Gibberellins in the embryonic axes of tall and dwarf beans and their changes with initial growth

Ten gibberellin-like activities were detected in the dry embryonicaxes of tall (cv. Kentucky Wonder) and dwarf (cv. Masterpiece)beans (Phaseolus vulgaris L.) using the lettuce hypocotyl assayof thin-layer chromatograms; 2 in the non-acidic ethyl acetatefraction (NEI, NEII), 3 in the acidic ethyl acetate fraction(AEI, AEII, AEIII), 2 in the non-acidic n-butanol fraction (NBI,NBII) and 3 in the acidic n-butanol fraction (ABI, ABII, ABIII).There was no qualitative difference in these gibberellins betweenthe tall and dwarf axes, but all, particularly AEIII, NBII andABIII as the main gibberellins in the axes, were contained muchmore abundantly in the tall axes. In both axes the gibberellinactivities of most fractions decreased during germination.Theamounts of some gibberellins in tall axes without cotyledonswere greater than those in axes with cotyledons at 48–72hr of germination. Neither AMO-1618 nor CCC caused significantreduction in the levels of the gibberellins. Axis growth inthe early germinating period depended on the gibberellins storedin the axis, itself. (Received November 26, 1974; )     

Variations in Endogenous Gibberellins in Developing Bean Seeds II. Changes Induced in Acidic and Neutral Fractions by GA(1)

Immature (8-mm), medium mature (11-mm), and mature green (16- and 17-mm) bean seeds (Phaseolus vulgaris L. cv. Kentucky Wonder and Bountiful) were incubated in gibberellin A₁ solutions for 24 hours at 20°. Extracts from the seeds were separated into nonacidic, acidic ethyl acetate, and acidic butanol fractions. These were chromatographed. The eluates of the chromatograms were tested on Progress No. 9 dwarf peas grown under red light. The level of neutral gibberellin-like substances remained unchanged in immature seed, but they increased markedly in mature green seeds. Coincident with increased levels of the neutral substances, there were significant decreases in acidic ethyl acetate-soluble gibberellin-like substances, including applied GA₁, and in 1 acidic butanol-soluble gibberellin-like substance. Seed incubation in GA₁ brought about increased activity of substance B-II in immature and medium mature seeds. The level of butanol-soluble gibberellin-like substance B-I in seeds of any size was not affected by incubation in GA₁. Considering the marked increases in activity induced in the neutral fraction and the decreases in activity of certain eluates from the chromatograms of the acidic fractions, it was concluded that the neutral fraction may serve as a reserve form of gibberellins in the dry seed. The acidic ethyl acetate substances and substance B-II may be required for normal development of the bean seed.     

Levels of gibberellin-like substances and their possible transport in developing dwarf and normal cultivars of pea (Pisum sativum L.)

The level of gibberellin-like substances was determined in the cotyledons and axis of developing seedlings of dwarf (Little Marvel) and normal (Tall Telephone) cultivars of pea (Pisum sativum L.). The effect of cotyledon removal with GA₃ application on growth was also examined. Greater levels of gibberellin-like substances were observed in the cotyledons of the normal cultivar than the dwarf. This was particularly evident in the cotyledons during the early stages of seedling growth. Subsequently there was a decline in GA levels in the cotyledons. This was coincidental with a rise in GA content in the axis with markedly greater levels in the normal than the dwarf cultivar. Decotyledonated dwarf and normal plants supplied with GA were much taller than the decotyledonated controls. This observation along with those of the gibberellin levels in the cotyledons and axis, provided circumstantial evidence that there may be translocation of gibberellins from the cotyledons to the axis.     

Natural growth inhibitors associated with the cotyledon effect in light-grown dwarf and tall beans

The contents of non-acidic, acidic and bound growth inhibitorsin hypocotyls and cotyledons were compared between dark- andlight-grown dwarf and tall beans by means of thin-layer chromatographyand bioassay. In the non-acidic fraction, one major inhibitoryactivity appeared on the chromatogram, but its Rf zone was differentbetween hypocotyls and cotyledons. In both the acidic and boundinhibitor fractions, one major inhibitory activity appearedat the Rf zone corresponding to ABA. The ABAIike substance whichwas the major inhibitor in the hypocotyl was more abundant inlight-gorwn than dark-grown ones, especially in the dwarf variety,but light irradiation did not cause its transport from cotyledonsto the hypocotyl. A larger amount of bound ABA-like substance,which was the major inhibitor in the cotyledon, was presentin the dwarf than the tall variety regardless of the light condition.Cotyldon-enhanced photoinhibition of hypocotyl growth couldnot be explained by the levels of the xanthoxinand ABA-likeinhibitors, or the transport of these free inhibitors from cotyledons. (Received September 8, 1977; )     

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.     

