The identification and metabolism of GA9 and its metabolites in seed coats and shoots of pea,line G2

[³H]gibberellin A₉ was applied to shoots or seed parts of G2 pea to produce radiolabeled metabolites. These were used as markers during purification for the recovery of endogenous GA₉ and its naturally occurring metabolites. GA₉ and its metabolites were purified by HPLC, derivatized and examined by GC-MS. Endogenous GA₉, GA₂₀, GA₂₉ and GA₅₁ were identified in pea shoots and seed coats. GA₅₁-catabolite and GA₂₉-catabolite were also detected in seed coats. GA₇₀ was detected in seed coats following the application of 1 g of GA₉. Applied [³H]GA₉ was metabolized through both the 13-hydroxylation and 2-hydroxylation pathways. Labeled metabolites were tentatively identified on the basis of co-chromatography on HPLC with endogenous compounds identified by GC-MS. In shoots [³H]GA₅₁ and [³H]GA₅₁-catabolite were the predominant metabolites after 6 hrs, but by 24 hrs there was little of these metabolites remaining, while [³H]GA₂₉-catabolite and an unidentified metabolite predominated. In seed coats [³H]GA₅₁ was the initial product, later followed by [³H]GA₅₁-catabolite and an unidentified metabolite (different from that in shoots), with lesser amounts of [³H]GA₂₀, [³H]GA₂₉ and [³H]GA₂₉-catabolite. [³H]GA₇₀ was a very minor product in both cases. [³H]GA₉ was not metabolized by pea cotyledons.Edited by T.J. Gianfagna.Author for correspondence     

Comparison of gas chromatography-mass spectrometry,radioimmunoassay and bioassay for the quantification of gibberellin A9 in norway spruce (Picea abies (L.) Karst.)

Quantitative estimates of gibberellin A₉ in Norway spruce extracts obtained by gas chromatography-mass spectrometry, radioimmunoassay (RIA_ and bioassay were compared after successive purifications of the extracts. The extracts were assayed in several dilutions with and without the addition of standard gibberellin A₉, thus showing the effect of extract components on the response of the assays. Radioimmunoassay produced estimates comparable to gas chromatography-mass spectrometry after one purification step by high-performance liquid chromatography. Extracts purified by polyvinylpyrrolidone-column chromatography and solvent partitioning but not high-performance liquid chromatography resulted in inaccurate RIA estimates. The performance of the RIA could be monitored by logit-log transformations of the standard curve and extract dilution curve and by calculating the slope of the standard addition curve. It was, however, not possible to correct for the interference caused by extract components by the standard addition procedure. Quantifications by Tan-ginbozu dwarf-rice bioassay were accurate, but a large and unpredictable variation makes it unsuitable for quantitative determinations.Abbreviations FW fresh weight - GA₉ gibberellin A₉ - GA₉–Me methylated GA₉ - GC-MS gas chromatography-mass spectrometry - HPLC high performance liquid chromatography - MID multiple-ion detection - RIA radioimmunoassay     

Biomolecule-functionalized magnetic nanoparticles for flow-through quartz crystal microbalance immunoassay of aflatoxin B<Subscript>1</Subscript>

A flow-through quartz crystal microbalance (QCM) immunoassay method has been developed based on aflatoxin B₁ antibody (anti-AFB₁)-functionalized magnetic core-shell Fe₃O₄/SiO₂ composite nanoparticles (bionanoparticles) in this study. To construct such an assay protocol, anti-AFB₁, as a model protein, was initially covalently immobilized onto the Fe₃O₄/SiO₂ surface, and then the functionalized nanoparticles were attached to the surface of the QCM probe with an external magnet. The binding of target molecules onto the immobilized antibodies decreased the sensor’s resonant frequency, and the frequency shift was proportional to the AFB₁ concentration in the range of 0.3–7.0 ng/ml. The regeneration of the developed immunosensor was carried out via attaching or detaching the external magnet from the detection cell. In addition, the selectivity, reproducibility, and stability of the proposed immunoassay system were acceptable. Compared with the conventional ELISAs, the proposed immunoassay system was simple and rapid without multiple labeling and separation steps. Importantly, the proposed immunoassay method could be further developed for the immobilization of other antigens or biocompounds.     

