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PubMed1989

An immunological approach to gibberellin purification and quantification.

Gibberellin (GA) specific, high-affinity monoclonal antibodies have been used to assay the GA content of various plant tissues and to purify selected GAs by immunoaffinity chromatography. These immunological techniques may not stand alone as a general method of GA analysis. The results of this study indicate, however, that in conjunction with gas chromatography-mass spectrometry for positive GA identification, radioimmunoassay and immunoaffinity chromatography are extremely powerful tools for purifying and quantifying GAs from plant tissues.

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PubMed1989

Effect of Temperature on Gibberellin (GA) Responsiveness and on Endogenous GA(1) Content of Tall and Dwarf Wheat Genotypes.

Near-isogenic wheat (Triticum aestivum L.) lines differing in height-reducing (Rht) alleles were used to investigate the effects of temperature on endogenous gibberellin (GA) levels and seedling growth response to applied GA(3). Sheath and lamina lengths of the first leaf were measured in GA treated and control seedlings, grown at 11, 18, and 25 degrees C, of six Rht genotypes in each of two varietal backgrounds, cv Maris Huntsman and cv April Bearded. Endogenous GA(1) levels in the leaf extension zone of untreated seedlings were determined by gas chromatography-mass spectrometry with a deuterated internal standard in the six Maris Huntsman Rht lines grown at 10 and 25 degrees C. Higher temperature increased leaf length considerably in the tall genotype, less so in the Rht1 and Rht2 genotypes, and had no consistent effect on the Rht1+2, Rht3 and Rht2+3 genotypes. In all genotypes, endogenous GA(1) was higher at 25 degrees C than at 10 degrees C. At 10 degrees C the endogenous GA(1) was at a similar level in all the genotypes (except Rht2+3). At 25 degrees C it increased 1.6-fold in the tall genotype, 3-fold in Rht1 and Rht2, 6-fold in Rht3, and 9-fold in Rht1+2. Likewise, the genotypic differences in leaf length were very conspicuous at 25 degrees C, but were only slight and often unsignificant at 11 degrees C. The response of leaf length to applied GA(3) in the Rht1, Rht2, and Rht1+2 genotypes increased significantly with lowering of temperature. These results suggest the possibility that the temperature effect on leaf elongation is mediated through its effect on the level of endogenous GA(1) and that leaf elongation response to endogenous or applied GAs is restricted by the upper limits set by the different Rht alleles.

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PubMed1989

Gibberellins in Embryo-Suspensor of Phaseolus coccineus Seeds at the Heart Stage of Embryo Development.

Gibberellins (GAs) in suspensors and embryos of Phaseolus coccineus seeds at the heart stage of embryo development were analyzed by combined gas chromatography-mass spectrometry (GC-MS). From the suspensor four C(19)-GAs, GA(1), GA(4), GA(5), GA(6), and one C(20) GA, GA(44), were identified. From the embryo, five C(19)-GAs GA(1), GA(4), GA(5), GA(6), GA(60) and two C(20) GAs, GA(19) and GA(44) were identified. The data, in relation to previous results, suggest a dependence of the embryo on the suspensor during early stages of development.

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PubMed1990

Gibberellins and Stem Growth as Related to Photoperiod in Silene armeria L.

Stem growth and flowering in the long-day plant Silene armeria L. are induced by exposure to a minimum of 3 to 6 long days (LD). Stem growth continues in subsequent short days (SD), albeit at a reduced rate. The growth retardant tetcyclacis inhibited stem elongation induced by LD, but had no effect on flowering. This indicates that photoperiodic control of stem growth in Silene is mediated by gibberellins (GA). The objective of this study was to analyze the effects of photoperiod on the levels and distribution of endogenous GAs in Silene and to determine the nature of the photoperiodic after-effect on stem growth in this plant. The GAs identified in extracts from Silene by full-scan combined gas chromatography-mass spectrometry (GC-MS), GA(12), GA(53), GA(44), GA(17), GA(19), GA(20), GA(1), GA(29), and GA(8), are members of the early 13-hydroxylation pathway. All of these GAs were present in plants under SD as well as under LD conditions. The GA(53) level was highest in plants in SD, and decreased in plants transferred to LD conditions. By contrast, GA(19), GA(20), and GA(1) initially increased in plants transferred to LD, and then declined. Likewise, when Silene plants were returned from LD to SD, there was an increase in GA(53), and a decrease in GA(19), GA(20), and GA(1) which ultimately reached levels similar to those found in plants kept in SD. Thus, measurements of GA levels in whole shoots of Silene as well as in individual parts of the plant suggest that the photoperiod modulates GA metabolism mainly through the rate of conversion of GA(53). As a result of LD induction, GA(1) accumulates at its highest level in shoot tips which, in turn, results in stem elongation. In addition, LD also appear to increase the sensitivity of the tissue to GA, and this effect is presumably responsible for the photoperiodic after-effect on stem elongation in Silene.

