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PubMed1972

Effect of light and gibberellin on ribonucleic Acid species of pea stem tissues as studied by deoxyribonucleic Acid-ribonucleic Acid hybridization.

The ability of gibberellin and light to alter gene transcription in dwarf pea (Pisum sativum L., var. Progress No. 9) stem issues has been investigated by means of DNA/RNA hybridization-competition techniques. Distinct changes in hybridizable RNA are caused by a 24-hour pretreatment of the seedlings with light, but no changes in RNA were detected up to 50 hours after treatment of the seedlings with gibberellin. Gibberellin is similar to auxin in its ability to induce stem growth without causing detectable changes in hybridizable RNA.

Journal Article↗

PubMed1977

Responses of enzymically isolated aleurone cells of oat to gibberellin a(3).

Oat (Avena sativa L.) aleurone layer cells (spheroplasts) were isolated by maceration of the aleurone layer with a mixture of commercially available cellulase and pectinase. About 20% of the cells present in intact layers were released as spheroplasts and 79 +/- 9% of the spheroplast population was viable as judged by methylene blue staining. The spheroplasts became disorganized in solutions containing less than 0.4 md-mannitol. When the spheroplasts were incubated for 48 hours, total activities of acid p-nitrophenyl phosphatase and acid proteinase increased and protein levels decreased. These changes were not effected by gibberellin A(3). Isolated aleurone layers incubated under the same conditions as the spheroplasts showed reduced responses to gibberellin A(3). It is concluded that the necessary presence of an osmoticum limits the value of spheroplasts as a system for studying the mechanism of action of gibberellin A(3) in the aleurone cell.

Journal Article↗

PubMed1981

In vitro gibberellin a(4) binding to extracts of cucumber hypocotyls.

Cucumber hypocotyls were extracted and the extract centrifuged at 100,000g to yield a supernatant or cytosol fraction. Binding of [(3)H]-gibberellin(4) (GA(4)) to soluble macromolecular components present in the cytosol was demonstrated at 0 C by Sephadex chromatography. Binding assays performed with cytosol that had been preheated or incubated with protease, DNase, RNase, or phospholipase A or C indicated that heat and protease treatments disrupted the binding, which suggests that binding occurred to a protein. Equilibrium dialysis of a protein-enriched fraction prepared by ammonium sulfate precipitation also indicated binding of [(3)H]GA(4) to macromolecular components. [(3)H]GA(4) binding was pH-sensitive, saturable, reversible, and significantly affected by biologically active gibberellins, but not by inactive gibberellins or other plant hormones such as indoleacetic acid, abscisic acid, or kinetin. Thin layer chromatography indicated that [(3)H]GA(4), and not a metabolite, was the species bound. A kinetic analysis indicated that specific binding of [(3)H]GA(4) was due to a single class of binding sites having an estimated K(d) of 10(-7) molar and a concentration of 0.8 x 10(-12) moles gram(-1) fresh weight or 0.4 x 10(-12) moles milligram(-1) soluble protein.

Journal Article↗

PubMed1981

Differential compartmentation of gibberellin a(1) and its metabolites in vacuoles of cowpea and barley leaves.

The metabolism and efflux of gibberellin A(1) (GA(1)) taken up by leaves of cowpea (Vigna sinensis cv. Blackeye pea No. 5), as well as the distribution of GA(1) metabolites in the protoplasts and vacuoles of cowpea and barley (Hordeum vulgare L. cv. Numar), were studied.GA(1) is metabolized rapidly in cowpea leaf discs to products tentatively identified as gibberellin A(8) (GA(8)) and gibberellin A(8) glucoside (GA(8)-glu). After labeling leaf discs with [(3)H]GA(1) for 1 hour, the release of radioactivity from the leaf was followed. Over a 12-hour period, the level of radioisotope in the tissue declined to about 35% of the original, after which no further release was observed. At this time, almost all of the radioactivity remaining in the leaf was GA(8)-glu, while most of the radioactivity which had been released was unmetabolized GA(1).Mesophyll protoplasts and vacuoles were isolated from cowpea and barley leaves previously fed [(3)H]GA(1). These protoplasts retain the ability to metabolize GA(1), indicating that neither the leaf structure nor the cell wall is necessary for this metabolism. A higher proportion of GA(8)-glu was found in the vacuoles relative to the entire protoplasts. The results obtained suggest that GA(1) metabolites are preferentially compartmentalized in the vacuoles relative to GA(1).