The partial purification and characterization of a gibberellin C-20 hydroxylase from immature Pisum sativum L. seeds

A gibberellin (GA) C-20 hydroxylase that catalyses the conversion of GA₅₃ to GA₄₄ was purified from developing pea embryos by ammonium-sulfate precipitation, gel filtration and anion-exchange column chromatography. The purification was about 270-fold and 15% of the enzymic activity was recovered. The relative molecular mass was 44000 by Sephadex G-200 gel filtration. The apparent Michaelis constant was 0.7 M and the isoelectric point was 5.6–5.9. The enzymic activity was optimal at pH 7.0 2-Oxoglutarate and ascorbate were required for activity. Low concentrations of Fe²⁺ stimulated the reaction, but externally added Fe²⁺ was not essential, even in the most purified preparation. Catalase and bovine serum albumin also stimulated. Dithiothreitol preserved the activity during purification but was not needed during incubation. In fact, the simultaneous presence of dithiothreitol and Fe²⁺ in the incubation mixture was inhibitory to the purified enzyme. The cofactor requirements are typical for those of 2-oxoglutarate-dependent dioxygenases.When the incubation time was long enough, GA₅₃ was converted to both GA₄₄ and GA₁₉. The proportions of these two products remained constant throughout the purification, but this does not necessarily mean that their formations is catalysed by a single enzyme. Sodium dodecyl sulfatepolyacrylamide gel electrophoresis showed that the final preparation contained several proteins. Although the most prominent protein band was located within the range expected for the enzyme on the grounds of its molecular weight, this band did not represent the enzyme, since it separated from the GA C-20 hydroxylase activity on ultrathin-layer isoeletric focusing.Abbreviation BSA bovine serum albumin - DEAE diethylaminoethyl - DTT dithiothreitol - EDTA ethylenediamine-tetraacetic acid - GA_n gibberellin A_n - HPLC high-performance liquid chromatography - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - Tris 2-amino-2-(hydroxymethyl)-1,3-propanediol     

Determination of femtomol quantities of gibberellic acid by radioimmunoassay

A highly sensitive and specific radioimmunoassay which allows the detection of as little as 5 fmol (2 pg) of gibberellic acid (GA₃) in crude plant extracts is described. Antisera of high affinity and titer were obtained by immunizing rabbits with a conjugate of carboxyl-coupled GA₃ and bovine serum albumin. [¹²⁵I]Gibberellic acid-[N-(p-hydroxybenzyl) putrescine]amide of high specific activity, used as the immunotracer, is readily displaced by gibberellic acid methyl ester but not by free gibberellic acid. Thus, methylation of extracts prior to analysis is required. The assay is very specific; besides GA₃, only the closely related GA₇ is highly immunoreactive. Various gibberellins, related compounds, as well as other classes of plant hormones do not interfere with the assay. Levels of immunoreactive gibberellins (GA₃, GA₇) in actively growing tissues, among them cell suspension cultures of 33 different species, were determined.Abbreviations ABA abscisic acid - GC-MS gas chromatography-mass spectroscopy - TLC thin-layer chromatography - GA gibberellin Part 17 in the Series: Use of Immunoassay in Plant Science     

Enzyme-amplified enzyme-linked immunosorbent assay for gibberellin A4 based on the NAD-dependent redox cycle

An antiserum against gibberellin A₄ (GA₄) raised in rabbits and its partially purified antibodies were used to develop radioimmunoassay (RIA) and indirect enzyme-linked immunosorbent assays (ELISAs) for GA₄. Of three immunoassays tested, an ELISA based on the NAD-dependent redox cycle (enzyme-amplified ELISA) had highest sensitivity. Levels of methylated GA₄ detected by this most sensitive method ranged from 0.1 fmol/assay (3.5 fg/assay) to 0.1 pmol/assay (3.5 pg/assay) suggesting applicability of this method to the detection of gibberellins in purified plant extracts.     

Partial Purification and Characterization of Gibberellin 3{beta}-hydroxylase from Immature Seeds of Phaseolus vulgaris L.

