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PubMed1994

Gibberellin-induced changes in the translatable mRNA populations of stamens and shoots of gibberellin-deficient tomato.

The gib1 mutant of tomato (Lycopersicon esculentum Mill.) is deficient in endogenous gibberellins and exhibits phenotypes including extreme dwarfism, reduced germination, and abnormal flower development, which are reversed by the application of gibberellic acid (GA3). Previous work has demonstrated that, in stamens of the gib1 mutant, pollen mother-cell development arrests at the premeiotic G1 stage (Jacobsen and Olszewski 1991, Plant Physiol. 97, 409-414). Following GA3 treatment of developmentally arrested flowers, pollen mother-cell development resumes and is synchronous. The present study examines gibberellin-induced changes in the translatable mRNA populations of developmentally arrested stamens and of vegetative shoots of the gib1 mutant. Following rescue of developmentally arrested stamens by treatment with GA3, we consistently detected increases and decreases in the abundance of 14 and 20 in-vitro translation products, respectively. Some of these changes were first detected 8 h post treatment and therefore represent the first changes observed in stamens whose development has been rescued by GA3 treatment. In vegetative gib1 shoots, the abundance of 13 in-vitro translation products decreased within 6-24 h after GA3 treatment. However, no in-vitro translation products that increased in abundance after GA3 treatment were detected.

Gene Expression↗

PubMed1986

An improved enzymatic synthesis of labeled gibberellin A12-aldehyde and gibberellin A12.

The biochemical synthesis of labeled gibberellin A12-7-aldehyde (GA12ald) and and gibberellin A12 (GA12) from labeled R,S-mevalonic acid (MVA) using cell-free extracts from pumpkin (Cucurbita maxima) endosperm has been improved over the previously reported procedure. Three major improvements were developing a one-step HPLC procedure to isolate GA12ald and GA12 in radiochemically pure form; adjusting the pH of the reaction mix to pH 6.9 which increased the GA12ald/GA12 ratio over that at pH 7.8 by ca. 18-fold while reducing the combined yield of these two compounds by less than 17%; and developing a technique that permitted sampling of the fruits without interrupting growth; this doubled the fraction of extracts with "high activity." Conversion of MVA into GA12ald or GA12 displayed Michaelis-Menten kinetics with half-maximal synthesis at 0.4 mM MVA. Four-hour incubations afforded the highest yields from 0.25 mM MVA. Up to 15 and 7% of the MVA was incorporated into GA12ald and GA12, respectively. About one-quarter of the extracts incorporated at least 10% of the 0.25 mM MVA into GA12ald. One pumpkin fruit can provide sufficient endosperm to synthesize ca. 0.6 mumol of GA12ald.

Cell-Free System↗

PubMed2003

Epitope mapping of gibberellin to the anti-gibberellin A(4) monoclonal antibody by saturation transfer difference NMR spectroscopy.

Saturation transfer difference (STD) NMR spectroscopy is a promising tool for rapid screening, identifying ligands that interact with a target protein, and characterizing the epitopes of the ligands. Gibberellins (GAs) are a class of plant hormones and form a large family consisting of more than 120 members. A few of them, called "active" GAs, are considered to be perceptible to a receptor that remains unknown. We applied STD NMR spectroscopy to detect the binding activity and identify the binding epitope of gibberellin A(3) (GA(3)) that is recognized by monoclonal antibody 4-B8(8)/E9. This is one of the antibodies that can mimic a GA receptor in the manner of recognition of active GAs. The information on the binding epitope, obtained by STD NMR, was in good agreement with that shown by analyzing the crystal structure of the antibody-GA(4) complex. This suggests that STD NMR spectroscopy would be very useful to characterize the interaction between GAs and such binding proteins as GA-catabolic enzymes and receptors.

Antibodies, Monoclonal↗

PubMed2000

Identification of endogenous gibberellins in strawberry, including the novel gibberellins GA123, GA124 and GA125.

Extracts of carboxylic acids from immature fruits of strawberry (Fragaria x ananassa Duch. cv. Elsanta) were analysed for gibberellins by combined gas chromatography-mass spectrometry. The following previously characterised gibberellins were identified by comparison of their mass spectra and Kovats retention indices (KRIs) with those of standards or published data: GA1, GA3, GA5, GA8, GA12, GA17, GA19, GA20, GA29, GA44, GA48, GA49, GA53, GA77, GA97, GA111 and GA112. Evidence for endogenous 1-epi GA61 (GA119) and 11alpha-OH-GA12 was also obtained. In addition, a number of putative GAs were detected. Of these, three were shown to be 12alpha-hydroxy-GA53, 12alpha-hydroxy-GA44, and 12alpha-hydroxy-GA19 by comparison with authentic compounds prepared by rational synthesis, and have been allocated the descriptors GA123, GA124 and GA125, respectively.

