PARAMETERS OF FILAMENT ELONGATION IN IPOMOEA NIL (CONVOLVULACEAE)

It has been thought for some time that morning glory filaments elongate in response to changes in concentrations of gibberellins (Murakami, 1973), but many other aspects of their growth have remained unstudied. In the present work, the interacting roles of gibberellin and ethylene in filament growth were examined. Filaments elongated ten-fold by epidermal cell elongation accompanied by ten-fold increases in fresh and dry weight. Applied gibberellins could stimulate filament growth in vitro, but gibberellin biosynthesis inhibitors had no effect. The putative gibberellin action inhibitor, ancymidol, reduced growth but the inhibition could be removed by blocking ethylene biosynthesis. Stimulators of the ethylene biosynthesis pathway and applied ethylene precursor (ACC) strongly inhibited filament elongation; ethylene biosynthesis inhibitors elicited as much growth as applied gibberellin. The filaments produced little ethylene at the time of the onset of growth. While the filaments produced ethylene rapidly before and after growth initiation, the closed flower bud had a relatively constant level of ethylene. It seems likely that in situ production of ethylene negatively influences filament growth.     

Role of plant growth substances in MS33-controlled stamen filament growth in Arabidopsis

The rapid growth of stamen filaments just before flower anthesis in Arabidopsis thaliana does not occur in the male sterile33 ( ms33 , formerly known as msZ ) mutant. ms33 filaments were approximately 40% shorter than the wild type (WT), and there was corresponding reduction in the epidermal cell length of filaments. This suggests that MS33 controls the final cell-elongation phase of filament growth. Both low temperatures and gibberellic acid (GA₃) restored filament and cell growth in intact ms33 flowers, but these treatments only had a small promotive effect on WT filaments. Decapitation experiments involving the removal of the anther had the opposite effect on WT and ms33 filaments; growth was inhibited in WT, but was increased in ms33 filaments. In young stamen primordia cultured in vitro, filament growth was less in WT, but more in ms33 , than in respective in vivo produced filaments. Plant growth substances (PGSs), GA₃ and indole-3-acetic acid (IAA) were promotive, zeatin had no effect, and abscisic acid (ABA) and ethrel inhibited filament growth in both intact and decapitated WT and ms33 filaments. Together these observations suggest that MS33 is activated immediately before anthesis and that the MS33 product either regulates temporal biosynthesis of gibberellins (GAs) and/or IAA or makes the filament tissue sensitive to these PGSs, which in turn trigger cell elongation and filament growth. The data also suggest that ms33 mutant anthers contain a relatively high ratio of growth inhibitors to promoters, which inhibits epidermal cell elongation and filament growth.     

Regulation of Gibberellin Biosynthesis in Gibberella fujikuroi

Gibberellin production by Gibberella fujikuroi started only after the nitrogen source was depleted and ceased upon its renewal. Nitrogen repression of gibberellin biosynthesis is not an indirect effect of the growth arrest that follows the depletion of an essential nutrient because gibberellins were not produced upon depletion of phosphate. Mycelia produced gibberellins when suspended in a glucose solution. Production ceased some time after depletion of glucose and resumed upon its readdition. Under certain conditions, the gibberellin production rate was inversely proportional to the glucose concentrations. The specific regulation of gibberellin biosynthesis by the nitrogen source imposes a revision of the concept that gibberellins are secondary metabolites whose production is triggered by imbalance or cessation of growth.     

Tannins as gibberellin antagonists

Fourteen chemically defined hydrolyzable tannins and six impure mixtures of either condensed or hydrolyzable tannins were found to inhibit the gibberellin-induced growth of light-grown dwarf pea seedlings. The highest ratio of tannins to gibberellic acid tested (1000: 1 by weight) inhibited from 80 to 95% of the induced growth for all tannins tested except for two monogalloyl glucose tannins which inhibited only 50% of the induced growth. The lowest ratio tested (10: 1) inhibited the induced growth by less than 25% except for the case of terchebin where 50% inhibition was found. The inhibition of gibberellin-induced growth was found to be completely reversed by increasing the amount of gibberellin in three cases tested. Tannins alone did not inhibit endogenous growth of either dwarf or nondwarf pea seedlings. Eight compounds related to tannins, including coumarin, trans-cinnamic acid, and a number of phenolic compounds were also tested as gibberellin antagonists. Most of these compounds showed some inhibition of gibberellin-induced growth, but less than that of the tannins. At the highest ratio (1000: 1) the greatest inhibition was 55%; at the lowest ratio (10: 1) no more than 17% was observed. These compounds did not inhibit endogenous growth, and the inhibition of gibberellin-induced growth could be reversed by increasing the amount of gibberellin in two cases tested.     

