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.     

Abscisic acid and the accumulation, biological activity and metabolism of four derivatives of [3H]gibberellin A1 in barley aleurone layers

Tritiated GA₁ and four of its synthetic derivatives were studiedin relation to their biological activity, uptake and metabolismby barley aleurone layers. Incubation was done in the presenceand absence of ABA. Tentative identification of some of themetabolites was made by TLC and GLC radiocounting of the metaboliteand its acid hydrolyzed derivative. Only GA₁ promoted -amylase synthesis. Uptake ranged from 20to 42%, varying with the derivative. ABA enhanced uptake of[³H]GA₁ and [³H]pseudoGA₁ and inhibited uptake of [³H]ketoGA₁the Wagner-Meerwein rearrangement product of [³H]GA₁ Uptakeof [³H]GA₁ methyl ester ([³H]GA₁-Me) and [³H]dihydroGA₁ wasunaffected by ABA. [³H]GA₁ was converted to an amphoteric GA₁ derivative ([³H]amphoGA₁)and [³H]GA₁-glycosyl ester. GA₁-Me was metabolized to four products,all of them GA₁ derivatives, including an apparent amphotericGA₁ derivative. DihydroGA₁ was quite stable; only one metabolitewas produced in sufficient yield to analyze. This product didnot cochromatograph with either of the expected acid hydrolyzedepimers of [³H]dihydroGA₁. [³H]ketoGA₁ was readily metabolizedto one product, probably the glycoside. [³H]pseudoGA₁ remainedessentially unmetabolized. Metabolism of all compounds testedwas not dramatically affected by ABA. Surprisingly, no metabolitesfrom hydroxylation at the 2-position were found. ¹ Present address: Monsanto Agricultural Co., 800 N. LindberghBlvd., St. Louis, MO 63166, U.S.A. (Received January 31, 1977; )     

C]GA(12)-Aldehyde, [C]GA(12), and [H]- and [C]GA(53) Metabolism by Elongating Pea Pericarp

Gibberellins (GAs) are either required for, or at least promote, the growth of the pea (Pisum sativum L.) fruit. Whether the pericarp of the pea fruit produces GAs in situ and/or whether GAs are transported into the pericarp from the developing seeds or maternal plant is currently unknown. The objective of this research was to investigate whether the pericarp tissue contains enzymes capable of metabolizing GAs from [¹⁴C]GA₁₂-7-aldehyde ([¹⁴C]GA₁₂ald) to biologically active GAs. The metabolism of GAs early in the biosynthetic pathway, [¹⁴C]GA₁₂ and [¹⁴C]GA₁₂ald, was investigated in pericarp tissue isolated from 4-day-old pea fruits. [¹⁴C]GA₁₂ald was metabolized primarily to [¹⁴C]GA₁₂ald-conjugate, [¹⁴C]GA₁₂, [¹⁴C]GA₅₃, and polar conjugate-like products by isolated pericarp. In contrast, [¹⁴C]GA₁₂ was converted primarily to [¹⁴C]GA₅₃ and polar conjugate-like products. Upon further investigations with intact 4-day-old fruits on the plant, [¹⁴C]GA₁₂ was found to be converted to a product which copurified with endogenous GA₂₀. Lastly, [²H]GA₂₀ and [²H]GA₁ were recovered 48 hours after application of [²H]- and [¹⁴C]GA₅₃ to pericarp tissue of intact 3-day-old pea fruits. These results demonstrate that pericarp tissue metabolizes GAs and suggests a function for pericarp GA metabolism during fruit 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.     

The influence of dwarfing interstocks on the distribution and metabolism of xylem-applied [h]gibberellin a(4) in apple

The influence of an interstock of the dwarfing cultivar M9 and the nondwarfing cultivar MM115 on the distribution and metabolism of labeled gibberellic acid A₄ ([³H]GA₄) of high specific radioactivity (5.18 × 10¹⁰ becquerel per millimole) applied to the xylem of the rootstock in grafted apple (Malus × domestica Borkh.) trees was compared. Free [³H] GA-like metabolites of [³H]GA₄, including putative GA₁, GA₂, GA₃, and GA₃₄, as well as various ³H-putative GA glucosyl conjugates were detected in stem segments from both cultivars. M9 interstocks reduced the total uptake of [³H]GA₄ and decreased the proportion of ³H metabolites transported to the shoots and leaves of scions. The M9 interstock tissue and adjacent rootstock and scion tissue retained a much greater amount and a higher proportion of the label than did comparable tissue of the nondwarfing MM115 interstock. In addition, the amount and proportion of free [³H]GAs was higher, and the proportion of putative [³H]GA glucosyl conjugates lower, in M9 interstocks compared to MM115. These effects of the dwarfing interstock on GA distribution and metabolism indicate a significant role for GAs in any satisfactory explanation of the dwarfing mechanism in apple.     

