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

RESEARCH PAPER

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

Petr Tarkowski1,*, Danuse Tarkowska2, Ondrej Novak2, Snjezana Mihaljevic3, Volker Magnus3,

Miroslav Strnad2 and Branka Salopek-Sondi3,†

1 Umea Plant Science Centre, Department of Forest Genetics and Plant Physiology, Umea, Sweden2 Laboratory of Growth Regulators, Palacky University and IEB ASCR, Olomouc, Czech Republic3 Rudjer Boskovic Institute, Zagreb, Croatia

Received 12 September 2005; Accepted 9 March 2006

Abstract

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 contin-

ues 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-(D2-isopentenyl)adenine, and their ribosides were

identified by tandem mass spectrometry. Single ion

monitoring in the presence of deuterated internal stand-

ards demonstrated the additional presence of the corres-

ponding riboside-59-monophosphates, O-glucosides,

and 9-glucosides, and afforded quantitative data on

the whole set of endogenous cytokinins. Fruit cytoki-

nins 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 changemarkedly in the period covered,

but the level of N6-(D2-isopentenyl)adenine-type cyto-

kinins appeared to increase slightly and transiently

during the greening phase. The perianths of unpolli-

nated or depistillated flowers, which survived, but did

not pass through the complete greening process, con-

tained significantly less cytokinins than observed in

fruit-bearing flowers. This suggests that perianth green-

ing 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

It is believed that the photosynthetic system adapts, bothanatomically and functionally, to the rate of assimilate con-sumption by metabolic ‘sinks’, such as fruits and storagetubers (Brenner, 1987), but the complexity of most whole-plant systems imposes practical limits on detailed mecha-nistic studies. A simple model system can be found in theflowers of the Christmas rose (Helleborus niger L.,Ranunculaceae), a herbaceous, winter-green, perennialnative to south-eastern Europe, which is also widely grownas an ornamental. In mild winters, the flowers may indeedappear at Christmas time, resembling wild roses withrespect to size and colour (white to pink). After pollination,the showy elements of the Christmas rose perianth (usually

* Present address: Department of Biochemistry, Faculty of Science, Palacky University, Slechtitelu 11, CZ-783 71 Olomouc, Czech Republic.y To whom correspondence should be addressed. E-mail: [email protected]: DZ, dihydrozeatin; DZ9G, dihydrozeatin 9-glucoside; DZOG, dihydrozeatin O-glucoside; DZR, dihydrozeatin riboside; DZRMP, dihydrozeatinriboside-59-monophosphate; DZROG, dihydrozeatin riboside O-glucoside; f. wt., fresh weight; HPLC, high-performance liquid chromatography; iP, N6-(D2-isopentenyl)adenine; iP9G, N6-(D2-isopentenyl)adenine 9-glucoside; iPR, N6-(D2-isopentenyl)adenine riboside alias N6-(D2-isopentenyl)adenosine; iPRMP,N6-(D2-isopentenyl)adenine riboside-59-monophosphate alias N6-(D2-isopentenyl)adenosine-59-monophosphate; LC–MS, liquid chromatography–massspectrometry; 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-59-monophosphate; ZROG, trans-zeatin riboside O-glucoside.

Journal of Experimental Botany, of 11

doi:10.1093/jxb/erj190

ª The Author [2006]. Published by Oxford University Press [on behalf of the Society for Experimental Biology]. All rights reserved.For Permissions, please e-mail: [email protected]

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characterized as sepals) develop a functional photosynth-etic system and persist until fruit ripening (Salopek-Sondiet al., 2000; Aschan and Pfanz, 2003). Comparable pro-cesses 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; Gronegress, 1974; Chaves dasNeves and Pais, 1980a, b) but, in these species, leaves arepresent at the same time to carry out the bulk of photo-synthesis. In the Christmas rose, the overwintering leavesare often pressed to the ground by snow and covered withdebris. They also senesce during fruit ripening, and theappearance of the new generation of leaves can be delayedby drought and low temperatures. Apart from the stores inthe roots, the green perianth thus represents the mostreliable, if not the only, source of assimilates for thedeveloping seeds. Notably, flowers ignored by pollinatorssurvive about as long as their fertilized neighbours, but onlyassume a faint greenish tinge (unpublished observations).The greening process is also arrested when the developingfruit are removed (Salopek-Sondi et al., 2000). It is resumedwhen the sepals are treated with cytokinins (Salopek-Sondiet al., 2002). Endogenous cytokinins could thus play arole in the greening process, and indeed preliminary anal-yses by high-performance liquid chromatography (HPLC)suggested dramatic quantitative changes during fruitdevelopment (Salopek-Sondi et al., 2002). This promptedthe analysis of the endogenous cytokinins in Christmasrose fruit and sepals using a liquid chromatography–massspectrometry (LC–MS) approach which provides more re-liable identifications and more complete quantitativedata. In fruits, cytokinins tend to be concentrated in theseeds, changing in kind and quantity as endospermand embryo development proceed (Morris, 1997;Emery et al., 1998, 2000). In the species studied so far,

these processes proceed to completion before seed rip-ening. In contrast, in Helleborus, embryo maturation isstill in progress when the seeds are released by the motherplant, and this also appears to be the case in manyperennial ornamentals. Cytokinin housekeeping during thedevelopment of this type of seeds is addressed here forthe first time. This also appears to be the first detailed reporton cytokinins in the perianth during its entire life cycle.

