Gibberellins: a phytohormonal basis for heterosis in maize.

Four commercially important maize parental inbreds and their 12 F(1) hybrids were studied to investigate the role of the phytohormone gibberellin (GA) in the regulation of heterosis (hybrid vigor). All hybrids grew faster than any inbred. In contrast, all inbreds showed a greater promotion of shoot growth after the exogenous application of GA(3). Concentrations of endogenous GA(1), the biological effector for shoot growth in maize, and GA(19), a precursor of GA(1), were measured in apical meristematic shoot cylinders for three of the inbreds and their hybrids by gas chromatography-mass spectrometry with selected ion monitoring; deuterated GAs were used as quantitative internal standards. In 34 of 36 comparisons, hybrids contained higher concentrations of endogenous GAs than their parental inbreds. Preferential growth acceleration of the inbreds by exogenous GA(3) indicates that a deficiency of endogenous GA limits the growth of the inbreds and is thus a cause of inbreeding depression. Conversely, the increased endogenous concentration of GA in the hybrids could provide a phytohormonal basis for heterosis for shoot growth.

Synthesis and bioactivity of 6alpha- and 6beta-hydroxy analogues of castasterone.

The reduction of castasterone with sodium in ethanol produced chiefly the known 6alpha-hydroxy stereoisomer, whereas reduction with sodium orohydride in methanol afforded mainly the novel 6beta-epimer. Both compounds showed variable bioactivity through four separate assays via the rice leaf lamina inclination bioassay. However, when treated with an appropriate statistical program to remove outliers, the averaged results clearly indicated that the two 6-hydroxy epimers possess comparable and significant bioactivity, which is, however, lower than that of castasterone or brassinolide. When applied together with 1000 ng of the auxin, indole-3-acetic acid (IAA), the activity of both the 6alpha and 6beta hydroxy epimers was enhanced by ca. one order of magnitude across a wide range of doses.

Bioactivity of 25-hydroxy-, 26-hydroxy, 25,26-dihydroxy- and 25,26-epoxybrassinolide.

The bioactivity of 25-hydroxybrassinolide, (25S)- and (25R)-26-hydroxybrassinolide, (25S)- and (25R)-25,26-dihydroxybrassinolide, and of (25R)-25,26-epoxybrassinolide was tested in the rice leaf lamina inclination assay. The 25- and (25S)-26-hydroxy derivatives are known metabolites of the naturally-occurring phytohormone brassinolide, whereas the other compounds are novel, but closely related, congeners. When tested alone, all showed either no activity or only weak activity at relatively high doses. When coapplied with indole-3-acetic acid (IAA), an auxin that synergizes the effects of brassinosteroids, enhanced bioactivity was observed for each compound. However, even when applied together with IAA, none of the compounds proved more bioactive than brassinolide with or without IAA. We conclude from these results that enzymatic hydroxylation of endogenous brassinolide at C-25 and/or C-26 does not enhance brassinosteroid activity, and so does not comprise an activation pathway in brassinolide biosynthesis. Instead, these hydroxylations result in modest to appreciable metabolic deactivation.

Endogenous gibberellins in immature seeds of Prunus persica L.: identification of GA(118), GA(119), GA(120), GA(121), GA(122) and GA(126).

The endogenous gibberellins in immature seeds of Prunus persica were analyzed by gas chromatography-mass spectrometry. Eleven known gibberellins, GA(3), GA(9), GA(17), GA(19), GA(30), GA(44), GA(61), GA(63), GA(87), GA(95) and GA(97) were identified. Additionally, several hitherto unknown gibberellins were detected and their putative structures were verified by synthesis of the authentic gibberellins. These gibberellins were then assigned trivial numbers, e.g. 1alpha-hydroxy GA(20) (GA(118)), 1alpha-hydroxy GA(9) (GA(119)), 1,2-didehydro GA(9) (GA(120)), 1,2-didehydro GA(70) (GA(121)), 1,2-didehydro GA(69) (GA(122)) and 1,2-didehydro GA(77) (GA(126)). GA(118) and GA(119) were the first 1alpha-hydroxy gibberellins identified from higher plants. The above profile of 1,2-didehydro gibberellins suggests that 1,2-dehydrogenation might occur prior to 3beta-hydroxylation in biosynthesis of GA(3), GA(30) and GA(87) in immature seeds of P. persica.