Gibberellin 3/ß-hydroxylase,a 2-oxoglutarate-dependentdioxygenase that catalyzes the hydroxylation of GA₂₀ to GA₁,was purified 313-fold from immature seeds of Phaseolus vulgarisL. The mol wt of the enzyme was estimated to be 42,000 by gelfiltration HPLC and SDS-polyacrylamide gel electrophoresis.The enzyme exhibited maximum activity at pH 7.7. The Km valuesfor [2,3-³H]GA20 and [2,3-³H]GA, were 0.29µu and 0.33µm, respectively. The enzyme requires 2-oxoglutarate asa cosubstrate; the K_m value for 2-oxoglutarate was 250µMusing [³H]- GA₂₀ as a substrate. Fe²⁺ and ascorbate significantlyactivated the enzyme at all purification steps, while catalaseand BSA activated the purified enzyme only. The enzyme was inhibitedby divalent cations Mn²⁺, Co²⁺, Ni²⁺, Cu²⁺, Zn²⁺, Cd²⁺ and Hg²⁺.3ß-Hydroxylation of [³H]- GA₂₀ was also inhibitedby non-radioactive GA₅, GA₉,GA₁₅, GA₂₀ and GA₄₄. The possiblesite of 3ß-hydroxylation in gibberellin biosynthesisis discussed in terms of the substrate specificity of partiallypurified gibberellin 3ß-hydroxylase. (Received February 29, 1988; Accepted June 3, 1988)     

Enzymic glucosylation of gibberellins

Starting from the well-known conversion of exogenously applied free gibberellic acid (GA₃) to its 3(O)-glucoside by intact immature fruits of runner beans (Phaseolus coccineus L.), a protein fraction has been prepared from this plant material possessing glucosylating activity towards GAs. This glucosyltransferase is located in the pericarp only and utilizes preferably UDP-glucose as a sugar donor. The product formed enzymically from GA₃ and UDP-glucose could be identified by derivatization and comparison with the authentic compound to be GA₃-3(O)-glucoside. Among 15 native or chemically modified GAs, the enzyme glucosylates only GA₃ and to a lower extent GA₇ and GA₃₀, indicating a high enzyme specificity with regard to the A ring of gibberellins. The physiological significance of the enzymic GA₃-3(O)-glucoside formation inPhaseolus coccineus is not clear, since this glucoside is not known to be endogenous in this plant. The enzyme preparation did not glucosylate substances of phenolic structure, such as hydroquinone, aesculetin, and quercetin. Glucosylation of GA₃ was achieved also by enzyme preparations fromVigna sinensis and from cell suspension cultures ofDigitalis purpurea. A number of other plant materials showed no activity.Gibberellins 100. For part 99 see Liebisch et al. 1984a.     

Identification of gibberellin A4 in Pisum sativum L. and the effects of applied gibberellins A9, A4, A5 and A3 on the le mutant

Gibberellin A₄ (GA₄) was identified for the first time in the garden pea (Pisum sativum) L.), by gas chromatography-mass spectrometry. However, in wild-type shoots the level of GA₄ was only about 6% of the level of GA₁, and it is therefore unlikely that GA₄ plays a major role per se in the control of pea stem elongation. In shoots of the le mutant, GA₄ was not detected, while the level of GA₉ was approximately twice that found in the wild-type. The le mutation also markedly reduced the elongation response to applied GA₉. It appears, therefore, that in Pisum the le mutation blocks the 3-hydroxylation of GA₉ to GA₄, in addition to the 3-hydroxylation of GA₂₀ to GA₁. In contrast, the le mutation did not reduce the response to applied GA₅, suggesting the step GA₅ to GA₃ is not catalysed by the enzyme controlled by the Le gene. The step GA₅ to GA₃ was confirmed in peas by metabolite analysis after treatment with deuterated GA₅.     

Enhancement of somatic embryogenesis frequency by gibberellic acid in fennel

The effect of GA₃ on somatic embryogenesis from petiole fragments excised from micropropagated fennel plantlets was studied. Explants were maintained for 4 weeks on an induction medium containing, 2,4-d and kinetin and were then transferred to a medium devoid of these growth regulators to allow embryo development. The addition of autoclaved or filter-sterilized GA₃ to the induction medium or to the embryo development medium increased the number of embryogenic explants. No positive effect was observed when GA₃ was added to the micropropagation medium of the mother plantlets. Gibberellic acid also counteracted the inhibiting effect of continuous light on the number of embryogenic explants. Moreover, the embryogenic frequency of petiole explants from several genotypes previously considered as poorly reacting was highly enhanced by GA₃.Abbreviations BA 6-benzyladenine - 2,4-d 2,4-dichlorophenoxyacetic acid - GA₃ Gibberellic acid - IAA Indole-3-acetic acid - IBA Indole-3-butyric acid     

Gibberellins and antheridiogen on sex in Blechnum spicant L.

