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 [3H]gibberellin A4 in somatic suspension cultures of anise

The native gibberellin A₄ (GA₄) was fed as [1, 2-³H]GA₄ (1.3 Ci/mmol) to anise somatic cultures maintained either at a proembryo-like stage with 2,4-dichlorophenoxyacetic acid (2,4-D), or allowed to undergo embryogenic development on a - 2,4-D medium. Proembryos, although only 20% of the dry wt of embryos, absorbed 1.4-times more [³H]GA₄/g dry wt than embryos. The [³H]GA₄ was metabolized to GA₁ and GA₈, and at least six conjugates [GA₄-glucoside (GA₄-G), GA₄ glucosyl ester (GA₄-GE), GA₁-0(3)-G, GA₁-0(13)-G, GA₁-GE and a GA₈-glucosyl conjugate]. The major metabolite was GA₄-G at each of two, 204 and 348 hr harvests (56–71 %), with GA₈-G increasing from < 1 % to 13 % with harvest time. The percentage and amount of GA₄-GE was highest at 204 hr (2% and 8 %, for embryos and proembryos, respectively), dropping to < 1 % at 348 hr, thereby indicating hydrolysis (e.g. reversible conjugation). Embryos had reduced amounts and percentages of biologically active GA₄ and GA₁, and most of their conjugates, but increased amounts and percentages of GA₈ and its conjugate(s). This finding is consistent with the hypothesis (based on present and past work) that high levels of biologically active GAs, especially GA₁, inhibit somatic embryogenesis in anise and carrot. The auxin, 2,4-D, may thus derive, at least in part, its ability to maintain the proembryo-like stage by inhibiting oxidative metabolism and conjugation of biologically active GAs.     

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.     

Formation of GA20 glucosyl conjugates in seedlings ofVicia faba

[³H]GA₂₀ (1)¹, fed toVicia faba seedlings, was converted to [³H]GA₂₀ glucosyl ester (5) and [³H]GA₂₀-13-0-glucoside (6). The GA₂₀ glucosyl ester (5) was identified by HPLC-RC and by GC-MS of GA₂₀-Me formed by transesterification of (5). The [³H]GA₂₀-Me was crystallized to constant specific radioactivity with authentic GA₂₀-Me. On HPLC-RC the GA₂₀-13-0-glucoside (6) was shown to have the same retention time as an authentic sample. Subsequent enzymic hydrolysis gave a product with an HPLC retention time identical to that of authentic GA₂₀ (1).     

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.     

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.     

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.     

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.     

Biological activity of some synthetic gibberellin glucosyl esters

Four gibberellin (GA₁, GA₃, GA₄ and GA₃₇) glucosyl esters were synthesized and found to be as active as their respective free acids in the rice seedling bioassay. The rapid hydrolysis of the glucosyl esters in rice seedlings was demonstrated by feeding experiments with glucosyl esters of [³H]GA₁ and [³H]GA₄.     

Heterosis and the metabolism of gibberellin A20 in sorghum

The correlation between gibberellin (GA) metabolism and growth rate was investigated using two Sorghum bicolor inbred lines, Hegari and AT×623, and their heterotic F₁ hybrid. Previous studies have demonstrated that this hybrid is taller and has substantially greater shoot dry weights and leaf areas than either parental inbred. [³H]GA₂₀ was applied to the leaf whorl of seedlings and after 24 hours, plants were harvested and separated into roots, shoot cylinders containing the apical meristems, and leaf blades. Chromatographic analyses of metabolites indicated the conversions of [³H]GA₂₀ to [³H]GA_{1,8 and 29}. The conversion of [²H]GA₂₀ to [²H]GA₁ was demonstrated by gas chromatography-selected ion monitoring (GC-SIM). Putative glucosyl conjugates of all of the [³H]GAs were also produced and GA₈ was identified by GC-SIM following enzymic cleavage of the putative [³H]GA₈ glucosyl conjugate fraction. Comparing the genotypes, [³H]GA₂₀ metabolism was more rapid in the shoot cylinders of the hybrid than in the shoot cylinders from inbreds. In the hybrid samples, there was a three-fold increase in the putative conjugate(s) of [³H]GA₁ which was the principal metabolite, and increased production of [³H]GA₈ and the putative conjugates of [³H]GA₂₉ and [³H]GA₈. Conversely, levels of the remaining precursor, [³H]GA₂₀, and its putative conjugate(s) were reduced in the hybrid. The rate of GA₂₀ metabolism was thus positively correlated with growth rate across these sorghum genotypes. This correlation supports a promotive role of GA in the regulation of shoot growth and in the expression of heterosis (hybrid vigor) in sorghum.     

