In-vivo binding of radioactive gibberellins in dwarf pea shoots

Summary When shoots of 6-day-old, dark-grown peas were excised 30 mm below the apex and floated on a solution of radioactive gibberellin A ₁ (³H-GA₁) or radioactive gibberellin A₅ (³H-GA₅), more radioactivity accumulated in the apical part of the stem which responds to GA than in the basal, unresponsive region. The accumulation of ³H-GA₁ was, however, less pronounced than the accumulation of ³H-GA₅. GA derivatives of very low biological activity were not taken up preferentially by the apical region of the stem. Light, which lowers the responsiveness of dwarf peas to GA₁ and particularly to GA₅, also reduced the accumulation of these GAs in the apical part of the stem. Sections from the GA-responsive region were able to retain a higher level of GA₅ than sections from the non-responsive, basal region. The accumulation and retention of GA in the hormone-responsive tissue may be due to binding of the hormone to specific GA receptors.This work was supported by the United States Atomic Energy Commission under Contract AT (11-1)-1338.     

On the uptake, metabolism and retention of [h] gibberellin a(1) by barley aleurone layers at low temperatures

Barley (c.v. Himalaya) aleurone layers were incubated in [³H]gibberellin A₁ (GA₁) at low temperatures. At 3 and 4 C, ³H-activity was steadily accumulated in aleurone layers, and this accumulation was correlated with significant [³H]GA₁ metabolism. At 1 and 1.5 C, metabolism could not be detected, and at these temperatures aleurone layers equilibrated with the [³H]GA₁ concentration in the incubation medium. At equilibrium, the total amount of ³H-activity per unit volume in the aleurone layers was higher than in the incubation medium. Aleurone layers incubated at 0.5 C for 72 hours with [³H]GA₁ in the presence of saturating levels of carrier GA₁ consistently retained lower levels of ³H-activity than when incubated in [³H]GA₁ alone. The retention of [³H]GA₁ was unaffected by saturating levels of carrier GA₈. GA₁ retained by barley aleurone layers that were incubated at 0.5 C for 72 hours was able to induce α-amylase synthesis when aleurone layers were subsequently washed and transferred to a gibberellin-free medium at 25 C.     

Metabolism of Tritiated Gibberellin A(9) by Shoots of Dark-grown Dwarf Pea, cv. Meteor

Tritium-labeled gibberellin A₉ (³H-GA₉) was metabolized by etiolated shoots of dwarf pea (Pisum sativum cv. Meteor) to GA₂₀, GA₁₀, 2,3-dihydro-GA₃₁, and a number of highly polar, acidic GA-like substances. Identifications were made by gasliquid radiochromatography and combined gas chromatography-mass spectrometry. Kinetic studies showed that GA₃₀ and 2,3-dihydro-GA₃₁ were produced within 5 hours following ³H-GA₉ application to pea shoots. The polar GA-like substances were produced between 5 and 10 hours after ³H-GA₉ application. Levels of GA₁₀ increased with time, and since no GA₁₀ was produced during the purification procedures, GA₁₀ was, in all probability, produced from ³H-GA₉ within the plant tissue. The radioactive interconversion products produced by pea from ³H-GA₉ have chromatographic properties similar to biologically active GA-like substances present in etiolated shoots of dwarf pea. Large scale applications of ³H-GA₉ with very low specific activity to etiolated pea shoots showed that the radioactivity of the interconversion products was correlated exactly with biological activity as assayed by dwarf rice (Oryza sativa cv. Tan-ginbozu).     

Uptake and metabolism of 3H-Gibberellin A1 by barley aleurone layers: Response to abscisic acid

Summary When barley aleurone layers were incubated with ³H-Gibberellin A₁ (³H-GA₁), the hormone was converted to ³H-GA-X (not identified), ³H-GA₈ and two other compounds tentatively identified as ³H-GA₁-glucoside, and ³H-GA₈-glucoside. Uptake and metabolism of the ³H-GA₁ were markedly enhanced by simultaneous treatment with abscisic acid (ABA). Uptake of ³H-GA₁ from the medium containing ABA was linear over a 24-h period, whereas in the absence of ABA, uptake of ³H-GA₁ leveled off after 5 h. After 24 h, aleurones treated with ³H-GA₁ and ³H-GA₁ plus ABA, had taken up 9 and 24%, respectively, of the original ³H-GA₁ provided. Metabolism of ³H-GA₁ proceeded at a linear rate in the presence of ABA. The amount of ³H-GA₁-metabolites which had accumulated by the end of a 24-h incubation appeared to be linearly correlated to the logarithm of the ABA concentration. Gibberellins A₈ and-A₈-glucoside did not reverse GA₁-enhanced synthesis of -amylase.     

