Metabolism of Tritiated Gibberellins in d-5 Dward Maize: II. [H]Gibberellin A(1), [H]Gibberellin A(3), and Related Compounds

After 30 minutes of incubation of young leaf sections of d-5 maize (Zea mays L.) in [³H]gibberellin A₁ ([³H]GA₁), the metabolite [³H]GA₈ was present in significant amounts, with a second metabolite, [³H]GA₈-glucose ([³H]GA₈-glu), appearing soon after. A third [³H]GA₁ metabolite, the polar uncharacterized conjugate [³H]GA₁-X, took more than 1 hour to appear. The protein synthesis inhibitor cycloheximide inhibited the production of all [³H]GA₁ metabolites, indicating a possible protein synthesis requirement for [³H]GA₁ metabolism.     

Gibberellins and Gravitropism in Maize Shoots : Endogenous Gibberellin-Like Substances and Movement and Metabolism of [3H]Gibberellin A20

[³H]Gibberellin A₂₀ (GA₂₀) of high specific radioactivity (49.9 gigabecquerel per millimole) was applied equilaterally in a ring of microdrops to the internodal pulvinus of shoots of 3-week-old gravistimulated and vertical normal maize (Zea mays L.), and to a pleiogravitropic (prostrate) maize mutant, lazy (la). All plants converted the [³H]GA₂₀ to [³H]GA₁⁻ and [³H]GA₂₉-like metabolites as well as to several metabolites with the partitioning and chromatographic behavior of glucosyl conjugates of [³H]GA₁, [³H]GA₂₉, and [³H]GA₈. The tentative identification of these putative [³H]GA glucosyl conjugates was further supported by the release of the free [³H]GA moiety after cleavage with cellulase. Within 12 hours of the [³H]GA₂₀ feed, there was a significantly higher proportion of total radioactivity in lower than in upper halves of internode and leaf sheath pulvini in gravistimulated normal maize. Further, there was a significantly higher proportion of putative free GA metabolites of [³H]GA₂₀, especially [³H]GA₁, in the lower halves of normal maize relative to upper halves. The differential localization of the metabolites between upper and lower halves was not apparent in the pleiogravitropic mutant, la. Endogenous GA-like substances were also examined in gravistimulated maize shoots. Forty-eight hours after gravistimulation of 3-week-old maize seedlings, endogenous free GA-like substances in upper and lower leaf sheath and internode pulvini halves were extracted, chromatographed, and bioassayed using the `Tanginbozu' dwarf rice microdrop assay. Lower halves contained consistently higher total levels of GA-like activity. The qualitative elution profile of GA-like substances differed consistently, upper halves containing principally a GA₂₀-like substance and lower halves containing mainly GA₁-like and GA₁₉-like substances. Gibberellins A₁ (10 nanograms per gram) and A₂₀ (5 nanograms per gram) were identified from these lower leaf sheath pulvini by capillary gas chromatography-selected ion monitoring. Results from all of these experiments are consistent with a role for GAs in the differential shoot growth that follows gravitropism, although the results do not eliminate the possibility that the redistribution of GAs results from the gravitropic response.     

Thermoinductive Regulation of Gibberellin Metabolism in Thlaspi arvense L. : I. Metabolism of [H]-ent-Kaurenoic Acid and [C]Gibberellin A(12)-Aldehyde

