Gibberellin-like Substances From Vegetative Tissue of a Conifer, Arizona Cypress

Vegetative shoots of Arizona cypress (Cupressus arizonica Greene) were found to contain an estimated 40 to 70 μg of gibberellin-like activity per kg. Based on elution patterns of silicic acid and celite partition columns, mobilities on thin layer chromatograms and specificity of the cucumber, d-3 dwarf maize, dwarf pea, and barley half seed bioassays it was possible to determine that the tissue contained at least 5 acidic, ethyl acetate-soluble gibberellin-like substances. The major one would appear to be GA₃. In addition, GA₉, GA₄, and/or GA₇-like compounds, and 2 unidentified gibberellin-like substances are present.     

LOSS OF GIBBERELLINS BY LEACHING FROM STEMS AND FOLIAGE OF CHRYSANTHEMUM MORIFOLIUM ‘PRINCESS ANNE’

Structurally complex metabolites such as carbohydrates, organic acids, and amino acids are leached from plants by rain and dew. Applied GA₃ (1 mg/ml) absorbed by a 5-cm stem segment of Chrysanthemum morifolium and translocated to a single attached leaf was leached from the leaf with distilled water. To detect endogenous gibberellins in plant leachates, Chrysanthemum morifolium ‘Princess Anne’ plants were leached under 8-hr and 18-hr photoperiods. The leachates were collected and extracted at pH 8.2 with methylene chloride to*** remove inhibitors and at pH 3.0 to remove gibberellins. The extract containing the gibberellins was fractionated on a silica gel column by an eluting phase of 0 to 100% ethyl acetate-n-hexane solutions. The presence of gibberellin-like substances was confirmed by their elution pattern from the silica gel column and by two bioassays, the lettuce hypocotyl and the Rumex bioassays. The results suggest that substances similar to GA_1–3 and GA₅ were leached from plants growing under an 18-hr photoperiod. The leachate from plants growing under an 8-hr photoperiod contained gibberellin-like substances similar to GA_1–3 and GA₆.     

The role of endogenous gibberellin-like substances and inhibitors in the growth of pea internodes

Third internodes or whole stems of 7-days old etiolated pea plants were extracted and the content of gibberellin-like substances and inhibitors has been determined. Extracts were found to contain four or five different gibberellin-like substances, some of which are chromatographically similar to GA₃. The content of gibberellins has been high in young internodes and decreased along with the internodes elongation. Brief red light irradiation brings about quantitative changes in gibberellin content, depending also on the length of internodes. The extracts contain acidic and neutral inhibitors which interfere with the response to GA₃. The content of the inhibitors does not seem to be affected by the ageing of internodes or by the light treatment.     

Isolation and Characterization of Gibberellins in Calonyction aculeatum and Structures of Gibberellins A30, A31, A33 and A34

Four novel gibberellins GA₃₀, GA₃₁, GA₃₃, GA₃₄, five known gibberellins GA₈, GA₁₇ (free acid and its monomethyl ester), GA₁₉, GA₂₇, GA₂₉ and the gibberellin-like substances were isolated from immature seeds of evening-glory (Calonyction aculeatum). The structures of GA₃₀, GA₃₁, GA₃₃ and GA₃₄ were elucidated as IX, XVIII, XXII and XXXIV, respectively. The three gibberellin-like substances were partially characterized.     

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.     

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.     

Endogenous gibberellin- and cytokinin-like substances in cultured shoot tissues of apple,Malus pumila cv. Jonathan,in relation to adventitious root formation

The frequencies of adventitious root formation in vitro of isolated shoots from bud cultures of apple (Malus pumila cv. Jonathan) after 1, 7 and 31 subcultures (weeks 5, 29 and 109 after the initial culture) were 5, 78 and 95% respectively. Endogenous gibberellin-like substances (GA) were extracted, chromatographed on SiO₂ partition columns, and assayed on dwarf rice (Oryza sativa cv. Tan-ginbozu). The levels of GA in shoots from the 1st, 7th and 31st subcultures were 40, 19 and 14 ng GA₃ eq./g dry weight of tissue, respectively, a trend which suggests an inverse relationship between endogenous GA level and rooting ability. This is consistent with the fact that applied GA₃ inhibits rooting in apple and many other species. The major peak of GA activity eluted coincidentally with GA₁/GA₃/GA₁₉. Endogenous cytokinin-like substances (CK) were chromatographed on paper and assayed with soybean hypocotyl sections. In contrast to the decrease in GA activity, CK activity increased 1.5–2.7 fold in the later subcultures (cytokinin activity per shoot, however, declined).     