Journal Article↗

PubMed1986

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 (GA(20)), the product (GA(1)), 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 GA(1) under red irradiation and in darkness. The lele plants grew in response to GA(20) and steviol in darkness but showed a much smaller response when red irradiated. The Le plants responded to GA(20) 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 3beta-hydroxylation of GA(20) to GA(1) in genotype lele is due to a Pfr-induced blockage in the expression of that activity.

Journal Article↗

PubMed1987

Identification of endogenous gibberellins from oilseed rape.

Oilseed rape (Brassica napus, canola variety ;Westar') plants were grown in greenhouse conditions and shoots were harvested during the final stages of shoot elongation. Leaves and immature pods were removed and the remaining stem tissue was extracted and purified. The extract was chromatographed on sequential, step-eluted silica gel partition and reverse-phase C(18) HPLC columns, and gibberellin (GA)-like substances were detected using the ;Tan-ginbozu' dwarf rice microdrop assay. Purified fractions showing GA-like activity were analyzed by capillary gas chromatography-mass spectrometry (GC-MS) and GC-selected ion monitoring (GC-SIM). Gibberellins A(1), A(3), and iso-A(3) were identified by full spectrum GC-MS with GA(1) being the most abundant GA in the stem tissue. Gibberellins A(19) and A(20) were identified by GC-SIM and are logical precursors of the GA(1).

Journal Article↗

PubMed1988

Gibberellins and Light Regulated Petiole Growth in Thlaspi arvense L.

Petiole growth in Thlaspi arvense L. was stimulated when a basic 8 hour photoperiod (4.20 milliwatts per square centimeter) was extended with low intensity light (0.16 milliwatt per square centimeter) from incandescent lamps. The day length extension was effective only when the light contained high proportions of far red light. Exogenous gibberellin A(3) (GA(3)) could partially substitute for the promotive effect of the extended photoperiod. Moreover, the GA biosynthesis inhibitor 2-chlorocholine chloride inhibited the increase in petiole growth induced by the extended photoperiod. However, evidence was obtained indicating that gibberellins do not mediate the effect of the extended photoperiod. First, petiole growth was greater in plants receiving both exogenous GA(3) and a day length extension than the sum of the effects of the two treatments alone. Second, petioles were sensitive to exogenous GA(3) only during the early stages of leaf development, whereas mature (but not senescent) leaves continued to respond to an extension of the photoperiod. Third, the cellular basis for growth induced by extending the photoperiod was different from that observed with GA(3). It was concluded that light and gibberellins are both important in the overall regulation of petiole growth, but act through independent mechanisms.

Journal Article↗

PubMed1988

Endogenous Gibberellins from Sporophytes of Two Tree Ferns, Cibotium glaucum and Dicksonia antarctica.