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.     

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).     

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.     

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.     

Gibberellin A(3) Is Biosynthesized from Gibberellin A(20) via Gibberellin A(5) in Shoots of Zea mays L

[17-¹³C,³H]-Labeled gibberellin A₂₀ (GA₂₀), GA₅, and GA₁ were fed to homozygous normal (+/+), heterozygous dominant dwarf (D8/+), and homozygous dominant dwarf (D8/D8) seedlings of Zea mays L. (maize). ¹³C-Labeled GA₂₉, GA₈, GA₅, GA₁, and 3-epi-GA₁, as well as unmetabolized [¹³C]GA₂₀, were identified by gas chromatography-selected ion monitoring (GC-SIM) from feeds of [17-¹³C, ³H]GA₂₀ to all three genotypes. ¹³C-Labeled GA₈ and 3-epi-G₁, as well as unmetabolized [¹³C]GA₁, were identified by GC-SIM from feeds of [17-¹³C, ³H]GA₁ to all three genotypes. From feeds of [17-¹³C, ³H]GA₅, ¹³C-labeled GA₃ and the GA₃-isolactone, as well as unmetabolized [¹³C]GA₅, were identified by GC-SIM from +/+ and D8/D8, and by full scan GC-MS from D8/+. No evidence was found for the metabolism of [17-¹³C, ³H]GA₅ to [¹³C]GA₁, either by full scan GC-mass spectrometry or by GC-SIM. The results demonstrate the presence in maize seedlings of three separate branches from GA₂₀, as follows: (a) GA₂₀ → GA₁ → GA₈; (b) GA₂₀ → GA₅ → GA₃; and (c) GA₂₀ → GA₂₉. The in vivo biogenesis of GA₃ from GA₅, as well as the origin of GA₅ from GA₂₀, are conclusively established for the first time in a higher plant (maize shoots).     

Metabolism of [3H]gibberellin A4 in somatic suspension cell cultures of carrot

The native gibberellin A₄ (GA₄), in radioactive form ([1,2-³H]GA₄, 1.06 Ci/mmol), was fed to carrot somatic cell cultures (suspension and immobilized cell systems) and its metabolism over a 48 hr period was investigated. It was found that the [³H]GA₄ was metabolized to at least two GAs, [³H]GA₁ and [³H]GA₈, six GA glucosyl conjugates, [³H]GA₁-0(3)-glucoside, [³H]GA₁-0(13)-glucoside, [³H]GA₁-glucosyl ester, [³H]GA₄-glucoside, [³H]GA₄-glucosyl ester, a [³H]GA₈ glucosyl conjugate(s) and a previously unknown [³H]GA₁ glucosyl conjugate ([³H]GA₁-0(3,13)-diglucoside-like compound). The GA₁-diglucoside-like compound was found only in extracts of cells and was present in significant amounts (33 % of total extractable radioactivity). All other metabolites were present in both cells and medium. For extracts of the medium, no differences between the suspension and immobilized cultures existed in types of [³H]GA₄ metabolites although quantitative differences were apparent.     

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.     

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.     

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     

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.     

Metabolism of [1,2-3H]gibberellin A4 by epicotyls and cell-free preparations from Phaseolus coccineus L. seedlings

Cell-free systems were prepared from germinating seed and seedlings of Phaseolus coccineus. Gibberellin A₄ (GA₄)-metabolising activity was detected in vitro using preparations from roots, shoots and cotyledons of germinating seed, but only up to 24 h after imbibition. Cell-free preparations from cotyledons converted [³H]GA₄ to GA₁, GA₃₄, GA₄-glucosyl ester and a putative O-glucoside of GA₃₄, and, in addition converted [³H]GA₁ to GA₈. Preparations from embryo tissues contained 2-hydroxylase activity, converting [³H]GA₄ to GA₃₄ and [³H]GA₁ to GA₈.The presence of GA-metabolising enzymes was also indicated by in-vivo feeds of [³H]GA₄ to epicotyls of intact 4-d-old seedlings, which resulted in the accumulation of GA₁, GA₈, GA₃-3-O-glucoside, GA₄-glucosyl ester, GA₈-2-O-glucoside and a putative O-glucoside of GA₃₄. Gibberellin A₁ was the first metabolite detected, 15 min after application of [³H]GA₄, but after 24 h most of the label was associated with GA₈-2-O-glucoside. Over 90% of the recovered radioactivity was found in the shoot. Within the shoot, movement was preferentially acropetal, and was not dependent upon metabolism of the applied [³H]GA₄.Abbreviations DEAE diethylaminoethyl - GA_n gibberellin A_n - GPC gel permeation chromatography - HPLC-RC high performance liquid chromatography-radio counting - S-1 1000·g supernatant - UDP uridine 5-diphosphate     

Gibberellins and Heterosis in Maize : II. Response to Gibberellic Acid and Metabolism of [H]Gibberellin A(20)