The role of gibberellins (GAs) in determining sex in the gametophyte of the fern Blechnum spicant L. was studied through (a) the effect of exogenous GA₄₊₇ and GA₃ (b) quantitation of the endogenous levels of GA_1, GA₃, GA₄, GA₇, GA₉, and GA₂₀ in male and female gametophytes, and (c) the effect of flurprimidol, a GAs biosynthesis inhibitor of the steps of oxidation of ent-kaureno to ent-kaurenoic acid. Our results show that GA₄₊₇ had a slight effect of inducing either male or female sexual organs, antheridia and archegonia, respectively. The endogenous GAs content was not significantly different between sexes, but the GA₄, GA₇, and GA₂₀ levels were raised above those of the other GAs in both sexes. Neither antheridiogen biosynthesis nor antheridia formation was inhibited by flurprimidol. Gametophytes regenerated from homogenized mature gametophytes (HG) show a different physiological behavior than spore-derived gametophytes. In the first case, gametophytes are males and synthesize antheridiogen before they attain maturity, in contrast to what occurs in spore-derived gametophytes which are females and synthesize antheridiogen when mature.     

Interconversion of gibberellin A4 to gibberellins A1 and A34 by dwarf rice,cultivar Tan-ginbozu

Summary Interconversion of GA₄ to GA₁ and GA₃₄ occurred within 24 h of application of 1,2-[³H]-GA₄ to seedlings of dwarf rice, cv. Tan-ginbozu. Identification was made by direct comparison of the trimethylsilyl ether derivatives of the methyl esters of Silica-gel partition-column fractions on gas-liquid radiochromatography with derivatized GA₁ and GA₃₄ standards on three columns: 2% QF-1, 2% SE-30, and 1% XE-60. GA₂, an artifact of the purification and chromatography system, may also be formed by the plant. The conversions from GA₄ to GA₁ and GA₃₄ are single hydroxylations. At least two unidentified radioactive products were also formed by the plant. Interconversions were in the order of 0.3 to 0.8% of applied [³H]-GA₄.Abbreviations GA gibberellin - GLC gas-liquid chromatography - GLRC gasliquid radiochromatography - TMSMe trimethylsilyl ether of methyl ester - TLC thin-layer chromatography     

Enzymic glucosylation of gibberellins

Starting from the well-known conversion of exogenously applied free gibberellic acid (GA₃) to its 3(O)-glucoside by intact immature fruits of runner beans (Phaseolus coccineus L.), a protein fraction has been prepared from this plant material possessing glucosylating activity towards GAs. This glucosyltransferase is located in the pericarp only and utilizes preferably UDP-glucose as a sugar donor. The product formed enzymically from GA₃ and UDP-glucose could be identified by derivatization and comparison with the authentic compound to be GA₃-3(O)-glucoside. Among 15 native or chemically modified GAs, the enzyme glucosylates only GA₃ and to a lower extent GA₇ and GA₃₀, indicating a high enzyme specificity with regard to the A ring of gibberellins. The physiological significance of the enzymic GA₃-3(O)-glucoside formation inPhaseolus coccineus is not clear, since this glucoside is not known to be endogenous in this plant. The enzyme preparation did not glucosylate substances of phenolic structure, such as hydroquinone, aesculetin, and quercetin. Glucosylation of GA₃ was achieved also by enzyme preparations fromVigna sinensis and from cell suspension cultures ofDigitalis purpurea. A number of other plant materials showed no activity.     

Gibberellin metabolism in excised lettuce hypocotyls: Evidence for the formation of gibberellin A1 glucosyl conjugates

The properties of the water-soluble metabolites of [³H]gibberellin A₁ ([³H]GA₁) from lettuce (Lactuca sativa L.) hypocotyls were compared with those of authentic samples of gibberellin (GA) glucosyl esters and ethers. Partitioning against l-butanol at high and low pH was not an efficient method of differentiating between ester and ether conjugates of GA₁ or GA₃. Extraction into l-butanol at pH 2.5 was, however, useful as a group purification step. Gel-filtration on acrylamide indicated a mean molecular weight of ca. 600 for the polar material and high-voltage electrophoresis separated two compounds (LH 1 and LH 2) with differing charge properties. Both metabolites incorporated ¹⁴C from glucose and ³H from GA₁. Subsequent enzymatic hydrolysis of LH 1 released material with identical properties to [¹⁴C]glucose together with a second uncharacterised component. Feeding with [³H]GA₁ methyl ester greatly reduced the formation of LH 1 but not LH 2. The metabolites were provisionally identified as GA₁-glucosyl ester (LH 1) and GA₁-glucosyl ether (LH 2).Abbreviations GA gibberellin - LH1 GA₃-glucosyl ester - LH2 GA₁-glucosyl ether - HVE high voltage paper electrophoresis - TLC thin-layer chromatography     