Hormonal regulation of germination and early seedling development in Acer pseudoplatanus (L.)

Summary Dormancy of intact sycamore (Acer pseudoplatanus) seeds was broken by chilling (5°C) for several weeks in moist conditions. Treatment of unchilled seeds with kinetin induced some germination, but gibberellin was ineffective. This stimulation by kinetin was not suppressed by the added presence of abscisic acid during incubation.The chilling requirement of intact seeds was eliminated by removal of the testa, and the naked embryos developed with no morphological abnormalities. During early growth of isolated embryos in the light, two distinct developmental processes were recognised. One involved initial elongation of the radicle accompanied by geotropic curvature and was stimulated by kinetin but not by gibberellin, while the other involved unrolling of the cotyledons, which was accelerated by gibberellin but much less by kinetin. Abscisic acid strongly suppressed both developmental processes when applied alone, inhibited cotyledon expansion in the presence of gibberellin, but failed to overcome the promotory effects of kinetin on radicle growth. Experiments with CCC indicated that under natural conditions the unrolling of the cotyledons is dependent upon endogenous gibberellin. Radicle growth of isolated embryos was unimpaired by incubation in the dark, but cotyledon expansion of water incubated embryos was poor, and although it was accelerated by gibberellin, the responses in all treatments were slower than in the corresponding light grown samples.It is suggested that endogenous cytokinins are primary factors in the initiation of radicle growth, while gibberellins are important in cotyledon expansion. Abscisic acid appears to have an inhibitory role in both processes, and the interactions of these regulators in the control of germination and development are discussed.     

The relationship between gibberellin and floral stimulus inBryophyllum daigremontianum

Summary The long-short-day plantBryophyllum daigremontianum initiates flower buds both upon change from long to short day and after gibberellin application in short day only at night temperatures of 11° and 15°C, but not at 19°C.Flowering of receptor scions in long day or short day takes place just as easily when the donor stocks have been induced by the shift from long day to short day or by gibberellin treatment in short day. Leaves taken from gibberellin-induced plants can also function as donors, even better so than photoperiodically induced leaves. Receptor scions induced by gibberellin-treated donors can in turn induce other vegetative scions (indirect induction).Flower formation induced by the change from long day to short day as well as by gibberellin treatment in short day is always associated with an increased length of newly formed internodes.It is concluded that gibberellin and the floral stimulus are not identical, but that gibberellin is a factor which normally limits production of the floral stimulus inBryophyllum under short days, and that the shift from long day to short results in an increase of the gibberellin level in the plant.With 5 Figures in the TextThis work was in part supported by the National Science Foundation, grants G-16408 and G-17483.     

Enhancement by Low pH of Gibberellin Effects on Dormant Celery Seeds and Embryoless Half-Seeds of Barley

The action of gibberellins on celery (Apium graveolens L.) seed germination and the release of reducing sugars from barley (Hordeum vulgare L.) seed endosperm were enhanced by decreasing the pH of the incubation solution to below the pKa point. In most cases, low pH was obtained by mixing the solution with weak acids such as succinic acid 2.2 dimethylhydrazide (SADH), 2-chloroethylphosphonic acid (CEPA), or citric acid. However, lowering the pH of the gibberellin solution with strong acid (HCl) also increased markedly the activity of low concentrations of GA_4/7. The synergistic action of ethylenediaminetetraacetic acid (EDTA) with gibberellin was not dependent on the pH level of the incubation solution. The response of celery seeds to gibberellins was increased when their distal ends were removed: solution pH and EDTA had no effect on this response. The possible explanations of the synergism between low pH compounds and gibberellins are discussed.     

A role for gibberellic acid in orienting microtubules and regulating cell growth polarity in the maize root cortex

The role of gibberellins and cortical microtubules in determining the polarity of cell growth in the root cortex of maize (Zea mays L.) was examined. Inhibition of gibberellin biosynthesis, either naturally through mutation (d5 mutant) or by means of chemicals such as 2S,3S paclobutrazol, caused thickening of root apices and increased their starch content. Immunofluorescence microscopy of cortical microtubules, coupled with a comparison of cell widhts, lengths and shapes, indicated that the meristem and immediate post-mitotic zone were the targets of gibberellin deficiency. Cortical cells in these regions were impaired in their ability to develop highly ordered transversal arrays of cortical microtubules. Consequently, the cells became wider and shorter. Application of gibberellic acid re-established the arrangements of cortical microtubules and the polarity of cell growth characteristic for roots having normal levels of gibberellins, it also decreased the starch content. These results indicate that gibberellins are morphogenetically active substances, not only in shoots but also in roots of maize.Abbreviations CMT cortical microtubule - GA gibberellin - GA₃ gibberellic acid - MT microtubule - PIG postmitotic isodiametric growth The authors acknowledge the support to F.B. from the Royal Society (London UK). We also thank Dr. J. Lenton (University of Bristol, Long Ashton Research Station) who kindly supplied us with 2S,3S paclobutrazol and grains of the GA-deficient d5 mutant of maize.     

