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Immature pumpkin (Cucurbita maxima) seeds contain gibberellin (GA) oxidases with unique catalytic properties resulting in GAs of unknown function for plant growth and development. Overexpression of pumpkin GA 7-oxidase (CmGA7ox) in Arabidopsis (Arabidopsis thaliana) resulted in seedlings with elongated roots, taller plants that flower earlier with only a little increase in bioactive GA4 levels compared to control plants. In the same way, overexpression of the pumpkin GA 3-oxidase1 (CmGA3ox1) resulted in a GA overdose phenotype with increased levels of endogenous GA4. This indicates that, in Arabidopsis, 7-oxidation and 3-oxidation are rate-limiting steps in GA plant hormone biosynthesis that control plant development. With an opposite effect, overexpression of pumpkin seed-specific GA 20-oxidase1 (CmGA20ox1) in Arabidopsis resulted in dwarfed plants that flower late with reduced levels of GA4 and increased levels of physiological inactive GA17 and GA25 and unexpected GA34 levels. Severe dwarfed plants were obtained by overexpression of the pumpkin GA 2-oxidase1 (CmGA2ox1) in Arabidopsis. This dramatic change in phenotype was accompanied by a considerable decrease in the levels of bioactive GA4 and an increase in the corresponding inactivation product GA34 in comparison to control plants. In this study, we demonstrate the potential of four pumpkin GA oxidase-encoding genes to modulate the GA plant hormone pool and alter plant stature and development.

Long day (LD) exposure of rosette plants causes rapid stem/petiole elongation, a more vertical growth habit, and flowering; all changes are suggestive of a role for the gibberellin (GA) plant growth regulators. For Arabidopsis (Arabidopsis thaliana) L. (Heynh), we show that enhancement of petiole elongation by a far-red (FR)-rich LD is mimicked by a brief (10 min) end-of-day (EOD) FR exposure in short day (SD). The EOD response shows red (R)/FR photoreversibility and is not affected in a phytochrome (PHY) A mutant so it is mediated by PHYB and related PHYs. FR photoconversion of PHYB to an inactive form activates a signaling pathway, leading to increased GA biosynthesis. Of 10 GA biosynthetic genes, expression of the 20-oxidase, AtGA20ox2, responded most to FR (up to a 40-fold increase within 3 h). AtGA20ox1 also responded but to a lesser extent. Stimulation of petiole elongation by EOD FR is reduced in a transgenic AtGA20ox2 hairpin gene silencing line. By contrast, it was only in SD that a T-DNA insertional mutant of AtGA20ox1 (ga5-3) showed reduced response. Circadian entrainment to a daytime pattern provides an explanation for the SD expression of AtGA20ox1. Conversely, the strong EOD/LD FR responses of AtGA20ox2 may reflect its independence of circadian regulation. While FR acting via PHYB increases expression of AtGA20ox2, other GA biosynthetic genes are known to respond to R rather than FR light and/or to other PHYs. Thus, there must be different signal transduction pathways, one at least showing a positive response to active PHYB and another showing a negative response.

We investigated the effect of overexpressing a pumpkin gibberellin (GA) 20-oxidase gene encoding an enzyme that forms predominantly biologically inactive products on GA biosynthesis and plant morphology in transgenic lettuce (Lactuca sativa cv Vanguard) plants. Lettuce was transformed with the pumpkin GA 20-oxidase gene downstream of a strong constitutive promoter cassette (El2-35S-Omega). The transgenic plants in which the pumpkin gene was detected by polymerase chain reaction were dwarfed in the T(2) generation, whereas transformants with a normal growth phenotype did not contain the transgene. The result of Southern-blot analysis showed that the transgene was integrated as a single copy; the plants segregated three dwarfs to one normal in the T(2) generation, indicating that the transgene was stable and dominant. The endogenous levels of GA(1) and GA(4) were reduced in the dwarfs, whereas large amounts of GA(17) and GA(25), which are inactive products of the pumpkin GA 20-oxidase, accumulated in these lines. These results indicate that a functional pumpkin GA 20-oxidase is expressed in the transgenic lettuce, resulting in a diversion of the normal pathway of GA biosynthesis to inactive products. Furthermore, this technique may be useful for controlling plant stature in other agricultural and horticultural species.