Field pennycress (Thlaspi arvense L.) is a winter annual crucifer with a cold requirement for stem elongation and flowering. In the present study, the metabolism of exogenous [²H]-ent-kaurenoic acid (KA) and [¹⁴C]-gibberellin A₁₂-aldehyde (GA₁₂-aldehyde) was compared in thermo- and noninduced plants. Thermoinduction greatly altered both quantitative and qualitative aspects of [²H]-KA metabolism in the shoot tips. The rate of disappearance of the parent compound was much greater in thermoinduced shoot tips. Moreover, there was 47 times more endogenous KA in noninduced than in thermoinduced shoot tips as determined by combined gas chromatography-mass spectrometry (GC-MS). The major metabolite of [²H]-KA in thermoinduced shoot tips was a monohydroxylated derivative of KA, while in noninduced shoot tips, the glucose ester of the hydroxy KA metabolite was the main product. Gibberellin A₉ (GA₉) was the only GA in which the incorporation of deuterium was detected by GC-MS, and this was observed only in thermoinduced shoot tips. The amount of incorporation was small as indicated by the large dilution by endogenous GA₉. In contrast, thermo- and noninduced leaves metabolized exogenous [²H]-KA into GA₂₀ equally well, although the amount of conversion was also limited. These results are consistent with the suggestion (JD Metzger [1990] Plant Physiol 94: 000-000) that the conversion of KA in to GAs is under thermoinductive control only in the shoot tip, the site of perception for thermoinductive temperatures in field pennycress. There were essentially no differences in the qualitative or quantitative distribution of metabolites formed following the application of [¹⁴C]-GA₁₂-aldehyde to the shoot tips of thermo- or noninduced plants. Thus, the apparent thermoinductive regulation of the KA metabolism into GAs is probably limited to the two metabolic steps involved in converting KA to GA₁₂-aldehyde.     

Gibberellin Metabolism in Maize (The Stepwise Conversion of Gibberellin A12-Aldehyde to Gibberellin A20

The stepwise metabolism of gibberellin A12-aldehyde (GA12-aldehyde) to GA20 is demonstrated from seedling shoots of maize (Zea mays L.). The labeled substrates [13C,3H]GA12-aldehyde, [13C,3H]GA12, [14C4]GA53, [14C4/2H2]GA44, and [14C4/2H2]GA19 were fed individually to dwarf-5 vegetative shoots. Both [13C,3H]GA12-aldehyde and [13C,3H]GA12 were also added individually to normal shoots. The labeled metabolites were identified by full-scan gas chromatography-mass spectrometry and Kovats retention indices. GA12-aldehyde was metabolized to GA53-aldehyde, GA12, GA53, GA44, and GA19; GA12 was metabolized to 2[beta]-hydroxy-GA12, GA53, 2[beta]-hydroxyGA53, GA44, 2[beta]-hydroxyGA44, and GA19; GA53 was metabolized to GA44, GA19, GA20, and GA1; GA44 was metabolized to GA19; and GA19 was metabolized to GA20. These results, together with previously published data from this laboratory, document the most completely defined gibberellin pathway for the vegetative tissues of higher plants.     

Effect of Abscisic Acid on Uptake and Metabolism of [H]Gibberellin A(1) and [H]Pseudogibberellin A(1) by Barley Half-seeds

Uptake and metabolism of 1,2-[³H]gibberellin A₁ ([³H]GA₁, I) and its 3-hydroxy epimer ([³H]pseudoGA₁, II) by barley (Hordeum vulgare L.) half-seeds were measured after 24 hours of incubation, in the presence or absence of abscisic acid in the media. Uptake of both compounds was enhanced by abscisic acid, and abscisic acid enhanced the extent of metabolism of [³H]GA₁. However, [³H]pseudoGA₁ was not metabolized, even in the presence of abscisic acid. The significance of the stereo-chemistry of the 3-hydroxyl position is discussed.     

