed. Some seeds had aborted (probably due to incomplete pollination), as seen most clearly in the fully grown follicles. (B) Seeds sampled at the same developmental stages plus a ripe seed after rehydration. Helleborus seeds bear an elaiosome (arill) which was removed from the ripe seed to prevent fungus infestation during the rehydration process. The dimensions of the fields of the grid used as a background in (A) and (B) are 1 mmx1 mm.

 
Selected features of the anatomy of developing Helleborus seeds are shown in Fig. 2. Following Schiffner (1891), the principal storage tissue has so far been claimed to be the endosperm, but no supporting documentation came to our attention. In accord with a recent comparative study (Endress and Igersheim, 1999) on the gynoecium of the basal eudicots (including the Ranunculaceae), it is therefore proposed here to attribute the most prominent tissue in Helleborus seeds to the nucellus (or a ‘pseudonucellus’ derived from the epidermal layers of the embryo sac), with only ~20% of its overall volume occupied by the enclosed endosperm. The latter was in its liquid (nuclear) phase during sepal greening and mostly cellular at seed ripening (Fig. 2). The embryo started developing during sepal greening to reach an early cotyledonary stage by the time the seeds were shed. Its development appears to continue during several months of after-ripening in moist soil, at summer temperatures (Hartmann and Kester, 1975; Lockhart and Albrecht, 1987). A further 3 months at +4 °C were then required for germination.


Figure 2
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  		Fig. 2 Longitudinal sections through seeds of Helleborus niger L. at two developmental stages. Left: toluidine blue-stained section prepared when the sepals started to turn green. Presented is the micropylar end of the nucellus (surrounded by integumental tissue which is not completely shown) enclosing the liquid endosperm. The intensely stained, large cell visible at the micropylar end of the endosperm is part of the embryo at a very early developmental stage. The length of the scale bar corresponds to 100 µm. Middle: toluidine blue-stained section through the same region taken at seed ripening. The now cellular endosperm encloses an embryo at its early cotyledonary stage with its radicle up (i.e. pointing toward the micropyle). The length of the scale bar corresponds to 200 µm. Right: ripe unstained seed in about the same orientation with the tiny embryo at the upper tip. The total length of the seed shown was 4 mm.

 
Identification of the cytokinin types present in Helleborus flowers
To gain qualitative insight into the set of cytokinins present in Helleborus fruit and sepals, plant extracts were subjected to a verified fractionation and purification procedure (see Materials and methods) and the isolates obtained were O-propionylated and analysed by LC–MS/MS. The results unequivocally demonstrated the presence of the free bases and ribosides of zeatin (Z), dehydrozeatin (DZ), and N6-({Delta}2-isopentenyl)adenine (iP). The mass spectra of mono-O-propionyl zeatin, mono-O-propionyl dihydrozeatin, tetra-O-propionyl zeatin riboside, and tri-O-propionyl N6-({Delta}2-isopentenyl)adenine riboside are shown in Fig. 3; those of tetra-O-propionyl dihydrozeatin riboside and iP, which are not presented, were also free of contaminantions, and showed analogous fragmentation patterns. Generally, all cytokinins form [M+H]+ quasi-molecular ions. These were at m/z 578 for tetra-O-propionyl dihydrozeatin riboside and at m/z 204 for iP; the values for the other cytokinins identified are shown in Fig. 3. The important fragments of cytokinin bases correspond to fragmentation of the side chain, with ions at m/z 136 (adenine) and 148. O-propionyl-zeatin also shows ions at m/z 220 and 202, corresponding to protonated Z and loss of water from the latter. In addition, loss of ammonia from the fragment at m/z 202 results in an ion at m/z 185. O-propionyl-dihydrozeatin shows a similar fragmentation pattern with ions at m/z 222 and 204. In the mass spectra of cytokinin ribosides, the presence of O-propionylated ribose is indicated by a fragment at m/z 301 (charged tri-O-propionyl ribose). Further elimination of two equivalents of propionic acid yields m/z 153, and additional loss of a methylketene (56) results in a fragment at m/z 97. The mass spectra were recorded at retention times corresponding to the retention times of appropriate synthetic standards.