Materials and methods

Plant material

Flowers of the Christmas rose (H. niger L. ssp. niger sensu Damboldtand Zimmermann, 1965) were collected at their natural habitat in themountain forests of Gorski kotar, Croatia (altitude, 800 m; dominanttree species, Abies alba L., Fagus silvatica L., and Picea abies L.).The six stages of the normal developmental cycle sampled forcytokinin analysis are shown in Table 1. Two stages of anthesis wererecognized. The (‘proterogynous’) flowers first passed through theirfemale phase during which the stigmata were receptive and theimmature anthers were arranged in a ring at the base of the clusterof carpels. The male phase began with the elongation of the fila-ments, and ended with anther abortion. Fruit development and theaccompanying metamorphosis of the perianth were monitored atfour subsequent stages: ‘initial perianth greening’ (sepals with firstgreenish tinge, barely noticeable fruit growth); ‘advanced perianthgreening’ (light green sepals, fruit weight twice that in late anthesis);‘perianth greening complete’ (chlorophyll content in the sepalsentering the stationary phase, fruit weight three times that in lateanthesis); and ‘2–3 weeks before seed ripening’ (sepals green, fruitfully grown, weight ;12 times that in late anthesis).

Non-pollinated flowers (same carpel size as in late anthesis) andflowers depistillated at bud opening were collected when the seeds offertilized flowers at the same location were 2–3 weeks beforeripening.

For cytokinin analysis, the perianths and the clusters of carpels ordeveloping fruit were separated, immediately frozen, and kept in

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

Description Perianth Fruit cluster

Coloura Fresh weight (g)c Length (mm)b Fresh weight (g)c

Anthesis, female phased White 1.12 Ovaries: 9 0.18Styles: 6

Anthesis, male phasee White 1.31 Ovaries: 10 0.26Styles: 9

Initial perianth greening Greenish-white 1.00 Bodies: 10 0.26Beaks: 10

Advanced perianth greening Light green 1.17 Bodies: 15 0.53Beaks: 10

Perianth greening complete Green 1.25 Bodies: 16 0.79Beaks: 11

2–3 weeks before seed ripening Green 1.41 Bodies: 26 3.10Beaks: 13

a Exposure to intense light induces red pigmentation causing ‘white’ flowers to apear rose and ‘green’ sepals to be tinged with purple in a rangeof shades.

b Arithmetic means (SE 65–10%) for random samples of 10 fruit clusters. The bodies of the follicles (derived from the ovaries) and the beaks (derivedfrom the styles) were measured separately.

c Arithmetic means (SE 610–15%) for the samples (n=30–65) from which aliquots for cytokinin analysis were taken.d First days after bud opening: stigmata receptive, stamina immature, anthers short, nectaries mostly erect.e Stigmata dry, anthers elongated, pollen mature, nectaries spreading.

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sealed plastic bags, at �80 8C, until work-up. Stamens and nectarieswere discarded.

Cytokinin identification

Cytokinins were extracted and separated, essentially as outlined byAstot et al. (1998). Frozen plant material (;10 g fresh weight) wasground with a mortar and pestle in liquid nitrogen and extractedovernight in methanol–chloroform–formic acid–water (12:5:1:2, byvol.) (Bieleski, 1964). Passing the extract, in sequence, througha cation (SCX-cartridge) and an anion [DEAE-Sephadex combinedwith an SPE(C18)-cartridge] exchanger afforded fraction 1 contain-ing the cytokinin free bases, ribosides, and glucosides, and fraction2 containing the riboside-59-monophosphates. Both fractions werepurified further by immunoaffinity chromatography based on genericmonoclonal anticytokinin antibodies, but fraction 2 was first treatedwith alkaline phosphatase. In fraction 1, the O-glucosides did notbind to the immunoaffinity column. The effluent was thus treatedwith b-glucosidase and the hydrolysate was rechromatographed onthe immunoaffinity column to yield the O-glucoside fraction (whichactually 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.) (Astotet al., 1998), evaporated in vacuo and stored at �20 8C until furtheranalysis.