Screening the foods of an endangered parrot, the kakapo (Strigops habroptilus), for oestrogenic activity using a recombinant yeast bioassay.

In recent years the possibility of environmental oestrogens affecting the reproduction of vertebrates has become an issue of both public and scientific interest. Although the significance of such chemicals remains controversial there is clear evidence that, in some contexts, environmental oestrogens can influence the fertility of vertebrates. Highly endangered species represent a situation in which even modest reductions in the fertility of key individuals may have implications for the survival of the entire species. This paper reports the screening of both natural and supplementary foods of the kakapo (Strigops habroptilus), a critically endangered New Zealand nocturnal parrot, for oestrogenic activity using a recombinant yeast based bioassay. Low levels of oestrogenic activity were detected in one of the 'chick-raising' foods, but no oestrogenic activity was detected in the adult supplementary foods. The oestrogenicity of a range of phytochemicals possibly associated with the kakapo natural diet was also examined. Two such phytochemicals, podocarpic acid and its reduced derivative podocarpinol, showed weak oestrogenic activity (approximately 10(-6) and 10(-4) of the activity of 17-beta-oestradiol, respectively).

Flowering of Chrysanthemum under non-inductive long days by gibberellins and N(6)-benzyladenine.

The flowering and inflorescence development of Chrysanthemum morifolium cv. Pink Champagne under non-inductive long days was promoted by exogenous application of GA5, GA3, GA4+GA7 or GA9 in combination with the cytokinin, BA. The combination of BA and GA5 was highly effective, BA and GA3 moderately effective. Applications of the GAs alone or BA alone also resulted in some flowering, with GA3 and GA5 being most effective. In general, the effects of GA and BA were synergistic, and the concentration of both growth substances was a limiting factor with regard to the number of plants flowering under long days. Only the concentration of GA was a limiting factor for inflorescence development, however. Simultaneous application of indole-3-acetic acid reduced inflorescence development in most treatments. Development to the stage of anthesis was in no case effected.

Endogenous Gibberellins of Pine Pollen: II. Changes during Germination of Pinus attenuata, P. coulteri, and P. ponderosa Pollen.

The endogenous gibberellins (GAs) of pollen of Pinus attenuata, P. coulteri, and P. ponderosa were bioassayed at hour 0, 3, 15, 24, 48 and 72 of germination. Dormant pollen showed relatively high GA activity throughout the elution spectrum (i.e. ranging from relatively nonpolar to highly polar). The maximum GA activity was obtained at hour 15 in more polar regions and especially in the zone corresponding to GA(3) (for P. attenuata estimated as 250 micrograms of GA(3)/kilogram pollen). It is probable that the "nonpolar" GAs present in high quantities in dormant pollen and in early stages of germination were converted to "more polar" GAs as germination progressed. The amount of all GAs decreased after hour 15 of germination and by hour 72 no GAs could be detected. Among the species tested P. attenuata showed the highest over-all GA activity.

Changes of Endogenous Gibberellin-like Substances with Sex Reversal of the Apical Inflorescence of Corn.

In developing apical meristems of corn, the level of acidic, ethyl acetate-soluble gibberellin (GA)-like substances increased to a maximum of 108 micrograms GA(3)-equivalents per kilogram dry weight of tissue at inflorescence initiation, and then fell rapidly. At anthesis, only a trace (0.2 microgram per kilogram) of GA-like activity remained in the apical (male) inflorescences, whereas moderate activity (32 micrograms per kilogram), mostly of a nonpolar nature, was present in lateral, female, inflorescences.A sex reversal of the apical inflorescence, from male to female, was elicited by reducing the ambient light intensity. Higher levels of GA-like substances, particularly those eluting from a SiO(2) partition column in the nonpolar region, were observed at all harvests in the reverting meristems; levels increased to 180 micrograms per kilogram at inflorescence initiation, then dropped to 122 micrograms per kilogram in the apical (female), reverted meristems. This increase in endogenous GA-like activity with reversion to the female inflorescence is consistent with observations that (a) reversion can be obtained with exogenous application of GA(3) and (b) maleness is enhanced in GA-deficient mutants of maize. Endogenous GAs may thus play a key role in the control of sexuality of corn.

