Gibberellin biosynthetic pathway and the physiologically active gibberellin in the shoot ofCucumis sativus L.

[²H₂]Gibberellin A₂₄ (GA₂₄) and [²H₄]-GA₉ were applied to the apices of normal-type cucumber (Cucumis sativus L. cv. Yomaki) seedlings treated with uniconazole, an inhibitor of GA biosynthesis. The metabolites from these feeds were identified by full-scan gas chromatography-mass spectrometry (GC-MS) to confirm the conversions of [²H₂]GA₂₄ to [²H₂]GA₉ and of [²H₄]GA₉ to [²H₄]GA₄. The results show that GA₄ is biosynthesized from GA₂₄ via GA₉. In a cucumber hypocotyl elongation bioassay using cv. Yomaki, prohexadione (DOCHC), an inhibitor of 2-oxoglutaratedependent dioxygenase, inhibited the hypocotyl elongation caused by application of GA₉, while DOCHC enhanced the elongation caused by application of GA₄. These results indicate that GA₄ is a physiologically active GA and that the activity of GA₉ is due to its conversion to GA₄ in cucumber shoots.     

Endogenous gibberellins in the shoots of normal- and bush-typeCucumis sativus L.

Endogenous gibberellins (GAs) in the shoots of normal- (cv. Yomaki, YO) and bush-type (cv. Spacemaster, SP) cultivars of cucumber (Cucumis sativus L.) grown under natural conditions were analyzed. From both YO and SP grown for 40 days, after sowing, a series of C-13-H GAs including GA₄, GA₉, GA₁₅, GA₂₄, GA₂₅, GA₃₄, and GA₅₁ were identified by gas chromatography-mass spectrometry (GC-MS; full scan). In addition to the above GAs, GA₁₂ and GA₇₀ were similarly identified from both YO and SP grown for 61 days after sowing. The endogenous levels of GA₄ and GA₉, which are highly active in promoting cucumber hypocotyl elongation, were quantified by GC-selected ion monitoring (GC-SIM) using [²H₂]GA₄ and [²H₄]GA₉ as internal standards. No remarkable difference in terms of endogenous levels of GA_4/9 was observed between YO and SP in both growth stages (40 and 61 days after sowing).     

Effect of exo-16,17-dihydro-gibberellin A5 on gibberellin A20 metabolism in seedlings of dwarf rice (Oryza sativa L. cv. Tan-ginbozu)

Several of the 16,17-dihydro gibberellins (GAs) inhibit elongation in a variety of species. In a study of their mechanism of action we have investigated the effect of exo-16,17-dihydro-Ga₅ (diHGA₅) on the metabolism of GA₂₀ in dwarf rice (Oryza sativa cv. Tan-ginbozu). A mixture of [³H]- and [³H]-GA₂₀ (100 ng per plant) was applied in microdrops to 4 d old seedlings which were harvested 72 h later. Concurrent treatment with diHGA₅ at 100 ng or 333 ng per plant reduced GA₂₀-induced elongation of the second leaf sheath by 41–66%. There was a concomitant reduction in the amount of [²H₂]GA₁ present at harvest, measured by gas chromatography-mass spectrometry-selected ion monitoring. The [²H₂]GA₂₉ content was also reduced. There was no clear effect of diHGA₅ on the total radioactivity recovered, or on conversion of the [³H]GA₂₀ to putative [³H]GA conjugates, or on the amount of [²H₂]GA₂₀ found. No free [²H₂]GA₈ was detected. In other experiments there was little effect of diHGA₅ on elongation induced by treatment with GA₁. We conclude that diHGA₅ inhibited GA₂₀-induced elongation in dwarf rice shoots at least partly by reducing the 3-hydroxylation of GA₂₀ to GA₁.Abbreviations diHGA₅ = exo- 16, 17-dihydro-gibberellin A₅ - GA = gibberellin - GC-MS-SIM = gas chromatography-mass spectrometry-selected ion monitoring     

Ethylene-Mediated Regulation of Gibberellin Content and Growth in Helianthus annuus L