Fruit↗

PubMed1994

Expression cloning of a gibberellin 20-oxidase, a multifunctional enzyme involved in gibberellin biosynthesis.

In the biosynthetic pathway to the gibberellins (GAs), carbon-20 is removed by oxidation to give the C19-GAs, which include the biologically active plant hormones. We report the isolation of a cDNA clone encoding a GA 20-oxidase [gibberellin, 2-oxoglutarate:oxygen oxidoreductase (20-hydroxylating, oxidizing) EC 1.14.11.-] by screening a cDNA library from developing cotyledons of pumpkin (Cucurbita maxima L.) for expression of this enzyme. When mRNA from either the cotyledons or the endosperm was translated in vitro using rabbit reticulocyte lysates, the products contained GA12 20-oxidase activity. A polyclonal antiserum was raised against the amino acid sequence of a peptide released by tryptic digestion of purified GA 20-oxidase from the endosperm. A cDNA expression library in lambda gt11 was prepared from cotyledon mRNA and screened with the antiserum. The identity of positive clones was confirmed by the demonstration of GA12 20-oxidase activity in single bacteriophage plaques. Recombinant protein from a selected clone catalyzed the three-step conversions of GA12 to GA25 and of GA53 to GA17, as well as the formation of the C19-GAs, GA1, GA9, and GA20, from their respective aldehyde precursors, GA23, GA24, and GA19. The nucleotide sequence of the cDNA insert contains an open reading frame of 1158 nt encoding a protein of 386 amino acid residues. The predicted M(r) (43,321) and pI (5.3) are similar to those determined experimentally for the native GA 20-oxidase. Furthermore, the derived amino acid sequence includes sequences obtained from the N terminus and two tryptic peptides from the native enzyme. It also contains regions that are highly conserved in a group of non-heme Fe-containing dioxygenases.

Amino Acid Sequence↗

PubMed2001

Azospirillum spp. metabolize [17,17-2H2]gibberellin A20 to [17,17-2H2]gibberellin A1 in vivo in dy rice mutant seedlings.

Azospirillum spp. are endophytic bacteria with beneficial effects on cereals--effects partially attributed to gibberellin production by the microorganisms. Azospirillum lipoferum and Azospirillum brasilense inoculated to rice dy mutant reversed dwarfism in seedlings incubated with [17,17-2H2]GA20 with formation of [17,17-2H2]GA1, showing the in vivo capacity to perform the 3beta-hydroxylation. When prohexadione-Ca, an inhibitor of late steps in gibberellin biosynthesis, was added to the culture medium, no complementation was observed and no [17,17-2H2]GA1 was produced. The latter suggests that the bacterial operating enzyme may be a 2-oxoglutarate-dependent dioxygenase, similar to those of plants.

Azospirillum↗

PubMed2005

Tissue-specific localization of gibberellins and expression of gibberellin-biosynthetic and signaling genes in wood-forming tissues in aspen.

Bioactive gibberellins (GAs) are known regulators of shoot growth and development in plants. In an attempt to identify where GAs are formed, we have analyzed the expression patterns of six GA biosynthesis genes and two genes with predicted roles in GA signaling and responses in relation to measured levels of GAs. The analysis was based on tangential sections, giving tissue-specific resolution across the cambial region of aspen trees (Populus tremula). Gibberellin quantification by GC/MS-SRM showed that the bioactive GA1 and GA4 were predominantly located in the zone of expansion of xylem cells. Based on co-localization of the expression of the late GA biosynthesis gene GA 20-oxidase 1 and bioactive GAs, we suggest that de novo GA biosynthesis occurs in the expanding xylem. However, expression levels of the first committed GA biosynthesis enzyme, ent-copalyl diphosphate synthase, were high in the phloem, suggesting that a GA precursor(s) may be transported to the xylem. The expression of the GA signaling and response genes DELLA-like1 and GIP-like1 coincided well with sites of high bioactive GA levels. We therefore suggest that the main role of GA during wood formation is to regulate early stages of xylem differentiation, including cell elongation.

Gene Expression Regulation, Developmental↗

PubMed1998

Effect of Gibberellin Biosynthesis Inhibitors on Native Gibberellin Content, Growth and Floral Initiation in Sorghum bicolor.