The use of nondwarf rice (Oryza sativa L.) cultivars treated with uniconazole as test plants for gibberellin (GA) bioassay instead of Tan-ginbozu dwarf rice variant was investigated. The sensitivity of six nondwarf rice cultivars to GAs was increased substantially by treatment of the seeds with uniconazole. The minimum detectable dose of a GA in the nondwarf cultivars treated with uniconazole was 1- to 1/10-fold of that in the nontreated Tanginbozu and 3- to 10-fold of that in uniconazole-treated Tanginbozu. The relative activity of several GAs on treated nondwarf rice cultivars was not largely different from that to Tan-ginbozu. Considering that seeds of nondwarf rice are available commercially, the nondwarf rice seedling assay would be useful as a simple assay for systematic analysis of GAs, and also as a routine teaching tool in high schools and universities.

A procedure using two small preparative columns (in sequence) of C(18) reverse phase Bondapak B material with methanolic extracts of plant tissue (Pisum sativum L., Malus domestica Borkh., Pimpinella anisum L.) yields two fractions: (i) gibberellin (GA) precursors, and (ii) free GA/GA methyl esters (GA-Me)/GA glucosyl conjugates. The discrete separation of (iii) free GA/GA-Me from (iv) GA glucosyl conjugates is then accomplished by a combination of differential solvent solubility and SiO(2) partition chromatography. All fractions are almost pigment free, and appreciable dry weight purification was accomplished for the GA precursor and free GA/GA-Me fractions. Solvent volumes can be kept low, no buffer salts are introduced, and each fraction (i, iii, iv) can be subjected directly to preparative or analytical reverse phase C(18) high performance liquid chromatography without recourse to solvent partitioning, and often without further purification.

Under controlled environment and/or field conditions, vegetative growth (height, internode length, leaf area, shoot dry weight, grain yield) was greater in an F(1) maize hybrid than in either parental inbred. Endogenous gibberellin (GA)-like substances in apical meristem cylinders were also higher in the hybrid than in either inbred, both on a per plant and per gram dry weight basis. There were no apparent qualitative differences in GA-like substances, however. Levels of GA-like substances in all genotypes were highest prior to tassel initiation. Chromatographic comparisons of the GA-like substances and authentic standards of GA native to maize on gradient-eluted SiO(2) partition and reverse-phase C(18) high-pressure liquid chromatography columns are described. No consistent differences in abscisic acid levels of the three genotypes were observed. This correlation of heterosis for endogenous GA-like substances with heterosis for growth suggests that amounts of endogenous GA may be related to hybrid vigor in maize.

Immature seeds of apricot (Prunus armeniaca L.) were fed the native gibberellin A(5) (GA(5)) as 1- and 1,2-[(3)H]GA(5) (5.3 Curies per millimole to 16 milliCuries per millimole) at doses (42 nanograms to 10.6 micrograms per seed) 2 to 530 times the expected endogenous level. After 4 days of incubation, seeds were extracted and free [(3)H]GA-like metabolites were separated from the highly H(2)O-soluble [(3)H]metabolites. For high specific activity feeds the retention times (Rts) of radioactive peaks were compared with Rts of authentic GAs on sequential gradient-eluted --> isocratic eluted reversed-phase C(18) high performance liquid chromatography (HPLC) -radiocounting (RC). From high substrate feeds (530 and 230 x expected endogenous levels) HPLC-RC peak groupings were subjected to capillary gas chromatography-selected ion monitoring (GC-SIM), usually six characteristic ions. The major free GA metabolites of [(3)H] GA(5) were identified as GA(1), GA(3), and GA(6) by GC-SIM. The major highly water soluble metabolite of [(3)H]GA(5) at all levels of substrate GA(5) had chromatographic characteristics similar to authentic GA(1)-glucosyl ester. Expressed as a percentage of recovered radioactivity, low substrate [(3)H]GA(5) feeds (2 x expected endogenous level) yielded a broad spectrum of metabolites eluting at the Rts where GA(1), GA(3), GA(5) methyl ester, GA(6), GA(22), GA(29) (17, 14, 1.6, 7, 1.1, 0.5%, respectively) and GA glucosyl conjugates of GA(1), GA(3), GA(5), and GA(8) (33, 11, 1, 0.1%, respectively) elute. Metabolites were also present at Rts where GA glucosyl conjugates of GA(6) and GA(29) would be expected to elute (8 and 0.1%, respectively). Only 5% of the radioactivity remained as GA(5). Increasing substrate GA(5) levels increased the proportion of metabolites with HPLC Rts similar to GA(1), GA(6), and especially GA(1) glucosyl ester, primarily at the expense of metabolites with HPLC Rts similar to GA(3), GA(3)-glucosyl ester, and a postulated conjugate of GA(6). There was evidence that high doses of substrate GA(5) induced new metabolites which often, but not always, differed from GA(1), GA(3), and GA(6) in HPLC Rt. These same metabolites, when analyzed by GC-SIM yielded m/e ions the same as the M(+) and other characteristic m/e ions of the above GAs, albeit at differing GC Rt and relative intensities.