Elongation of hypocotyls of sunflower can be promoted by gibberellins (GAs) and inhibited by ethylene. The role of these hormones in regulating elongation was investigated by measuring changes in both endogenous GAs and in the metabolism of exogenous [³H]- and [²H₂]GA₂₀ in the hypocotyis of sunflower (Helianthus annuus L. cv Delgren 131) seedlings exposed to ethylene. The major biologically active GAs identified by gas chromatography-mass spectrometry were GA₁, GA₁₉, GA₂₀, and GA₄₄. In hypocotyls of seedlings exposed to ethylene, the concentration of GA₁, known to be directly active in regulating shoot elongation in a number of species, was reduced. Ethylene treatment reduced the metabolism of [³H]GA₂₀ and less [²H₂]GA₁ was found in the hypocotyls of those seedlings exposed to the higher ethylene concentrations. However, it is not known if the effect of ethylene on GA₂₀ metabolism was direct or indirect. In seedlings treated with exogenous GA₁ or GA₃, the hypocotyls elongated faster than those of controls, but the GA treatment only partially overcame the inhibitory effect of ethylene on elongation. We conclude that GA content is a factor which may limit elongation in hypocotyls of sunflower, and that while exposure to ethylene results in reduced concentration of GA₁ this is not sufficient per se to account for the inhibition of elongation caused by ethylene.     

A mutant gene that increases gibberellin production in brassica

A single gene mutant (elongated internode [ein/ein]) with accelerated shoot elongation was identified from a rapid cycling line of Brassica rapa. Relative to normal plants, mutant plants had slightly accelerated floral development, greater stem dry weights, and particularly, increased internode and inflorescence elongation. The application of the triazole plant growth retardant, paclobutrazol, inhibited shoot elongation, returning ein to a more normal phenotype. Conversely, exogenous gibberellin A₃ (GA₃) can convert normal genotypes to a phenotype resembling ein. The content of endogenous GA₁ and GA₃ were estimated by gas chromatography-selected ion monitoring using [²H]GA₁, as a quantitative internal standard and at day 14 were 1.5- and 12.1-fold higher per stem, respectively, in ein than in normal plants, although GA concentrations were more similar. The endogenous levels of GA₂₀ and GA₁, and the rate of GA₁₉ metabolism were simultaneously analyzed at day 7 by feeding [²H₂]GA₁₉ and measuring metabolites [²H₂]GA₂₀ and [²H₂]GA₁ and endogenous GA₂₀ and GA₁, with [²H₅]GA₂₀ and [²H₅]GA₁ as quantitative internal standards. Levels of GA₁ and GA₂₀ were 4.6- and 12.9-fold higher, respectively, and conversions to GA₂₀ and GA₁ were 8.3 and 1.3 times faster in ein than normal plants. Confirming the enhanced rate of GA₁ biosynthesis in ein, the conversion of [³H]GA₂₀ to [³H]GA₁ was also faster in ein than in the normal genotype. Thus, the ein allele results in accelerated GA₁ biosynthesis and an elevated content of endogenous GAs, including the dihydroxylated GAs A₁ and A₃. The enhanced GA production probably underlies the accelerated shoot growth and development, and particularly, the increased shoot elongation.     

Identification of endogenous gibberellins, and metabolism of tritiated and deuterated GA4, GA9 and GA20, in Scots pine (Pinus sylvestris) shoots