Barley endosperm bioassay for gibberellins. I. Parameters of the response system

Parameters of the bioassay based on the gibberellin-induced reducing sugar release of barley endosperm were investigated. Procedures for the rapid handling and processing of up to several hundred treatments without loss in sensitivity of the test are described, and the effects of variations in many aspects of the bioassay were assessed.

In general, the variations in varieties, techniques, additives, conditions, and even gibberellins, all illustrate the stability, sensitivity, and adaptability of the hormone-induced response and emphasize its utility as a gibberellin bioassay.

     

Gibberellins and early growth in seedlings of Phaseolus vulgaris

Summary 1. Amounts of gibberellin extractable from young bean seedlings using phosphate buffer, and following acid hydrolysis, or protease treatment, have been examined and compared with those obtained using methanol extraction. 2. Considerable differences in the amounts of gibberellins extractable are found for different batches of material. The level of extracted gibberellin is less for dry seeds and for 1-day old seedlings than for seedlings 4 days old. Before germination, the amounts of free, buffer-soluble, gibberellins are low, but these rise rapidly in the postgermination period. 3. The appearance of large amounts of free gibberellins in the cotyledons, believed to represent the conversion of bound forms, is not dependent upon the presence of the embryonic axis, since removal of this at planting has only a small effect on the amount of gibberellin extractable at day 4. There is evidence for synthesis of new gibberellin in the intact seedling by day 4. 4. amylase activity in the cotyledons also develops in the absence of the embryo, but the high levels of activity shown by intact seedlings are not reached in cotyledons cultured in absence of the embryo.     

The Effect of the Plant Growth Retardants Amo 1618 and CCC on Gibberellin Production in Fusarium monili-forme: Light Stimulated Biosynthesis of Gibberellins

Through the use of a single gene dwarf mutant of Zea mays L., dwarf-1, the interaction of growth retardants with gibberellin biosynthesis was studied in Fusarium monitiforme. It was demonstrated that the growth retardants 2-isopropyl-4-dimcthylamine-5-methyphenyl-1-piperidine-cai'boxylate methyl chloride (Amo 1618) and (2-chloroethyl) trimethylammonium chloride (CCC) are more effective inhibitors of gibberellin biosynthesis in cultures maintained under continuous illumination. Light grown cultures produced significantly more biologically active gibberellin-like materials than dark grown cultures. Stock cultures exposed to light also promoted the subsequent biosynthesis of gibberellins in the dark. Chromatographical analysis of the soluble gibberellins extracted from the culture medium revealed that large amounts of chromatographically detectable A₃ and A₇ were produced in light cultures with only A₇ produced in the dark. Light also induced a greater incorporation of acelate-2-¹⁴C into the gibberellins A₇, A₃ and an unidentified gibberellin. Growth returdants occasionally caused a complete disappearance of chromatographically detectable gibberellins in the dark; however, in the light at no concentration tested was it possible to detect the complete disappearance of gibberellin-like material. A₃ was always detectable. Like higher plants, different strains of F. moniliforme exhibit variation which makes them more or less sensitive to the growth retardants. This variation is interpreted to mean that there may be more than one pathway leading to the synthesis of the gibberellins.     

Actin-activation of unphosphorylated gizzard myosin

The effect of light chain phosphorylation on the actin-activated ATPase activity and filament stability of gizzard smooth muscle myosin was examined under a variety of conditions. When unphosphorylated and phosphorylated gizzard myosins were monomeric, their MgATPase activities were not activated or only very slightly activated by actin, and when they were filamentous, their MgATPase activities could be stimulated by actin. At pH 7.0, the unphosphorylated myosin in the presence of ATP required 2-3 times as much Mg2+ for filament formation as did the phosphorylated myosin. The amount of stimulation of the unphosphorylated myosin filaments depended upon pH, temperature, and the presence of tropomyosin. At pH 7.0 and 37 degrees C and at pH 6.8 and 25 degrees C, the MgATPase activity of filamentous, unphosphorylated, gizzard myosin was stimulated 10-fold by actin complexed with gizzard tropomyosin. These tropomyosin-actin-activated ATPase activities were 40% of those of the phosphorylated myosin. Under other conditions, pH 7.5 and 37 degrees C and pH 7.0 and 25 degrees C, even though the unphosphorylated myosin was mostly filamentous, its MgATPase activity was stimulated only 4-fold by tropomyosin-actin. Thus, both unphosphorylated and phosphorylated gizzard myosin filaments appear to be active, but the cycling rate of the unphosphorylated myosin is less than that of the phosphorylated myosin. Active unphosphorylated myosin may help explain the ability of smooth muscles to maintain tension in the absence of myosin light chain phosphorylation.     

