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First published online August 26, 2005; 10.1104/pp.105.064162 Plant Physiology 139:213-223 (2005) © 2005 American Society of Plant Biologists Gibberellin Biosynthesis in Developing Pumpkin Seedlings1,2Institut für Pflanzenbiologie der Technischen Universität Braunschweig, D–38106 Braunschweig, Germany
A gibberellin (GA) biosynthetic pathway was discovered operating in root tips of 7-d-old pumpkin (Cucurbita maxima) seedlings. Stepwise analysis of GA metabolism in cell-free systems revealed the conversion of GA12-aldehyde to bioactive GA4 and inactive GA34. Highest levels of endogenous GA4 and GA34 were found in hypocotyls and root tips of 3-d-old seedlings. cDNA molecules encoding two GA oxidases, CmGA20ox3 and CmGA3ox3, were isolated from root tips of 7-d-old LAB150978-treated seedlings. Recombinant CmGA20ox3 fusion protein converted GA12 to GA9, GA24 to GA9, GA14 to GA4, and, less efficiently, GA53 to GA20, and recombinant CmGA3ox3 protein oxidized GA9 to GA4. Transcript profiles were determined for four GA oxidase genes from pumpkin revealing relatively high transcript levels for CmGA7ox in shoot tips and cotyledons, for CmGA20ox3 in shoot tips and hypocotyls, and for CmGA3ox3 in hypocotyls and roots of 3-d-old seedlings. Transcripts of CmGA2ox1 were mainly found in roots of 7-d-old seedlings. In roots of 7-d-old seedlings, transcripts of CmGA7ox, CmGA20ox3, and CmGA3ox3 were localized in the cap and the rhizodermis by in situ hybridization. We conclude that hypocotyls and root tips are important sites of GA biosynthesis in the developing pumpkin seedling.
Gibberellins (GAs) are signaling molecules that regulate and integrate developmental processes during the entire life cycle of higher plants, including shoot elongation and root development (Richards et al., 2001  ; Olszewski et al., 2002; Fu and Harberd, 2003; Reid et al., 2004; Sun, 2004).
GA biosynthetic pathways are of considerable complexity (for review, see Hedden and Kamiya, 1997
Most of the genes coding for enzymes of the GA biosynthetic pathway have been isolated and characterized and our understanding is increasing about their tissue-cell specificity during plant development (for review, see Sponsel and Hedden, 2004 ). Genes encoding GA oxidases from pumpkin are summarized in Table I. Localization of the transcripts of GA biosynthetic genes indicates complex tissue- and developmental-specific expression patterns in many species (Yamaguchi et al., 2001). Endogenous GAs have frequently been identified in shoots and occasionally in roots of higher plants (for review, see MacMillan, 2002). In seedlings, GA oxidase genes are mainly expressed in rapidly developing tissues, including shoot and root tips (for review, see Olszewski et al., 2002). Recently, a growing body of evidence supports the hypothesis that GAs are produced close to or at their site of action (for review, see Sponsel and Hedden, 2004), which might still include unknown transport mechanisms (Fleet and Sun, 2005). There is some indication that GAs derived from cotyledons play a role in cell division during tissue reunion in the cortex of hypocotyls, as has been shown for cucumber and tomato (Asahina et al., 2002). It has been shown that GAs promote root growth in Arabidopsis by inducing the degradation of the DELLA proteins RGA (repressor of gal-3) and GAI (GA insensitive) and that other hormones are involved in regulating this action (Achard et al., 2003; Fu and Harberd, 2003; Fleet and Sun, 2005). However, direct evidence for GA biosynthesis occurring in roots is missing.
To address this question and investigate potential sites of GA biosynthesis in pumpkin seedlings, endogenous GA levels were analyzed and a GA biosynthetic pathway in root tips of 7-d-old pumpkin seedlings was identified. Characteristic genes of this pathway were isolated, including those coding for GA 20-oxidase (CmGA20ox3) and GA 3-oxidase (CmGA3ox3; Table I). GA oxidase transcripts were quantified in different parts of developing pumpkin seedlings by competitive reverse transcription (RT)-PCR and localized in roots and, more specifically, in root tips by in situ hybridization.