The control of flower initiation by gibberellin in Helianthus annuus L. (sunflower), a non-photoperiodic plant

The involvement of gibberellins in the control of flowering of sunflower was studied by direct application of GA₃ to the apex of the plants, analysis of the endogenous levels of gibberellin-like substances at different plant ages, and indirectly by the application of paclobutrazol, an inhibitor of gibberellin synthesis. GA₃ speeded-up flower initiation and floral apex development. The time of GA₃ application was more critical than the amount of GA₃ applied. The endogenous levels of gibberellin-like compounds increased significantly by day 15 after sowing. The application of paclobutrazol markedly delayed floral initiation and this effect was also depedent on plant age. Both GA₃ and paclobutrazol had their greatest effects between 10 and 20 days after sowing suggesting that an increase in gibberellins in that time period plays a role in floral initiation.     

Growth-promoting activity of some selectively modified gibberellins

Synthetic gibberellin analogues derived mainly from GA₃ were tested for their growth-promoting activity in three standard bioassay systems (dwarf pea, cucumber and lettuce). The highest potency in all the tests was displayed by 13-O-methyl-GA₃, while in the case of 7-homo-GA₃ a large decrease in activity was observed. No obvious correlation could be found between the partitioning of acidic analogues between ethyl acetate and water and their biological potencies. The results of the bioassays appear to be compatible with the assumption that it is the spatial correspondence between an active gibberellin (such as GA₃ or GA₇) and a specific receptor that plays the prime role in the growth response.     

Shoot elongation in Lathyrus odoratus L.: Gibberellin levels in light- and dark-grown tall and dwarf seedlings

The levels of gibberellin A₁ (GA₁), GA₂₀, GA₁₉, GA₈, GA₂₉ and GA₈₁ (2-epiGA₂₉) were measured in tall (L-) and dwarf (ll) sweet-pea plants grown in darkness and in light. In both environments the apical portions of dwarf plants contained less GA₁; GA₈ and GA₁₉, but more GA₂₀, GA₂₉, and GA₈₁ than did those of tall plants. It is concluded that the partial block in 3β-hydroxylation of GA₂₀ to GA₁ is imposed by allele l in darkness as well as in the light. Furthermore, darkness does not appear to enhance elongation in sweet pea by increasing GA₁ levels. The reduction of the pool size of GA₁₉ in dwarf plants supports recent theories on the regulation of GA biosynthesis, formulated on the basis of observations in monocotyledonous species. Darkness results in decreased GA₂₀, GA₂₉, and GA₈₁ levels in the apical portions of tall and dwarf plants and possible reasons for this are discussed.     

Gibberellin and Inhibitor Content during Iris Bulb Development

Concentrations of gibberellin-like substances and inhibitors were determined in different organs from Iris hollandica cv. Prof. Blaauw during cold treatment and during growth in the greenhouse. Barley-aleurone, dwarf pea and Avena coleoptile straight growth bioassays were used to determine the activity. Developing bulblets contained the highest level of gibberellin activity followed by that in floral organs. During the development of the plants quantitative changes occurred both in the non-acidic, free, and bound pools of gibberellins. Two kinds of inhibitors, “acidic” and “non-acidic”, have been found in the extracts. Most of the non-acidic type were present in the roots. The acidic inhibitors were present in scales and sheath-leaves and disappeared with cold treatment.     

Evidence for Phytochrome Regulation of Gibberellin A(20) 3beta-Hydroxylation in Shoots of Dwarf (lele) Pisum sativum L

The effect of light on the dwarfing allele, le, in Pisum sativum L. was tested as the growth response to gibberellins prior to or beyond the presumed block in the gibberellin biosynthetic pathway. The response to the substrate (GA₂₀), the product (GA₁), and a nonendogenous early precursor (steviol) was compared in plants bearing the normal Le and the deficient lele genotypes in plants made low in gibberellin content genetically (nana lines) or by paclobutrazol treatment to tall (cv Alaska) and dwarf (cv Progress) peas. Both genotypes responded to GA₁ under red irradiation and in darkness. The lele plants grew in response to GA₂₀ and steviol in darkness but showed a much smaller response when red irradiated. The Le plants responded to GA₂₀ and steviol in both light and darkness. The red effects on lele plants were largely reversible by far-red irradiation. It is concluded that the deficiency in 3β-hydroxylation of GA₂₀ to GA₁ in genotype lele is due to a Pfr-induced blockage in the expression of that activity.     