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PubMed1990

In Vivo Gibberellin Biosynthesis in Endosperm of Sechium edule Sw. Seeds.

Biosynthesis of gibberellins (GAs) was studied in vivo in endosperms of Sechium edule Sw. Exogenous ent-[(14)C]kaurene was metabolized into four major products: GA(12), GA(4), GA(7) and 16, 17-dihydro-16-hydroxy-GA(15) alcohol glucoside. Other minor metabolites were also observed including ent-kaurenol and ent-kaurenal. Conversion of ent-[(14)C]kaurene to ent-kaurenol glucoside by endosperm cell-free preparations in the presence of UDPG was observed. However, the finding was not confirmed in in vivo studies and is probably artifactual. Overall evidence coming from the analysis of endogenous GAs and in vitro and in vivo biosynthetic studies are discussed in relation to the possible existence in the Sechium seeds of a different route, along with the known pathway, branching from ent-kaurene or ent-7-alpha-hydroxykaurenoic acid and this also leading to biologically active GAs.

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PubMed1990

A mutant gene that increases gibberellin production in brassica.

A single gene mutant (elongated internode [ein/ein]) with accelerated shoot elongation was identified from a rapid cycling line of Brassica rapa. Relative to normal plants, mutant plants had slightly accelerated floral development, greater stem dry weights, and particularly, increased internode and inflorescence elongation. The application of the triazole plant growth retardant, paclobutrazol, inhibited shoot elongation, returning ein to a more normal phenotype. Conversely, exogenous gibberellin A(3) (GA(3)) can convert normal genotypes to a phenotype resembling ein. The content of endogenous GA(1) and GA(3) were estimated by gas chromatography-selected ion monitoring using [(2)H]GA(1), as a quantitative internal standard and at day 14 were 1.5- and 12.1-fold higher per stem, respectively, in ein than in normal plants, although GA concentrations were more similar. The endogenous levels of GA(20) and GA(1), and the rate of GA(19) metabolism were simultaneously analyzed at day 7 by feeding [(2)H(2)]GA(19) and measuring metabolites [(2)H(2)]GA(20) and [(2)H(2)]GA(1) and endogenous GA(20) and GA(1), with [(2)H(5)]GA(20) and [(2)H(5)]GA(1) as quantitative internal standards. Levels of GA(1) and GA(20) were 4.6- and 12.9-fold higher, respectively, and conversions to GA(20) and GA(1) were 8.3 and 1.3 times faster in ein than normal plants. Confirming the enhanced rate of GA(1) biosynthesis in ein, the conversion of [(3)H]GA(20) to [(3)H]GA(1) was also faster in ein than in the normal genotype. Thus, the ein allele results in accelerated GA(1) biosynthesis and an elevated content of endogenous GAs, including the dihydroxylated GAs A(1) and A(3). The enhanced GA production probably underlies the accelerated shoot growth and development, and particularly, the increased shoot elongation.

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PubMed1990

Biosynthetic Origin of Gibberellins A(3) and A(7) in Cell-Free Preparations from Seeds of Marah macrocarpus and Malus domestica.

Cell-free preparations from seeds of Marah macrocarpus L. and Malus domestica L. catalyzed the conversion of gibberellin A(9) (GA(9)) and 2,3-dehydroGA(9) to GA(7); GA(9) was also metabolized to GA(4) in a branch pathway. The preparation from Marah seeds also metabolized GA(5) to GA(3) in high yield; GA(6) was a minor product and was not metabolized to GA(3). Using substrates stereospecifically labeled with deuterium, it was shown that the metabolism of GA(5) to GA(3) and of 2,3-dehydroGA(9) to GA(7) occurs with the loss of the 1beta-hydrogen. In cultures of Gibberella fujikuroi, mutant B1-41a, [1beta,2beta-(2)H(2)]GA(4), was metabolized to [1,2-(2)H(2)]GA(3) with the loss of the 1alpha- and 2alpha-hydrogens. These results provide further evidence that the biosynthetic origin of GA(3) and GA(7) in higher plants is different from that in the fungus Gibberella fujikuroi.