Gibberellin A(1) (GA(1)), 3-epi-GA(1), GA(4), GA(9), 11alpha-hydroxyGA(12), 12alpha-hydroxyGA(12), GA(15), GA(17), GA(19), GA(20), GA(25), GA(37), GA(40), GA(58), GA(69), GA(70), and GA(71) have been identified from Kovats retention indices and full scan mass spectra by capillary GC-MS analyses of purified extracts from sporophytes of the tree fern, Cibotium glaucum. Abscisic acid, dihydrophaseic acid, an epimer of 4'-dihydrophaseic acid, and the epimeric ent-6alpha, 7alpha, 16alpha, 17-(OH)(4) and ent-6alpha, 7alpha, 16beta, 17-(OH)(4) derivatives of ent16, 17-dihydrokaurenoic acid, in addition to the epimeric 16alpha, 17- and 16beta, 17-dihydroxy-16, 17-dihydro derivatives of GA(12), were also identified in extracts of C. glaucum. An oxodihydrophaseic acid and a hydroxydihydrophaseic acid were also detected. In extracts of sporophytes of Dicksonia antarctica, GA(4), GA(9), 12alpha- and 12beta-hydroxyGA(12), GA(15), GA(25), and GA(37) were identified by the same criteria, as well as abscisic acid, phaseic acid, 8'-hydroxymethylabscisic acid and dihydrophaseic acid. This is the first time that GA(40) has been identified in a higher plant; it is also the first report of the natural occurrence of the two gibberellins, 11alpha- and 12beta-hydroxyGA(12). The total gibberellin (GA) content in C. glaucum (tall) was at least one order of magnitude greater than that of D. antarctica (dwarf) based on total ion current response in GC-MS and bioassay data. Abscisic acid was a major component of D. antarctica and the oxodihydrophaseic acid was a major component of C. glaucum.

Journal Article↗

PubMed1988

Monoclonal antibodies to 13-deoxy-gibberellins.

The production and characterization of two high affinity rat monoclonal antibodies to 13-deoxy-gibberellins is described. Hybrid myelomas were derived from rats immunized with an immunogenic keyhole limpet hemocyanin-gibberellin conjugate, linked at carbon-3 to gibberellin A(4) via a hemisuccinate bridge. The selected monoclonal antibodies were characterized by a competitive radioimmunoassay.

Journal Article↗

PubMed1991

Characterization of the Arrest in Anther Development Associated with Gibberellin Deficiency of the gib-1 Mutant of Tomato.

The role of gibberellins in flower bud development was investigated by studying the gib-1 mutant of tomato, Lycopersicon esculentum. This gibberellin-deficient mutant initiates flower buds, but floral development is not completed unless the mutant is treated with gibberellin. Treatment with other plant growth regulators does not induce normal flower development. Development of gib-1 flower buds, as measured by progress toward anthesis, ceases at a bud length of 2.5 millimeters; however, increase in size of the bud continues. Buds between 2.5 and 3.7 millimeters are developmentally arrested but still are capable of developing normally after treatment with gibberellic acid. Anthers of these developmentally arrested buds contain pollen mother cells that are in the G1 phase of premeiotic interphase. Following treatment of developmentally arrested buds with gibberellic acid, premeiotic DNA synthesis and callose accumulation in pollen mother cells are evident by 48 hours posttreatment, and within 66 hours, prophase I of meiosis- and meiosis-related changes in tapetum development are observable.

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PubMed1997

Mendel's stem length gene (Le) encodes a gibberellin 3 beta-hydroxylase.

We describe the isolation of the Le gene of pea, which controls internode elongation and originally was described by Mendel. Heterologous screening of a pea cDNA library yielded a partial clone that was 61% identical to coding regions of the putative Arabidopsis gibberellin 3 beta-hydroxylase gene, GA4. DNA gel blot analysis with this cDNA revealed a HindIII restriction fragment length polymorphism between pea isolines differing at Mendel's Le locus. Genomic clones of the GA4-related gene were isolated from the Le and le isolines. Polymerase chain reaction combined with restriction fragment length polymorphism analysis were used to show that the gene mapped to the Le locus. A cDNA containing a complete open reading frame of the pea GA4-related gene was amplified by polymerase chain reaction from each isoline. Recombinant expression in Escherichia coli demonstrated that the product of the Le cDNA was a gibberellin 3 beta-hydroxylase that is able to convert GA20 to the bioactive GA1. Substantially reduced levels of gibberellin 3 beta-hydroxylase activity were measured, after expression of the le cDNA, by using identical methods. This reduced activity was associated with an alanine-to-threonine substitution in the predicted amino acid sequence of the enzyme near its proposed active site.