Endogenous gibberellin transport and biosynthesis in relation to geotropic induction of excised sunflower shoot-tips

Summary Surgical experiments on Helianthus annuus and Phaseolus multiflorus seedlings involving the application of auxin and gibberellin to decapitated plants, suggested that internode extension growth occurs under the controlling influence of apically synthesised gibberellin rather than auxin. Studies were made of diffusible gibberellins from sunflower apical buds in relation to geotropic stimulation. Approximately ten times as much gibberellin was obtained from lower than from upper tissues of horizontal shoot-tips, whereas approximately equal quantities were obtained from the two halves of upright tips. Evidence was obtained suggesting both lateral transport of gibberellin in the young internode, and also enhanced gibberellin synthesis in buds maintained in a horizontal position during the collection of diffusible gibberellins into agar. The results are discussed in relation to current concepts of the role of auxin in geotropism.     

The Pathway of Assembly of Intermediate Filaments from Recombinant α-Internexin

The pathway of filament assembly from the neuronal intermediate filament α-intermexin was investigated. Optimal assembly occurred in solutions of pH 6.5 to 7 and moderate ionic strength at 37°C. Short filaments formed upon dialysis at 24°C, which elongated further when incubated at 37°C. Soluble forms of α-internexin were characterized by analytical ultracentrifugation and electron microscopy. In 10 mM Tris, pH 8, conditions that favor formation of tetramers and other small oligomers for other intermediate filament proteins, α-internexin formed 10.5 S particles, apparently unit-length half-filaments in the form of rods 10.6 nm in diameter and 68 nm long. Dialysis vs the same buffer with added 10 mM NaCl yielded 16 S rods, probably unit-length filaments, of the same length but 13.0 nm in diameter. At 50 mM NaCl, rods about 13 nm in diameter and heterogeneous in length were observed in electron micrographs, apparently formed from longitudinal annealing of unit-length rods. The results favor a model of assembly in which coiled coil dimers aggregate laterally to form first “unit-length half-filaments” (Herrmann, H., and Aebi, U. (1998)Curr. Opin. Struct. Biol.8, 177–185) and then “unit-length filaments,” which subsequently elongate by annealing.     

THE ROLES OF AUXIN,ETHYLENE, AND ACID GROWTH IN FILAMENT ELONGATION IN GAILLARDIA GRANDIFLORA (ASTERACEAE)

Filament elongation and the role of auxin in this process in Gaillardia grandiflora was investigated. Filament elongation in vivo occurred just prior to anthesis and was accompanied by cell elongation and fresh weight increase. Filaments isolated and exposed to auxin in vitro grew more rapidly than controls and their growth was comparable to that of filaments in vivo. Furthermore, the natural auxin content of disc flowers (determined by double-standard isotope dilution analyses) increased just prior to anthesis and filament elongation. These results imply that auxin controls filament elongation. Applied ethylene slightly promoted filament elongation in vitro, and ethylene production of the flowers (determined by gas chromatography) slightly increased prior to filament growth. Fusicoccin and acidic buffers also stimulated elongation of isolated filaments. Thus, the role of auxin in controlling filament elongation in Gaillardia may involve stimulation of ethylene biosynthesis and acid growth.     

Regulation of Internodal Extension in Avena Shoots by the Inflorescence, Nodes, Leaves, and Intercalary Meristem

It has been found in recent studies that the inflorescence and nodes (node-pulvini) are the primary sources for native gibberellins in the Avena shoot, and that GA₃ is the predominant gibberellin in the inflorescence. In the present work, linear growth of next-to-last internode is drastically reduced by removal of the inflorescence and last leaf. This growth is completely abolished when the nodes are also excised. It is restored fully by the addition of GA₃ when the nodes are present, and restored only partially when the nodes are deleted. Internodal growth in Avena stem segments with basal node present is also restored by native GA₃-like substances extracted from Avena inflorescences and partially purified by silica gel partition column chromatography. Evidence from these studies, taken in toto, indicates that the inflorescence, nodes, and leaves supply gibberellins, leaves supply substrate, and nodes modulate the gibberellin growth response in next-to-last Avena internodes.     