Radioactive gibberellin a(5) and its metabolism in dwarf peas

Radioactive gibberellin A₅ (³H-GA₅) was synthesized from gibberellic acid. When it was applied to dwarf peas grown in the dark, an average of 3% was converted to another acid gibberellin within 48 hours. The biological activity of the metabolite did not account for the response to applied GA₅. GA₅ is therefore assumed to be biologically active per se.³H-GA₅ did not appear to form a stable complex with a macromolecule in pea shoots. When injected into dwarf pea pods, ³H-GA₅ was readily metabolized by maturing seed to more water-soluble substances and to two other acidic compounds. This metabolism continued even throughout germination of the seed without reconversion of the metabolites to GA₅. It is concluded that “bound” GA₅ plays no part in the germination of dwarf pea seeds.     

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     

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     

Incorporation of 3H-Gibberellin A20 in roots of G2 peas

Following application of ³H-Gibberellin A₂₀ (GA₂₀) to roots of G2 pea seedlings and homogenization of the roots, about 3% of the radioactivity in the tissue could be precipitated from a 30,000 × g supernatant with trichloroacetic acid (TCA) (soluble fraction) while about 5% of the radioactivity pelleted at 30,000 × g (particulate fraction). The radioactivity in the particulate fraction was soluble in sodium dodecyl sulfate (SDS), but was not dialyzable and was insoluble in ethanol. Electrophoresis of the soluble fraction gave only one band of radioactivity, while that of the particulate fraction gave multiple bands. Acid hydrolysis of the soluble fraction released radioactivity that ran coincident with acid-treated GA₂₀ on silicic-acid column chromatography. The particulate fraction gave numerous radioactive peaks following acid hydrolysis, two of which were coincident with GA₂₀ and GA₂₉ (hydroxylation product of GA₂₀) on silicic acid chromatography. Treatment of the particulate and soluble fractions with RNase, DNase, and proteases showed a significant solubilization of radioactivity only with the proteases, suggesting that the GA is bound to a proteinaceous macromolecule. Complete proteolytic hydrolyis followed by thin layer chromatography showed 65% of the radioactivity from the soluble fraction running separately from free GAs or the individual amino acids; the particulate fraction gave mainly (60%) free GAs on enzymatic hydrolysis and much smaller amounts (17%) in a position separate from that of the GAs or amino acids. Binding of ³H-GA to protease-sensitive material was obtained with biologically active ³H-GA₂₀ and ³H-GA₁.     

Translocation and Intracellular Distribution of Tritiated Gibberellin A3

The localization of tritium-radioactivity in dwarf kidney bean plants (Phaseolus vulgaris) of ³H-gibberellm A₃(³H-GA₃) applied in a large quantity was investigated in advance of the study on GA₃ metabolism in this plant. Immediately after the application of ³H-GA₃, the radioactivity was distributed uniformly in the top of this plant; no further transportation of the radioactivity into the growing apical region from mature leaves and stems was the observed as the growth stage proceeded. An investigation on the intracellular localization of the radioactivity demonstrated that most part of the radioactivity was found in the cellular soluble fraction, while no radioactivity was detected in such subcellular particles as nuclei, mitochondria and microsomes. Examinations of the occurrence of GA₃ bound with such macromolecules as RNA and protein gave negative results.     

Identification of gibberellin A9 methyl ester as a natural substance regulating formation of reproductive organs in Lygodium japonicum

Prothallia of Lygodium japonicum (Thunb.) Sw. were aseptically cultured under white light in a mineral solution. Solvent fractionation of the resultant culture medium and subsequent preparative thinlayer chromatography yielded a fraction that induced antheridium formation and inhibited archegonium formation. Combined gas chromatography-selected ion monitoring analysis of this fraction confirmed the presence of gibberellin A₉ methyl ester (GA₉-me) as an antheridiogen and an inhibitor of archegonium formation. Exogenously applied [³H]GA₉ was rapidly converted to [³H]GA₉-me in the prothallial tissue. Authentic GA₉-me was active to 10^-10M in antheridium formation and to 10^-9M in the inhibition of archegonium formation.Abbreviations GAs gibberellins - GA_n gibberellin A_n - GA_n me, gibberellin A_n methyl ester - TLC thin-layer chromatography - GCSIM Combined gas chromatography-selected ion monitoring     

