Uptake and metabolism of radioactive gibberellins by barley aleurone layers

Summary When aleurone layers were treated with labeled gibberellin A₁ (³H-GA₁), gibberellin A₅ (³H-GA₅) and the methyl ester of ³H-GA₅ (³H-GA₅-ME), radioactivity was accumulated by the tissue for a period of 20–30 h. After this time, radioactivity was released into the medium. Concomitantly, ribonuclease was also liberated by the tissue. The radioactivity accumulated by aleurone layers was associated with polar metabolites of the respective GAs, and the extent of extent of accumulation was a function of the degree of GA metabolism (GA₅-ME>GA₅>GA₁). Accumulation of radioactivity was inhibited in the cold and by the metabolic poisons NaF and dinitrophenol. This was thought to be due to inbition of GA metabolism. The accumulation of ³H-GA₁ in aleurone tissue did not reach saturation when unlabeled GA₃ up to 10^-2 M was added to the incubation medium.Abbreviations GA gibberellin - GA₅ ME, gibberellin A₅ methyl ester - RNase ribonuclease     

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.     

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.     

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).     

Preparation of radioactive gibberellin a(1) and its metabolism in dwarf peas

Gibberellin A₁-3,4-³H was prepared by selective catalytic reduction of gibberellic acid with a mixture of tritium and hydrogen. ³H-GA₁ was applied at physiological concentrations to dwarf peas and the metabolism of the hormone was investigated. ³H-GA₁ was converted to an acidic, biologically active compound. Radioactive but biologically inactive compounds were also found in the neutral fraction and could not be converted to acidic gibberellins by hydrolysis. No attachment of gibberellin to any macromolecular fraction was evident.     

Photoperiod modification of [C]gibberellin a(12) aldehyde metabolism in shoots of pea, line g2

In G2 peas (Pisum sativum L.) apical senescence occurs only in long days (LD), and indeterminate growth is associated with elevated gibberellin (GA) levels in the shoot in short days (SD). Metabolism of GA₁₂ aldehyde was investigated by feeding shoots grown in SD or LD with [¹⁴C]GA₁₂ aldehyde through the cut end of the stem for 0.5 to 6 hours in the light and analyzing the tissue extract by high performance liquid chromatography. More radioactive products were detected than can be accounted for by the two GA metabolic pathways previously known to be present in peas. Three of the major products appear to be GA conjugates, but an additional pathway(s) of GA metabolism may be present. The levels of putative C₂₀ GAs, [¹⁴C]GA₅₃, [¹⁴C]GA₄₄, [¹⁴C]GA₁₉, and/or [¹⁴C] GA₁₇, were all elevated in SD as compared to LD. Putative [¹⁴C]GA, was slightly higher in LD than in SD. Putative [¹⁴C]GA₅₃ was a major metabolite after 30 minutes of treatment in SD but had declined after longer treatment times to be replaced by elevated levels of putative [¹⁴C] GA₄₄ and [¹⁴C]GA_19/17. Metabolism of GA₂₀ was slow in both photoperiods. Although GA₂₀ and GA₁₉ are the major endogenous GAs as determined by gas chromatography-mass spectrometry, putative [¹⁴C]GA₂₀ and [¹⁴C]GA₁₉ were never major products of [¹⁴C]GA₁₂ aldehyde metabolism. Thus, photoperiod acts in G2 peas to change the rate of GA₅₃ production from GA₁₂ aldehyde, with the levels of the subsequent GAs on the 13-OH pathway being determined by the amount of GA₅₃ being produced.     

Ontogenetic variation in levels of gibberellin A1 in Pisum

The levels of the biologically active gibberellin (GA), GA₁, and of its precursor, GA₂₀, were monitored at several stages during ontogeny in the apical portions of isogenic tall (Le) and dwarf (le) peas (Pisum sativum L.) using deuterated internal standards and gas chromatography-selected ion monitoring. The levels of both GAs were relatively low on emergence and on impending apical arrest. At these early and late stages of development the internodes were substantially shorter than at intermediate stages, but were capable of large responses to applied GA₃. Tall plants generally contained 10–18 times more GA₁ and possessed internodes 2–3 times longer than dwarf plants. Further, dwarf plants contained 3–5 times more GA₂₀ than tall plants. No conclusive evidence for the presence of GA₃ or GA₅ could be obtained, even with the aid of [²H₂]GA₃ and [²H₂]GA₅ internal standards. If GA₃ and GA₅ were present in tall plants, their levels were less than 0.5% and 1.4% of the level of GA₁, respectively. Comparison of the effects of gene le on GA₁ levels and internode length with the effects of ontogeny on these variables shows that the ontogenetic variation in GA₁ content was sufficient to account for much of the observed variation in internode length within the wild-type. However, evidence was also obtained for substantial differences in the potential length of different internodes even when saturating levels of exogenous GA₃ were present.Abreviations GA_n gibberellin A_n We thank Noel Davies, Omar Hasan, Leigh Johnson, Katherine McPherson and Naomi Lawrence for technical help, Professor L. Mander (Australian National University, Canberra) for deuterated GA standards and the Australian Research Council for financial assistance.     