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PubMed1990

Selection and Characterization of a Gibberellin-Deficient Mutant of Thlaspi arvense L.

Field pennycress (Thlaspi arvense L.) is a winter annual weed with a cold requirement for reproductive development. Previous work in this laboratory has demonstrated that the bolting aspect (rapid stem growth) of reproductive development is mediated by gibberellins (GA). The present paper describes the selection and characterization of a mutant lacking the capacity for thermoinduced stem growth. Seeds of an inbred field pennycress line (CR(1)) were treated with the chemical mutagen ethyl methane sulfonate, germinated, and allowed to produce seed. Plants derived from these seeds were screened for reduced stem growth. A mutant line, EMS-141, in which the lack of stem growth can be fully overcome with exogenous GA(3), was selected for further analysis. Other phenotypic abnormalities exhibited by the mutant line include reduced petiole growth, slightly delayed floral initiation, and failure of flowers to develop fully. These are also reversed with exogenous GA(3). Evidence is presented indicating that all of the alterations in growth and development exhibited by EMS-141 are conferred by a recessive mutation of a single nuclear gene. Through quantitative analysis of endogenous GA and GA precursors and a comparison of the abilities of various compounds to restore normal growth when applied to plants of EMS-141, the physiological basis for the mutant phenotype was determined to be the result of highly reduced endogenous GA levels. Moreover, the affected site in GA biosynthesis appears to be the accumulation of ent-kaurene, probably at the level of ent-kaurene synthase. The relative abilities of exogenous GA and GA precursors to restore normal growth of petioles and stems are compared, and the results are used to make inferences on the functions of the two different pathways of GA metabolism that exist in field pennycress.

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PubMed1990

Light-Stimulated Gibberellin Biosynthesis in Gibberella fujikuroi.

Gibberellins (GAs) are a group of plant growth hormones that were first isolated from the fungus Gibberella fujikuroi. The biosynthesis of GA in liquid cultures of the fungus has been examined using high-performance liquid chromatography and combined gas chromatography-mass spectrometry. GA(3) was the predominant GA in well-aerated cultures. GA(4) and GA(7), intermediates in GA(3) biosynthesis, accumulated in cultures with low levels of dissolved oxygen, but were not detectable in more highly aerated cultures. Light stimulated the production of GA(3) in G. fujikuroi cultures grown from young stock cultures. Cell-free enzyme studies revealed a significant stimulation in the levels of kaurenoic acid oxidation in cultures grown in the light in comparison with those grown in the dark. However, measurements of the relative rates of [(14)C]mevalonic acid incorporation into kaurene showed no effect of light on this early part of the pathway. Preliminary experiments indicated that blue light is most effective in enhancing kaurenoic acid oxidation.

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PubMed1991

Phenotypic characterization of lettuce dwarf mutants and their response to applied gibberellins.

Four monogenic, recessive dwarf mutants of lettuce (Lactuca sativa L.), previously isolated from a population induced by ethyl methanesulfonate, were compared with the normal genotype (E-1) for plant height, weight, leaf area, as well as hypocotyl length and root length. These nonallelic dwarfs (dwf1, dwf2, and dwf3) exhibited reduced hypocotyl length, smaller, dark green leaves, and reduced stem length. Another mutant, dwf2, allelic with dwf2, exhibited an intermediate phenotype. Epidermal cells on hypocotyls and mature leaves were counted for both normal E-1 and dwf2 plants. The total number of epidermal cells per unit area for hypocotyls and for leaves from these plants was very similar, implying the dwarf's smaller size was due to an inhibition of cell expansion and not due to decreased cell divisions. Both dwarf and normal hypocotyls elongated normally in response to exogenous gibberellin A(3) (GA(3)). In the rosette stage, only E-1 and dwf2 responded similarly to lower concentrations of GA(3), while the other dwarfs required higher concentrations to respond. Hypocotyls of dwf2 and E-1 elongated equally with applied ent-kaurenol, ent-kaurenoic acid, GA(53)-aldehyde, GA(53), GA(19), GA(20), and GA(1) indicating that the biochemical block in dwf2 occurs at a very early step in the GA-biosynthetic pathway.

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PubMed1991

The endogenous gibberellins of dwarf mutants of lettuce.