Metabolism of [3H]gibberellin A5 in developing pharbitis nil seeds

The native gibberellin A₅ (GA₅), as [1-³H]GA₅ (3.2 Ci/mmol) was fed to seed capsules (0.58 μCi/capsule) of Pharbitis nil cv Violet at the 2-week stage of development, and its metabolism in the seeds was investigated after 43 hr. Extractable radioactivity in free GA metabolites was 38%, with 56% in GA glucosyl conjugate-like substances. Only 2.5% of the extractable radioactivity remained as [³H]GA₅. Tentative identifications, based on comparisons with authentic standards after sequential chromatography on silica gel partition column → gradient-eluted C₁₈ HPLC → isocratic-eluted C₁₈ HPLC-radiocounting (RC), showed that [³H]GA₅ was converted to at least six free GAs, GA₁, GA₃, GA₆, GA₈, GA₂₂, GA₂₉, a GA₅ methyl ester-like metabolite, and at least twelve GA glucosyl conjugate-like substances, GA₅-glucoside (GA₅-G), GA₅-glucosyl ester (GA₅-GE), GA₁-O(3)-G, GA₁-O(13)-G, GA₁-GE, GA₃-O(3)-G, GA₃-O(13)-G, GA₃-GE, GA₆-G or GE, GA₈-O(2)-G, GA₂₂-G or GE and GA₂₉-O(2)-G. After lower specific activity feeds of [1,2-³H]GA₅ (74 mCi/mmol; 0.1 μCi/capsule) at approximately the same stage of development, the presence of GA₁, GA₃, GA₅, GA₆, GA₈ and GA₂₉ was further confirmed by sequential (after C₁₈ HPLC-RC) capillary gas chromatography-selected ion monitoring (GC-SIM), using six characteristic ions. However, for GA₂₂ only a trace of the parent ion was present at the appropriate retention time.     

The role of endogenous gibberellins in the formation of α-amylase by aleurone layers of germinating barley caryopses

Using sensitive and selective immunological assays we have shown that in germinating caryopses of Hordeum vulgare L. cv. Himalaya, the level of gibberellin A₄ (GA₄) rises approximately 18-to 20-fold shortly (2–4 h) before -amylase activity increases. Gibberellin A₄ is the predominant immunoreactive gibberelin during these developmental stages and reaches a peak amount of approximately 9 pmol per caryopsis about 48 h after imbibition. Isolated aleurone layers produce GA₄ in the presence of an exogenous gibberellin, such as GA₁, which is not a biosynthetic precursor for GA₄. Experiments with inhibitors of gibberellin biosynthesis indicate that gibberellin synthesis is required in this tissue for the induction of -amylase. The inductive effect of exogenously applied GA₁ is indirect and appears to be mediated by GA₄. Embryos form predominantly GA₁; however, very little of this material is released by isolated embryos into the incubation medium. The results presented make it unlikely that the role of the embryo in the process of -amylase induction in aleurone layers is to provide gibberellins or gibberellin precursors.Abbreviations ABA abscisic acid - GA gibberellin - GA₃ gibberellic acid - RIA radioimmunoassay - TLC thin-layer chromatography     

Metabolism of H-Gibberellin A(1) and H-Gibberellin A(4) by Phaseolus coccineus Seedlings

[³H]-Gibberellin A₁ (GA₁) and ³H-GA₄ were applied separately to Phaseolus coccineus seedlings grown under red light. ³H-GA₁ was converted to a compound with gas-liquid radiochromatography retention times identical to those of GA₈. ³H-GA₄ underwent conversion to at least three metabolites, none of which corresponded to GA_1-38. The rate of metabolism of ³H-GA₄ was significantly higher than that of ³H-GA₁.     

Transport and metabolism of h-gibberellin a(1) in dioecious cucumber seedlings

The transport of ³H-GA₁ through hypocotyl segments of cucumber (Cucumis sativus L.) was found to be nonpolar. The transport of ³H-GA₁ was increased by pretreatment with relatively high concentrations of either IAA or Ethephon (2-chloroethylphosphonic acid). Hypocotyl segments from plants of a gynoecious genotype transported more ³H-GA₁ than those of an androecious. The metabolism of ³H-GA₁ in hypocotyl segments was neither related to the sex genotype of the cucumber plant nor influenced by pretreatment with Ethephon. The primary metabolite of GA₁ was suggested to be GA₈. Two other suspected metabolites were not identified. Differences in the endogenous GA of gynoecious and androecious plants could not be accounted for by transport differences.     