Internode length in pisum. Mutants lk,lka, and lkb do not accumulate gibberellins

The levels of gibberellin A₁ (GA₁), GA₈, GA₁₉, GA₂₀, GA₂₉, and GA₄₄ in the short Pisum sativum L. mutants lk, lka, and lkb, and comparable wild-type plants, were determined by gas chromatography-selected ion monitoring (GC-SIM) using ²H or ¹³C internal standards. The mutants possessed similar GA₁ levels to wild-type plants, consistent with their classification as GA-sensitivity rather than GA-synthesis mutants. However, these mutants differ from certain sensitivity mutants in other species, in which substantial accumulation of GA₁ occurs. The results suggest that if the proposed feedback model for the regulation of GA synthesis occurs in peas it is not the reduced growth per se that is the trigger for elevated levels of C₁₉ GAs. The results are also consistent with the hypothesis that in those GA-sensitivity mutants which do not accumulate C₁₉ GAs, the biochemical lesion may be well down the transduction pathway which leads from GA₁ reception to stem elongation.     

Fate of radioactive gibberellin a(1) in maturing and germinating seeds of peas and Japanese morning glory

Radioactive gibberellin A₁ (³H-GA₁) was injected into excised fruits of peas and Japanese morning glory. These were then grown in sterile culture to maturity and the label was followed in the seeds during further development and subsequent germination. During development of both pea and morning-glory seeds a large part of the radioactivity became associated with the aqueous fraction, while another part of the ³H-GA₁ was converted into 2 new, acidic, biologically active compounds, designated X₁ and X₂. A relatively small part of the neutral compounds could be converted back to ³H-GA₁, X₁, and X₂ by means of mild acid hydrolysis. During germination of pea and morning-glory seeds, part of the bound compounds was released in the form of ³H-GA₁, X₁ and X₂ while, particularly during rapid seedling growth, a further conversion of ³H-GA₁, mainly to X₁, took place. In pea seedlings, growth during the first 2 to 3 days after imbibition was not affected by Amo-1618, an inhibitor of gibberellin biosynthesis. This, in conjunction with the findings on the interconversions between free and bound ³H-GA₁ suggests that, at least in peas, early seedling growth may at least partly be regulated by gibberellins released from a bound form which was formed during seed development.     

Extractable and Diffusible Gibberellins From Light- and Dark-grown Pea Seedlings

Gibberellins were obtained from light- and dark-grown peas by solvent extraction and agar diffusion. Both A₅- and A₁-like gibberellins were obtained by extraction; however, by diffusion only the A₁-like gibberellin was found. There was no significant quantitative difference in the levels of diffusible or extractable gibberellin obtained from light- and dark-grown tall and dwarf peas. Several possible explanations for the discrepancy between diffusible and extractable gibberellin were investigated. Of these, only I was supported by experimental evidence, namely, that GA₅ can be converted to GA₁.     

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.     

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.     

Molecular cloning and analysis of a pea cDNA that is expressed in darkness and very rapidly induced by gibberellic acid

Using cDNA representational difference analysis (cDNA RDA), we isolated a cDNA named GDA-1 from a cDNA library constructed with mRNA from short-day (SD) grown G2 pea apical tissue. The amino acid sequence deduced from GDA-1 shares partial identity with the B2 protein which is expressed during embryogenesis of carrot cells. Northern analysis showed that GDA-1 mRNA is abundant in SD-grown G2 pea apical buds. In long-day (LD) conditions, there was almost no detectable GDA-1 mRNA. When LD-grown G2 peas were kept in continuous darkness for 24 h, the GDA-1 mRNA content reached a level equivalent to about 50% of that in the SD samples. On the other hand, when SD-grown peas were transferred into the light for 24 h, the amount of hybridizable GDA-1 mRNA dropped to the same as that of LD-grown plants. GDA-1 expression was found to be independent of flower initiation time. GA₃ application in vitro resulted in rapid accumulation of GDA-1 mRNA in LD-grown G2 pea apical buds, which is compatible with its delaying effect on apical senescence. Time-course experiments revealed that GDA-1 is induced within 15 min of GA₃ application. Exogenous GA₃ did not influence the expression of GDA-1 in SD-grown G2 peas. Since both photoperiod and GA induce the expression of GDA-1, we speculate that they may activate similar signal transduction pathways in G2 peas. Our work also shows that photoperiod may change the efficiency of gibberellin perception by plants. Received: 27 March 1998 / Accepted: 2 June 1998     

Transport of gibberellin a(1) in cowpea membrane vesicles

The permeability properties of gibberellin A₁ (GA₁) were examined in membrane vesicles isolated from cowpea hypocotyls. The rate of GA₁ uptake was progressively greater as pH decreased, indicating that the neutral molecule is more permeable than anionic GA₁. Membrane vesicles used in this study possessed a tonoplast-type H⁺-translocating ATPase as assayed by MgATP-dependent quenching of acridine orange fluorescence and methylamine uptake. However, GA₁ uptake was not stimulated by MgATP. At concentrations in excess of 1 micromolar, GA₁, GA₅, and GA, collapsed both MgATP-generated and artifically imposed pH gradients, apparently by shuttling H⁺ across the membrane as neutral GA. The relatively high permeability of neutral GA and the potentially detrimental effects of GA in uncoupling pH gradients across intracellular membranes supports the view that GA₁ accumulation and compartmentation must occur by conversion of GA₁ to more polar metabolites.     