After the application of 2.36 Curies per millimole [2,3-(3)H]gibberellin A(20) (GA(20)) to 21-day-old maize (Zea mays L., hybrid CM7 x CM49) plants, etiolated maize seedlings, or maturing maize cobs, a number of (3)H-metabolites were observed. The principal acidic (pH 3.0), ethyl acetate-soluble metabolite was identified as [(3)H]GA(1) on the basis of co-chromatography with standard [(3)H]GA(1) on SiO(2) partition, high resolution isocratic elution reverse phase C(18) high performance liquid chromatography and gas-liquid chromatography radiocounting. Two other acidic metabolites were identified similarly as [(3)H]GA(8) and C/D ring-rearranged [(3)H]GA(20), although gas-liquid chromatography radiocounting was not performed on these metabolites. Numerous acidic, butanol-soluble (e.g. ethyl acetate-insoluble) metabolites were observed with retention times on C(18) high performance liquid chromatography radiocounting similar to those of authentic glucosyl conjugates of GA(1) and GA(8), or with retention times where conjugates of GA(20) would be expected to elute. Conversion to [(3)H]GA(1) was greatest (23% of methanol extractable radioactivity) in 21-day-old maize plants. In etiolated maize seedlings, the C/D ring-rearranged [(3)H]GA(20)-like metabolite was the major acidic product, while conversion to [(3)H]GA(1) was low.

Gibberellins [(3)H]GA(4) (1.33 Curies per millimole) and [(3)H]GA(20) (2.36 Curies per millimole) were injected into the shanks of maize (Zea mays L.) cobs during rapid grain filling and mature seeds were subsequently harvested. Extracts of mature, dry seeds from 1980 feeds yielded only 20 to 30% of the (3)H radioactivity in acidic, ethyl acetate-soluble form, and this was principally associated with the precursor, with lesser amounts of the major metabolite, [(3)H]GA(1) (putative identification based on sequential SiO(2) partition, and gradient-eluted reverse-phase C(18) high performance liquid chromatography [HPLC]). Most of the radioactivity in the dry seeds was associated with compounds having partition characteristics of, and co-chromatographing on, sequential SiO(2) partition and reverse-phase HPLC with glucosyl conjugates of the precursors (GA(4) or GA(20)) and their probable major metabolite (GA(1)). The majority of conjugate associated with the precursor GA(4) eluted coincidental with GA(4) glucoside. Subsequent acid or enzymic hydrolysis (beta-glucosidase or cellulase) yielded the free GAs, putative identification being based on isocratic HPLC of each (3)H-labeled conjugate --> hydrolysis --> isocratic HPLC of the (3)H-labeled hydrolysate. Upon imbibition of the seeds, radioactivity associated with the conjugate fraction decreased; concomitantly, statistically significant increases in levels of free [(3)H]GA-like compounds were observed. Although the specific ratios of GA-like and GA-glucosyl conjugate-like substances varied substantially across years, hybrids, and even, in different plants from the same hybrid, this ;reversible conjugation' (i.e. apparent conjugation during seed maturation followed by release of the GA moiety during germination), was reproducible for [(3)H]GA(20) in seed from two maize hybrids produced over 2 years.

