ࡱ> Z\Y@VFbjbjFF 4J,,RRRRRRRfD,f_%(*****===$$$$$$$$&R($QR=9===$RR**%=R*R*$=$ RR!* CuQj!v",/%0_%!)),!ffRRRR)R!=======$$ffd cffGibberellin biosynthesis in developing fruit of the Christmas rose (Helleborus niger L.) V. Magnus1*, B. Ayele2, S. Mihaljevi1, D. M. Reinecke2, J. A. Ozga2 and B. Salopek-Sondi1 1Department of Molecular Biology, Ruer Boakovi Institute, Bijeni ka cesta 54, HR-10000 Zagreb, Croatia, 2Department of Agricultural, Food and Nutritional Science, University of Alberta, T6G 2P5, Canada, *corresponding author (magnus@irb.hr) Introduction The perianth of the Christmas rose (Helleborus niger L.), which is white or rose at anthesis, turns increasingly green as the seeds start developing. This process stops when the fruit are removed, but can be reinitiated by treatment with cytokinins and gibberellins (Salopek-Sondi et al. 2002). We have already shown that endogenous cytokinin levels in the fruit rise enormously, during early seed development and then remain nearly constant throughout the maturation phase (Tarkowski et al. 2006), suggesting a role for cytokinins in the induction and maintenance of the photosynthetic apparatus in the perianth. Here we address the question of whether the endogenous gibberellins show similar concentration dynamics. Materials and methods Carpels of (proterogyneous) flowers in their female and male phases (0.17 and 0.25 g FW) and three stages of developing fruit (0.52, 0.77 and 2.95 g FW) were collected in the 'Gorski kotar' region of Croatia. The freeze-dried plant material was extracted with 80% aqueous methanol containing butylated hydroxytoluene as an antioxidant and defined amounts of [2H2]GA1, [2H2]GA3, [2H2]GA4 and [2H2]GA7 as internal standards. Non-gibberellin plant constituents were removed by a series of purification steps including solvent partition, solid-phase extraction, and high-performance liquid chromatography (HPLC). For separation and identification, the gibberellins were converted to volatile methyl ester trimethylsilyl ethers and subjected to gas chromatography-mass spectrometry-selected ion monitoring. The criteria for identification were (1) the retention times relative to a series of alkane standards (Kovts retention indices), as observed in the final gas chromatographic step, and (2) the presence of the respective molecular ions and two (per GA) characteristic fragment ions, as revealed by selected ion monitoring. Quantification was accomplished by comparing the peak areas in ion chromatograms for the molecular ions of the endogenous gibberellins (i. e. their volatile derivatives) and the corresponding deuterated internal standards. To obtain H. niger GA20ox cDNA, RNA was extracted according to Southerton et al. (1998) from different tissues of Christmas rose flowers, followed by amplification by RT-PCR using SuperScript"! II RNase H- Reverse Transcriptase and Taq Polymerase Platinum (Invitrogen) according to the manufacturer's protocols. Degenerate primers for putative GA 20-oxidase were designed at highly conserved motifs, based on nucleotide sequences available in the NCBI database. The RT-PCR products were gel-purified and ligated into the pGEM-T-Easy vector (Promega). Sequencing of the cDNA clones was performed at the DNA sequencing facility at the Ruer Boakovi Institute. Results and discussion To gain first insight into gibberellin physiology, in the Christmas rose, we screened pistils and developing fruit for GA1, GA3, GA4, and GA7, which are the major bioactive GAs in other plant species (Sponsel and Hedden 2004). All four gibberellins were detected, but with widely different concentration dynamics (Table 1). Their total quantities per flower (i.e. per fruit cluster) increased about ten-fold, from late anthesis to the most advanced stages of fruit development examined. While GA3 and GA7 remained minor gibberellins throughout development, GA4 started to accumulate markedly during initial perianth greening; GA1 accumulation followed when perianth greening entered the stationary phase. It is thus tempting to infer that a fraction of the gibberellins produced in the fruit is exported to the sepals to participate in the induction and maintenance of the photosynthetic apparatus. Gibberellins also stimulated chlorophyll formation in other experimental systems, as first noted for regreening orange fruit (Coggins et al. 1960). Table 1: GA1, GA3, GA4 and GA7 