The application of gibberellin A_4/7 (GA_4/7) to the stem of previous-year (1-year-old) terminal shoots of Scots pine (Pinus sylvestris) seedlings has been observed to stimulate cambial growth locally, as well as at a distance in the distal current-year terminal shoot, but the distribution and metabolic fate of the applied GA_4/7, as well as the pathway of endogenous GA biosynthesis in this species, has not been investigated. As a first step, we analysed for endogenous GAs and monitored the transport and metabolism of labelled GAs 4, 9 and 20. Endogenous GAs from the elongating current-year terminal shoot of 2-year-old seedlings were purified by column chromatography and high-performance liquid chromatography and analysed by combined gas chromatography-mass spectrometry (GC-MS). GAs 1, 3, 4, 9, 12 and 20 were identified in the stem, and GAs 1, 3 and 4 in the needles, by full-scan mass spectrometry (GAs 1, 3, 4, 9 and 12) or selected-ion monitoring (GA₂₀) and Kovats retention index. Tritiated and deuterated GA₄, GA₉ or GA₂₀ were applied around the circumference at the midpoint of the previous-year terminal shoot, and metabolites were extracted from the elongating current-year terminal shoot, the application point, and the 1-year-old needles and the cambial region above and below the application point. After purification, detection by liquid scintillation spectrometry and analysis by GC-MS, it was evident that, for each applied GA, unmetabolised [²H₂]GA and [³H]radioactivity were present in every seedling part analysed. Most of the radioactivity was retained at the application point when [³H]GA₉ and [³H]GA₂₀ were applied, whereas the largest percentage of radioactivity derived from [³H]GA₄ was recovered in the current-year terminal shoot. It was also found that [²H₂]GA₉ was converted to [²H₂]GA₂₀ and to both [²H₂]GA₄ and [²H₂]GA₁, [²H₂]GA₄ was metabolised to [²H₂]GA₁, and [²H₂]GA₂₀ was converted to [²H₂]GA₂₉. The data indicate that for Pinus sylvestris shoots (1) GAs applied laterally to the outside of the vascular system of previous-year shoots not only are absorbed and translocated extensively throughout the previous-year and current-year shoots, but also are readily metabolised, (2) the GA metabolic pathways found are closely related to the endogenous GAs identified, and (3) GA₉ metabolism follows two distinctly different routes: in one, GA₉ is converted to GA₁ through GA₄, and in the other it is converted to GA₂₀, which is then metabolised to GA₂₉. The results suggest that the late 13-hydroxylation pathway is an important route for GA biosynthesis in shoots of Pinus sylvestris, and that the stimulation of cambial growth in Scots pine by exogenous GA_4/7 may be due to its conversion to GA₁, rather than to it being active per se.     

Stem elongation and gibberellins in alpine and prairie ecotypes of Stellaria longipes

The potential for gibberellins (GAs) to control stem elongation and itsplasticity (range of phenotypic expression) was investigated inStellaria longipes grown in long warm days. Gibberellinmetabolism and sensitivity was compared between a slow-growing alpine dwarfwithlow stem elongation plasticity and a rapidly elongating, highly plastic prairieecotype. Both ecotypes elongated in response to exogenous GA₁,GA₄ or GA₉, but surprisingly, the alpine dwarf wasrelatively unresponsive to GA₃. Endogenous GA₁,GA₃, GA₄, GA₅, GA₈, GA₉and GA₂₀ were identified and quantified in stem tissue harvested atcommencement, middle and end of the period of most rapid elongation. Theconcentration of GAs which might be expected to promote shoot elongation washigher during rapid elongation than toward its end for both ecotypes. Whilethere was a trend for certain GAs (GA₃, GA₄,GA₉, GA₂₀) to be higher in stems of the alpine ecotypeduring rapid elongation, that result does not explain the slower growth of thealpine ecotype and the faster growth of the prairie ecotype under a range ofconditions. To determine if the two ecotypes metabolized GA₂₀differently, plants were fed [²H]- or[³H]-GA₂₀. The metabolic products identified included[²H₂]-GA₁, -GA₈, -GA₂₉,-GA₆₀, -3-epi-GA₁, GA₁₁₈(-1-epi-GA₆₀) and -GA₇₇. The concentration of[²H₂]-GA₁ also did not differ between the twoecotypes and metabolism of [²H₂]- or[³H]-GA₂₀ was also similar. In the same experiments thepresence of epi-GA₁, GA₂₉, GA₆₀,GA₁₁₈ and GA₇₇ was indicated, suggesting that these GAsmay also occur naturally in S. longipes, in addition tothose described above. Collectively, these results suggest that while stemelongation within ecotypes is likely regulated by GAs, differences in GAcontent, sensitivity to GAs (GA₃ excepted), or GA metabolism areunlikely to be the controlling factor in determining the differences seen ingrowth rate between the two ecotypes under the controlled environmentconditionsof this study. Nevertheless, further study is warranted especially underconditions where environmental factors may favour a GA:ethylene interaction.     