Gibberellins, Endogenous and Applied, in Relation to Flower Induction in the Long-Day Plant Lolium temulentum

Changes in endogenous gibberellin-like substances (GAs) and related compounds in the shoot apices of Lolium temulentum during and after flower induction by one long day was examined for plants grown in three consecutive years. The total GA level in the shoot apical tissue was high (up to 42 micrograms per gram dry weight, or 3 × 10⁻⁵ molar GA₃ equivalents), increasing several-fold on the day after the long day and then declining. Of the many GA-like substances present, the putative polyhydroxylated components—with HPLC retention times between those of GA₈ (three hydroxyls) and GA₃₂ (four hydroxyls), and accounting for about a quarter of the total GA activity—were most consistent and striking in their changes. Their level in the apices increased 3- to 5-fold on the day after the long day and then subsided. When various GAs were applied to plants in noninductive short days, flower initiation was induced by several, most notably by GA₃₂, GA₅, 2,2-dimethyl GA₄, GA₃, and GA₇. GA₃₂ was most like one long day in eliciting a strong flowering response while having little effect on stem growth, whereas GA₁ had the opposite effect. It is suggested that highly hydroxylated C-19 GAs may play a central role in the induction of flowering in this long-day plant.     

Gibberellin-like Substances from the Developing Banana Fruit

The occurrence of 2 gibberellin-like substances was demonstrated in the developing banana fruit, Musa sapientum, Linn. Chemical and biological evidence led to the tentative identification of the 2 compounds as GA₇ and GA_x (previously isolated from citrus fruits). Support for such identification was obtained from thin layer chromatography, gradient elution column chromatography, spectrofluorometry, the dwarf maize test, and the cucumber hypocotyl test. Significance of the GA_x -designated compound increased since it is believed to occur in the fungus Fusarium moniliforme, Sheld. in addition to 2 different species of higher plants. It does not resemble any of the known gibberellins as far as chromatography is concerned.     

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

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

Abscisic Acid and Gibberellin-in Three Ungrafted Apple (Malus sylvestris) Rootstock Clones

ABA and gibberellin-like substances were extracted from roots, shoots, and leaves of most dwarfing M9, semi-dwarfing MM 106, and least dwarfing MM111 apple rootstock plants. Among tissues, the roots contained the largest and the shoots the smallest amounts of all the phytohormones studied. Plants of M9 contained the highest and MM111 the lowest ABA-like substances in all tissues tested. Gibberellin₄₊₇-like compounds were at the highest levels in the roots of MM111 and lowest in the roots of M9, whereas GA₃-like substance was in the reverse order. All three gibberellin-like compounds were highest in the shoots and leaves of MM111 and lowest in the respective tissues of M9 clone. Role of these phytohormones in the dwarfing mechanism is discussed.     

Isolation and Characterization of Gibberellins in Mature Seeds of Phaseolus vulgaris

The change of endogenous gibberellin in the germinated seeds of Phaseolus vuigaris cv. Kentucky Wonder was investigated. Based on this result, endogenous gibberellins in the mature seeds were extensively investigated to result in the isolation of GA₁, GA₈, GA₈ glucoside, AB-II (unknown gibberellin-like substance) and glucosyl esters of GA₁, GA₄, GA₃₇ and GA₃₈.     

Gibberellin-like activity in pea seedlings during growth under red,blue and white light

The influence of blue, red and white light and gibberellic acid (GA₃) on gibberellin-like activity in tissue extracts of leaves, stems and roots was investigated during growth of pea seedlings (Pisum salivum L. cv. Bördi). Higher GA-like activity was found in leaves and stems of pea plants that were growing in blue light than in those under red or white light. Patterns of change of activity were different in leaves, stems and roots, and in GA₃-treated plants.     

Identification and quantitative analysis of gibberellins inCitrus

Nine gibberellins (GAs) have been identified from tissues of Valencia orange (Citrus sinensis Osbeck) using gas chromatography—mass spectrometry and gas chromatography-selected ion monitoring of high-performance liquid chromatography (HPLC)-fractionated extracts. These GAs are GA₁, GA₃, GA₈, GA₁₉, GA₂₀, GA₂₉, 3-epi-GA₁, 2-epi-GA₂₉, and iso-GA₃. Selected-ion monitoring and stable-isotope dilution assays have been used to estimate levels of some of these GAs in vegetative and reproductive tissues. GA₂₉ was found to be the most abundant GA measured. GA₁ was found in all samples examined, and there was always less 3-epi-GA₁ than GA₁. GA₂₀ was present in most extracts. Leaves of developing inflorescence shoots contained six times more GA₂₉ than did leaves of comparable vegetative shoots. Levels of GA₂₉ increased during the early stages of fruit development. GA₂₀ may be more abundant in growing fruitlets than in those about to abscise; however, there were no consistent differences in the relative amounts of the other GAs. No major differences were found between tissues of immature seeded and seedless fruit, and developing seeds did not contain high levels of any of the GAs measured. It is concluded that seed-produced GAs are not essential for normal fruit development in Valencia orange.     

Physiological effects of gibberellic Acid. X. The release of gibberellin-like substances by germinating barley embryos

Barley embryos, completely free from endosperm, were excised from germinating grain at various times and allowed to diffuse into an aqueous medium for varying lengths of time. At the end of this time, the embryos and ambient solutions were separately extracted. Gibberellin-like activity in the extracts was determined with the barley endosperm bioassay using seed from the same variety, harvest and treatment schedule as was employed for the embryo diffusion experiments. Gibberellin-like substances were released by embryos throughout the 60 hour germination period, though at no time during this period could sufficient activity be extracted from the embryos themselves to account for the observed release. Solvent partitioning and chromatography identified at least one major acidic component migrating at an Rf similar to that of GA₃.