The gibberellin (GA) content of E-1, a tall genotype of early flowering lettuce (Lactuca sativa L.), and of three selected GA-responsive dwarfs, dwf1, dwf2, and dwf2(1), has been determined using (13)C-labeled internal standards and gas chromatographymass spectrometry (GC-MS). In the shoots of the E-1 parent, GA(1), 3-epi-GA(1), GA(3), GA(5), GA(8), GA(19), GA(20), GA(29), and GA(53) were identified by full scan GC-MS and Kovats retention indices. Purification by immunoaffinity chromatography selective for 13-hydroxy GAs, was necessary for GA identification. Relative to the parent E-1, the concentrations of GA(1), GA(8), GA(20), and GA(29) in the shoots of dwf2 plants were reduced to about 10% and in shoots of dwf2(1) plants to less than 50%. In dwf1 the levels of GA(1), GA(8), and GA(29) were also reduced to less than 50% of the parent E-1, but the level of GA(20) was fivefold higher than in E-1. Plant height was correlated with the endogenous levels of GA(1) and GA(8).

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PubMed1991

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.

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PubMed1991

Implication of Gibberellins in Head Smut (Sporisorium reilianum) of Sorghum bicolor.

The head smut fungus, Sporisorium reilianum ([Kuhn] Landon and Fullerton), was shown to reduce plant height in infected Sorghum bicolor ([L.] Moench) plants. The major reductions occurred in the internodes nearest the panicle and were more severe in naturally infected than in inoculated plants. Less affected plants developed reproductively sterile panicles, and eventually smutted panicles developed phyllodied growths which progressed into leafy shoots. Extracts of smutted, sterile, and healthy (control) panicles of field-grown plants exhibited gibberellin (GA)-like activity in the dwarf rice bioassay. When extracts were purified and assayed with deuterium-labeled GA standards by gas chromatography-mass spectrometry-selected ion monitoring (GC-MS-SIM), GA(1), GA(3), GA(19), GA(20), and GA(53) were detected based on coelution with the standards, identical Kovats retention index values, and matching ion masses and relative abundances for three major ions. In addition, based on published Kovats retention index values, ion masses, and relative abundance values, GA(4), GA(7), GA(8), GA(14), GA(29), and GA(44) were tentatively identified. Quantitative analysis revealed that panicles of healthy control plants contained from 60 to 100% higher total concentrations of GAs than panicles of smutted plants. These comparisons were most striking for the early 13-hydroxylation pathway precursors GA(53), GA(44), and GA(19) but not for GA(20). Extracts of S. reilianum sporidia and culture medium exhibited GA-like bioactivity, and GA(1) and GA(3) were detected based on GC-MS-SIM assay with (2)H-labeled internal standards. Quantitative analysis of these GAs showed increasing concentrations from 4 to 7 to 10 days of culture and a decline at 20 days. This is the first GC-MS-SIM detection of GAs in a non-Ascomycete fungus, and the disease symptoms and quantitative data suggested that fungal infection may interfere with biosynthesis of GAs by the host plant.

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PubMed1991

Effects of the gibberellin biosynthetic inhibitor uniconazol on mutants of Arabidopsis.

Using the gibberellin (GA) biosynthetic inhibitor Uniconazol, we determined that det1, a mutant that no longer requires light to be germinated, still requires GA synthesis for germination. This result suggests that dark inhibition of germination in Arabidopsis may be due to inhibition of GA synthesis by the DET1 gene product in mature wild-type seeds. Similar experiments with mutants that lack seed dormancy due to a reduced sensitivity to abscisic acid (abi) have shown that abi1 and abi3 no longer require GA for germination. Furthermore, by shifting wild-type seeds to inhibitor at 6-hour intervals during imbibition, we determined that GA synthesis is only required during the first 24 hours of the imbibition process to reverse abscisic acid-induced dormancy in Arabidopsis.

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PubMed1991

Identification of Gibberellins in Spinach and Effects of Light and Darkness on their Levels.