Gibberelic acid-mediated activation of monophenolase in de-embryonated half-seeds of wheat (Triticum Aestivum)

Stimulation of monophenolase activity was observed when de-embryonated prewashed half-seeds of wheat were imbibed in a solution of gibberellic acid (GA₃, 10^t?5 M). Crude extracts, prepared from GA₃-treated half-seeds, showed ca a two-fold increase in monophenolase activity over the controls at pH 6.6, while a dramatic rise in enzyme activity (seven- to nine-fold) was observed at pH 9.0. The partially purified (NH₄)₂SO₄ fraction precipitate (30–50% saturation) also showed enhancement of enzyme activity at pH 9.0 in GA₃-treated half-seeds, while in controls, there was negligible activity at this pH. Administration of five amino acid analogues (1 mM each) to half-seeds showed no significant inhibition of GA₃-stimulated monophenolase activity, but proved very effective in decreasing (86% inhibition) the GA₃-induced amylase activity. This indicated that the hormone-regulated monophenolase activity was not dependent on de novo protein synthesis. Treatment of half-seeds with GA₃ modified the monophenolase and altered the electrophoretic pattern. The enzyme was relatively thermostable at 55° and the pH optimum was shifted from pH 7.0 to 9.0. In addition, the GA₃-treated half-seeds showed relatively high stability of enzyme activity in the presence of (NH₄)₂S0₄ ions. These alterations in the GA₃-stimulated nomophenolase suggest there is activation of preformed enzyme molecules. The appearance of slow-migrating multiple forms on acrylamide gels, in response to GA₃ treatment, is probably due to the association of fast-migrating forms. Such oligomerization could result in a conformational change leading to enzyme activation. This may be an adaptive mechanism so that the enzyme can function with varying temperature, pH and ionic strength during early stages of seed germination.     

GA3和Spd对杜鹃开花期光合特性和抗氧化系统的影响

为探讨GA_3和Spd对杜鹃(Rhododendron simsii)开花花期和开花品质的影响,研究了外源GA_3和Spd对杜鹃开花期光合特性和抗氧化系统的变化。结果表明,外源GA_3对花期有显著的提前作用,Spd对花期有明显的延迟作用,但两者均使花期延长、花径增大且成花率提高。GA_3和Spd处理提高了花期叶片的光合色素含量和净光合速率(Pn)、气孔导度(Gs)和胞间CO_2浓度(Ci);GA_3处理提高了叶片的蒸腾速率(Tr),而Spd使叶片的Tr下降,两者均有效缓解了末花期叶绿素含量的下降。GA_3和Spd处理显著降低了花瓣MDA含量,提高了抗氧化酶SOD、POD和CAT活性,并减缓了末花期SOD的下降,有效延缓了衰老进程,延长花期。以1 600 mg L~(–1) GA_3和0.10 mmol L~(-1) Spd处理效果较好,能有效提高杜鹃花的观赏品质。     