It is concluded that the endogenous gibberellin-like substance(s) originates within the embryo during germination, and that the release of this substance(s) is temporally consistent with, and quantitatively sufficient to account for the in vivo endosperm mobilization response syndrome. A gibberellin-like substance is undoubtedly the endosperm mobilizing hormone.

     

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.     

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     

Metabolism of Tritiated Gibberellins A(4) and A(9) in Norway Spruce, Picea abies (L.) Karst. : Effects of a Cultural Treatment Known to Enhance Flowering

Shoots of mature grafted propagules of Picea abies (L.) Karst. metabolized [³H]gibberellin A₄ (GA₄) to at least 14 acidic substances, two of which were tentatively identified by gas-liquid radiochromatography as GA₂ (possibly an artifact) and GA₃₄. [³H]GA₉ was converted into a number of metabolites, one of which was chromatographically similar to, but not identical with, GA₄. Metabolism was maximally 61 and 57% over 48 hours for GA₄ and GA₉, respectively, and was correlated with the rate of change (i.e. increase followed by decrease) in endogenous GA-like substances as shoot elongation progressed. Propagules covered with a clear plastic film, a treatment which promotes flowering, metabolized [³H]GA₄ more slowly than did control plants in the open. Inasmuch as a GA_4/7 mixture can also promote flowering in P. abies, the retarded metabolism of [³H]GA₄ may reflect the manner in which trees under plastic metabolize endogenous GA-like substances. If so, then the stimulating effect of this cultural treatment on flowering may come about through an increased level of endogenous, less polar GA-like substances.     

Gibberellins and leaf expansion in near-isogenic wheat lines containing Rht1 and Rht3 dwarfing alleles

In near-isogenic lines of winter wheat (Triticum aestivum L. cv. Maris Huntsman) grown at 20° C under long days the reduced-height genes, Rht1 (semi-dwarf) and Rht3 (dwarf) reduced the rate of extension of leaf 2 by 12% and 52%, respectively, compared with corresponding rht (tall) lines. Lowering the growing temperature from 20° to 10° C reduced the rate of linear extension of leaf 2 by 2.5-fold (60% reduction) in the rht3 line but by only 1.6-fold (36% reduction) in the Rht3 line. For both genotypes, the duration of leaf expansion was greater at the lower temperature so that final leaf length was reduced by only 35% in the rht3 line and was similar in the Rht3 line at both temperatures. Seedlings of the rht3 (tall) line growing at 20° C responded positively to root-applied gibberellin A₁ (GA₁) in the range 1–10 μM GA₁; there was a linear increase in sheath length of leaf 1 whereas the Rht3 (dwarf) line remained unresponsive. Gibberellins A_{1, 3, 4, 8, 19, 20, 29, 34, 44} and ₅₃ were identified by full-scan gas chromatography-mass spectrometry in aseptically grown 4-d-old shoots of the Rht3 line. In 12-d-old seedlings grown at 20° C, there were fourfold and 24-fold increases in the concentration of GA₁ in the leaf expansion zone of Rht1 and Rht3 lines, respectively, compared with corresponding rht lines. Although GA₃ was present at a similar level to GA₁ in the rht3 (tall) line it accumulated only fivefold in the Rht3 (dwarf) line. The steady-state pool sizes of endogenous GAs were GA₁₉ ? GA₂₀ = GA₁ in the GA-responsive rht3 line whereas in the GA non-responsive Rht3 line the content of GA₁₉≈ GA₂₀ ? GA₁. It is proposed that one of the consequences of GA₁ action is suppression of GA₁₉-oxidase activity such that the conversion of GA₁₉ to GA₂₀ becomes a rate-limiting step on the pathway to GA₁ in GA-responsive lines. In the GA-non-responsive Rht lines it is suggested that GA₁₉ oxidase is not downregulated to the same extent and GA₁ accumulates before the next rate-limiting step on the pathway, its 2β-hydroxylation to GA₈. The steady-state pool sizes of GA_{19, 20, 1, 3} and ₈ were similar in developmentally equivalent tissues of the rht3 (tall) line growing at 10° C and 20° C despite a 2.5-fold difference in the rate of leaf expansion. In contrast, in the Rht3 (dwarf) line, the extent of accumulation of GA₁ reflected the severity of the phenotype at the two temperatures with slower growing tissues accumulating less, not more, GA₁. These results are interpreted as supporting the proposed model of regulation of the GA-biosynthetic pathway rather than previous suggestions that GA₁ accumulates in GA-insensitive dwarfs as a consequence of reduced growth rates.     

Reinvestigation of apple-seed gibberellins

Summary Re-investigation of the gibberellins in apple seed has established the presence of GA₄ and GA₇ and of traces of GA₉. The fraction, previously considered to be GA₇ by TLC, contains small amounts of GA₄ and traces of GA₉; and the fraction, characterized as GA₄ by TLC, contains traces of GA₇ and GA₉. However the present results do not change the previous conclusions that during the stratification of apple seed the level of GA₇ remains fairly constant while that of GA₄ increases.