The endogenous gibberellin (GA) content of spinach (Spinacia oleracea) was reinvestigated by combined gas chromatography-mass spectrometry analysis. The 13-hydroxy GAs: GA(53), GA(44), GA(19), GA(17), GA(20), GA(5), GA(1), GA(29), and GA(8); the non-3, 13-hydroxy GAs: GA(12), GA(15), GA(9), and GA(51); and the 3beta-hydroxy GAs: GA(4), GA(7), and GA(34), were identified in spinach extracts by comparing full-scan mass spectra and Kovats retention indices with those of reference GAs. In addition, spinach plants contained GA(7)-isolactone, 16,17-dihydro-17-hydroxy-GA(53), GA(29)-catabolite, 3-epi-GA(1), and 10 uncharacterized GAs with mass spectra indicative of mono- and dihydroxy-GA(12), monohydroxy-GA(25), dihydroxy-GA(24), and dihydroxy-GA(g). The effect of light-dark conditions on the GA levels of the 13-hydroxylation pathway was studied by using labeled internal standards in selected ion monitoring mode. In short day, the GA levels were higher at the end of the light period than at the end of the dark period. Levels of GAs at the end of each short day were relatively constant. During the first supplementary light period of long day treatment, GA(53) and GA(19) declined dramatically, GA(44) and GA(1) decreased slightly, and GA(20) increased. During the subsequent high-intensity light period, the GA(20) level decreased and the levels of GA(53), GA(44), GA(19), and GA(1) increased slightly. Within 7 days after the beginning of long day treatment, similar patterns for GA(53) and GA(19) occurred. Furthermore, when these plants were transferred to darkness, an increase in the levels of GA(53) and GA(19) was observed. These results are compatible with the idea that in spinach, the flow through the GA biosynthetic pathway is much enhanced during the high-intensity light period, although GA turnover occurs also during the supplementary period of long day, both effects being responsible for the increase of GA(20) and GA(1) in long day.

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PubMed1992

Nondwarf rice seedling bioassay for gibberellins.

The use of nondwarf rice (Oryza sativa L.) cultivars treated with uniconazole as test plants for gibberellin (GA) bioassay instead of Tan-ginbozu dwarf rice variant was investigated. The sensitivity of six nondwarf rice cultivars to GAs was increased substantially by treatment of the seeds with uniconazole. The minimum detectable dose of a GA in the nondwarf cultivars treated with uniconazole was 1- to 1/10-fold of that in the nontreated Tanginbozu and 3- to 10-fold of that in uniconazole-treated Tanginbozu. The relative activity of several GAs on treated nondwarf rice cultivars was not largely different from that to Tan-ginbozu. Considering that seeds of nondwarf rice are available commercially, the nondwarf rice seedling assay would be useful as a simple assay for systematic analysis of GAs, and also as a routine teaching tool in high schools and universities.

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PubMed1992

The Distribution of Gibberellins in Vegetative Tissues of Pisum sativum L. : I. Biological and Biochemical Consequences of the le Mutation.

The concentrations of endogenous gibberellin (GA) 1, 5, 8, 19, 20, and 29 in the component tissues of maturing tall (Le) and dwarf (le) pea (Pisum sativum) plants have been determined. The following conclusions were drawn from the data obtained: (a) GA(20) and its metabolites accumulate only in the growing regions of Le and le plants; (b) the le mutation is biochemically expressed in all immature tissues of the dwarf plants; (c) the quantitative composition of the GA metabolites in the various immature tissues is variable; (d) the total GA concentration in apical buds, unexpanded leaves, and tendrils is considerably higher than in GA(1)-responsive stem tissue; and (e) there is very little GA accumulation of the inactive 2beta-hydroxylated GAs (GA(8) and GA(29)) in either the mature vegetative tissues or the roots of pea plants.

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PubMed1992

Gibberellin A(1) Biosynthesis in Pisum sativum L. : II. Biological and Biochemical Consequences of the le Mutation.

A comparative study of the metabolism of radiolabeled gibberellin (GA) 1, 19, and 20 in isolated vegetative tissues of isogenic Le and le pea (Pisum sativum) plants incubated in vitro with the appropriate GA substrate is described. The results of this study provide evidence that the enzymes involved in the latter stages of GA biosynthesis are spatially separated within the growing pea plant. Apical buds were not apparently involved in the production of bioactive GA(1) or its immediate precursors. The primary site of synthesis of GA(20) from GA(19) was immature leaflets and tendrils, and the synthesis of bioactive GA(1) and its inactive catabolite GA(8) occurred predominantly in stem tissue. GA(29), the inactive catabolite of GA(20), was produced to varying extents in all the tissues examined. Little or no difference was observed in the ability of corresponding Le and le tissues to metabolize radiolabeled GA(1), GA(19), or even GA(20). During a fixed period of 24 hours, stems of plants carrying the le mutation produced slightly more [(3)H]GA(1) (and [(3)H]GA(29)) than those of Le plants. It has been concluded that the le mutation does not lie within the gene encoding the GA(20) 3beta-hydroxylase protein.

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