Reversible conjugation of gibberellins in situ in maize

Gibberellins [³H]GA₄ (1.33 Curies per millimole) and [³H]GA₂₀ (2.36 Curies per millimole) were injected into the shanks of maize (Zea mays L.) cobs during rapid grain filling and mature seeds were subsequently harvested. Extracts of mature, dry seeds from 1980 feeds yielded only 20 to 30% of the ³H radioactivity in acidic, ethyl acetate-soluble form, and this was principally associated with the precursor, with lesser amounts of the major metabolite, [³H]GA₁ (putative identification based on sequential SiO₂ partition, and gradient-eluted reverse-phase C₁₈ high performance liquid chromatography [HPLC]). Most of the radioactivity in the dry seeds was associated with compounds having partition characteristics of, and co-chromatographing on, sequential SiO₂ partition and reverse-phase HPLC with glucosyl conjugates of the precursors (GA₄ or GA₂₀) and their probable major metabolite (GA₁). The majority of conjugate associated with the precursor GA₄ eluted coincidental with GA₄ glucoside. Subsequent acid or enzymic hydrolysis (β-glucosidase or cellulase) yielded the free GAs, putative identification being based on isocratic HPLC of each ³H-labeled conjugate → hydrolysis → isocratic HPLC of the ³H-labeled hydrolysate. Upon imbibition of the seeds, radioactivity associated with the conjugate fraction decreased; concomitantly, statistically significant increases in levels of free [³H]GA-like compounds were observed. Although the specific ratios of GA-like and GA-glucosyl conjugate-like substances varied substantially across years, hybrids, and even, in different plants from the same hybrid, this `reversible conjugation' (i.e. apparent conjugation during seed maturation followed by release of the GA moiety during germination), was reproducible for [³H]GA₂₀ in seed from two maize hybrids produced over 2 years.     

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.     

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

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

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.     

Lack of influence of photoperiod on the metabolism of gibberellin A20 in Salix pentandra

The influence of photoperiod on the metabolism of GA₂₀ in Salix pentandra was studied by feeding [³H]-GA₂₀ to seedlings which had been grown previously under long day (LD) or short day (SD) conditions. After 48 h in LD or SD, metabolites were separated on sequential, silica gel partition columns and reversed-phase C₁₈ HPLC. The principal metabolite co-chromatographed with [³H]-GA₁ and this conversion was confirmed by feeding [²H]-GA₂₀, which was converted to [²H]-GA₁ as identified by gas chromatography-selected ion monitoring. Chromatographic evidence also indicated the minor conversion of [³H]-GA₂₀ to [³H]-GA₈ (via [³H]-GA₁) and trace conversion to [³H]-GA₂₉ (GAs A_1.8,20.29 are native in Salix). Ethyl acetate-insoluble [³H] metabolites were formed and could be cleaved by cellulase to release putative [³H]-GA₂₀ and [³H]-GA₁ suggesting the conversion to glucosyl conjugates of these GAs. Metabolism of [³H]-GA₂₀ was slightly more rapid in plants previously grown under LD than SD, an effect which reflected the generally increased shoot growth under LD. However, altering the photoperiod after [³H]-GA₂₀ addition had only a slight effect on the metabolism of [³H]-GA₂₀ in Salix seedlings. This indicates that the conversion of GA₂₀ to GA₁ is not a controlling step in the photoperiodic regulation of growth cessation in Salix.     

Metabolic formation and occurrence of gibberellin A1-3-0-β-D-glucopyranoside in immature fruits of Phaseolus coccineus L.

Endogenous pools of presumptive gibberellin (GA) glucosyl conjugates of Phaseolus coccineus were metabolically labelled by feeding of [³H]GA₁ to immature fruits. The [³H]GA₁ glucoside fraction was isolated and the main constituent tentatively identified by enzymic hydrolysis, ion exchange chromatography and elution volume on HPLC-RC as GA₁-3-0--D-glucopyranoside.     