Endogenous GAs of Developing Pumpkin Seedlings
In preliminary experiments, seedlings were treated for 7 d with inhibitor of GA biosynthesis 1-(4-trifluormethyl)-2-(1,2,4-triazolyl-(1))-3-(5-methyl-1,3-dioxan-5-yl)-propen-3-ol (LAB150978), with GA4, and with both LAB150978 and GA4 (Table II). LAB150978 serves as an inhibitor of ent-kaurene oxidase, an enzyme that catalyzes early steps of the GA biosynthetic pathway (Jung et al., 1986
Endogenous GAs were quantified from embryos of dry pumpkin seeds and from different parts of 3-, 5-, and 7-d-old pumpkin seedlings by gas chromatography-mass spectrometry (GC-MS) single ion monitoring (Fig. 2). Levels of precursors of the early 3  -hydroxylated pathway (GA14, GA37, and GA36) were low in all tissues (<0.1 ng g–1 fresh weight; data not shown) with the exception of embryos from dry seeds where GA14 and GA36 levels were 1.9 and 0.6 ng g–1 fresh weight, respectively. Embryos of pumpkin seeds contain very high levels of the tricarboxylic acids GA25 and GA17, as well as catabolic GA8 (Fig. 2, A and B). High levels of the latter two GAs persist during the first 7 d of seedling development (Fig. 2, D, F, and H). Levels of 13-hydroxylated GAs decreased during the first 7 d of seedling development, with the exception of the plant hormone GA1, which was found at low levels in roots and root tips of 7-d-old seedlings (Fig. 2, D, F, and H).
Precursors of the nonhydroxylated pathway were mainly found in shoot and root tips and in the hypocotyls, with decreasing levels in seedlings getting older (Fig. 2, C, E, and G). Highest levels of the plant hormone GA4 were identified in root tips and hypocotyls of 3-d-old seedlings, which decrease at later stages of development (Fig. 2, C, E, and G). Catabolic GA34 was found in the embryo and in early stages of seedling development, particularly in hypocotyls and root tips. These results indicate that mainly hypocotyls and root tips of developing seedlings contain relatively high concentrations of bioactive GAs compared to the other tissues. In addition, hypocotyl elongation was maximal between days 3 and 5, while the primary root length doubled approximately every second day between days 3 and 7 of seedling development (Fig. 2, left).
GA biosynthetic pathways were investigated in cell-free systems prepared from root tips of untreated seedlings and of seedlings that have been treated with LAB150978. The identities of the products were confirmed by full-scan GC-MS (Table III). From 14C-GA12-aldehyde 14C-GA12 was formed, indicating the presence of GA 7-oxidase activity. Conversion was higher in root tips of untreated seedlings than in LAB150978-treated seedlings. 14C-GA12 was converted to 14C-GA15, and 14C-GA24 was metabolized to 14C-GA9, indicating the presence of GA 20-oxidase activity different from the one previously identified in developing pumpkin seeds (Table III; Lange et al., 1994
Cloning and Expression of GA Oxidase Genes from Root Tips of 7-d-Old Pumpkin Seedlings
The GA biosynthetic pathway discovered in root tips requires 20-oxidases and 3-oxidases with different catalytic properties to the ones identified previously from developing pumpkin seeds (Lange, 1998
Catalytic properties of recombinant CmGA20ox3 (GA 20-oxidase) and CmGA3ox3 (GA 3-oxidase) proteins were investigated by expression of the respective cDNA molecules in pUC18 in Escherichia coli NM522 (Table IV). Cell lysates of recombinant CmGA20ox3 protein, prepared from the full-length clone, oxidized 14C-GA12 to -GA15 and 14C-GA24 to -GA9 in standard enzyme assays (data not shown). GA 20-oxidation activity highly increased in cell lysates prepared from the predicted open reading frame (ORF) of the CmGA20ox3 clone (designated CmGA20ox3 ORF) and, therefore, was used for further characterization (Table IV). Recombinant CmGA20ox3 ORF protein is capable of oxidizing and subsequently removing the C-20 position of 14C-labeled substrates GA12, GA24, GA14, and, less efficiently, GA53. Of all GAs tested, recombinant CmGA3ox3 protein oxidizes only 14C-GA9 at the C-3
Transcript Analysis of GA Oxidases in Developing Pumpkin Seedlings
Expression levels of mRNA were quantified for GA oxidase-encoding genes in different tissues from 3-, 5-, and 7-d-old pumpkin seedlings by competitive RT-PCR (Fig. 3). The primers used were based on the sequence of CmGA7ox and CmGA2ox1 cDNAs that were originally isolated from pumpkin endosperm and embryo, respectively (Lange, 1997