Vernalization and Gibberellin Physiology of Winter Canola (Endogenous Gibberellin (GA) Content and Metabolism of [3H]GA1 and [3H]GA20

Winter canola (Brassica napus cv Crystal) is an oilseed crop that requires vernalization (chilling treatment) for the induction of stem elongation and flowering. To investigate the role of gibberellins (GAs) in vernalization-induced events, endogenous GA content and the metabolism of [3H]GAs were examined in 10-week vernalized and nonvernalized plants. Shoot tips were harvested 0, 8, and 18 d postvernalization (DPV), and GAs were purified and quantified using 2H2-internal standards and gas chromatography-selected ion monitoring. Concentrations of GA1, GA3, GA8, GA19, and GA20 were 3.1-, 2.3-, 7.8-, 12.0-, and 24.5-fold higher, respectively, in the vernalized plants at the end of the vernalization treatment (0 DPV) relative to the nonvernalized plants. Thermoregulation apparently occurs prior to GA19 biosynthesis, since vernalization elevated the concentration of all of the monitored GAs. [3H]GA20 or [3H]GA1 was applied to the shoot tips of vernalized and nonvernalized plants, and after 24 h, plants were harvested at 6, 12, and 15 DPV. Following high-performance liquid chromatography analyses, vernalized plants showed increased conversion of [3H]GA20 to a [3H]GA1-like metabolite and reduced conversion of [3H]GA1 or [3H]GA20 to polar 3H-metabolites, putative glucosyl conjugates. These results demonstrate that vernalization influences GA content and GA metabolism, with GAs serving as probable regulatory intermediaries between chilling treatment and subsequent stem growth.     

Effect of Photoperiod on Metabolism of [H] Gibberellins A(1), 3-epi-A(1), and A(20) in Agrostemma githago L

To determine whether daylength influences the rate of metabolism of gibberellins (GAs) in the long-day (LD) rosette plant Agrostemma githago L., [³H]GA₂₀ and [³H]GA₁ were applied under short day (SD) and LD. Both were metabolized faster under LD than under SD. [³H]GA₂₀ was metabolized to a compound chromatographically identical to 3-epi-GA₁. [³H]GA₁ was metabolized to two acidic compounds, the major metabolite having chromatographic properties similar to, but not identical with GA₈. [³H]3-epi-GA₁ applied to plants under LD was metabolized much more slowly than was [³H]GA₁, and formed a very polar metabolite which did not partition into ethyl acetate at pH 2.5. Very polar metabolites were also formed after the feeds of [³H]GA₂₀ and [³H]GA₁. It was not possible to characterize these very polar compounds further because of their apparent instability. The results obtained suggest that in Agrostemma GA₂₀ is the precursor of 3-epi-GA₁, but there is at present no evidence indicating the precursor of GA₁.     

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.     

Metabolism of [3H] Gibberellin A5 in Cell Suspension Cultures of Pharbitis nil

Gibberellin A₅ (GA₅), a native GA of immature seeds of Pharbitis nil, was fed to Pharbitis nil cell suspension cultures as [C-l, ³H] GA₅ (3.1 Ci/mmol), and its metabolism over a 48 hr period was investigated. Radioactivity in free GA metabolites was 13.1%, with 79.9% in GA glucosyl conjugate-like metabolites. Only 7.0% of the radioactivity remained as [³H] GA₅. Tentative identifications were based on comparison with retention times of authentic free GAs and/or glucosyl conjugates after sequential chromatography on Si gel partition column → gradient-eluted C18 HPLC-radiocounting (RC) → isocratic-eluted C18 HPLC-RC, and showed that [³H] GA₅ was converted to [³H] GA₁ (2%), [³H] GA₃ (4%), [³H] GA₆ (2%), [³H] GA₂₂ (1%) and their glucosyl conjugates, and also to [³H] GA₈ glucoside, and [³H] GA₅ glucosyl conjugates. The major conjugate-like substances were [³H] GA₁ and [³H] GA₃ glucosyl esters, at 15% and 34%, respectively, of the total extractable radioactivity.     