Participation of gibberellin in the control of apical dominance in soybean and redwood.

Loss of apical dominance in soybeans and redwood was increased when the plants were treated with the growth retardant AMO-1618. Simultaneous application of gibberellin reduced the number of elongating buds and promoted growth of the first or second uppermost axillary bud, thus restoring apical dominance. It is concluded that gibberellin participates in the expression of apical dominance.

Interconversion of gibberellin A4 to gibberellins A 1 and A 34 by dwarf rice, cultivar Tan-ginbozu.

Interconversion of GA4 to GA1 and GA34 occurred within 24 h of application of 1,2-[(3)H]-GA4 to seedlings of dwarf rice, cv. Tan-ginbozu. Identification was made by direct comparison of the trimethylsilyl ether derivatives of the methyl esters of Silica-gel partition-column fractions on gas-liquid radiochromatography with derivatized GA1 and GA34 standards on three columns: 2% QF-1, 2% SE-30, and 1% XE-60. GA2, an artifact of the purification and chromatography system, may also be formed by the plant. The conversions from GA4 to GA1 and GA34 are single hydroxylations. At least two unidentified radioactive products were also formed by the plant. Interconversions were in the order of 0.3 to 0.8% of applied [(3)H]-GA4.

Ethylene-Mediated Regulation of Gibberellin Content and Growth in Helianthus annuus L.

Elongation of hypocotyls of sunflower can be promoted by gibberellins (GAs) and inhibited by ethylene. The role of these hormones in regulating elongation was investigated by measuring changes in both endogenous GAs and in the metabolism of exogenous [(3)H]- and [(2)H(2)]GA(20) in the hypocotyis of sunflower (Helianthus annuus L. cv Delgren 131) seedlings exposed to ethylene. The major biologically active GAs identified by gas chromatography-mass spectrometry were GA(1), GA(19), GA(20), and GA(44). In hypocotyls of seedlings exposed to ethylene, the concentration of GA(1), known to be directly active in regulating shoot elongation in a number of species, was reduced. Ethylene treatment reduced the metabolism of [(3)H]GA(20) and less [(2)H(2)]GA(1) was found in the hypocotyls of those seedlings exposed to the higher ethylene concentrations. However, it is not known if the effect of ethylene on GA(20) metabolism was direct or indirect. In seedlings treated with exogenous GA(1) or GA(3), the hypocotyls elongated faster than those of controls, but the GA treatment only partially overcame the inhibitory effect of ethylene on elongation. We conclude that GA content is a factor which may limit elongation in hypocotyls of sunflower, and that while exposure to ethylene results in reduced concentration of GA(1) this is not sufficient per se to account for the inhibition of elongation caused by ethylene.

Metabolism of [(3)H]Gibberellin A 20 by plants of Bryophyllum daigremontianum under long- and short-day conditions.

[(3)H]Gibberellin A20 ([(3)H]GA20), a native gibberellin of this plant, was injected into mature leaves of Bryophyllum daigremontianum (Hamet et Perr.) Berg. under long- and short-day conditions. It was converted, in order of decreasing yields, to GA29, 3-epi-GA1 (pseudo GA1), C/D-ring-rearranged GA20, and two minor, unidentified metabolites. Identifications were made by gas-liquid chromatography with radioactive monitoring using three different phases. Metabolism to 3-epi-GA1 was greater under short days, particularly in the treated leaf pair, although the absolute amount of GA29 was greater than that of 3-epi-GA1 under both photoperiods. The levels of radioactive metabolites in the shoots above the treated leaf pair gradually increased over a 51-day period, GA29 reaching 5 times the contten of 3-epi-GA1.