For analysis, the samples were redissolved in 1% aqueous formicacid and subjected to LC–MS/MS. The chromatographic separationwas performed on a capillary column (150 mm30.3 mm; LCPackings, Amsterdam, The Netherlands) packed with SymmetryC18 packing material, particle size 3.5 lm (Waters). The eluent,applied at a flow rate of 4 ll 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 effluentwas introduced into a Micromass Quattro Ultima triple-stage quad-rupole mass spectrometer via an electrospray ion source (capillaryvoltage, +2.9 kV; cone voltage, +70 V; source temperature, 90 8C;desolvation temperature, 120 8C; cone gas flow, 120 l h�1; desolvationgas 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 asfollows (Novak et al., 2003). Aliquots of plant material (1 g) wereprocessed by adding the following internal standards at the extractionstage: trans-[2H5]zeatin, trans-[2H5]zeatin riboside, trans-[2H5]zeatin9-glucoside, [2H3]dihydrozeatin, [2H3]dihydrozeatin riboside,[2H3]dihydrozeatin 9-glucoside, [2H6]isopentenyladenine, [2H6]iso-pentenyladenine riboside, [2H6]isopentenyladenine 9-glucoside,trans-[2H5]zeatin O-glucoside, trans-[2H5]zeatin riboside O-glucoside, trans-[2H5]zeatin riboside-59-monophosphate, [2H3]dihy-drozeatin riboside-59-monophosphate, and [2H6]isopentenyladenineriboside-59-monophosphate (OlChemIm Ltd, Olomouc, CzechRepublic). After fractionation and purification as for cytokinin iden-tification (but no propionylation), the samples were subjected toHPLC on a reversed-phase column (150 mm32.1 mm; particle size,5 lm) (Symmetry C18, Waters) operated at 30 8C. The componentsof 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 ll min�1. Using post-column splitting (1:1), the eluent was simultaneously introduced intoa diode array detector (Waters PDA 996) and the electrospray source(source temperature, 100 8C; capillary voltage, +3.0 kV; conevoltage, +20 V; desolvation temperature, 250 8C) of a single-stagequadrupole mass spectrometer (ZMD 2000, Micromass, Manchester,

UK). Nitrogen was used as both the desolvation gas (400 l h�1) andthe cone gas (50 l h�1). Quantification was done by single ion moni-toring of the quasi-molecular ([M+H]+) ions of the plant cytokininsand the corresponding deuterated internal standards.

Results and discussion

Perianth and fruit development

The metamorphosis of the sepals in pollinated Helleborusflowers has already been documented in detail (Salopek-Sondi et al., 2000, 2002). In brief, during anthesis, onlythe guard cells contain chloroplasts, resulting in bulkchlorophyll levels below 1 lg g�1 fresh weight (f. wt.).After fertilization, chloroplasts form in the entire peri-anth, eventually affording chlorophyll concentrations of;350 lg g�1 f. wt. At the same time, the interior of thesepals assumes a sponge-like structure, due to the expan-sion of intercellular spaces. Cell divisions were not noticed;cell expansion was limited, and so was overall sepalgrowth. The small weight gain during the assimilatoryphase shown in Table 1 was supported by further obser-vations through several growth seasons (Salopek-Sondiet al., 2000, 2002, and unpublished data). The weightfluctuations observed in the present samples during anthe-sis and ‘initial sepal greening’ (Table 1) are not typicaland appear to reflect intraspecific variability in flower size.

Simultaneously with perianth greening, the fruit clustersstarted to grow. Characteristic developmental stages of thefollicles (Fig. 1A) and the seeds (Fig. 1B) are illustrated inFig. 1. Interestingly, fruit growth was not restricted to theovaries, but included the styles which developed intoelongated beaks (Table 1). Most fruit weight was gainedwhile the photosynthetic capacity of the perianth was fullyestablished. The ratio between pericarp and seed weightdepended on seed set (more or less successful pollination).In several samples analysed 3–4 weeks before ripening, upto two-thirds of the overall fruit weight was in the pericarp.

Selected features of the anatomy of developing Helle-borus seeds are shown in Fig. 2. Following Schiffner(1891), the principal storage tissue has so far been claimedto be the endosperm, but no supporting documentation cameto our attention. In accord with a recent comparative study(Endress and Igersheim, 1999) on the gynoecium of thebasal eudicots (including the Ranunculaceae), it is thereforeproposed here to attribute the most prominent tissue inHelleborus seeds to the nucellus (or a ‘pseudonucellus’derived from the epidermal layers of the embryo sac), withonly ;20% of its overall volume occupied by the enclosedendosperm. The latter was in its liquid (nuclear) phaseduring sepal greening and mostly cellular at seed ripen-ing (Fig. 2). The embryo started developing during sepalgreening to reach an early cotyledonary stage by the timethe seeds were shed. Its development appears to continueduring several months of after-ripening in moist soil, atsummer temperatures (Hartmann and Kester, 1975; Lockhart

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and Albrecht, 1987). A further 3 months at +4 8C werethen required for germination.