Gibberellins (GAs) A(1), A(5), and A(29) were identified, and also GA(32) was confirmed, as endogenous GAs of immature seeds (3-4 weeks after anthesis, 0.25-0.5 gram fresh weight) of apricot (Prunus armeniaca L.) based on capillary gas chromatography (GC), retention time (Rt), and selected ion monitoring (SIM), in comparison with authentic standards. Fractions subjected to GC-SIM were purified and separated using sequential solvent partitioning --> paper chromatography --> reverse phase C(18) high performance liquid chromatography (HPLC) --> bioassay on dwarf rice cv Tan-ginbozu. Two other peaks of free GA-like bioactivity (microdrop and immersion dwarf rice assays) were eluted from C(18) HPLC at Rts where GA(4/7) and GA(8) (or other GAs with similar structures) would elute. Also, three unidentified GA glucoside-like compounds (based on bioactivity on the immersion assay, and no bioactivity on the microdrop assay) were noted. There were very high amounts of GA(32) (112 ng of GA(3) equivalents per gram fresh weight), and minor amounts (0.5 ng of GA(3) equivalents) for each of GA(1) and GA(5), respectively, based on the microdrop assay.

Endogenous gibberellin (GA)-like substances were examined in suspension cultures of somatic embryos of a hybrid grape (Vitis vinifera x Vitis rupestris) during embryogenesis, and in mature embryos chilled at 4 degrees C, and subsequently incubated at 26 degrees C with and without abscisic acid (ABA). The extract was separated into a nonpolar fraction (would contain GA-precursors); a fraction that would contain free GAs; and a highly H(2)O-soluble fraction (would contain GA glucosyl conjugates and very polar free GAs). Quantitation after SiO(2) partition chromatography was accomplished by microdrop and immersion dwarf rice bioassays. As embryogenesis developed, the free and highly H(2)O-soluble GA-like substances, expressed on a dry weight basis, decreased (however, they increased on a per embryo basis). Chilling at 4 degrees C for 1 week greatly increased activity of free GA-like substances (per g dry weight and per embryo), it then declined over the next three weeks of chilling. Activity (per g dry weight and per embryo) in the H(2)O-soluble fraction declined throughout chilling. Activity in the GA-precursor fraction, however, increased steadily with chilling (per g dry weight and per embryo). Incubation at 26 degrees C after chilling enhanced activity in the free GA and H(2)O-soluble fractions (per g dry weight and per embryo), but activity in the GA-precursor fraction dropped dramatically. Incubation at 26 degrees C with (+/-) ABA after chilling prevented germination and maintained high activity for GA precursors and less polar free GAs and low activity in the polar free GA and H(2)O-soluble fractions.Kaurene and kaurenoic acid were characterized in the GA-precursor fraction of chilled embryos by gas-liquid chromatography-mass spectrometry (GLC-MS). The existence of GA(4) and GA(9) in ABA-treated, chilled embryos was also confirmed by GLC-MS.

Based on detection and quantitation by bioassay, endogenous gibberellin-like substances (GAs) and cytokinins (CKs) in Pinus radiata D. Don buds during sequential shoot initiation shift from less polar to more polar forms (GAs) and from conjugated to free forms (CKs). As the terminal bud moves from the production of "short shoots" (needle fascicles) to "long shoots" (lateral branches or female conebuds), a more polar GA appears while a glucoside-conjugate of zeatin riboside is reduced, and zeatin riboside levels increase markedly.Permethyl derivatives of the two highly active CK fractions were examined by capillary gas chromatography-mass spectrometry after separation by C(18) reverse phase high performance liquid chromatography. The mass spectra indicated the presence of: 9-beta-d-ribofuranosyl-6-(4-hydroxy-3-methyl-but-2-enylamino)purine (zeatin riboside) and 9-[hexosyl(probably glucosyl)-beta-d-ribofuranosyl]-6-(4-hydroxy-3-methyl-but-2- enylamino)purine (a glycoside of zeatin riboside in which the glycosyl moiety is attached directly to the ribosyl moiety at an unknown position).