Gibberellin-like substances in root exudate of Vitis vinifera

Summary The levels of gibberellin (GA)-like activity in the root exudate of two seedless varieties of Vitis vinifera were examined by the barley endosperm assay, and compared with levels determined for other parts of the plant. That activity was due to GA-like substances was confirmed with dwarf-5 corn.When acidic, ethyl acetate soluble GA-like substances from sap and leaf extracts were chromatogrammed on thin layers of silica gel in chloroform/ethyl acetate/formic acid (50:50:1), activity moved to the same Rf as GA₃ and GA₁ (Rf 0.05–0.25). However, substances inhibitory to the barley endosperm assay were detected in both sap and leaf extracts. In the above solvent system the inhibitor(s) co-chromatogrammed with a GA₁/GA₇ mixture, and with abscisin II. The GA-like activity co-chromatogrammed with GA₃ on paper developed in isopropanol/ammonia/water (10:1:1).Calculations on the rate of gibberellin movement from the roots seemed to be compatible with levels of activity in the leaves, although these levels could also be a reflection of the general gibberellin level in the plant.The relevance of the findings is discussed.     

Exogenous GA3 Application Can Compensate the Morphogenetic Effects of the GA-Responsive Dwarfing Gene Rht12 in Bread Wheat

The most common dwarfing genes in wheat, Rht-B1b and Rht-D1b, classified as gibberellin-insensitive (GAI) dwarfing genes due to their reduced response to exogenous GA, have been verified as encoding negative regulators of gibberellin signaling. In contrast, the response of gibberellin-responsive (GAR) dwarfing genes, such as Rht12, to exogenous GA is still unclear and the role of them, if any, in GA biosynthesis or signaling is unknown. The responses of Rht12 to exogenous GA₃ were investigated on seedling vigour, spike phenological development, plant height and other agronomic traits, using F_2∶3 and F_3∶4 lines derived from a cross between Ningchun45 and Karcagi-12 in three experiments. The application of exogenous GA₃ significantly increased coleoptile length and seedling leaf 1 length and area. While there was no significant difference between the dwarf and the tall lines at the seedling stage in the responsiveness to GA₃, plant height was significantly increased, by 41 cm (53%) averaged across the three experiments, in the GA₃-treated Rht12 dwarf lines. Plant height of the tall lines was not affected significantly by GA₃ treatment (<10 cm increased). Plant biomass and seed size of the GA₃-treated dwarf lines was significantly increased compared with untreated dwarf plants while there was no such difference in the tall lines. GA₃-treated Rht12 dwarf plants with the dominant Vrn-B1 developed faster than untreated plants and reached double ridge stage 57 days, 11 days and 50 days earlier and finally flowered earlier by almost 7 days while the GA₃-treated tall lines flowering only 1–2 days earlier than the untreated tall lines. Thus, it is clear that exogenous GA₃ can break the masking effect of Rht12 on Vrn-B1 and also restore other characters of Rht12 to normal. It suggested that Rht12 mutants may be deficient in GA biosynthesis rather than in GA signal transduction like the GA-insensitive dwarfs.     

A SEARCH FOR SOME ANG1OSPERM HORMONES AND THEIR METABOLITES IN CAULERPA PASPALOIDES (CHLOROPHYTA)1

The best available methods were used to search for an-giosperm hormones in the green alga Caulerpa paspa-loides (Bory) Greville. Solvent partitioning of methanol extracts led to acidic ethyl acetate fractions that it were run through high-performance liquid chromatography. Each of the resulting 25 fractions was divided in half so that one half was tested in the dwarf rice or lettuce hypocotyl assays for gibberellins and the other half was methylated and silylated for capillary gas chromatography combined with computerized mass spectrometry. By comparing algal mass spectra with spectra in the extensive library of mass spectra and associated Kovats Retention Indices that MacMillan's Bristol group has amassed, indole-3-acetic acid's presence in the alga was confirmed and dioxindole-3-acetic acid was found. However, despite the presence of several peaks of gibberellin-like activity in the bioassays, no gibberellin or gibberellin metabolite was found in the thousands of full mass spectra examined. The gibberellin-like activity in the acidic ethyl acetate fractions was therefore presumably not from any gibberellin known from vascular plants.     

Ein Beitrag zur Analyse der Gibberelline in Samen von Pisum sativum L.

Summary Unripe seeds of Pisum sativum were analyses for gibberellins and gibberellin-like substances. Forty kg of seeds were extracted with methanol, the methanol was evaporated and the residue distributed between water and ethylacetate, and between water and butanol at different pH-values. The acidic ethylacetate and the butanol fractions were separated by thin layer chromatography. In the acidic ethylacetate fraction a new substance tentatively named gibberellin A_x was isolated by preparative thin layer chromatography. In different solvent mixtures this substance migrates closely behind gibberellin A₃. It is biological active in a dwarf pea and in dwarf maize mutant bioassays. IR and fluorescense spectra point to a gibban structure. Furthermore gibberellin A₅ was found to be the main gibberellin in pea seeds. It was identified by its R_f-values and the R_f-values of its methylester in different solvent systems as well as by its characteristic relative biological activity in three biological test systems. Most probably gibberellin A₆ is also present in the pea seeds, but because the amounts detected were small, no clear evidence can be presented.In the butanol phase two gibberellin-like substances could be separated by thin layer chromatography.