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.     

Metabolism of Tritiated Gibberellin A(20) in Immature Seeds of Dwarf Pea, cv. Meteor

Tritium-labeled gibberellin A₂₀ ([³H]GA₂₀) was applied via the pedicel to immature pods and seeds of dwarf peas and three harvests were made at days 5, 10, and 23 (mature) after application. Of the five metabolites of [³H]GA₂₀, the three in highest yield were GA₂₉, an α,β-unsaturated ketone, and a compound (B), whose structure was only tentatively assigned. The metabolic sequence GA₂₀ → GA₂₉ → compound B → the ketone was indicated. The amount of [³H]GA₂₉ in both seeds and pods was highest at day 5 and declined to its lowest level at maturity. The amount of the [³H]ketone in the seed increased with time to its highest level at maturity. It is suggested that compound B and the ketone represent the major pathway of catabolism of GA₂₉, a 2β-hydroxylated GA of low biological activity, and that the ketone is not metabolized, or only slowly metabolized, during seed maturation.     

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.     

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     

Ethylene-induced stem growth of deepwater rice is correlated with expression of gibberellin- and abscisic acid-biosynthetic genes

Ethylene decreases the content of endogenous abscisic acid (ABA) and increases the level of bioactive gibberellin A₁ (GA₁) in the submerged internodes of deepwater rice. During partial submergence, internodes of deepwater rice undergo rapid elongation as a result of ethylene accumulation in the internodal lacunae. In anin vitro experiment using stem sections from deepwater rice, treatment with 5 μL L^-1 ethylene promoted stem growth by up to 3.2-foId times over air treatment. Expression patterns were analyzed for genes that encode GA- and ABA-biosynthesis enzymes to determine any possible molecular basis for the changes observed in GA₁ and ABA contents as a result of ethylene action. Expression of theOsGA20ox2 andOsGA20ox4 genes, which encode GA 20-oxidase, and of theOsGA3ox2 gene, which encodes the enzyme that converts GA₂₀ to CA₁, was up-regulated, whereas that of three ABA-biosynthetic genes —OsNCED1, OsNCED2, andOsNCEDS-was down-regulated in the presence of ethylene. These results indicate that GA and ABA contribute equally to the submergence-or ethylene-induced stem elongation of deepwater rice via the coordinated and opposite regulation of biosynthesis.     

Internode length in Pisum

The influence of the Na and Le genes in peas on gibberellin (GA) levels and metabolism were examined by gas chromatographic-mass spectrometric analysis of extracts from a range of stem-length genotypes fed with [¹³C, ³H]GA₂₀. The substrate was metabolised to [¹³C, ³H]GA₁, [¹³C, ³H]GA₈ and [¹³C, ³H]GA₂₉ in the immature, expanding apical tissue of all genotypes carrying Le. In contrast, [¹³C, ³H]GA₂₉ and, in one line, [¹³C, ³H]GA₂₉-catabolite, were the only products detected in plants homozygous for the le gene. These results confirm that the Le gene in peas controls the 3-hydroxylation of GA₂₀ to GA₁. Qualitatively the same results were obtained irrespective of the genotype at the Na locus. In all Na lines the [¹³C, ³H]GA₂₀ metabolites were considerably diluted by endogenous [¹²C]GAs, implying that the metabolism of [¹³C, ³H]GA₂₀ mirrored that of endogenous [¹²C]GA₂₀. In contrast, the [¹³C, ³H]GA₂₀ metabolites in na lines showed no dilution with [¹²C]GAs, confirming that the na mutation prevents the production of C₁₉-GAs. Estimates of the levels of endogenous GAs in the apical tissues of Na lines, made from the ¹²C:¹³C isotope ratios and the radioactivity recovered in respective metabolites, varied between 7 and 40 ng of each GA per plant in the tissue expanded during the 5 d between treatment with [¹³C, ³H]GA₂₀ and extraction. No [¹²C]GA₁ and only traces of [¹²C]GA₈ (in one line) were detected in the two Na le lines examined. These results are discussed in relation to recent observations on dwarfism in rice and maize.Abbreviations GA_n gibberellin A_n - GC-MS gas chromatography-mass spectrometry - HPLC high-pressure liquid chromatography