in pistils and developing fruit of Helleborus niger. Developmental stageGA1GA3GA4GA7ng/gFW ng/flowerng/gFWng/flowerng/gFWng/flowerng/gFWng/flowerAnthesis, female phase0.50.10.40.140.36.80.80.1Anthesis, male phase17.44.31.40.321.75.30.40.1Initial perianth greening0.40.20.50.234.516.20.70.3Perianth greening complete7.45.90.70.524.119.00.40.32 weeks before seed ripening18.248.00.41.024.664.80.10.3 The concentration of GA1 had a sharp concentration maximum during the male phase of anthesis and, after a minimum during initial perianth greening, increased again towards the end of seed ripening. The GA4 concentration peaked during the female phase of anthesis, passed through a second, less explicit, maximum during initial perianth greening, to level off towards seed ripening. A similar pattern was, for instance, observed in orange ovaries, in which GA4 levels were high before bud opening and during anthesis to decline after petal drop, while GA1 passed through a maximum at anthesis (Talon et al. 1992). The difference between the two plant systems is: orange flowers self-pollinate before bud opening in Helleborus flowers, the pistils are receptive (female phase) before the anthers shed their pollen (male phase). Thus, the period 'before bud opening' in orange, with its GA4-maximum, corresponds to the 'female phase' in Helleborus. The period of 'anthesis' in orange flowers, when GA1 levels peak, thus follows pollination, as does the 'male phase' in Christmas rose flowers. During advanced seed development, GA-levels declined rapidly in commonly studied species (Pharis and King 1985). They remained high in Helleborus, apparently due to the fact that embryo development is extremely slow, barely reaching the early cotyledonary stage when the seed is released from the ripe fruit. To explore the possible role of tissue-specific GA biosynthesis in seed development and perianth greening, a partial GA 20-oxidase cDNA clone was isolated from developing Helleborus seeds by RT-PCR using a pair of degenerate primers. Sequence analysis identified a clone designated HnGA20ox (ca. 640-bp long) as a partial sequence of a GA 20-oxidase encoding a 212 amino acid polypeptide that is closely related to highly conserved regions of GA 20-oxidases previously isolated from Citrus cv. (accession no. AJ250187, 78 % identity), potato (accession no. AJ291453, 76 % identity), Populus sp. (accession no. AJ001326, 74 % identity), bean (accession no. U70530, 74 % identity), pumpkin (accession no. 308480, 73 % identity) and pea (accession no. PSU58830, 69 % identity). References Coggins C.V. jr., Hield H.Z., Garber M.J. (1960). Journal of the American Society of Horticultural Science 76, 193-198 Pharis R.P. and King R.W. (1985) Annual Review of Plant Physiology 36, 517-568. Salopek-L  8 : B d f h j     ! > E H l @ R 㯦~h\EmH sH h@mH sH hnhmH sH h mH sH hCmH sH hC6mH sH hCh iXmHsHh&h iXH*ht htH*h&mHsH h&H* h iXH* h&>*h iXh&6mH sH h&mH sH h&h .h '($If$5<<$If^`5a$gdWugd2:VFR W ] m x     A E c -hoy    !"#$'(%Hh)h_ h9;]H* h9;]H*h9;]h h\EhMh iXhhh iXmH sH hnhmH sH h@mH sH hCmH sH hJmH sH h\EmH sH hmH sH =;x .:h.\df@.4l˿˺˶˶˲؛ h;U?H*h;U?hwah>mHsHhwamHsHhwaht} h>H*hIj h2h>6h> h26h2h iXh h hl6h_h\Ehlh9;]h)h 9Zjw{/;?@Vx&'(+167;<@AHIm}ʼ hKhKhNhKH* hKH*hKhNhK5CJ hK5CJhWuhWu6h iX hN6 hH*hhNh];hWuh2:hV hZH*hZ hj6hZhj6hjh;U?h@3   !$;?@CDGHKLPQTUXY\x{|㶫򠜠hhK hrhKhrhrmH sH  hrhr hr5hhK5 hV5hrhr5hrhK5mH sH hrhK5 hK5CJ hK5hKhKH*=6!$5<<$If^`5a$kd$$Ifl4r >q#330q#4 lalp2 !%&;@DHFf\$If $Ifgdb$IfFf$If <<$IfgdHLQUY]^x|Ff&$IfFfA $If $Ifgdb   -34E F r !!!!""""""#k####h\mH sH h`zh\hZF hWu6hV hWuH*hhWu6hWuhKZhhKK #&& 'p'BCD4ETFVFgd2:Ff$If$IfFf ## $$$#$@$J$$$$$$$3%O%x%%%%%%^&i&j&m&&&&&&&' '/'A'b'o'p'(BhB𺲺𲪢yuskh@mHsHUh@ hZFhZF hZF6hZFhB h6hh iXhKZhWuh\mH sH hmH sH hVmH sH hV6mH sH hsSmH sH hVhWu6mH sH hWu6mH sH hVmH sH hk|mH sH hWumH sH h`zmH sH )Sondi B., Kova  M., Prebeg T. and Magnus V. (2002). Journal of Experimental Botany 53, 1949-1957. Southerton S.G., Marshall H., Mouradov A., Teasdale R.D. (1998). Plant Physiology 118, 365-372. Sponsel V. and Hedden P. (2004) In: Davies P.J. (Ed.) Plant hormones. Biosynthesis, signal transduction, action. Kluwer, Dordrecht: 63-94. Talon M., Zacarias L., Primo-Millo E. (1992) Plant Physiology 99, 1575-1581. Tarkowski P., Tarkowsk D., Novk O., Mihaljevi S., Magnus V., Strnad M. and Salopek-Sondi B. (2006). 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