Competition for in vitro [h]gibberellin a(4) binding in cucumber by substituted phthalimides: comparison with in vivo gibberellin-like activity

Certain N-substituted phthalimides (NSPs) have gibberellin (GA)-like activity in a number of GA bioassays. The interaction between representative NSPs and a protein fraction from cucumber (Cucumis sativus L.) hypocotyls that has GA-binding characteristics consistent with those expected of GA receptors was studied. Analysis of in vitro equilibrium saturation data indicated the presence of only one class of high affinity [³H]GA₄ binding sites (K_d ~ 30 nanomolar, n = 0.25 picomole per milligram of protein). In the presence of 6 or 60 micromolar 1-[3-chlorophthalimido]-cyclohexanecarboximide (AC-94,377), the K_d for [³H]GA₄ increased, whereas the maximum number of saturable [³H]GA₄ binding sites did not change significantly. The dissociation of [³H]GA₄ from its binding sites was complex and was best described by a bi-exponential equation. AC-94,377 did not affect the rates of [³H]GA₄ dissociation from its binding sites. These results implied that AC-94,377 and [³H]GA₄ compete for binding to the same sites. A correlation was observed between the activity of over 20 NSPs in the cucumber hypocotyl bioassay and their in vitro affinity for the GA binding sites. Our observations lend further support to the notion that certain GA binding proteins in cucumber cytosol are GA receptors and also provide a molecular explanation for the GA-like in vivo activity of some NSPs.     

Light intensity, gibberellin content and the resolution of shoot growth in Brassica

The role of gibberellins (GAs) in the regulation of shoot elongation is well established but the phytohormonal control of dry-matter production is poorly understood. In the present study, shoot elongation and dry-matter production were resolved by growing Brassica napus L. seedlings under five light intensities (photon flux densities) ranging from 25 to 500 μmol m⁻² s⁻¹. Under low light, plants were tall but produced little dry weight; as light intensity was increased, plants were progressively shorter but had increasing dry weights. Endogenous GAs in stems of 16- and 17-d-old plants were analyzed by gas chromatography-selected ion monitoring with [²H₂] internal standards. The contents of GAs increased dramatically with decreasing light intensity: GA₁, GA₃, GA₈ and GA₂₀ were 62, 15, 16 and 32 times higher, respectively, under the lowest versus highest light intensities. Gibberellin A₁₉ was not measured at 25 μmol m⁻² s⁻¹ but was 9␣times greater in the 75 compared to 500 μmol m⁻² s⁻¹ treatment. Shoot and hypocotyl lengths were closely positively correlated with (log) GA concentration (for example: r ² = 0.93 for GA₁ and hypocotyl length) but shoot dry matter was negatively correlated with GA concentration. The application of gibberellic acid (GA₃) produced elongation of plants grown under high light, indication that their low level of endogenous GA was limiting shoot elongation. Although endogenous GA₂₀ showed the greatest influence of light treatment, metabolism of [³H]GA₂₀ and of [³H]GA₁ was only slightly influenced by light intensity, suggesting that neither 2β- nor 3β-hydroxylation were points of metabolic regulation. The results of this study indicate that GAs control shoot elongation but are not directly involved in the regulation of shoot dry weight in Brassica. The study also suggests a role of GAs in photomorphogenesis, serving as an intermediate between light condition and shoot elongation response. Received: 18 June 1998 / Accepted: 29 July 1998     

Function and Substrate Specificity of the Gibberellin 3β-Hydroxylase Encoded by the Arabidopsis GA4 Gene