Identification of the cytokinin types present inHelleborus flowers

To gain qualitative insight into the set of cytokinins presentin Helleborus fruit and sepals, plant extracts were subjectedto a verified fractionation and purification procedure (seeMaterials and methods) and the isolates obtained were O-propionylated and analysed by LC–MS/MS. The resultsunequivocally demonstrated the presence of the free basesand ribosides of zeatin (Z), dehydrozeatin (DZ), andN6-(D2-isopentenyl)adenine (iP). The mass spectra ofmono-O-propionyl zeatin, mono-O-propionyl dihydrozea-tin, tetra-O-propionyl zeatin riboside, and tri-O-propionylN6-(D2-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. Thesewere at m/z 578 for tetra-O-propionyl dihydrozeatin ribo-side and at m/z 204 for iP; the values for the othercytokinins identified are shown in Fig. 3. The importantfragments of cytokinin bases correspond to fragmentationof 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 thelatter. In addition, loss of ammonia from the fragment at m/z202 results in an ion at m/z 185. O-propionyl-dihydrozeatinshows a similar fragmentation pattern with ions at m/z222 and 204. In the mass spectra of cytokinin ribo-sides, the presence of O-propionylated ribose is indi-cated by a fragment at m/z 301 (charged tri-O-propionylribose). Further elimination of two equivalents of pro-pionic acid yields m/z 153, and additional loss of a methyl-ketene (56) results in a fragment at m/z 97. The massspectra were recorded at retention times corresponding tothe retention times of appropriate synthetic standards.

In addition to the above major cytokinins, ions whichmay belong to cis-zeatin riboside appeared in somesamples, at the appropriate retention times, but completedaughter ion spectra with acceptable signal-to-noise ratioscould not be obtained, possibly due to low levels of therespective cytokinin. The extraction and sample purifica-tion procedure used is also suitable for the isolation andidentification of aromatic cytokinins. Those were, how-ever, 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 modifiedfractionation procedure for the plant extracts, permittedquantification of the cytokinins present in Helleborusflowers. The data for six stages of their life cycle aresummarized in Tables 2 (pistils and developing fruit) and3 (sepals). The analyses were repeated for three series ofsamples (2–4 replicates per sample) and mostly agreedwithin the error limits indicated in the tables. Only theresults for iP in the sepals varied too much to state definitenumerical values (arithmetic means), but were clusteredaround a concentration of 0.2 pmol g�1 f. wt., throughoutthe life cycle of the perianth. The method used was previ-ously tested with authentic standards and in plant samples,including extensive validation by enzyme immunoassayusing specific anti-cytokinin antibodies (Novak et al., 2003).

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 contributeslightly more than 1% to the overall cytokinin concentra-tion; elsewhere they were less abundant or undetectable.

Fig. 1. Fruit and seed growth in Helleborus niger L. (glutaraldehyde-fixed material). (A) Follicles split lengthwise at the followingdevelopmental stages (from left to right): white flower, male phase(i.e. shortly after fertilization), initial perianth greening, perianthgreening complete, and 2–3 weeks before ripening. The upper halfof the follicle wall was removed. Some seeds had aborted (probably dueto incomplete pollination), as seen most clearly in the fully grown fol-licles. (B) Seeds sampled at the same developmental stages plusa ripe seed after rehydration. Helleborus seeds bear an elaiosome(arill) which was removed from the ripe seed to prevent fungus in-festation during the rehydration process. The dimensions of the fields ofthe grid used as a background in (A) and (B) are 1 mm31 mm.


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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 thesepals started to turn green. Presented is the micropylar end of the nucellus (surrounded by integumental tissue which is not completely shown) enclosingthe liquid endosperm. The intensely stained, large cell visible at the micropylar end of the endosperm is part of the embryo at a very early developmentalstage. The length of the scale bar corresponds to 100 lm. Middle: toluidine blue-stained section through the same region taken at seed ripening. Thenow cellular endosperm encloses an embryo at its early cotyledonary stage with its radicle up (i.e. pointing toward the micropyle). The length ofthe scale bar corresponds to 200 lm. Right: ripe unstained seed in about the same orientation with the tiny embryo at the upper tip. The total length of theseed shown was 4 mm.

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-(D2-isopentenyl)adenosine. The fragmentation pattern is indicated in the structuralformulae.


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

Cytokinin Quantity

Anthesis: femalephase

Anthesis: malephase

Initial perianthgreening

Advanced perianthgreening

Perianth greeningcomplete

2–3 weeks beforeseed ripening

pmolg�1 f. wt.

pmolf. c.�1a

pmolg�1 f. wt.

pmolf. c.�1a

pmolg�1 f. wt.

pmolf. c.�1a

pmolg�1 f. wt.

pmolf. c.�1a

pmolg�1 f. wt.

pmolf. c.�1a

pmolg�1 f. wt.