Figure 3
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  		Fig. 3 Daughter ion mass spectra (electron spray ionization, positive ions) of (A) O-propionyl trans-zeatin, (B) O-propionyl-dihydrozeatin, (C) tetra-O-propionyl-trans-zeatin riboside, and (D) tri-O-propionyl-N6-({Delta}2-isopentenyl)adenosine. The fragmentation pattern is indicated in the structural formulae.

 
In addition to the above major cytokinins, ions which may belong to cis-zeatin riboside appeared in some samples, at the appropriate retention times, but complete daughter ion spectra with acceptable signal-to-noise ratios could not be obtained, possibly due to low levels of the respective cytokinin. The extraction and sample purification procedure used is also suitable for the isolation and identification of aromatic cytokinins. Those were, however, not detected in Christmas rose fruit and sepals.

Quantitative cytokinin relationships—general features
Mass spectrometric analysis, in the presence of deuterium-labelled internal standards, combined with a slightly modified fractionation procedure for the plant extracts, permitted quantification of the cytokinins present in Helleborus flowers. The data for six stages of their life cycle are summarized in Tables 2 (pistils and developing fruit) and 3 (sepals). The analyses were repeated for three series of samples (2–4 replicates per sample) and mostly agreed within the error limits indicated in the tables. Only the results for iP in the sepals varied too much to state definite numerical values (arithmetic means), but were clustered around a concentration of 0.2 pmol g–1 f. wt., throughout the life cycle of the perianth. The method used was previously tested with authentic standards and in plant samples, including extensive validation by enzyme immunoassay using specific anti-cytokinin antibodies (Novák et al., 2003).


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  		Table 2 Cytokinin levels in pistils and developing fruit of Helleborus niger

 
In both the perianth and the developing fruit, the 9-glucosides played a negligible role: only in one sample (fruit 2–3 weeks before ripening) did they contribute slightly more than 1% to the overall cytokinin concentration; elsewhere they were less abundant or undetectable. Also, while LC–MS (single ion monitoring) data suggested the presence of picomolar concentrations of cis-zeatin cytokinins in some samples, no consistent pattern could be established. Therefore the focus will be on the interconvertible forms of Z, DZ, and isopentenyladenine cytokinins: free bases, ribosides, riboside-5'-monophosphates, and O-glucosides.

Cytokinin dynamics in developing fruit
The overall cytokinin levels in the carpels (Table 2) decreased slightly as anthesis proceeded from the female to the male phase, to rise dramatically during fruit ripening. This was mainly due to a transient increase in the concentrations of trans-zeatin riboside (ZR) and dihydrozeatin riboside (DZR), with maxima at about the time when sepal greening was complete. During later fruit development, the corresponding free bases increased on account of their ribosides, leaving overall cytokinin levels almost constant until 2–3 weeks before seed ripening, the most advanced developmental stage investigated. At anthesis, DZ-type cytokinins were slightly more abundant than their zeatin analogues. During fruit development, this proportion was inverted, with Z and derivatives attaining up to seven times higher concentrations.