Highest transcript levels of CmGA7ox, CmGA20ox3, and CmGA3ox3 were identified in 3-d-old seedlings (Fig. 3, A–C, E–G, and I–K): CmGA7ox transcripts were found mainly in shoot tips and cotyledons of 3-d-old seedlings, with moderate levels in hypocotyls and root tips (Fig. 3A), CmGA20ox3 transcript levels were highest in shoot tips and hypocotyls (Fig. 3B), and CmGA3ox3 transcripts were mostly detectable in hypocotyls (Fig. 3C). Moderate transcript levels of CmGA7ox, CmGA20ox3, and CmGA3ox3 were visible in root tips of seedlings 5 d after imbibition (Fig. 3, E–G). In contrast, CmGA2ox1 transcript levels increased in roots from day 3 to day 7 after imbibition of the seedling (Fig. 3, D, H, L). Highest CmGA2ox1 transcript levels were found in roots, together with moderate levels in shoot and root tips. Additionally, transcript levels of three previously cloned GA oxidase genes from developing pumpkin seeds (Lange, 1997 ; Lange et al., 1997; Frisse et al., 2003) were analyzed in untreated 5-d-old seedlings and either were not detectable (CmGA20ox1, CmGA20ox2, and CmGA3ox1) or were identified at very low levels in root tips (CmGA3ox2, 0.02 µg transcript per gram of total RNA; data not shown). The impact of LAB150978 treatment on transcript levels was analyzed for four GA oxidase genes in 7-d-old seedlings. Transcript levels of CmGA7ox and CmGA2ox1 did not alter significantly between treated and untreated plants (Fig. 4, A and D). However, transcript levels of CmGA20ox3 and of CmGA3ox3 genes were low in general and, after LAB150978 treatment, increased dramatically in all parts of the seedling (Fig. 4, B and C).
Expression patterns for transcripts of four GA oxidases were investigated by in situ hybridization in root tips of 7-d-old pumpkin seedlings. CmGA7ox, CmGA20ox3, and CmGA3ox3 transcripts were localized in the cap of the root and the rhizodermis (Fig. 5, A–C and E–G). However, only weak signals for CmGA2ox1-encoding transcripts were detectable in root tips by in situ hybridization (Fig. 5, D and H). No differences in expression pattern of these transcripts were detectable between 3- and 7-d-old plants and between untreated and LAB150978-treated plants by in situ hybridization. However, the intensity of the signals obtained for CmGA20ox3 and CmGA3ox3 were stronger in LAB150978-treated plants (data not shown).
In pumpkin, GA biosynthetic pathways have been elucidated mainly in cell-free systems prepared from developing seeds (Lange et al., 1993a , 1993b; for review, see Graebe, 1987). These pathways are still active in mature pumpkin seeds even 36 h after imbibition. However, they disappear rapidly in later stages of the developing seedling (Lange et al., 1997). The main products formed via those seed-specific pathways in pumpkin are GAs containing C-20 carboxylic acids (e.g. GA25 and GA17; Fig. 1). In fact, the mature embryo (as well as the testa; data not shown) contains large quantities of those tricarboxylic GAs that have no known physiological function (Fig. 2).
There is growing evidence for the presence of root-based GA biosynthesis from many plant species, including pumpkin (Yamaguchi et al., 1996
GA metabolic studies demonstrate that the products resulting from GA 20-oxidase and GA 3-oxidase activities are different in developing pumpkin seeds and seedlings. We isolated two cDNA molecules, CmGA20ox3 and CmGA3ox3, from root tip poly(A)+ RNA of 7-d-old LAB150978-treated pumpkin seedlings, encoding GA 20-oxidase and 3-oxidase, respectively. The broad specificity of the recombinant CmGA20ox3 protein indicates that a single enzyme is capable of catalyzing alternative GA biosynthetic pathways (Fig. 1). Unlike CmGA20ox1 and CmGA20ox2 proteins from developing seeds, recombinant CmGA20ox3 protein exhibits strong decarboxylation activities and converts GA precursors GA12, GA14, and GA53 to their respective C19-GAs. Recombinant CmGA3ox3 protein, however, reveals more stringent substrate specificity than CmGA3ox1 or CmGA3ox2 protein from developing pumpkin seeds (Lange et al., 1997
Sites of bioactive GA synthesis seem to be tissues containing rapidly expanding cells (Silverstone et al., 1997