cDNA corresponding to the GA4 gene of Arabidopsis thaliana L. (Heynh.) was expressed in Escherichia coli, from which cell lysates converted [¹⁴C]gibberellin (GA)₉ and [¹⁴C]GA₂₀ to radiolabeled GA₄ and GA₁, respectively, thereby confirming that GA4 encodes a GA 3β-hydroxylase. GA₉ was the preferred substrate, with a Michaelis value of 1 μm compared with 15 μm for GA₂₀. Hydroxylation of these GAs was regiospecific, with no indication of 2β-hydroxylation or 2,3-desaturation. The capacity of the recombinant enzyme to hydroxylate a range of other GA substrates was investigated. In general, the preferred substrates contained a polar bridge between C-4 and C-10, and 13-deoxy GAs were preferred to their 13-hydroxylated analogs. Therefore, no activity was detected using GA₁₂-aldehyde, GA₁₂, GA₁₉, GA₂₅, GA₅₃, or GA₄₄ as the open lactone (20-hydroxy-GA₅₃), whereas GA₁₅, GA₂₄, and GA₄₄ were hydroxylated to GA₃₇, GA₃₆, and GA₃₈, respectively. The open lactone of GA₁₅ (20-hydroxy-GA₁₂) was hydroxylated but less efficiently than GA₁₅. In contrast to the free acid, GA₂₅ 19,20-anhydride was 3β-hydroxylated to give GA₁₃. 2,3-Didehydro-GA₉ and GA₅ were converted by recombinant GA4 to the corresponding epoxides 2,3-oxido-GA₉ and GA₆.Dwarf mutants with reduced biosynthesis of the GA plant hormones have been valuable tools in studies of the function of these compounds (Ross, 1994). In Arabidopsis thaliana, mutations at six loci (GA1-GA6) that result in reduced GA biosynthesis have been identified (Koorneef and van der Veen, 1980; Sponsel et al., 1997), and three of these loci have recently been cloned. The GA1 locus was isolated by genomic subtraction (Sun et al., 1992) and shown by heterologous expression in Escherichia coli to encode the enzyme that cyclizes geranylgeranyl diphosphate to copalyl diphosphate (Sun and Kamiya, 1994). This enzyme was formerly referred to as ent-kaurene synthase A but has been renamed copalyl diphosphate synthase (Hedden and Kamiya, 1997; MacMillan, 1997). The GA5 locus was shown to correspond to one of the GA 20-oxidase genes (Xu et al., 1995), the products of which catalyze the conversion of GA₁₂ to GA₉ and GA₅₃ to GA₂₀ (Phillips et al., 1995; Xu et al., 1995). GA 20-oxidases are 2-oxoglutarate-dependent dioxygenases that are encoded by small multigene families, members of which are differentially expressed in plant tissues (Phillips et al., 1995; Garcia-Martinez et al., 1997).The GA4 locus was isolated by T-DNA tagging and, on the basis of the derived amino acid sequence, was also shown to encode a dioxygenase (Chiang et al., 1995). Several lines of evidence indicate that the GA4 gene encodes a GA 3β-hydroxylase. Shoots of a ga4 mutant, all alleles of which are semidwarf, contained reduced concentrations of the 3β-hydroxy GAs GA₁, GA₄, and GA₈ compared with the Landsberg erecta wild type, whereas levels of immediate precursors to these GAs were elevated (Talon et al., 1990). Furthermore, metabolism of [¹³C]GA₂₀ to [¹³C]GA₁ was substantially less in the mutant than in the wild type (Kobayashi et al., 1994). In the present paper we confirm by functional expression of its cDNA in E. coli that GA4 encodes a GA 3β-hydroxylase. In addition, we determine the substrate specificity of recombinant GA4 using a number of C₂₀- and C₁₉-GAs and show by kinetic analysis that the enzyme has a higher affinity for GA₉ than for GA₂₀, which is consistent with the non-13-hydroxylation pathway predominating in Arabidopsis (Talon et al., 1990).     

Effects of 3-Methylgibberellin Analogs on Gibberellin 3β-Hydroxylases and Plant Growth

Six 3-methylgibberellin analogs were synthesized, and their effects on the GA 3β-hydroxylases from immature seeds of Phaseolus vulgaris and Cucurbita maxima, and/or on the growth of dwarf rice (Oryza sativa L. cv. Tan-ginbozu) and cucumber (Cucumis sativus L. cv. Spacemaster) were investigated. 3-Methyl-GA₅ and 2, 3-didehydro-3-methyl-GA₉· inhibited the conversion of [2, 3-³H2]GA₉ to [2-³H]GA₄ by GA 3β-hydroxylases from both P. vulgaris and C. maxima at 3 μM and higher. Their C/D-ring-rearranged isomers, 2, 3-didehydro-3-methyl-DGC and 16-deoxo-2, 3-didehydro-3-methyl-DGC, inhibited 3β-hydroxylation by the enzyme from P. vulgaris threefold more strongly than the non-C/D-ring-rearranged compounds, but exhibited no effect on 3β-hydroxylation by the enzyme from C. maxima. In a dwarf rice seedling assay, 3-methyl-GA₅ and 2, 3-didehydro-3-methyl-GA₉ promoted shoot elongation at doses of 300 ng/plant and higher, and 3α-methyl-GA₁ and 3α-methyl-GA₄ at doses of 30 ng/plant and higher. In contrast 2, 3-didehydro-3-methyl-DGC inhibited shoot growth to half that of the control at a dose of 300 ng/plant, and 16-deoxo-2, 3-didehydro-3-methyl-DGC showed no effect on growth. In a cucumber seedling assay, 3α-methyl-GA₄ promoted hypocotyl elongation at doses of 300 ng/plant and higher. The other C-3 methyl compounds showed no effect on the hypocotyl elongation of cucumber seedlings.     