pmolf. c.�1a

Z 0.4660.05 0.08 0.4460.08 0.11 0.4360.16 0.11 9.9760.43 5.30 35.8365.42 28.43 63.6266.58 197.01ZOG 0.0060.00 0.00 0.2060.03 0.05 0.0860.00 0.02 1.4260.29 0.76 7.1861.28 5.70 37.1762.50 115.11ZR 1.2160.23 0.21 1.0960.26 0.28 0.67960.15 0.21 30.7962.08 16.35 127.06617.14 100.82 72.3569.44 224.07ZROG 0.3060.05 0.05 0.3860.06 0.10 0.5960.00 0.15 2.3860.66 1.26 10.8161.53 8.57 40.7362.91 126.13ZRMP 3.0660.45 0.54 2.1660.54 0.55 1.8560.39 0.48 47.2861.44 25.10 51.7062.49 41.02 15.6060.26 48.30Z9G 0.0060.00 0.00 0.0060.00 0.00 0.0060.00 0.00 0.2160.01 0.11 0.7660.22 0.60 3.3060.92 10.21All trans-zeatin forms 5.0360.51 0.88 4.2760.61 1.09 3.7560.45 0.97 92.0562.67 48.89 233.33618.26 185.15 232.76612.17 720.83DZ 0.1360.01 0.02 0.0560.01 0.01 0.0660.00 0.01 0.3460.03 0.18 1.1060.30 0.87 2.0260.16 6.26DZOG 0.0060.00 0.00 0.0060.00 0.00 0.5060.09 0.13 0.7360.16 0.39 1.0860.27 0.86 1.9760.28 6.09DZR 4.8960.32 0.86 3.2260.40 0.82 1.5960.21 0.41 6.2160.39 3.30 16.2962.67 12.93 13.2160.38 40.92DZROG 1.6360.14 0.29 1.3760.12 0.35 0.8960.17 0.23 1.7560.25 0.93 3.8460.34 3.05 11.3761.12 35.22DZRMP 5.2160.46 0.92 3.4260.33 0.87 1.3160.13 0.34 12.5960.35 6.69 11.4561.15 9.09 6.7060.44 20.76DZ9G 0.0060.00 0.00 0.0060.00 0.00 0.0060.00 0.00 0.0060.00 0.00 0.0360.01 0.03 0.2960.04 0.92All dihydrozeatin forms 11.8660.57 2.09 8.0660.53 2.06 4.3460.31 1.12 21.6160.6 11.47 33.8062.96 26.82 35.5761.30 110.17iP 0.5060.05 0.09 0.2760.01 0.07 5.2061.24 1.34 3.2560.66 1.73 2.6560.35 2.10 1.9060.19 5.89iPR 1.4660.23 0.26 1.3560.52 0.34 0.7560.11 0.19 1.1860.20 0.63 0.7760.04 0.61 0.3560.03 1.08iPRMP 9.0660.90 1.59 3.4460.43 0.88 5.7060.68 1.47 32.7966.26 17.42 24.4062.58 19.36 4.1960.49 12.98iP9G 0.0060.00 0.00 0.0060.00 0.00 0.0260.00 0.00 0.0360.00 0.01 0.0660.02 0.05 0.0460.01 0.11All isopentenyladenineforms

11.0160.93 1.94 5.0660.68 1.29 11.6761.42 3.01 37.2666.29 19.79 27.8862.60 22.12 6.4860.52 20.06

Free cytokinins 1.0960.07 0.19 0.7660.08 0.19 5.6961.25 1.47 13.5660.81 7.20 39.5865.44 31.40 67.5466.59 209.1625Ribosides+ribotides 24.8861.19 4.37 14.6861.04 3.75 11.9960.84 3.10 130.8466.77 69.49 231.67617.75 183.83 112.4169.47 348.11O-Glucosides 1.9360.15 0.34 1.9660.13 0.50 2.0660.20 0.53 6.2760.78 3.33 22.9162.05 18.18 91.2464.01 282.559-Glucosides 0.0060.00 0.00 0.0060.00 0.00 0.0260.00 0.004 0.2460.01 0.13 0.8560.22 0.68 3.6360.92 11.24Total cytokinins 27.9161.21 4.91 17.3961.05 4.44 19.7661.52 5.10 150.9266.86 80.15 295.01618.68 234.07 274.81612.25 851.06

a f. c., fruit cluster; the cluster of carpels or developing fruit of a single flower.

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

Cytokinin Quantity

Anthesis: femalephase

Anthesis: malephase

Initial perianthgreening

Advanced perianthgreening

Perianth greeningcomplete

2–3 weeks beforeseed ripening

pmolg�1 f. wt.

pmolperianth�1

pmolg�1 f. wt.

pmolperianth�1

pmolg�1 f. wt.

pmolperianth�1

pmolg�1 f. wt.

pmolperianth�1

pmolg�1 f. wt.

pmolperianth�1

pmolg�1 f. wt.