As shown in Fig. 4, the cytokinins in Helleborus fruit sampled 3–4 weeks before ripening are mostly localized in the seeds. This is probably the case during most of fruit development, as suggested, for instance, by analogy with the morphologically similar legume fruits for which detailed data on cytokinin distribution during the entire developmental cycle are available (Emery et al., 1998, 2000). In the seed types which have been studied so far, such as cereal grains (Morris, 1997) and chick peas (Emery et al., 1998), cytokinin levels rise dramatically shortly after pollination, and drop abruptly as seed differentiation begins, thus correlating with cell division rates in the endosperm (Morris, 1997), in accordance with the stimulatory role of cytokinins in the cell division cycle (Roef and van Onckelen, 2004). In Helleborus, cytokinin levels also increase dramatically during early fruit development (while the sepals are going through the greening process). This coincides with rapid growth, not only of the endosperm, but also of the nucellus, both of which ultimately occupy >90% of the volume of the seed. Cell division rates in these tissues were not recorded, but should decrease to zero before, or when, the seeds reach their maximal size. This occurs 2–3 weeks before ripening (Fig. 1). Why is there no corresponding decrease in cytokinin levels? Is it because the embryo is still developing?


Figure 4
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  		Fig. 4 Pool sizes of zeatin and dihydrozeatin cytokinins in the seeds (S) and pericarps (P) of an individual fruit cluster (cluster of 4–7 follicles present in a single flower) collected 3–4 weeks before maturity. The respective amounts of isopentenyladenine cytokinins were below the detection limit except for iP, 0.10; iPR, 0.09; and iPRMP, 2.37 pmol in the seeds, and iP, 0.10 pmol in the pericarps. The values shown are the arithmetic means of two (seeds) to three (pericarps) independent analyses; sampling errors were in the same range as for the data shown in Table 2. Cytokinin concentrations can be calculated considering that the mean weight (n=43) of a fruit cluster was 1.88 g, of which 1.223 g was in the pericarps and 0.657 g in the seeds. The figure is based on fruit collected in a growth season for which material completely identical to that in Tables 1–3 was not available.

 
In somatic embryos, which are more easily accessible than zygotic embryos, the role of cytokinins appears to depend on the developmental stage, the type and concentration of the cytokinin, and, possibly, the plant species. In Corydalis yanhusuo W. T. Wang (Fumariaceae), a species with relatively close phylogenetic ties to Helleborus, Z or benzyladenine were required for embryo development from the globular to the cotyledonary stage (Sagare et al., 2000), with optimal results at a concentration of 500 ng ml–1 (~2250 pmol ml–1). This is close to the overall concentration of interconvertible cytokinins in Helleborus seeds 3–4 weeks before ripening (1357 pmol g–1; Fig. 4). In carrot (Daucus carota L.) cell suspension cultures, 1000 pmol ml–1 Z increased the rate of embryogenesis, affording about the same range of developmental stages as described above for Corydalis (Nomura and Komamine, 1985). When the embryos were induced on hypocotyl sections, exogenous cytokinins were not required. However, purine riboside inhibited embryogenesis and its effect could be compensated by simultaneous addition of as little as 100 pmol ml–1 ZR (Tokuji and Kuriyama, 2003). This is less than the concentration of this cytokinin which was found in Helleborus seeds 3–4 weeks before ripening (340 pmol g–1; Fig. 4).

It is therefore suggested that the high cytokinin levels during late seed development in Helleborus are related to the fact that the embryo is still developing towards the cotyledonary stage. In the model systems investigated so far (Morris, 1997; Emery et al., 1998, 2000), early embryogenesis and rapid endosperm proliferation are simultaneous processes, a fact which would rationalize the observation that there is only a single short cytokinin maximum during early seed development.

Cytokinin dynamics in the perianth
In the sepals, only the levels of N6-({Delta}2-isopentenyl)adenine riboside-5'-monophosphate (iPRMP) appeared to be linked to perianth greening, increasing by a factor of two to three as chlorophyll started to appear (Table 3). However, the changes were small in absolute terms, and further experiments may be required to confirm a causal relationship. The overall cytokinin concentration in the sepals decreased slightly after anthesis, showing only minor fluctuations during further development, and most individual cytokinins followed this general trend. However, the concentrations of Z and most of its derivatives increased slightly, but consistently, towards the end of the life cycle of the perianth and, taken together, eventually exceeded the pooled concentration of all DZ forms. Earlier in perianth development, the latter were up to three times more abundant than the total zeatin cytokinins. However, the DZ in the sepals was mostly in two bound forms: the riboside and its O-glucoside; the concentration of the free cytokinin barely exceeded the detection limit.