Endogenous Gibberellins of Pine Pollen: III. Conversion of 1,2-[H]GA(4) to Gibberellins A(1) and A(34) in Germinating Pollen of Pinus attenuata Lemm.

Gibberellins A(1) and A(34) (possibly A(2)) were found as products of metabolism of 1,2-[(3)H]GA(4) during germination of Pinus attenuata pollen. The conversion from GA(4) to GA(1) and GA(34) occurred as hydroxylations at atoms C-13 and C-2 of the ent-gibberellane skeleton, respectively. Percentage interconversion of the GA(4) absorbed was in the range of 0.15 to 0.43% for GA(1) and 1.54 to 3.22% for GA(34). Identifications were made on a gas-liquid chromatograph with radioactive monitoring by comparison with standards.

Ethylene, gibberellins, auxin and the apical control of branch angle in a conifer, Cupressus arizonica.

Decapitation, gibberellin A3, high light, their combination, and certain levels of indole-3-acetic acid increase ethylene evolution and also induce branch hyponasty (upturning) in seedlings of Cupressus arizonica Greene, the increase in ethylene preceding obvious hyponasty. Exogenous ethylene also causes branch hyponasty and branches of seedlings maintained in an atmosphere scavenged of ethylene by mercuric perchlorate grow downwards. It is concluded that ethylene may play a role in the apical control of branch angle in some conifers. The positive effect of ethylene in increasing branch hyponasty may be direct, or reflect changes in levels of endogenous auxin and/or gibberellin.

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 degrees is associated with changes in endogenous gibberellin-like substances (GAs), and in differential metabolism of applied [(3)H]GA(4) (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 GA(3)-like (polar) substance. Native GAs of Avena include GA(3), GA(4), and GA(7). Geostimulation enhanced the ratio of nonpolar to polar GAs for both halves of internodes, but tended to depress it for sheaths and nodepulvini.The disposition and metabolism of applied [(3)H]GA(4) confirmed the above trends for endogenous GAs regarding localization (e.g. up to 2-fold increases in [(3)H]GA(4) and acidic (3)H-metabolites in the lower halves, relative to upper halves). Also, metabolism into highly water-soluble (3)H-metabolites (biologically inactive conjugates?) was greater (up to 1.8-fold) in upper than in lower halves. The end result of such metabolic trends would be to reduce acidic (biologically active?) GAs in the upper half, while retaining them for a longer time in the lower half.Geotropically stimulated Avena shoots thus increase, within 24 hours, the levels of acidic GAs in the lower halves of the p-1 node-pulvinus and p-1 internode, the two plant parts responsible for the geostimulated growth.

Mutual Antagonism of Sulfur Dioxide and Abscisic Acid in Their Effect on Stomatal Aperture in Broad Bean (Vicia faba L.) Epidermal Strips.

Abscisic acid (ABA) was found to counteract the stomatal opening in Vicia faba L. caused by SO(2). The antagonism between SO(2) and ABA was mutual, and their combined effect depended upon which compound was in the greatest concentration. Stomatal apertures were monitored in detached epidermal strips floated in the light on aqueous solutions of SO(2) (sulfurous acid) and/or ABA in 0.01 molar sodium citrate buffer (pH 5.8). Low concentrations of sulfurous acid (10(-10) to 10(-7) molar) increased stomatal aperture, but concentrations greater than 10(-5) molar decreased it. A progressive decrease in aperture size occurred as ABA was increased from 10(-10) to 10(-5) molar.No evidence was found for a direct chemical reaction between the buffered sulfurous acid and ABA (exogenous or endogenous). Extractable, endogenous ABA in the strips remained relatively constant after exposure to several different concentrations of sulfurous acid. A technique for quantitating ABA from methanolic extracts of small samples of epidermis (20 milligrams dry weight) using reverse phase high performance liquid chromatography is described.