Two maize inbreds, CM7 and CM49, and CM7 × CM49, their F₁ hybrid (which displayed significant heterosis), were examined with regard to response to exogenous gibberellin A₃ (GA₃), and in their ability to metabolize GA₂₀, a native GA of maize. The leaf sheath elongation response to GA₃ was far greater for the imbreds than for their hybrid. The inbreds also displayed significant elongation of the leaf blades in response to GA₃, whereas the hybrid was unaffected. Promotion of cell division in the leaf sheath of CM7 and the hybrid was effected by GA₃, but no promotion of cell elongation was observed in CM49, even though significant leaf sheath elongation occurred. Shoot dry weight of both inbreds was significantly increased by GA₃, but response by the hybrid in this parameter was slight and variable. Root dry weight of CM7 was significantly increased by GA₃, but was unchanged in CM49 and the hybrid. Thus, inbred shoot dry weight increases effected by GA₃ were not at the expense of the root system. Rapid metabolism of [2,3-³H]GA₂₀ occurred in all genotypes, although genotypic differences were observed. The hybrid had the highest rates of metabolism to GA glucosyl conjugate-like substances. Oxidative metabolism was also fastest in the hybrid, followed by CM7, and slowest in CM49, the slowest-growing inbred. Thus, rate of GA₂₀ metabolism is under genetic control in normal (i.e. not dwarfed) maize genotypes. These results, taken together with previous reports that the hybrid has significantly enhanced levels of endogenous GA-like substances, suggest that GA play a role in the expression of heterosis in maize.     

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.     

Quantitation of gibberellins and the metabolism of [3H]gibberellin A1 during somatic embryogenesis in carrot and anise cell cultures

In a carrot (Daucus carota L.) cell line lacking the ability to undergo somatic embryogenasis, and in carrot and anise (Pimpinella anisum L.) cell lines in which embryogenesis could be regulated by presence or absence of 2,4-dichlorophen-oxyacetic acid (2,4-D), in the medium (+2,4-D=no embryogenesis,-2,4-D=embryo differentiation and development), the levels of endogenous gibberellin(s) (GA) were determined by the dwarfrice bioassay, and the metabolism of [³H]GA₁ was followed. Embryos harvested after 14 d of subculture in-2,4-D had low levels (0.2–0.3 g g^-1 dry weight) of polar GA (e.g. GA₁-like), but much (3–22 times) higher levels of less-polar GA (GA_4/7-like); GA₁, GA₄ and GA₇ are native to these cultures. Conversely, the undifferentiated cells in a non-embryogenic strain, and proembryos of an embryogenic strain (+2,4-D) showed very high levels of polar GA (2.9–4.4 g g^-1), and somewhat reduced levels of less-polar GA. Cultures of anise undergoing somatic embryo development (-2,4-D) metabolized [³H]GA₁ very quickly, whereas proembryo cultures of anise (+2,4-D) metabolized [³H]GA₁ slowly. The major metabolites of [³H]GA₁ in anise were tentatively identified as GA₈-glucoside (24%), GA₈ (15%), GA₁-glucoside (8%) and the ¹⁽¹⁰⁾GA₁-counterpart (2%). Thus, high levels of a GA₁-like substance and a reduced ability to metabolize GA₁ are correlated with the absence of embryo development, while lowered levels of GA₁-like substance and a rapid metabolism of GA₁ into GA₈ and GA-conjugates are correlated with continued embryo development. Exogenous application of GA₃ is known to reduce somatic embryogenesis in carrot cultures; GA₄ was found to have the same effect in anise cultures. Thus, a role (albeit negative) in somatic embryogenesis for a polar, biologically active GA is implied.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - GA gibberellin(s) or gibberellin-like substances - GC-RC gas chromatography-radiochromatogram counting - HPLC high-presare liquid chromatography - Rt retention time - TLC thinlaver chromatography     

Synthesis of [2H]gibberellins from steviol using the fungus Gibberella fujikuroi

[²H]Steviol (ent-13-hydroxykaur-16-en-19-oic acid) was synthesized from steviol acetate norketone (ent-13-acetoxy-16-oxo-17-norkauran-19-oic acid) by the Wittig reaction using (methyl-d₃)triphenylphosphonium bromide. A mixture of steviol analogs was produced containing from one to four ²H/molecule. [²H]Steviol was fed to strain LM-45-399 of the fungus Gibberella fujikuroi which was grown on synthetic medium (ICI, 0% N) in the presence of the growth retardant CCC. [²H]GA₁, [²H]GA₁₈, [²H]GA₂₃ and [²H]GA₅₃ were isolated from the fungal medium after 4 days. This strain converted steviol to 13-hydroxy GAs in the highest yields of the four Gibberella strains tested, and in amounts suitable for metabolic studies with higher plants.