pmolperianth�1

Z 0.2460.02 0.27 0.2960.02 0.38 0.2260.03 0.22 0.1960.01 0.22 0.6360.17 0.79 1.4960.11 2.09ZOG 0.1160.01 0.13 0.1760.02 0.23 0.1960.03 0.19 0.1660.01 0.19 0.2460.02 0.30 0.5260.04 0.73ZR 1.4460.21 1.62 1.1160.08 1.45 0.5560.05 0.55 0.3660.04 0.20 0.5760.08 0.72 1.2560.24 1.76ZROG 0.2860.05 0.32 0.4160.05 0.53 0.2060.04 0.20 0.2060.03 0.24 0.2560.01 0.31 0.6460.12 0.91ZRMP 1.8060.27 2.03 2.5960.35 3.39 1.5560.19 1.54 0.8960.13 1.05 1.3360.06 1.66 0.7460.16 1.04Z9G 0.0060.00 0.00 0.0060.00 0.00 0.0060.00 0.00 0.0060.00 0.00 0.0060.00 0.00 0.0060.00 0.00All trans-zeatin forms 3.8860.35 4.36 4.5860.37 5.98 2.7160.20 2.69 1.8060.14 2.11 3.0260.20 3.78 4.6460.34 6.53DZ 0.1060.02 0.11 0.0560.01 0.07 0.0660.01 0.06 0.0660.01 0.07 0.0460.01 0.05 0.0560.01 0.07DZOG 0.4660.06 0.51 0.4860.06 0.62 0.5560.10 0.55 0.5760.08 0.67 0.6260.14 0.78 0.7860.15 1.10DZR 4.7760.24 5.35 2.6760.17 3.48 2.0060.22 1.99 1.3360.07 1.56 1.3060.08 1.62 0.6960.09 0.97DZROG 1.0560.10 1.18 1.0360.07 1.34 1.2360.10 1.22 1.1860.07 1.39 1.6660.12 2.08 1.3960.16 1.95DZRMP 2.0660.40 2.32 3.3860.21 4.41 1.9460.34 1.93 2.2260.11 2.61 1.1860.19 1.47 0.3260.05 0.45D9G 0.0060.00 0.00 0.0060.00 0.00 0.0060.00 0.00 0.0060.00 0.00 0.0060.00 0.00 0.0060.00 0.00All dihydrozeatin forms 8.4360.48 9.48 7.6060.29 9.93 5.7860.43 5.75 5.3760.17 6.30 4.8060.28 6.00 3.2260.24 4.53iP 0.1660.02 0.18 0.1860.01 0.23 ***a ***a ***a ***a ***a ***a 0.1860.02 0.26iPR 0.9460.07 1.05 0.7260.03 0.94 0.5560.07 0.55 0.4360.04 0.51 0.4760.03 0.59 0.2860.04 0.39iPRMP 4.3160.35 4.85 3.2560.33 4.24 7.0761.50 7.07 9.1861.47 10.77 5.1160.84 6.38 4.1760.95 5.86iP9 0.0160.00 0.01 0.0260.00 0.02 0.0160.00 0.01 0.0260.00 0.02 0.0260.00 0.02 0.0660.00 0.08All isopentenyladenine forms 5.4260.36 6.09 4.1660.33 5.44 7.6361.50 7.63 9.6361.47 11.30 5.6060.84 6.99 4.6960.95 6.59Free cytokinins 0.5060.03 0.56 0.5260.02 0.68 0.2860.03 0.28 0.2560.01 0.29 0.6760.17 0.84 1.7260.11 2.42Ribosides + ribotides 15.3260.68 17.22 13.7260.56 17.91 10.4460.49 12.63 14.4161.48 16.70 9.9660.87 12.44 7.4561.00 10.47O-Glucosides 1.9060.13 2.14 2.0960.11 2.72 2.1760.15 3.37 2.1160.11 2.49 2.7760.19 3.47 3.3360.25 4.699-Glucosides 0.0160.00 0.01 0.0260.00 0.02 0.0160.00 0.07 0.0260.00 0.02 0.0260.00 0.02 0.0660.00 0.08Total cytokinins 17.7360.69 19.93 18.4060.57 21.33 12.9060.51 16.35 16.7961.48 19.50 13.4260.91 16.77 12.5661.04 17.66

a The results of the individual analyses were too divergent to justify calculation of arithmetic means, but suggested isopentenyladenine levels in the same range as in earlier and laterdevelopmental stages. The value 0.0060.00 was used when computing cumulative parameters (i.e. all iP forms, free and total cytokinins).

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Also, while LC–MS (single ion monitoring) data suggestedthe presence of picomolar concentrations of cis-zeatincytokinins in some samples, no consistent pattern could beestablished. Therefore the focus will be on the intercon-vertible forms of Z, DZ, and isopentenyladenine cyto-kinins: free bases, ribosides, riboside-59-monophosphates,and O-glucosides.