Metabolism of ent-Kaurene to Gibberellin A(12)-Aldehyde in Young Shoots of Normal Maize

Young shoots of normal maize (Zea mays L.) were used to determine both the stepwise metabolism of ent-kaurene to gibberellin A₁₂-aldehyde and the endogenous presence of the members in this series. Each of the five steps in the sequence was established by feeds of 17-¹³C, ³H-labeled kauranoids to cubes from the cortex of elongating internodes, to homogenates from the cortex of elongating internodes, and/or to homogenates from dark-grown seedlings. The ¹³C-metabolites were identified by Kovats retention indices (KRI) and full-scan capillary gas chromatography-mass spectrometry (GC-MS). Five substrates and the final product in this sequence were shown to be native by the isotopic dilution of 17-¹³C, ³H-labeled substrates added as internal standards to extracts obtained from elongating internodes. Evidence for the isotopic dilution was obtained by KRI and full-scan capillary GC-MS. Thus, we document the presence in young maize shoots of the metabolic steps, ent-kaurene → ent-kaurenol → ent-kaurenal → ent-kaurenoic acid → ent-7 α-hydroxykaurenoic acid → gibberellin A₁₂-aldehyde.     

l-Phenylalanine Ammonia-lyase (Maize): Partial Purification and Response to Gibberellic Acid and Cycloheximide of l-Phenylalanine and l-Tyrosine Ammonia-lyase Activities

Extracts of maize leaf sheath tissue deaminate both l-phenylalanine and l-tyrosine. The activities with both substrates are enhanced by treating the plant with gibberellic acid. Both activities decrease rapidly at the same rate when tissue is incubated in a moist atmosphere, and this decrease can be slowed by treatment with cycloheximide. The ratio of the activities was constant throughout a series of purification steps which included acetone and ammonium sulfate precipitation, and passage through an agarose column. The two activities could not be separated by isoelectric focusing. These results support our earlier conclusion that both activities occur at the same catalytic site.     

Endogenous Gibberellins of Pine Pollen: III. Conversion of 1,2-[H]GA(4) to Gibberellins A(1) and A(34) in Germinating Pollen of Pinus attenuata Lemm

Gibberellins A₁ and A₃₄ (possibly A₂) were found as products of metabolism of 1,2-[³H]GA₄ during germination of Pinus attenuata pollen. The conversion from GA₄ to GA₁ and GA₃₄ occurred as hydroxylations at atoms C-13 and C-2 of the ent-gibberellane skeleton, respectively. Percentage interconversion of the GA₄ absorbed was in the range of 0.15 to 0.43% for GA₁ and 1.54 to 3.22% for GA₃₄. Identifications were made on a gas-liquid chromatograph with radioactive monitoring by comparison with standards.     

Effects of Light, Abscisic Acid, and N-Benzyladenine on the Metabolism of [H]Gibberellin A(4) in Seeds and Seedlings of Lettuce, cv. Grand Rapids

Gibberellin A₄ (GA₄) can substitute for light in the germination of Grand Rapids lettuce seeds. Seeds imbibed in [³H]GA₄ do not convert this to other GAs prior to, or immediately following, visible germination: thus GA₄ alone can promote radicle expansion. Abscisic acid inhibited [³H]GA₄-induced germination, but did not significantly affect [³H]GA₄ uptake or metabolism during germination. ⁶N-benzyladenine overcame the inhibitory effect of abscisic acid and increased [³H]GA₄ uptake, although radicle emergence was delayed somewhat.     