Influence of daylength on gibberellin metabolism and stem growth in Silene armeria

Summary When radioactive gibberellin A₅ (³H-GA₅) was applied to the apices and surrounding young leaves of the long-day plant Silene armeria, it was partially converted to at least two other acidic substances. One of them was similar to GA₃ in chromatographic, but not in biological properties. The other metabolite was more polar than GA₃ and inactive in the dwarf d-5 corn assay.The rate of ³H-GA₅ conversion was influenced by the photoperiod under which Silene plants were grown. Exposure to 2 long days significantly increased ³H-GA₅ metabolism over that in control plants kept under short days. The increased conversion of ³H-GA₅ persisted for at least a few days after transferring Silene plants back from long to short days. Likewise, stem growth induced by long photoperiods continued for a considerable period of time under subsequent short days.Application of the growth retardant AMO-1618 to Silene reduced the levels of two endogenous GA-like substances, one of them with GA₅-like properties, more under long than under short days. These results indicate that long photoperiods, which induce flower formation and stem elongation in Silene, increase the turnover of endogenous gibberellins.     

The transport and metabolism of gibberellins A1 and A5 in excised segments from internodes of Phaseolus coccineus

Jacobs, W. P., Beall, F. D. and Pharis, R. P. 1988. The transport and metabolism of gibberellins A₁ and A₅ in excised segments from internodes of Phaseolus coccineus. -Physiol. Plant. 72: 529–534. The transport and metabolism of gibberellins (GAs) ([³H]-GA, and [³H]-GA₅) of high specific radioactivity were investigated in excised segments from young internodes of Phaseolus coccineus L. Both GA₁ and GA₅ are native to this species and present in shoot tissue. The segments, 5.1 mm long, were incubated for 6 h in the horizontal position with agar donor blocks containing the [³H]-GA on the morphological apical or basal ends and with plain agar receiver blocks on the opposite end. At the end of incubation, the individual agar blocks were analyzed immediately for total radioactivity, or both blocks and intervening tissue were frozen and freeze-dried for later chromatographic analysis. The movement of both [³H]-GA, and [³H]-GA₅ was found to be consistently without polarity. However, approximately 5-fold more [³H]-GA, than [³H]-GA₅ was transported through the Phaseolus segments into receivers when equal amounts were in the donors. The extractable radioactivity from receiver blocks was primarily that of the donor GA. No putative GA conjugates were found in any class of receivers, but more GA metabolites were found in the free acid fraction from acropetal than basipetal receivers. Chromatographic analysis by reversed phase C₁₈ high performance liquid chromatography of the tissue segments showed that [³H]-GA, was metabolized more than [³H]-GA₅. Tissue adjacent to receiver blocks contained not only the precursor GA from the donor, but also polar ‘free GA metabolites’ and putative GA glucosyl conjugates. These results provide evidence that GA., which is the known ‘effector’ GA for elongation in shoot tissue of several species, is more effectively transported than GA₅ (a known precursor of GA₁) or than GA₁s more polar metabolites.     

Growth and gibberellin-A1 metabolism in normal and gibberellin-insensitive (Rht3) wheat (Triticum aestivum L.) seedlings

Growth parameters were determined for tall (rht3) and dwarf (Rht3) seedlings of wheat (Triticum aestivum L.). Plant statures and leaf length were reduced by 50% in dwarfs but root and shoot dry weights were less affected. Leaves of dwarf seedlings had shorter epidermal cells and the numbers of cells per rank in talls and dwarfs matched the observed relationships in overall length. Talls grew at twice the rate of dwarfs (2.3 compared with 1.2 mm h^-1). [³H]Gibberellin A₁ ([³H]GA₁) was fed to seedlings via the third leaf and metabolism was followed over 12 h. Immature leaves of tall seedlings transferred radioactivity rapidly to compounds co-chromatographing with [³H]gibberellin A₈ ([³H]GA₈) and a conjugate of [³H]GA₈, whereas leaves of dwarf seedlings metabolised [³H]GA₁ more slowly. Roots of both genotypes produced [³H]GA₈-like material at similar rates. Isotopic dilution studies indicated a reduced 2-hydroxylation capacity in dwarfs, but parallel estimates of the endogenous GA pool size, obtained by radioimmunoassay, indicated a 12–15 times higher level of GA in the dwarf immature leaves. Dwarfing by the Rht3 gene does not appear to operate through enhanced, or abnormal metabolism of active gibberellins and the act of GA metabolism does not bear an obligate relationship to the growth response.Abbreviations GA_n gibberellin A_n - HPLC high-performance liquid chromatography     