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

Tritium-labeled gibberellin A(20) ([(3)H]GA(20)) 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 [(3)H]GA(20), the three in highest yield were GA(29), an alpha,beta-unsaturated ketone, and a compound (B), whose structure was only tentatively assigned. The metabolic sequence GA(20) --> GA(29) --> compound B --> the ketone was indicated. The amount of [(3)H]GA(29) in both seeds and pods was highest at day 5 and declined to its lowest level at maturity. The amount of the [(3)H]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 GA(29), a 2beta-hydroxylated GA of low biological activity, and that the ketone is not metabolized, or only slowly metabolized, during seed maturation.

Promotion of flowering in apple trees with gibberellin A4 and C-3 epi-gibberellin A 4.

The proportion of spurs flowering on apple trees (Malus domestica Borkh. cv Golden Delicious) displaying a high degree of alternate-year flowering was increased in the "off" year by gibberellin A4 (GA4) and C-3 epi-GA4 applied in the previous year. When applied 4.5 weeks after anthesis amounts of GA4 ranging from 3 to 300 µg per spur and 25 or 50 µg of C-3 epi-GA4 per spur were effective. Treatments with GA4 made seven weeks after anthesis were less effective. A combination of 30 µg GA4 and 30 µg zeatin (6-(4-hydroxy-3-methylbut-trans-2-enylamino)purine) promoted flowering at both treatment times, and tended to be more effective than GA4 alone.

Photocontrol of gibberellin metabolism in situ in maize.

Mature maize seeds were labeled with 10 to 100 pg per seed of [(3)H] gibberellins (GA) and [(3)H]GA glucosyl conjugate-like substances by feeding [(3)H]GA(20) of high specific activity (2.3 Curies per millimole) during seed maturation. The dry seeds, which contained 14% [(3)H]GA(20), 7% putative [(3)H]GA(1) and 78% [(3)H]GA glucosyl conjugate-like metabolites, were imbibed and germinated in the dark and under incandescent light. In both light and dark the proportion of [(3)H]GA conjugate-like metabolities declined (relative to that in the mature dry seeds) during imbibition and up to germination at hour 36. This decline was accompanied by increases in the proportions of [(3)H]GA(20) and putative [(3)H]GA(1) thereby indicating hydrolysis, which was greater in the dark than in the light. The proportions of [(3)H]GA conjugate-like substances in light-grown germinants were higher (121 and 141% of dark-grown) at 24 and 48 hour harvests and this statistically significant pattern was sustained up to 120 hours after imbibition. Conversely, the proportions of [(3)H]GA(20) and putative [(3)H]GA(1) were lower in the light-grown seedlings. Thus, during imbibition, hydrolysis (de-conjugation) of [(3)H]GA glucosyl conjugate-like substances apparently occurred, and occurred more rapidly in the dark than in the light. Subsequently, during germination the reformation of [(3)H]GA conjugate-like substances was less rapid in the dark than in the light. The observation that dark-imbibed seeds and dark-grown seedlings have higher proportions of putative free [(3)H]GAs, relative to [(3)H]GA conjugate-like substances, is consistent with the increased shoot elongation (etiolation) that occurs in dark-grown maize seedlings, and may indicate a homeostatic role for GAs and their conjugates in shoot elongation of maize germinants.

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 A(4) ([(3)H]GA(4)) of high specific radioactivity (5.18 x 10(10) becquerel per millimole) applied to the xylem of the rootstock in grafted apple (Malus x domestica Borkh.) trees was compared. Free [(3)H] GA-like metabolites of [(3)H]GA(4), including putative GA(1), GA(2), GA(3), and GA(34), as well as various (3)H-putative GA glucosyl conjugates were detected in stem segments from both cultivars. M9 interstocks reduced the total uptake of [(3)H]GA(4) and decreased the proportion of (3)H 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 [(3)H]GAs was higher, and the proportion of putative [(3)H]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.