CCC, uniconazol, ancymidol, prohexadione-calcium (BX-112), and CGA 163'935, which represent three groups of gibberellin (GA) biosynthesis inhibitors, were applied as a soil drench to Sorghum bicolor cultivars 58M (phyB-1, phytochrome B-deficient mutant) and 90M (phyB-2, equivalent phenotypically to wild type, PHYB, except for small differences in flowering dates). The inhibitors that block steps before GA12 (CCC, uniconazol, and ancymidol) lowered the concentrations of all endogenous early-C13alpha-hydroxylation pathway GAs found in sorghum: GA12, GA53, GA44, GA19, GA20, GA1, and GA8. In contrast, the inhibitors that block the conversion of GA20 --> GA1, (CGA 163'935 and BX-112) drastically reduced GA1 and GA8 levels, but they either did not change or caused accumulation of intermediates from GA12 to GA20. Combinations of pre-GA12 inhibitors and GA3 plus GA1 strongly reduced GAs other than GA1 and GA3. Each of these compounds inhibited shoot growth in both cultivars and delayed floral initiation in 90M. Floral initiation of 58M was also delayed by CCC, uniconazol, and ancymidol but not by CGA 163'935 and BX-112. This separation of shoot elongation from floral initiation in sorghum is novel. Both inhibition of shoot growth and delayed floral initiation were almost completely relieved by a mixture of GA3 and GA1 in both 58M and 90M. This observation, plus the much lower levels of endogenous GA3 than of GA1 observed in these experiments, implies that GA1 is the major endogenous GA active in shoot elongation. CGA 163'935 and BX-112 also failed to promote tillering in 58M, whereas inhibitors active before GA12 did so. The possibility that the GA20 --> GA1 inhibitors fail to block flowering and promote tillering in 58M because biosynthetic intermediates between GA12 and GA20 accumulate and/or because 58M is altered in GA metabolism in this same region of the biosynthetic pathway is discussed. Key Words. Gibberellin biosynthesis inhibitors-Phytochrome B-Flowering-Sorghum

Journal Article↗

PubMed1994

The Metabolism of Gibberellin A20 to Gibberellin A1 by Tall and Dwarf Mutants of Oryza sativa and Arabidopsis thaliana.

The purpose of this study was to demonstrate the metabolism of gibberellin A20 (GA20) to gibberellin A1 (GA1) by tall and mutant shoots of rice (Oryza sativa L.) and Arabidopsis thaliana (L.) Heynh. The data show that the tall and dx mutant of rice and the tall and ga5 mutant of Arabidopsis metabolize GA20 to GA1. The data also show that the dy mutant of rice and the ga4 mutant of Arabidopsis block the metabolism of GA20 to GA1. [17-13C,3H]GA20 was fed to tall and the dwarf mutants, dx and dy, of rice and tall and the dwarf mutants, ga5 and ga4, of Arabidopsis. The metabolites were analyzed by high-performance liquid chromatography and full-scan gas chromatography-mass spectrometry together with Kovats retention index data. For rice, the metabolite [13C]GA, was identified from tall and dx seedlings; [13C]GA1 was not identified from the dy seedlings. [13C]GA29 was identified from tall, dx, and dy seedlings. For Arabidopsis, the metabolite [13C]GA1 was identified from tall, ga5, and ga4 plants. The amount of [13C]GA1 from ga4 plants was less than 15% of that obtained from tall and ga5 plants. [13C]GA29 was identified from tall, ga5, and ga4 plants. [13C]GA5 and [13C]GA3 were not identified from any of the six types of plant material.

Journal Article↗

PubMed1972

Comparison of endogenous gibberellins and of the fate of applied radioactive gibberellin a(1) in a normal and a dwarf strain of Japanese morning glory.

The effect of application of GA(3) on hypocotyl growth, the endogenous GAs, and the metabolism of applied (3)H-GA(1) were investigated in relation to dwarfism and light-mediated growth inhibition in the normal (tall) strain Violet and the dwarf strain Kidachi of Japanese morning glory (Pharbitis nil). GA(3) applied in a wide concentration range (10(-9) to 10(-3)m) to 4-day-old seedlings caused great extension of the hypocotyls in light-grown plants of both the normal and the dwarf strain. However, the dwarf strain did not attain the same length as the normal one at any given GA(3) concentration, even when saturation was reached. Dark-grown plants of the dwarf strain responded to GA(3), although relatively much less than light-grown ones; dark-grown plants of the normal strain showed no GA(3) response at all.The levels of free GAs in both strains remained more or less constant in both dark- and light-grown plants up to 18 days after germination. The levels of bound GA in dark- as well as light-grown plants of both strains increased after germination, reached a maximum at the 9th day after germination, and then rapidly declined again. The period of increase coincided with rapid elongation of the hypocotyl and the expansion of the cotyledons.The dwarf strain, Kidachi, contained less endogenous gibberellins, particularly bound gibberellins, than the normal strain, Violet. Dark-grown plants of both strains contained less bound GAs than light-grown plants.Applied (3)H-GA(1) was metabolized to the same extent in both dwarfs and normals, on the one hand, and in both dark- and light-grown plants, on the other. This metabolism involved binding as well as breakdown of the (3)H-GA(1).