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  		Table 3 Cytokinin levels in the sepals of Helleborus niger during anthesis and fruit ripening

 
To verify the effect of developing seeds on cytokinin levels in the perianth, depistillated and unpollinated flowers were analysed (Fig. 5). The seedless flowers contained no ribotides, and the levels of most other cytokinins were also smaller than in the sepals of fruit-bearing flowers of the same physiological age (i.e. 2–3 weeks before seed ripening). The seedless flowers also weighed less and did not pass through a complete greening process. This indicates that cytokinins play a part in the normal life cycle of the perianth including the greening response following fertilization. So far, the impact of these hormones on the photosynthetic apparatus has mostly been investigated in model systems based on the prevention of chlorophyll loss as one of the symptoms of senescence (Richmond and Lang, 1957; Medford et al., 1989; Gan and Amasino, 1995), an analogy which now appears less straightforward than originally believed (Werner et al., 2001; Ananieva et al., 2004). The reverse process, cytokinin-mediated conversion of etioplasts into chloroplasts (Parthier, 1979), was, for instance, observed in a line of tobacco callus which grew well without external cytokinin but nevertheless required it for the formation of chloroplasts when the normally dark-grown tissue was transferred to the light (Stetler and Laetsch, 1965). Greening processes of this kind are akin to what happens in the Christmas rose perianth after fertilization when the leucoplasts present at anthesis develop into chloroplasts (Salopek-Sondi et al., 2000, 2002). Further research will show if the accompanying, comparatively small, changes in the relative abundance of individual cytokinins (in the perianth) are by themselves sufficient to trigger the formation of a photosynthetic apparatus, and to keep it functional during seed ripening. They may also be just one set of components in a complex signalling network.


Figure 5
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  		Fig. 5 Cytokinin concentrations in normal flowers bearing seeded fruit (N), in depistillated flowers (D), and in unfertilized flowers with small seedless fruit (U). The individual cytokinin forms are presented in the same way as in Fig. 4. The data shown are the arithmetic means of two (D and U) to eight (N) measurements; sampling errors were in the same range as for the data shown in Table 3. All flowers analysed were collected at about the same time after anthesis which corresponded to 2–3 weeks before seed ripening in the normally fertilized flowers. The average weight per perianth was 1.41 g (n=33) for fruit-bearing flowers, 0.970 g (n=5) for depistillated flowers, and 0.846 g (n=5) for intact, unfertilized flowers.

 
Concluding remarks
Seeds have long been known to be a rich source of cytokinins, but their origin has been a controversial issue. They were long thought to be synthesized in the root tips and transported to their sites of action via the xylem and the phloem (Letham, 1994), but recent data now demonstrate that this is an oversimplification (Faiss et al., 1997; Emery et al., 2000; Nordström et al., 2004). In particular, Lee et al. (1989) demonstrated that wheat (Triticum aestivum L.) ears cultured in vitro, in a hormone-free liquid medium, maintain a normal cytokinin pattern. Also, Miyawaki et al. (2004) found some of the genes encoding isopentenyltransferases involved in cytokinin biosynthesis expressed in immature seeds. It is thus plausible to assume that most of the cytokinins detected in Helleborus seeds are synthesized in situ. If so, two observations suggest that this preferentially occurs via iPRMP, i.e. (i) the latter is always the most abundant isopentenyladenine-cytokinin; and (ii) the dramatic increase of zeatin and DZ cytokinins during perianth greening is preceded by an (albeit less prominent) increase in the concentration of iPRMP. Also, as fruit approach maturity, iPRMP starts to decrease while zeatin and DZ cytokinins are still maintaining top levels.