The source of gibberellins in the parthenocarpic development of ovaries on topped pea plants

The role and source of gibberellins (GAs) involved in the development of parthenocarpic fruits of Pisum sativum L. has been investigated. Gibberellins applied to the leaf adjacent to an emasculated ovary induced parthenocarpic fruit development on intact plants. The application of gibberellic acid (GA₃) had to be done within 1 d of anthesis to be fully effective and the response was concentration-dependent. Gibberellin A₁ and GA₃ worked equally well and GA₂₀ was less efficient. [³H]Gibberellin A₁ applied to the leaf accumulated in the ovary and the accumulation was related to the growth response. These experiments show that GA applied to the leaf in high enough concentration is translocated to the ovary. Emasculated ovaries on decapitated pea plants develop without application of growth hormones. When [³H] GA₁ was applied to the leaf adjacent to the ovary a substantial amount of radioactivity accumulated in the growing shoot of intact plants. In decapitated plants, however, this radioactivity was mainly found in the ovary. There it caused growth proportional to the accumulation of CA₁. Application of LAB 150978, an inhibitor of GA biosynthesis, to decapitated plants inhibited parthenocarpic fruit development and this inhibition was counteracted by the application of GA₃ (either to the fruit, or the leaf adjacent to the ovary, or through the lower cut end of the stem). All evidence taken together supports the view that parthenocarpic pea fruit development on topped plants depends on the import of gibberellins or their precursors, probably from the vegetative aerial parts of the plant.Abbreviations FW flesh weight - GA_n gibberellin A_n - HPLC high-performance liquid chromatography     

An amphoteric conjugate of [h]gibberellin a(1) from barley aleurone layers

The major metabolite produced during incubation of [³H]gibberellin A₁ ([³H]GA₁) with barley aleurone layers is an amphoteric, water-soluble compound tentatively called [³H]ampho GA₁. Formation of [³H]ampho GA₁ in barley aleurones begins after a period of 2.5 hours. As judged by degradation studies as well as Sephadex column chromatography, GA₁ appears to be linked to a peptide; positions C-3 and C-7 were ruled out as conjugation sites.     

Metabolism of [H]Gibberellin A(5) by Immature Seeds of Apricot (Prunus armeniaca L.)

Immature seeds of apricot (Prunus armeniaca L.) were fed the native gibberellin A₅ (GA₅) as 1- and 1,2-[³H]GA₅ (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 [³H]GA-like metabolites were separated from the highly H₂O-soluble [³H]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 C₁₈ high performance liquid chromatography (HPLC) -radiocounting (RC). From high substrate feeds (530 and 230 × 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 [³H] GA₅ were identified as GA₁, GA₃, and GA₆ by GC-SIM. The major highly water soluble metabolite of [³H]GA₅ at all levels of substrate GA₅ had chromatographic characteristics similar to authentic GA₁-glucosyl ester. Expressed as a percentage of recovered radioactivity, low substrate [³H]GA₅ feeds (2 × expected endogenous level) yielded a broad spectrum of metabolites eluting at the Rts where GA₁, GA₃, GA₅ methyl ester, GA₆, GA₂₂, GA₂₉ (17, 14, 1.6, 7, 1.1, 0.5%, respectively) and GA glucosyl conjugates of GA₁, GA₃, GA₅, and GA₈ (33, 11, 1, 0.1%, respectively) elute. Metabolites were also present at Rts where GA glucosyl conjugates of GA₆ and GA₂₉ would be expected to elute (8 and 0.1%, respectively). Only 5% of the radioactivity remained as GA₅. Increasing substrate GA₅ levels increased the proportion of metabolites with HPLC Rts similar to GA₁, GA₆, and especially GA₁ glucosyl ester, primarily at the expense of metabolites with HPLC Rts similar to GA₃, GA₃-glucosyl ester, and a postulated conjugate of GA₆. There was evidence that high doses of substrate GA₅ induced new metabolites which often, but not always, differed from GA₁, GA₃, and GA₆ 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.