Competition for in vitro [h]gibberellin a(4) binding in cucumber by gibberellins and their derivatives

The gibberellin (GA) binding properties of a cytosol fraction from hypocotyls of cucumber (Cucumis sativus L. cv National Pickling) were examined using a DEAE filter paper assay, [³H]GA₄, and over 20 GAs, GA derivatives and other growth regulators. The results demonstrate structural specificity of the binding protein for γ-lactonic C-19 GAs with a 3 β-hydroxyl and a C-6 carboxyl group. Additional hydroxylations of the A, C, or D ring of the ent-gibberellane skeleton and methylation of the C-6 carboxyl impede or abolish binding affinity. Bioassay data are generally supported by the in vitro results but significantly GA₉ and GA₃₆, both considered to be precursors of GA₄ in cucumber, show no affinity for the binding protein. The results are discussed in relation to the active site of the putative GA₄ receptor in cucumber.     

Kinetics of growth retardant and hormone interactions in affecting cucumber hypocotyl elongation

The capacities of indole-3-acetic acid (IAA) and gibberellin A₃ (GA₃) to counteract the inhibitory effects of (2-chloroethyl) trimethylammonium chloride (CCC), 2-isopropyl-4-dimethylamino-5-methylphenyl-1-piperidinecarboxylate methyl chloride (Amo-1618), and N,N-dimethylaminosuccinamic acid (B-995) on hypocotyl elongation in light-grown cucumber (Cucumis sativus L.) seedlings were investigated. One μg of GA₃ applied to the shoot tip was sufficient to completely nullify the effect of 10 μg of Amo-1618 or 25 μg of B-995 applied simultaneously to the shoot tip, and 10 μg of GA₃ completely counteracted the effect of 10⁻³ m CCC added to the root medium. One μg of IAA counteracted the effect of 10⁻³ m CCC in the root medium, but IAA did not nullify the action of either Amo-1618 or B-995. Experiments were conducted using 2 growth retardants simultaneously, which indicated that Amo-1618 and CCC inhibit a common process, namely GA biosynthesis, essential to hypocotyl elongation. However, since the effect of CCC was overcome by applications of both GA and IAA, growth retardation resulting from treatment with CCC apparently is not due solely to inhibition of GA biosynthesis. B-995 did not interact additively with either Amo-1618 or CCC, which suggests that B-995 affects a process different from those affected by the other 2 retardants. Thus, while inhibition evoked by B-995 is reversible by applied GA, the action of B-995 does not appear to be inhibition of GA biosynthesis.     

Accumulation of Gibberellin A1 and the Metabolism of Gibberellin A9 to Gibberellin A1 in a Phaeosphaeria sp. L487 Culture

Gibberellin A₁ (GA₁), which was identified as the major GA from the GA-producing fungus Phaeosphaeria sp. L487, was accumulated in the culture with a maltose-yeast extract medium, its amount in the culture filtrate being about 50 mg per liter after a 3-week culture. The new fungal biosynthetic pathway to GA₁ from GA₉ via GA₄ was elucidated by feeding experiments with synthetic [17-²H₂]GA₉ and [17-²H₂]GA₄.     

Localization of Stem Elongation Control in Cucumis sativus L

Reciprocal grafts, and applications of gibberellin (GA) and indoleacetic acid (IAA) were used to localize the site of control for stem elongation in cucumber (Cucumis sativus L.). Dwarf and tall plants were reciprocally grafted to determine influence of stems and roots on stem elongation. At 21 days there were no significant differences in length between stems grafted to their own roots and those grafted to roots of the other type. GA₃, GA₄₊₇, and IAA were applied to seedlings with and without live apical buds. Seedlings with live apical buds responded to level of added GA, but not to added IAA. GA₄₊₇ was more effective than GA₃. Hypocotyls of tall plants responded more to both GA treatments than did those of the dwarves when both types had live apical buds. When either GA₄₊₇ or IAA was applied to seedlings with dead apical buds, elongation of the hypocotyl responded to level of the growth regulator, but there was no difference in response between the dwarf and tall plants.     