Cytokinin dynamics in developing fruit

The overall cytokinin levels in the carpels (Table 2) de-creased slightly as anthesis proceeded from the female to themale phase, to rise dramatically during fruit ripening. Thiswas mainly due to a transient increase in the concentrationsof trans-zeatin riboside (ZR) and dihydrozeatin riboside(DZR), with maxima at about the time when sepal green-ing was complete. During later fruit development, the corres-ponding free bases increased on account of their ribosides,leaving overall cytokinin levels almost constant until 2–3weeks before seed ripening, the most advanced developmen-tal stage investigated. At anthesis, DZ-type cytokinins wereslightly more abundant than their zeatin analogues. Duringfruit development, this proportion was inverted, with Z andderivatives attaining up to seven times higher concentrations.

As shown in Fig. 4, the cytokinins in Helleborus fruitsampled 3–4 weeks before ripening are mostly localizedin the seeds. This is probably the case during most offruit development, as suggested, for instance, by analogywith the morphologically similar legume fruits forwhich detailed data on cytokinin distribution during theentire developmental cycle are available (Emery et al.,

1998, 2000). In the seed types which have been studied sofar, such as cereal grains (Morris, 1997) and chick peas(Emeryetal.,1998),cytokininlevelsrisedramaticallyshortlyafter pollination, and drop abruptly as seed differentiationbegins, thus correlating with cell division rates in theendosperm (Morris, 1997), in accordance with the stimu-latory role of cytokinins in the cell division cycle (Roefand van Onckelen, 2004). In Helleborus, cytokinin levelsalso increase dramatically during early fruit development(while the sepals are going through the greening pro-cess). This coincides with rapid growth, not onlyof the endo-sperm, but also of the nucellus, both of which ultimatelyoccupy >90% of the volume of the seed. Cell division ratesinthese tissues were not recorded, but should decrease tozero before, or when, the seeds reach their maximalsize. This occurs 2–3 weeks before ripening (Fig. 1).Why is there no corresponding decrease incytokinin levels?Is it because the embryo is still developing?

In somatic embryos, which are more easily accessiblethan zygotic embryos, the role of cytokinins appears todepend on the developmental stage, the type and concen-tration of the cytokinin, and, possibly, the plant species. InCorydalis yanhusuo W. T. Wang (Fumariaceae), a specieswith relatively close phylogenetic ties to Helleborus, Z orbenzyladenine were required for embryo development fromthe 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 concentra-tion of interconvertible cytokinins in Helleborus seeds 3–4weeks before ripening (1357 pmol g�1; Fig. 4). In carrot(Daucus carota L.) cell suspension cultures, 1000 pmolml�1 Z increased the rate of embryogenesis, affording aboutthe same range of developmental stages as described abovefor Corydalis (Nomura and Komamine, 1985). When theembryos were induced on hypocotyl sections, exogenouscytokinins were not required. However, purine ribosideinhibited embryogenesis and its effect could be compen-sated by simultaneous addition of as little as 100 pmol ml�1

ZR (Tokuji and Kuriyama, 2003). This is less than the con-centration of this cytokinin which was found in Helleborusseeds 3–4 weeks before ripening (340 pmol g�1; Fig. 4).

It is therefore suggested that the high cytokinin levelsduring late seed development in Helleborus are related tothe fact that the embryo is still developing towards thecotyledonary stage. In the model systems investigatedso far (Morris, 1997; Emery et al., 1998, 2000), earlyembryogenesis and rapid endosperm proliferation aresimultaneous processes, a fact which would rationalizethe observation that there is only a single short cytokininmaximum during early seed development.

Cytokinin dynamics in the perianth

In the sepals, only the levels of N6-(D2-isopentenyl)adenineriboside-59-monophosphate (iPRMP) appeared to be linked

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 folliclespresent in a single flower) collected 3–4 weeks before maturity. Therespective amounts of isopentenyladenine cytokinins were below thedetection limit except for iP, 0.10; iPR, 0.09; and iPRMP, 2.37 pmol inthe seeds, and iP, 0.10 pmol in the pericarps. The values shown are thearithmetic 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 meanweight (n=43) of a fruit cluster was 1.88 g, of which 1.223 g was in thepericarps and 0.657 g in the seeds. The figure is based on fruit collectedin a growth season for which material completely identical to that inTables 1–3 was not available.


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to perianth greening, increasing by a factor of two to threeas chlorophyll started to appear (Table 3). However, thechanges were small in absolute terms, and further experi-ments may be required to confirm a causal relationship. Theoverall cytokinin concentration in the sepals decreasedslightly after anthesis, showing only minor fluctuationsduring further development, and most individual cytokininsfollowed this general trend. However, the concentrations ofZ and most of its derivatives increased slightly, butconsistently, towards the end of the life cycle of theperianth and, taken together, eventually exceeded thepooled concentration of all DZ forms. Earlier in perianthdevelopment, the latter were up to three times moreabundant than the total zeatin cytokinins. However, theDZ in the sepals was mostly in two bound forms: theriboside and its O-glucoside; the concentration of the freecytokinin barely exceeded the detection limit.