Amino Acid Sequence↗

PubMed1997

Partial purification and characterization of a gibberellin-binding protein from seedlings of Azukia angularis.

Little is known about mechanisms for the perception of gibberellin (GA), a phytohormone, by plants. A GA-binding protein (GBP) with receptor-like characteristics was partially purified from azuki bean (Azukia angularis) seedlings. Gibberellins that caused much elongation of azuki epicotyl sections showed strong binding affinity for the GBP, and their biosynthetic precursors and inactive metabolites had weak affinity for it. The apparent Kd value for GA4 was calculated to be 7 x 10(-10) M from Scatchard plots. The molecular weight of the GBP in the native state was estimated to be 25,000 by high-performance liquid chromatography with a gel-permeation column. The GBP may be one of the receptor candidates for GAs.

Carrier Proteins↗

PubMed1995

A gibberellin-stimulated ubiquitin-conjugating enzyme gene is involved in alpha-amylase gene expression in rice aleurone.

A ubiquitin-conjugating enzyme (UBC) gene, induced by gibberellin (GA) within an hour, was identified in rice (Oryza sativa) seeds by the mRNA differential display technique. GA inducibility was confirmed by RNA hybridization. A full-length UBC cDNA clone and a genomic clone have been isolated and sequenced. The deduced amino acid sequence shares a significant identity with several known UBC sequences, which are probably involved in the pathway responsible for degrading short-lived regulatory proteins. In vivo transient assays using the UBC gene promoter, joined to the luciferase cDNA as the reporter gene, showed that the sequence located between positions 231 and 159 upstream of the transcription start site of this promoter is crucial for GA-dependent activation of the luciferase cDNA. Finally, trans-activation experiments indicated that this UBC gene is involved in gibberellin-stimulated alpha-amylase gene expression.

Amino Acid Sequence↗

PubMed1996

The petunia homologue of tomato gast1: transcript accumulation coincides with gibberellin-induced corolla cell elongation.

Gibberellins (GAs) regulate petunia corolla pigmentation and elongation. To study this hormone's effect at the molecular level, we used the tomato gast1 gene as a probe to isolate a gibberellin-induced gene (gip) from petunia corollas. The deduced sequence of gip exhibited 82% identity with GAST1 protein and contained a short, highly hydrophobic N-terminal region. High levels of gip expression were detected in elongating corollas and young stem intemodes. When detached corollas were grown in vitro in sucrose medium, gip expression was strongly induced by gibberellic acid (GA3). GA3-induced gip expression in corollas was inhibited by abscisic acid (ABA). The expression of the gene was also induced by GA3 in detached young stem segments. Sucrose was not essential for GA-induced gip expression in corollas but enhanced its effect. In stems, on the other hand, sucrose inhibited the effect of the hormone. The results of the present work support the possible role of gip in GA-induced corolla and stem elongation.

Abscisic Acid↗

PubMed1996

Gibberellins regulate the abundance of RNAs with sequence similarity to proteinase inhibitors, dioxygenases and dehydrogenases.

In an effort to understand the molecular mechanism of gibberellin (GA) action, we have cloned and performed an initial characterization of three cDNAs (GAD1, 2, and 3) which correspond to RNAs that become less abundant by 2 h after treatment of tomato (Lycopersicon esculentum Mill.) shoot tissue with gibberellic acid (GA3). Treatment with either auxin or ethephon also decreases the abundance of all three of the GAD RNAs. The tomato ethylene-insensitive mutant, Nr, and the GA-deficient mutant, gib1, were used to show that GA or auxin regulation of GAD RNA abundance is not dependent on ethylene sensitivity, and that ethylene or auxin regulation is not dependent on normal levels of gibberellin biosynthesis. Treatment with abscisic acid (ABA) antagonizes the GA-induced suppression of the GAD1 and GAD2 RNAs. GAD1 is similar to type-II wound-inducible plant proteinase inhibitors. Like the well-characterized proteinase inhibitor II (pin II) of tomato, the GAD1 and GAD2 RNAs are wound inducible. Induction of pin II and GAD1 RNA in gib1 was found to require less-severe wounding than was required using wild-type plants or plants doubly mutant for gib1 and sit (the sit mutation causes ABA deficiency). The predicted GAD2 protein sequence is similar to 2-oxoglutarate-dependent dioxygenases while the predicted GAD3 protein sequence is similar to proteins belonging to the nonmetallo-short-chain alcohol-dehydrogenase family, especially the T ASSELSEED2 (TS2) gene of maize and bacterial hydroxysteroid dehydrogenases.