In root tips, however, transcript levels determined for pumpkin GA 20-oxidase (CmGA20ox1, CmGA20ox2, and CmGA20ox3) and GA 3-oxidase (CmGA3ox1, CmGA3ox2, and CmGA3ox3) genes do not correlate well with endogenous GA levels. GA oxidases form multigene families (Sponsel and Hedden, 2004
Plant Material, Enzyme, and Total RNA Preparations Seeds of pumpkin (Cucurbita maxima L. cv Riesenmelone, gelb genetzt) were sown in 100-mL pots containing 10 g of vermiculite moistened with 50 mL of 0.01% methanol, 0.01% methanol containing GA4 (10–6 M), or 0.01% methanol containing the plant growth retardant 1-(4-trifluormethyl)-2-(1,2,4-triazolyl-(1))-3-(5-methyl-1,3-dioxan-5-yl)-propen-3-ol (10–6 M; LAB150978; a gift from Dr. Rademacher, BASF Agricultural Center, Limburgerhof, Germany). Water and growth regulators were replenished 10 mL/d. Germination and growth of the seedlings occurred under an 18-h photoperiod at 24°C during the day and 18°C during the night. Light was supplied by Osram Powerstar HQI-T 400W/D daylight lamps, located 1 m above the plants, giving a photon fluence rate of 200 µE m–2 s–1. At the time of sampling, the plants were rinsed, dissected into shoot tips (the upper 5 mm of the stem), cotyledons, hypocotyl, roots, and root tips (the lower 2 mm of the primary and lateral roots) and immediately frozen in liquid N2 and stored at –80°C. The plant material was ground to a fine powder in liquid nitrogen with a mortar and pestle. Total RNA was isolated from the frozen powder (25 mg) with a NucleoSpin RNA plant kit, according to the manufacturer's instructions (Machery-Nagel), and treated with DNase I (Sigma; 10 units/µg total RNA) for 20 min at 37°C, followed by phenol-chloroform extraction. Total RNA was stored at –80°C and used for quantification of specific GA oxidase transcripts as described below. Cell-free enzyme preparations were obtained from the frozen powder (1 g) to which 200 mM Tris-HCl buffer (pH 7.9, as measured at 4°C) containing 10 mM dithiothreitol was added 1:1 (w/v). After thawing, the extract was centrifuged for 30 min at 40,000g and the supernatant was stored at –80°C.
Poly(A)+ RNA (5 µg) was extracted from root tips of 7-d-old pumpkin seedlings treated with the growth retardant LAB150978, using a mRNA isolation kit, according to the manufacturer's instructions (Fast Track 2.0; Invitrogen), and used for the preparation of an oligo(dT)-primed cDNA library in pBluescript SK+ using commercial kits (pBluescript II XR cDNA library construction kit; Stratagene). A cDNA plasmid library in Escherichia coli XL 10 GOLD of 3.4x105 independent cell-forming units was obtained and amplified, 95% of which contained inserts of a length between 800 to 2,000 bp, as shown by agarose gel electrophoresis of PCR products using pBluescript specific M13 primers.
Screening of the pBluescript SK+ cDNA library for GA 20-oxidase, 3-oxidase, and 2-oxidase genes was performed by a PCR-based cloning strategy (Frisse et al., 2003
DNA sequence analysis revealed that the cDNA inserts of the three clones were not in frame to the lac-promotor of the pBluescript vector. The pBluescript SK+ vector containing CmGA20ox3 cDNA was digested with XbaI and EcoRI, filling in recessed 3'-termini with Klenow fragment and then religated. The predicted ORF of the CmGA20ox3 clone was amplified by PCR (at an annealing temperature of 60°C), using sense primer 5'-NGAATTCAAACCAAACCATGCATGTCGTGAC-3' and antisense primer 5'-NGGATCCTTTTCCTCAGGCGAGGAAAAGTG-3', cut by EcoRI and BamHI digest and cloned into the appropriate cloning sites of pUC18 vector (and pBluescript SK– vector for preparation of riboprobes for in situ hybridization; see below). The cDNA of clone CmGA3ox3 was excised by XhoI and XbaI and recessed 3'-termini were filled in by Klenow fragment and subcloned into the SmaI site of pUC18. The plasmid vectors containing respective inserts were used to transform E. coli NM 522 cells. Protein induction and cell lysis were carried out as described by Lange (1997)
All 14C-labeled GAs were prepared as described elsewhere (Lange and Graebe, 1993