Investigations into the possible regulation of negative gravitropic curvature in intact Avena sativa plants and in isolated stem segments by ethylene and gibberellins

Using Avena sativa L. cv. Victory oat seedlings and excised p-1 stem segments (including the p-1 and p-2 internodes) the effect of exogenously supplied ethylene and the removal of ethylene on internodal extension and gravitropic bending was assessed. Similarly, the ability of the excised system to respond to gravistimulation was assessed in the presence of inhibitors of ethylene action (AgNO₃) and ethylene synthesis (3,5-diiodo-4-hydroxybenzoic acid and benzyl isothiocyanate; BITC). The production of ethylene from both intact and excised systems was also measured from 0 to 48 h after gravistimulation, relative to vertical controls. Although gravitropic curvature is initiated, and indeed enters the most rapid phase of upward bending during the first 6 h, there is no difference in ethylene production between vertical and geostimulated plants during this period. The ethylene production of gravistimulated plants rises sharply to a maximum at 24 h, then decreases steeply to almost the control level by 48 h, at which time the rate of upward curvature is diminishing. Neither the addition nor removal of ethylene, nor the addition of inhibitors affecting ethylene-action (AgNO₃) or synthesis (DIHB) influence gravitropic bending or internodal extension in excised segments. Although the ethylene synthesis inhibitor BITC showed down the rate of upward bending, this effect could not be reversed by addition of ethylene. We conclude that the burst in ethylene production that develops in leaf-sheath bases (pulvini) after they have started to curve upwards is not primary to the induction of curvature. We further suggest that ethylene has no major effect or role in the induction of upward bending after gravistimulation. The metabolism of high specific activity gibberellin A₁ ([³H]-GA₁) in the excised system was assessed during 1, 2 and 4 h of gravistimulation. Changes in endogenous GAs and GA metabolism have been shown previously to be correlated (at the later stages) with gravistimulated bending in intact Avena shoots. The excised segments ‘leaked’ free [³H]-GAs and [³H]-GA glucosyl conjugate-like substances into the bathing medium, and this was a confounding factor. Nevertheless, gravistimulated stem segments, and especially the bottom half of the segment, were significantly less leaky then vertical segments. Thus, just 1 h after gravistimulation, bottom segment halves retained 22% more precursor [³H]-GA₁, 36% more free [³H]-GA-like metabolites, and 48% more [³H]-GA glucosyl conjugate-like metabolites than vertical segments. In contrast, the 1 h gravistimulated top halves retained slightly less (1–4%) precursor [³H]-GA and free [³H]-GA metabolites, but 21% more [³H]-GA glucosyl conjugate-like radioactivity than vertical segments.     

Metabolism of tritiated gibberellin a(20) in maize

After the application of 2.36 Curies per millimole [2,3-³H]gibberellin A₂₀ (GA₂₀) to 21-day-old maize (Zea mays L., hybrid CM7 × CM49) plants, etiolated maize seedlings, or maturing maize cobs, a number of ³H-metabolites were observed. The principal acidic (pH 3.0), ethyl acetate-soluble metabolite was identified as [³H]GA₁ on the basis of co-chromatography with standard [³H]GA₁ on SiO₂ partition, high resolution isocratic elution reverse phase C₁₈ high performance liquid chromatography and gas-liquid chromatography radiocounting. Two other acidic metabolites were identified similarly as [³H]GA₈ and C/D ring-rearranged [³H]GA₂₀, although gas-liquid chromatography radiocounting was not performed on these metabolites. Numerous acidic, butanol-soluble (e.g. ethyl acetate-insoluble) metabolites were observed with retention times on C₁₈ high performance liquid chromatography radiocounting similar to those of authentic glucosyl conjugates of GA₁ and GA₈, or with retention times where conjugates of GA₂₀ would be expected to elute. Conversion to [³H]GA₁ was greatest (23% of methanol extractable radioactivity) in 21-day-old maize plants. In etiolated maize seedlings, the C/D ring-rearranged [³H]GA₂₀-like metabolite was the major acidic product, while conversion to [³H]GA₁ was low.     

The synthesis of [3H]gibberellin A3 and [3H]gibberellin A1 by the palladium-catalyzed actions of carrier-free tritium on gibberellin A3

Reaction of gibberellin A₃ (GA₃) with carrier-free tritium gas and 5% palladium on calcium carbonate as catalyst gave a complex mixture of products, several of which were isolated and identified. Three of the purified products are the radioactive forms of naturally occurring gibberellins: [³H]GA₃ (1), [³H]GA₁ (2) and [³H]tetrahydro GA₃ (4). Another substance was isolated and tentatively identified as [³H]16,17-dihydro GA₃ (3). GLC was used to determine the specific activities of 1 and 2. [³H]GA₃ likely arises from palladium catalysed nonspecific exchange of GA₃ alkane hydrogen atoms with tritium. [³H]GA₁ is also exchange labeled but most of its radioactivity is due to tritium addition to the C-1,2 olefinic bond of GA₃.     

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