To verify the effect of developing seeds on cytokininlevels in the perianth, depistillated and unpollinated flowerswere analysed (Fig. 5). The seedless flowers contained noribotides, and the levels of most other cytokinins were alsosmaller than in the sepals of fruit-bearing flowers of thesame physiological age (i.e. 2–3 weeks before seed ripen-ing). The seedless flowers also weighed less and did notpass through a complete greening process. This indicatesthat cytokinins play a part in the normal life cycle of theperianth including the greening response following fertil-ization. So far, the impact of these hormones on the pho-tosynthetic apparatus has mostly been investigated inmodel systems based on the prevention of chlorophyllloss as one of the symptoms of senescence (Richmondand Lang, 1957; Medford et al., 1989; Gan and Amasino,1995), an analogy which now appears less straightforwardthan originally believed (Werner et al., 2001; Ananievaet al., 2004). The reverse process, cytokinin-mediatedconversion of etioplasts into chloroplasts (Parthier, 1979),was, for instance, observed in a line of tobacco callus whichgrew well without external cytokinin but neverthelessrequired it for the formation of chloroplasts when thenormally dark-grown tissue was transferred to the light(Stetler and Laetsch, 1965). Greening processes of thiskind are akin to what happens in the Christmas roseperianth after fertilization when the leucoplasts present atanthesis develop into chloroplasts (Salopek-Sondi et al.,2000, 2002). Further research will show if the accom-panying, comparatively small, changes in the relativeabundance of individual cytokinins (in the perianth) areby themselves sufficient to trigger the formation of aphotosynthetic apparatus, and to keep it functional duringseed ripening. They may also be just one set of com-ponents in a complex signalling network.

Concluding remarks

Seeds have long been known to be a rich source ofcytokinins, but their origin has been a controversial issue.

They were long thought to be synthesized in the root tipsand transported to their sites of action via the xylem andthe phloem (Letham, 1994), but recent data now demon-strate that this is an oversimplification (Faiss et al., 1997;Emery et al., 2000; Nordstrom et al., 2004). In particu-lar, Lee et al. (1989) demonstrated that wheat (Triticum ae-stivum L.) ears cultured in vitro, in a hormone-freeliquid medium, maintain a normal cytokinin pattern. Also,Miyawaki et al. (2004) found some of the genes encod-ing isopentenyltransferases involved in cytokinin bio-synthesis expressed in immature seeds. It is thusplausible to assume that most of the cytokinins detectedin Helleborus seeds are synthesized in situ. If so, twoobservations suggest that this preferentially occurs viaiPRMP, i.e. (i) the latter is always the most abundantisopentenyladenine-cytokinin; and (ii) the dramatic increaseof zeatin and DZ cytokinins during perianth greening ispreceded by an (albeit less prominent) increase in theconcentration of iPRMP. Also, as fruit approach maturity,iPRMP starts to decrease while zeatin and DZ cytokininsare still maintaining top levels.

The significance of the changing patterns of cytokininforms during perianth and fruit development requiresfurther research. It has been widely assumed that only thefree bases have genuine hormonal activity, the ribosidesplay a role in long-range transport, the O-glucosides arestorage 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 cytokininreceptors with complex expression patterns (Maxwell andKieber, 2004) and, apparently, widely different substratespecificities. In Arabidopsis, for instance, one of the recep-tors (CRE1/AHK4) is specific for trans-zeatin, but anotherone (AHK3) also recognizes cis-zeatin and DZ as well ascytokinin ribosides and ribotides (Spichal et al., 2004).Similar differences in the substrate specificities of indi-vidual cytokinin receptors were also reported for maize(Yonekura-Sakakibara et al., 2004).

Does the obvious correlation of fruit growth and perianthgreening imply that cytokinins synthesized in the seedsare exported to the sepals? A complete answer is not yetpossible, but clearly defined cytokinin levels in the sepalsare required to induce the formation of a photosyntheticsystem and to keep it functional during seed filling, because(i) the perianth of seedless flowers, whether depistillatedat the bud stage or just ignored by pollinators at floweringtime, is cytokinin deficient and such flowers do not passthrough a complete greening process; and (ii) exogenouscytokinins stimulate chlorophyll accumulation in suchflowers (Salopek-Sondi et al., 2002). During advancedfruit development, cytokinin pool sizes are indeed muchlarger in the fruit than in the perianth (Tables 2, 3).However, when the greening process is initiated (‘initialperianth greening’), the perianth of an individual flowercontains a larger cytokinin pool than the fruit cluster


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of that same flower. Under these circumstances, thebiosynthetic machinery in the fruit would have to workpreferentially for the perianth. That this is not impossible isdemonstrated by an example from the developmentalphysiology of the pea (Pisum sativum L.) fruit. Indirectevidence shows convincingly that the seeds supply auxin tothe pericarp (Ozga and Reinecke, 2003), even at a stagewhen the latter is about finger-long and contains a signifi-cantly larger auxin pool than the pinhead-sized seeds(Magnus et al., 1997).

Acknowledgements

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

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

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