For quantitative determination of endogenous GAs, 2 g fresh weight of frozen plant tissue were spiked with 17, 17-d2-GA standards (2 ng each; from Professor L. Mander, Canberra, Australia) and pulverized under liquid nitrogen. Eighty percent methanol-water (8 mL) was added and the extract was stirred for 1 h at 4°C. After centrifugation, the pellet was re-extracted with methanol (4 mL) for 30 min and recentrifuged. The re-extraction procedure was repeated two times. The combined methanol extracts were evaporated to dryness and resuspended in water (3 mL), adjusted to pH 8.0 (1 M KOH). Solvent partition was performed using ethyl acetate (four times, 1 mL). The aqueous phase was adjusted to pH 3 (acetic acid) followed by solvent partition with ethyl acetate (four times, 1 mL). The combined ethyl acetate fractions were evaporated to dryness, redissolved in methanol (100 µL), and methylated with ethereal diazomethane (two times, 200 µL). The methyated samples were dried and redissolved in methanol (100 µL) and water (5 mL), adjusted to pH 3.2 (acetic acid). After loading onto a C18 cartridge (Waters), the cartridge was washed with water (10 mL), pH 3.2. Methylated GAs were eluted with methanol (6 mL), which was then dried. For purification by HPLC, the residues were redissolved in 200 µL methanol-water, pH 3.2 (1:1), and applied to a C18 reverse-phase column (10 cm long, 8 mm i.d., 4 µm particle size, Novapack liquid chromatography cartridge in a RCM100 radial compression system; Waters), which was eluted with a gradient from 25% methanol in water to 100% methanol in 40 min delivered by a two-pump HPLC system (models 501 and 510; Waters) at a flow rate of 1 mL min–1. Starting from 13.5 mL, 18 fractions were collected per run, each containing 2 mL eluate and dried. Dried HPLC fractions were redissolved in 2 µL N-methyl-N-trimethylsilyltrifluoracetamide (Macherey-Nagel). The derivatized samples were analyzed using a Turbo-Mass MS system (Perkin-Elmer) equipped with a Perkin-Elmer AutoSystem XL gas chromatograph. Samples (1–2 µL) injected into a SGE BPX5 capillary column (30 m long, 0.25 mm i.d., 0.25-µm film thickness; SGE) at an oven temperature of 60°C. The split value (30:1) was open after 1 min, after which the temperature was increased by 45°C min–1 to 220°C and then with 4°C min–1 to 300°C. The He inlet was pneumatic pressure controlled at a constant flow rate of 1.5 mL min–1 and the injector, transfer line, and source temperatures were 220°C, 280°C, and 240°C, respectively. Data were acquired in the selected ion monitoring mode after 5 min. The ions monitored for quantification of endogenous GAs were 270 and 272 (GA12-aldehyde), 300 and 302 (GA12), 239 and 241 (GA15), 314 and 316 (GA24), 270 and 272 (GA9), 284 and 286 (GA25), 284 and 286 (GA4), 506 and 508 (GA34), 207 and 209 (GA53), 207 and 209 (GA44), 374 and 376 (GA19), 418 and 420 (GA20), 492 and 494 (GA17), 506 and 508 (GA1), 594 and 596 (GA8), 416 and 418 (GA14), 432 and 434 (GA37), and 284 and 286 (GA36). Identification was confirmed on the basis of retention time and the co-occurrence of additional ions. Endogenous levels were calculated on the basis of peak areas, after corrections were made for the contribution of naturally occurring isotopes and for the presence of unlabeled GAs in the internal standards, where necessary.
Quantification of CmGA7ox transcripts was performed as described by Lange et al. (1997)
Sense and antisense riboprobes of full-length cDNAs of CmGA7ox (Lange, 1997 Sequence data from this article can be found in the GenBank/EMBL data libraries under accession numbers AJ315663 and AJ302040.
We thank Anja Liebrandt for technical assistance and Julia Niemeyer for help with RT-PCR. Received April 25, 2005; returned for revision June 13, 2005; accepted July 10, 2005.
1 This work was supported by the Deutsche Forschungsgemeinschaft (grant nos. La880/4–2 and La880/4–3). 
2 This paper is dedicated to Jan E. Graebe on the occasion of his 75th birthday.
3 Present address: Schleicher & Schuell BioScience GmbH, Postfach 1160, D–37582 Dassel, Germany. Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.105.064162. * Corresponding author; e-mail theo.lange{at}tu-bs.de; fax 49–531–3918180.
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ABSTRACT
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- and 13-hydroxylated gibberellins in a cell-free system from Cucurbita maxima endosperm and the identification of new endogenous gibberellins. Planta 189: 340–349