Identification of Pea Gibberellins by Studying [C]GA(12)-Aldehyde Metabolism

Experiments were designed to test the hypothesis that the labeled products recovered from plant tissue incubated with [¹⁴C]GA₁₂-7-aldehyde ([¹⁴C]GA₁₂ald) would serve as appropriate [¹⁴C]markers for the recovery of naturally-occurring gibberellins (GAs). The [¹⁴C]GA₁₂ald (about 200 millicuries per millimole) was synthesized from pumpkin endosperm using [4,5-¹⁴C]mevalonic acid. It was added to the adaxial surface of isolated pea cotyledons at 22 days after flowering. Products recovered after 0.5 and 4.0 hour incubations yielded four major peaks which were separated by high performance liquid chromatography (HPLC). These products were purified by multiple-column HPLC using on-line radioactivity detection. They were then added as [¹⁴C]markers to two unlabeled pea extracts. In general, preparative HPLC followed by further HPLC purification resulted in a single UV-absorbing peak co-eluting with each [¹⁴C]marker. These [¹⁴C] and UV-absorbing peaks were shown to contain GA₅₃, GA₄₄, GA₂₀, GA₁₉, and GA₁₇ by GC-MS. The finding of GA₅₃ is novel; all others have previously been found in pea. Endogenous GAs of pea were thus readily detected using [¹⁴C]GA₁₂ald metabolites as [¹⁴C]markers to recover naturally occurring GAs suggesting that the method may be applicable in detecting naturally occurring GAs in other species.     

Effects of Chilling and ABA on [H]Gibberellin A(4) Metabolism in Somatic Embryos of Grape (Vitis vinifera L. x V. rupestris Scheele)

Previous work has indicated that changes in gibberellin (GA) metabolism may be involved in chilling-induced release from dormancy in somatic embryos of grape (Vitis vinifera L. × V. rupestris Scheele). We have chilled somatic embryos of grape for 2, 4, or 8 weeks, then incubated them with [³H]GA₄ (of high specific activity, 4.81 × 10¹⁰ becquerel per millimole) for 48 hours at 26°C. Chilling had little effect on the total amount of free [³H]GA-like metabolites formed during incubation at 26°C, but did change the relative proportions of individual metabolites. The amount of highly water-soluble [³H] metabolites formed at 26°C decreased in embryos chilled for 4 or 8 weeks. The concentration of endogenous GA precursors (e.g., GA₁₂ aldehyde-, kaurene-, and kaurenoic acid-like substances) increased in embryos chilled for 4 or 8 weeks. Treatment with abscisic acid (ABA) (known to inhibit germination in grape embryos) concurrent with [³H]GA₄ treatment at 26°C, reduced the uptake of [³H] GA₄ but had little effect on the qualitative spectrum of metabolites. However, in the embryos chilled for 8 weeks and then treated with ABA for 48 hours at 26°C, there was a higher concentration of GA precursors than in untreated control embryos. Chilled embryos thus have an enhanced potential for an increase in free GAs through synthesis from increased amounts of GA precursors, or through a reduced ability to form highly water-soluble GA metabolites (i.e., GA conjugates or polyhydroxylated free GAs).     

Changes in Endogenous Gibberellins and the Metabolism of [H]GA(4) after Geostimulation in Shoots of the Oat Plant (Avena sativa)

The recovery from “lodging,” or bending over, by shoots of 42-day-old Avena sativa plants is controlled primarily by a negatively geotropic differential growth of the lower halves of the p-1 node-pulvinus and the base of the p-1 internode, relative to the upper halves. Although geostimulation causes a significant reduction in p-1 internode length, dry matter accumulation in the p-1 node-pulvinus is increased, apparently at the expense of the sheath. Recovery to an angle of 30° is associated with changes in endogenous gibberellin-like substances (GAs), and in differential metabolism of applied [³H]GA₄ (1.4 Curie per millimole). Although geostimulation depressed total GAs (relative to upright plant parts) to 0.40 and 0.13 for node-pulvini and sheaths, respectively, it increased them 2-fold for internodes. Within the plant part geostimulation increased GAs (relative to upper halves) 29- and 7-fold in lower halves of node-pulvini and internodes, respectively, but reduced GAs to 0.3 in lower halves of sheaths. At age 42 days a GA_4/7-like (nonpolar) substance predominates, with lesser amounts of a GA₃-like (polar) substance. Native GAs of Avena include GA₃, GA₄, and GA₇. Geostimulation enhanced the ratio of nonpolar to polar GAs for both halves of internodes, but tended to depress it for sheaths and nodepulvini.