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Mit Hilfe der Deutschen Forschungsgemeinschaft.     

Effect of Different Gibberellins on the Growth of the Hypocotyl

It has been stated earlier that hypocotyls of different plants show different growth response to added GA₃. It was suggested that this difference may be due to the requirement of some specific gibberellin. Hence hypocotyl growth response of three groups of plants has been studied with different gibberellins: group one showing no or insignificant growth response, group two showing 150–200 per cent growth response and group three showing 300–500 per cent growth response to added GA₃. Eight gibberellins were used, viz., GA₁, GA₂, GA₃, GA₄, GA₅, GA₇, GA₈ and GA₉, to test if this varying response is connected with the requirement of some specific gibberellin. In general, the results obtained do not favour this view. Iberis amara, a plant showing no response to added GA₃, Dianthus sp., a plant showing 150 to 200 per cent response and Lactuca satwa, Antirrhinum majus and Nicotiana tabacum, plants showing 300 to 500 per cent response, were promoted by all the gibberellins tested to a similar extent as by GA₃, with the exception of GA₈ which was inactive in most of the cases.     

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.     

Endogenous gibberellins and kauranoids identified from developing and germinating barley grain

Several gibberellins (GAs) and kauranoids were identified in extracts of barley (Hordeum vulgare) by combined capillary gas chromatography-mass spectrometry (GC-MS). A partially purified acidic ethyl acetate extract from 21-day postanthesis developing barley grain (cv. Proctor) contained GA₁ (trace), GA₄ (trace), GA₈ (trace), GA₁₂, GA₁₇, GA₂₀ (tentative) (trace), GA₂₅, GA₃₄, GA₄₈, 18-hydroxy-GA₄, 12-hydroxy-GA₉, and 18-hydroxy-GA₃₄ (tentative). A hydrolyzed butanol extract contained GA₁₇, GA₂₀, GA₄₈, and 18-hydroxy-GA₃₄ (tentative). An acidic ethyl acetate extract from 3-day-old germinating barley grain (cv. Maris Otter) contained GA₁, GA₃ (possibly a contaminant), GA₁₇, GA₁₉, GA₂₀, GA₃₄, GA₄₈, and 18-hydroxy-GA₃₄ (tentative). A hydrolyzed butanol extract contained GA₃₄, GA₄₈, and 18-hydroxy-GA₃₄ (tentative). In germinating grain, levels of all GAs were very low. Two hydroxylated kauranoic acids and a number of other kauranoids were also detected in the above extracts. 1-Hydroxylated GAs previously found in wheat were not found in barley in this study.This work has been reported in a poster demonstration (Gaskin et al. 1982).     

Synergism between Gibberellin and Auxin in the Growth of Intact Tomato

Gibberellin A_4&7 was more effective than gibberellic acid in increasing shoot elongation when applied to the apex of intact Lycopersicum esculentum seedlings of Tiny Tim, a dwarf cultivar, and Winsall, a tall cultivar. After 14 days, gibberellic acid and gibberellin A_4&7 stimulated growth of the dwarf more than the tall tomato. In tall tomato the application of indole-3-acetic acid alone (6.1 μg/plant) showed an inhibitory growth effect, but when applied with 17.5 μg per plant of gibberellic acid, it had a synergistic effect at 7 days but not at 14 days. When the auxin concentration was reduced to 0.61 μg per plant a synergistic effect was observed on tall plants at 7 and 14 days between indole-3-acetic acid and gibberellic acid. Application of gibberellin A_4&7 with auxin did not give a synergistic response in tall or dwarf tomato.     

Endogenous gibberellins of a radiation induced single gene dwarf mutant of bean

The distribution of endogenous gibberellins in Dwarf-1, a single gene dwarf mutant of Mexico 80-R red beans, was studied. Parallel extraction and fractionation of seeds of this mutant and those of a normal homozygous line followed by thin layer chromatography and bioassays using Rumex obtusifolius, wheat seed endosperm and dwarf bean plants revealed that a stem elongation control factor was contained in the non-acidic fraction from normal, but not from Dwarf-1, seeds. It was concluded that the single gene mutation causes a block either in gibberellin precursor formation or in production of a non-acidic fraction gibberellin-like substance.     

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