Gibberellins in Diffusates from Shoots of Apple Trees

Excised shoot apices, leaves and internodes from shoots of apple trees (Malus×domestica) give off gibberellins by diffusion on agar. A methanol extract of the agar was prepared, the extract separated on thin layer plates, and the gibberellin activity estimated by means of Rumex and lettuce hypocotyl bioassays. The largest amounts of gibberellin are found in diffusates from the shoot apex, the two upper leaves and the two upper internodes. Several promotive fractions are found on the chromatograms as well as growth inhibitors. Removal of young leaves retards elongation of the internodes. Probably gibberellins produced in young leaves exercise some control over this process. The growth regulators Alar and CCC also retard internode elongation. Diffusates from shoots treated with these substances were also examined. Preliminary results suggest that the amount of diffusible gibberellins from treated shoots is not reduced.     

Possible involvement of phosphoinositide-Ca2+ signaling in the regulation of alpha-amylase expression and germination of rice seed (Oryza sativa L.)

We have studied the effects of neomycin, a potent inhibitor of inositol phospholipid-specific phospholipase C (PLC), on the germination of rice seed and the gibberellin-induced expression of alpha-amylase in the aleurone layer and the scutellar tissues. It was shown that, in the absence of exogenous Ca2+, neomycin markedly reduced the germination speed and seedling growth of rice seeds and inhibited the gibberellin-induced expression of alpha-amylase in both secretory tissues. In addition, neomycin decreased the formation of inositol 1,4,5-trisphosphate (IP3) in the gibberellin-treated aleurone layer and the scutellar tissues. However, the inhibitory effects on the germination speed and the expression of alpha-amylase activity were overcome by supplementation of Ca2+. In addition, gibberellin elevated the level of IP3, and ABA prevented the gibberellin-induced formation of IP3, although ABA alone did not alter the IP3 level. The expression of a membrane-bound PLC molecule in rice aleurone layer was shown to be induced by gibberellin, and the gibberellin-induced expression of PLC was markedly delayed by treatment with ABA. These results strongly suggest that the phosphoinositide-Ca2+ signal transduction pathway may play an important role in the gibberellin-induced expression of alpha-amylase molecules closely related to the germination processes of rice seed.     

In vitro morphogenetic responses of Ilex paraguariensis nodal segments treated with different gibberellins and Prohexadione-Ca

One-bud nodal segments of Ilex paraguariensis (yerba mate) were cultured in vitro in a sugar-rich medium with different gibberellins or an inhibitor of their synthesis. Bud sprouting, shoot length (assessed as shoots of less or more than 5 mm) and bud abscission were evaluated after 45 d of culture in a growth chamber at 27±2 °C, with a 14 h photoperiod of white fluorescent light. There was a differential effect of the two types of gibberellins used; the double bond ring-A gibberellins (GA₃ and GA₇) inhibited shoot length, while the non double bond-ring A gibberellins (GA₁ and GA₄) stimulated shoots with a length of more than 5 mm. Prohexadione-Ca (Bx-112; a late step gibberellin biosynthesis inhibitor), at high doses, restrained bud sprouting up to 75%, but lower doses promoted shoot lengthening.     

Interaction of growth retardants,daylength, and gibberellins A19, A20, and A1 on shoot elongation in birch and alder

Gibberellins A₁₉, A₂₀, and A₁ were applied to seedlings of birch (Betula pubescens Ehrh.) and alder (Alnus glutinosa (L.) Gaertn.) in order to test their ability to counteract growth inhibition induced by growth retardants (ancymidol and BX-112) or short day (SD, 12 h) photoperiod. Ancymidol inhibits early and BX-112 inhibits late steps in gibberellin biosynthesis. BX-112 inhibited stem elongation in both species while ancymidol, applied as a soil drench, was effective in alder only. Growth retardants affected stem elongation mainly by inhibiting elongation of internodes. All three gibberellins were equally active when applied to seedlings treated with ancymidol; however, only GA₁ was able to counteract the growth inhibition induced by BX-112. SD-induced cessation of elongation growth in birch was counteracted by GA₁, and to some degree, by GA₂₀, while GA₁₉ was inactive. SD treatment did not induce cessation of apical growth in alder. These results are consistent with the hypothesis that of gibberellins belonging to the early C-13 hydroxylation pathway, GA₁ is the only active gibberellin for stem elongation.