The significance of the changing patterns of cytokinin forms during perianth and fruit development requires further research. It has been widely assumed that only the free bases have genuine hormonal activity, the ribosides play a role in long-range transport, the O-glucosides are storage forms, and the 7- and 9-glucosides are catabolites (Mok and Mok, 2001). This picture is changing, however, as all plant species studied so far contain several cytokinin receptors with complex expression patterns (Maxwell and Kieber, 2004) and, apparently, widely different substrate specificities. In Arabidopsis, for instance, one of the receptors (CRE1/AHK4) is specific for trans-zeatin, but another one (AHK3) also recognizes cis-zeatin and DZ as well as cytokinin ribosides and ribotides (Spichal et al., 2004). Similar differences in the substrate specificities of individual cytokinin receptors were also reported for maize (Yonekura-Sakakibara et al., 2004).

Does the obvious correlation of fruit growth and perianth greening imply that cytokinins synthesized in the seeds are exported to the sepals? A complete answer is not yet possible, but clearly defined cytokinin levels in the sepals are required to induce the formation of a photosynthetic system and to keep it functional during seed filling, because (i) the perianth of seedless flowers, whether depistillated at the bud stage or just ignored by pollinators at flowering time, is cytokinin deficient and such flowers do not pass through a complete greening process; and (ii) exogenous cytokinins stimulate chlorophyll accumulation in such flowers (Salopek-Sondi et al., 2002). During advanced fruit development, cytokinin pool sizes are indeed much larger in the fruit than in the perianth (Tables 2, 3). However, when the greening process is initiated (‘initial perianth greening’), the perianth of an individual flower contains a larger cytokinin pool than the fruit cluster of that same flower. Under these circumstances, the biosynthetic machinery in the fruit would have to work preferentially for the perianth. That this is not impossible is demonstrated by an example from the developmental physiology of the pea (Pisum sativum L.) fruit. Indirect evidence shows convincingly that the seeds supply auxin to the pericarp (Ozga and Reinecke, 2003), even at a stage when the latter is about finger-long and contains a significantly larger auxin pool than the pinhead-sized seeds (Magnus et al., 1997).


   Acknowledgements
 
The work presented was supported by grant no. MSM 6198959216 from the Ministry of Education, Youth, and Sports of the Czech Republic and by grants no. 0098080 and 0119111 from the Ministry of Science, Education, and Sports of the Republic of Croatia. We are indebted to E Yeung, University of Calgary, who helped us to understand the anatomy of developing Christmas rose seeds.


   Footnotes
 
* Present address: Department of Biochemistry, Faculty of Science, Palacky University, Slechtitelu 11, CZ-783 71 Olomouc, Czech Republic. Back


   Abbreviations
 
DZ, dihydrozeatin; DZ9G, dihydrozeatin 9-glucoside; DZOG, dihydrozeatin O-glucoside; DZR, dihydrozeatin riboside; DZRMP, dihydrozeatin riboside-5'-monophosphate; DZROG, dihydrozeatin riboside O-glucoside; f. wt., fresh weight; HPLC, high-performance liquid chromatography; iP, N6-({Delta}2-isopentenyl)adenine; iP9G, N6-({Delta}2-isopentenyl)adenine 9-glucoside; iPR, N6-({Delta}2-isopentenyl)adenine riboside alias N6-({Delta}2-isopentenyl)adenosine; iPRMP, N6-({Delta}2-isopentenyl)adenine riboside-5'-monophosphate alias N6-({Delta}2-isopentenyl)adenosine-5'-monophosphate; LC–MS, liquid chromatography–mass spectrometry; LC-MS/MS, liquid chromatography-tandem mass spectrometry; Z, trans-zeatin; Z9G, trans-zeatin 9-glucoside; ZOG, trans-zeatin O-glucoside; ZR, trans-zeatin riboside; ZRMP, trans-zeatin riboside-5'-monophosphate; ZROG, trans-zeatin riboside O-glucoside..


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
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