Gibberellin metabolism in excised lettuce hypocotyls: Response to GA9 and the conversion of [3H]GA9

Elongation growth and gibberellin (GA₉) metabolism in excised hypocotyls of lettuce (Lactuca sativa L. cv. Arctic) were investigated. Exogenously supplied GA₉ stimulates elongation of hypocotyl sections and this response is intermediate between that elicited by GA₁ or GA₂₀ and GA_4/7 mixture. Although uptake of radioactivity from [³H]GA₉ increases with time, this gibberellin does not accumulate in the tissue but is rapidly converted to a compound with HPLC properties resembling those of [³H]GA₂₀. After 2 h incubation in [³H]GA₉, the presumptive GA₂₀ represents 90% of the acidic ethyl acetate-soluble radioactivity in the tissue. Radioactivity is also associated with an acidic butanol-soluble fraction containing two components resolvable by HVE. The major component is similar in electrophoretic properties to a GA-glucosyl ether while the other compares to a GA-glucosyl ester. Conversion of [³H]GA₉ to its [³H]GA₂₀-like metabolite is reduced by addition of carrier GA₉ or GA_4/7 at concentrations as low as 1 M, while GA₁, GA₃ and L-proline are without effect. Formation of the GA₂₀-like compound can be blocked by the addition of 2,2-dipyridyl, and this inhibitory effect of dipyridyl can be reversed by addition of Fe²⁺. At 200 M dipyridyl, elongation growth as well as [³H]GA₉ metabolism are reduced by 80%. The relationship of the metabolism of GA₉ to the growth response is discussed.Abbreviations AB butanol-soluble - AE ethyl-acetate-soluble - GA gibberellin - GA₁, GA₄ gibberellin A₁, gibberellin A₄, etc. - TLC thin layer chromatography - HPLC high performance liquid chromatography - HVE high voltage electrophoresis     

Endogenous Gibberellins in Elongating Shoots of Clones of Salix dasyclados and Salix viminalis

Elongating shoots of rapidly growing clones of Salix viminalis L. (clone 683-4) and Salix dasyclados Wimm. (clone 908) harvested in early August were analyzed for endogenous gibberellins (GA). Distribution of GA-like activity, determined by Tan-ginbozu dwarf rice microdrop bioassay after reverse phase C₁₈ high performance chromatography, was similar for both species. For S. dasyclados, combined gas chromatography-selected ion monotoring (GC-SIM) yielded identifications of GA₁, GA₈, GA₁₉, GA₂₀, and GA₂₉. Identifications of GA₄ and GA₉ were also made using co-injections of known amounts of [17, 17-²H₂]GAs. By bioassay, the main activity was GA₁₉-like in both species. Gibberellin A₁, GA₁₉, and GA₂₀ concentrations were approximated by GC-SIM using co-injections of known amounts of [17,17-²H₂]GAs. Both bioassay and GC-SIM results indicated very high concentrations of GA₁₉ and GA₂₀ (about 6000 nanograms per kilogram fresh weight shoot tissue using GC-SIM: 800 ng using bioassay), compared to the concentration of GA₁ (about 130 nanograms per kilogram fresh weight using either GC-SIM or bioassay).     

Exogenously applied GA4 is converted to GA1 in seedlings of Salix

Gibberellin A₄ (GA₄) is biologically active in Salix pentandra and is able to induce stem elongation in seedlings grown under short day (SD) conditions, as well as in seedlings grown under long day (LD) conditions and treated with a growth retardant BX-112. [³H₂]GA₄ and [²H₂]GA₄ were applied to seedlings and leaf and stem explants of S. pentandra, and metabolites were studied using HPLC and GC-MS. After application of [³H₂]GA₄ to seedlings of S. pentandra, one of the three main radioactive metabolites in the free acid fraction had retention properties similar to GA₁. Using [²H₂]GA₄, this compound was identified by GC-MS with SIM as [²H₂]GA₁ both from short day-grown and BX-112-treated seedlings, as well as in leaf and stem explants. After injection of GA₄ into a mature leaf, GA₁ was mainly found in the elongating stem tissue. Thus, the possibility that the biological activity of GA₄ in Salix is due to its conversion to GA₁ cannot be excluded.     