外源GA3对紫斑牡丹幼苗叶片解剖结构与光合特性及内源激素含量的影响

以1年生紫斑牡丹幼苗为试验材料,采用不同浓度(0、100、300、500 mg/L)赤霉素(GA_3)喷施叶片处理,通过透射电镜、扫描电镜、光学显微镜观察幼苗叶片解剖结构,光合仪测定幼苗光合参数并以酶联免疫吸附法测叶片内源激素含量,探究外源GA_3对紫斑牡丹幼苗叶片解剖结构、光合特性和内源激素水平的影响。结果表明:(1)低浓度GA_3处理的紫斑牡丹叶肉细胞增大,栅栏组织外层细胞中叶绿体数量增加,高浓度GA_3处理则与之相反;GA_3处理叶片的栅栏组织/海绵组织比值(P/S)、组织结构紧密度(CTR)均下降,而其组织结构疏松度(SR)增加;GA_3处理的幼苗叶片的叶肉细胞内各叶绿体大小显著大于对照,随着GA_3处理浓度增加,紫斑牡丹叶肉细胞内叶绿体的体积趋于增大,类囊体垛叠凝聚逐渐松散,叶绿体上淀粉颗粒在300 mg/L GA_3处理中较明显;叶片气孔长度、宽度、气孔器大小、气孔开度和气孔密度随着GA_3浓度升高先升高后下降,同时叶片上表皮角质层厚度随GA_3浓度的升高而增加。(2)紫斑牡丹叶片净光合速率(P_n)、气孔导度(Cond)、蒸腾速率(T_r)、水分利用率(WUE)在100和300 mg/L GA_3处理下大都显著高于对照,且300 mg/L GA_3处理显著高于其余处理,而其在500 mg/L GA_3处理下显著低于对照。(3)紫斑牡丹叶片脱落酸(ABA)和吲哚乙酸(IAA)含量均在500 mg/L GA_3下显著高于对照,而在其余浓度处理下不同程度低于对照,叶片内源玉米素核苷(ZR)和GA_3含量均在300 mg/L GA_3处理下显著高于其余处理和对照,而其余处理相比对照均无显著变化;叶片的ZR/ABA、ZR/IAA、ZR/GA_3和(IAA+GA_3+ZR)/ABA比值都在300 mg/L GA_3处理下显著高于其他处理,叶片的IAA/ABA和ABA/GA_3比值均在500 mg/L GA_3处理下显著高于其他处理。研究发现,适宜浓度外源GA_3处理,能显著提高紫斑牡丹幼苗叶片光合速率、水分利用效率及蒸腾速率,调节植物体内源激素的含量及平衡,从而使叶片能合成较多有机物,促进幼苗生长。     

GA3-Modulated multiple forms of monophenolase in wheat seed

Multiple forms of monophenolase in wheat half-seeds were separated by molecular sieving on Sephadex G-200. A single molecular form of monophenolase was observed in control, while two multiple forms were present in GA₃-treated wheat half-seeds. A high MW (200 000 or above) multiple form (activity peak I) which eluted soon after the void volume was exclusively present in GA₃-treated half-seeds. The second activity peak (peak II) was a low MW (45 000) multiple form and its elution profile coincided in control and GA₃-treated wheat half-seeds. Both the multiple forms of monophenolase in GA₃-treated wheat half-seeds showed a pH optimum at 9.0, while the optimum enzyme activity of the control molecular form (peak II) was at pH 7.0. This indicated that the treatment of wheat half-seeds with GA₃ brought about a structural modification in monophenolase. The in vitro addition of trypsin enhanced the control of the molecular form of monophenolase but this treatment failed to alter the activity of multiple forms in GA₃-treated half-seeds. This differential response of monophenolase towards trypsin could be ascribed to a conformational change of the enzyme in hormone-treated half-seeds. Brief exposure of the enzyme preparation to urea (6 M) brought about an irreversible activation of monophenolase both in control and GA₃-treated wheat half-seeds.     

Seed effects on gibberellin metabolism in pea pericarp

Pea fruit (Pisum sativum L.) is a model system for studying the effect of seeds on fruit growth in order to understand coordination of organ development. The metabolism of ¹⁴C-labeled gibberellin A₁₂ (GA₁₂) by pea pericarp was followed using a method that allows access to the seeds while maintaining pericarp growth in situ. Identification and quantitation of GAs in pea pericarp was accomplished by combined gas chromatography-mass spectrometry following extensive purification of the putative GAs. Here we report for the first time that the metabolism of [¹⁴C]GA₁₂ to [¹⁴C]GA₁₉ and [¹⁴C]GA₂₀ occurs in pericarp of seeded pea fruit. Removal of seeds from the pericarp inhibited the conversion of radiolabeled GA₁₉ to GA₂₀ and caused the accumulation of radiolabeled and endogenous GA₁₉. Deseeded pericarp contained no detectable GA₂₀, GA₁, or GA₈, whereas pericarp with seeds contained endogenous and radiolabeled GA₂₀ and endogenous GA₁. These data strongly suggest that seeds are required for normal GA biosynthesis in the pericarp, specifically the conversion of GA₁₉ to GA₂₀.