Amino Acid Sequence↗

PubMed1994

Genetics and gibberellin production in Gibberella fujikuroi.

Gibberella fujikuroi (Fusarium moniliforme) is a complex group of plant pathogens. Some strains produce gibberellic acid and other gibberellins that promote growth and regulate various stages in plant development. The paper describes the research effort directed to development of genetic tools for this species. Furthermore the main features of the gibberellin biosynthetic pathway as established in Gibberella are described.

Crosses, Genetic↗

PubMed1999

Gibberellin and ethylene control endoreduplication levels in the Arabidopsis thaliana hypocotyl.

We have previously shown that endoreduplication levels in hypocotyls of Arabidopsis thaliana (L.) Heynh. are under negative control of phytochromes. In this study, the hormonal regulation of this process was analysed using a collection of A. thaliana mutants. The results show that two hormones in particular, gibberellin (GA) and ethylene, play distinct roles. Hypocotyl cells of the GA-deficient mutant ga1-11 grown in the dark did not elongate and showed a greatly reduced endoreduplication. Normal endoreduplication could be restored by supplying 10(-9) M of the gibberellin GA(4+7), whereas the restoration of normal cell growth required 100-fold higher concentrations. The GA-insensitive mutant gai showed reduced cell elongation but normal ploidy levels. We conclude that (i) GA(4+7) has a global positive effect on endoreduplication and (ii) that endoreduplication is more sensitive to GA(4+7) than cell elongation. Ethylene had a completely different effect. It induced an extra round of endoreduplication both in light- and dark-grown seedlings and acted mainly on discrete steps rather than having a global effect on endoreduplication. The genes EIN2 and CTR1, components of the ethylene signal transduction pathway were both involved in this process.

Arabidopsis↗

PubMed2004

Gibberellin inhibitors improve embryogenic tissue initiation in conifers.

Somatic embryogenesis (SE), the most promising technology to multiply high-value coniferous trees from advanced breeding and genetic engineering programs, is expected to play an important role in increasing productivity, sustainability, and uniformity of future forests in the United States. For commercial use, SE technology must work with a variety of genetically diverse trees. Initiation in loblolly pine (LP; Pinus taeda L.), our main focus species, is often recalcitrant for desirable genotypes. Initiation of LP, slash pine (SP; Pinus elliottii), Douglas-fir (DF; Pseudotsuga menziesii), and Norway spruce (NS; Picea abies) were improved through the use of paclobutrazol, a gibberellin synthesis inhibitor. Paclobutrazol was effective at concentrations ranging from 0.25 mg/l to 3.0 mg/l (0.85-10.2 microM) and optimal in LP at 1.0 mg/l. Using control media (no paclobutrazol) and 0.33-1.0 mg/l paclobutrazol, initiation percentages in LP, SP, DF, and NS were improved from 37.7% to 44.2% (across experiments), 19.3% to 28.5%, 16.9% to 23.7%, and 38.8% to 48.5%, respectively. Other gibberellin inhibitors such as flurprimidol, chlormequat-Cl, and daminozide also caused statistically significant increases in LP initiation when added to the medium at concentrations of 0.34, 10.0, and 1.0 microM, respectively. No detrimental effects on subsequent embryo development were observed when 29 new initiations from medium without GA inhibitor and 28 new initiations from medium containing paclobutrazol were tracked through culture capture, liquid culture establishment, cotyledonary embryo development, and germination.

Culture Techniques↗