Calcium and gibberellin-induced elongation of lettuce hypocotyl sections

The relationship between calcium ions and gibberellic acid (GA₃)-induced growth in the excised hypocotyl of lettuce (Lactuca sativa L.) was investigated. The short-term kinetics of growth responses were measured using a linear displacement transducer. Test solutions were added either as drops to the filter paper on which the hypocotyl stood (non-flow-past) or by switching solution flowing past the base of hypocotyl (flow-past), resulting in differences in growth behavior. Drops of CaCl₂ added at a high concentration (10 mM) inhibited growth within a few minutes. This inhibition was reversed by ethylenediaminetetraacetic acid (EDTA). Drops of EDTA or ethyleneglycol-bis(2-aminoethylether)-tetraacetic acid caused a rapid increase in growth rate. Growth induced by EDTA was not further promoted by GA₃. A continuous H₂O flow resulted in growth rates comparable to those in response to GA₃. Addition of CaCl₂ to the flow-past medium inhibited growth and this inhibition was reversed by a decrease in CaCl₂ concentration. The growth rate was found to be a function of CaCl₂ concentration. When a constant CaCl₂ concentration was maintained by the flow-past medium, a shift in pH from 5.5 to 4.25 had no obvious effect on hypocotyl elongation. Gibberellic acid was found to reverse the inhibitory effect of CaCl₂, causing an increase in growth rate similar to that found previously when GA₃ was added to hypocotyls grown in H₂O under non-flow-past conditions. We propose that gibberellin controls extension growth in lettuce hypocotyl sections by regulating the uptake of Ca²⁺ by the hypocotyl cells.Abbreviations EDTA ethylenediaminetetraacetic acid - EGTA ethyleneglycol-bis(2-aminoethylether)-tetraacetic acid - GA gibberellin - GA₃ gibberellic acid - IAA indole-3-acetic acid     

Gibberellins and parthenocarpic ability in developing ovaries of seedless mandarins

Satsuma (Citrus unshiu [Mak] Marc.) and Clementine (Citrus reticulata [Hort.] Ex. Tanaka, cv Oroval) are two species of seedless mandarins differing in their tendency to develop parthenocarpic fruits. Satsuma is a male-sterile cultivar that shows a high degree of natural parthenocarpy and a high fruit set. Seedless Clementine varieties are self-incompatible, and in the absence of cross-pollination show a very low ability to set fruit. The gibberellins (GAs) GA53, putative 17-OH-GA₅₃, GA₄₄, GA₁₇, GA₁₉, GA₂₀, GA₂₉, GA₁, 3-epi-GA₁, GA₈, GA₂₄, GA₉, and GA₄ have been identified from developing fruits of both species by full-scan combined gas chromatography-mass spectrometry. Using selected ion monitoring with [²H₂]- and [¹³C]-labeled internal standards, the levels of GA₅₃, GA₄₄, GA₁₉, GA₂₀, GA₁, GA₈, GA₄, and GA₉ were determined in developing ovaries at anthesis and 7 days before and after anthesis, from both species. Except for GA8, levels of the 13-hydroxy-GAs were higher in Satsuma than in Clementine, and these differences were more prominent for developing young fruits. At petal fall, Satsuma had, on a nanograms per gram dry weight basis, higher levels of GA₅₃ (10.4x), GA₄₄ (13.9x), GA₁₉ (3.0x), GA₂₀ (11.2x), and GA₁ (2.0x). By contrast, levels of GA₈ were always higher in Clementine, whereas levels of GA₄ did not differ greatly. Levels of GA₉ were very low in both species. At petal fall, fruitlets of Satsuma and Clementine contained 65 and 13 picograms of GA₁, respectively. At this time, the application of 25 micrograms of paclobutrazol to fruits increased fruit abscission in both varieties. This effect was reversed by the simultaneous applications of 1 microgram of GA₃. GA₃ alone improved the set in Clementine (13x), but had little influence on Satsuma. Thus, seedless fruits of the self-incompatible Clementine mandarin may not have adequate GA levels for fruit set. Collectively, these results suggest that endogenous GA content in developing ovaries is the limiting factor controlling the parthenocarpic development of the fruits.