Journal Article↗

PubMed1990

Comparison of Biological Activities of Gibberellins and Gibberellin-Precursors Native to Thlaspi arvense L.

Field pennycress (Thlaspi arvense L.) is a winter annual weed with a cold requirement for stem elongation and flowering. The relative abilities of several native gibberellins (GAs) and GA-precursors to elicit stem growth were compared. Of the eight compounds tested, gibberellin A(1), (GA(1)), GA(9), and GA(20) caused stem growth in noninduced (no cold treatment) plants. No stem growth was observed in plants treated with ent-kaurene, ent-kaurenol, ent-kaurenoic acid, GA(53), or GA(8). Moreover, of the biologically active compounds, GA(9) was the most active followed closely by GA(1). In thermoinduced plants (4-week cold treatment at 6 degrees C) that were continuously treated with 2-chlorocholine chloride to reduce endogenous GA production, GA(9) was the most biologically active compound. However, the three kaurenoid GA precursors also promoted stem growth in thermoinduced plants, and were almost as active as GA(20). No such increase in activity was observed for either GA([unk]) or GA(53). The results are discussed in relation to thermoinductive regulation of GA metabolism and its significance to the initiation of stem growth in field pennycress. It is proposed that thermoinduction results in increased conversion of ent-kaurenoic acid to GAs through the C-13 desoxy pathway and that GA(9) is the endogenous mediator of thermoinduced stem growth in field pennycress.

Journal Article↗

PubMed1990

Thermoinductive Regulation of Gibberellin Metabolism in Thlaspi arvense L. : I. Metabolism of [H]-ent-Kaurenoic Acid and [C]Gibberellin A(12)-Aldehyde.

Field pennycress (Thlaspi arvense L.) is a winter annual crucifer with a cold requirement for stem elongation and flowering. In the present study, the metabolism of exogenous [(2)H]-ent-kaurenoic acid (KA) and [(14)C]-gibberellin A(12)-aldehyde (GA(12)-aldehyde) was compared in thermo- and noninduced plants. Thermoinduction greatly altered both quantitative and qualitative aspects of [(2)H]-KA metabolism in the shoot tips. The rate of disappearance of the parent compound was much greater in thermoinduced shoot tips. Moreover, there was 47 times more endogenous KA in noninduced than in thermoinduced shoot tips as determined by combined gas chromatography-mass spectrometry (GC-MS). The major metabolite of [(2)H]-KA in thermoinduced shoot tips was a monohydroxylated derivative of KA, while in noninduced shoot tips, the glucose ester of the hydroxy KA metabolite was the main product. Gibberellin A(9) (GA(9)) was the only GA in which the incorporation of deuterium was detected by GC-MS, and this was observed only in thermoinduced shoot tips. The amount of incorporation was small as indicated by the large dilution by endogenous GA(9). In contrast, thermo- and noninduced leaves metabolized exogenous [(2)H]-KA into GA(20) equally well, although the amount of conversion was also limited. These results are consistent with the suggestion (JD Metzger [1990] Plant Physiol 94: 000-000) that the conversion of KA in to GAs is under thermoinductive control only in the shoot tip, the site of perception for thermoinductive temperatures in field pennycress. There were essentially no differences in the qualitative or quantitative distribution of metabolites formed following the application of [(14)C]-GA(12)-aldehyde to the shoot tips of thermo- or noninduced plants. Thus, the apparent thermoinductive regulation of the KA metabolism into GAs is probably limited to the two metabolic steps involved in converting KA to GA(12)-aldehyde.

Journal Article↗

PubMed2001

A new MFS-transporter gene next to the gibberellin biosynthesis gene cluster of Gibberella fujikuroi is not involved in gibberellin secretion.

The genes of the gibberellin (GA) biosynthesis pathway in Gibberella fujikuroi are organized in a gene cluster consisting of at least seven genes. Here we report the cloning and characterization of smt, a gene encoding a membrane transporter of the major facilitator super-family 1, which is located next to the GA gene cluster. Since pathway-specific transporters occur frequently in prokaryotic and fungal antibiotic and toxin clusters, smt was thought to be involved in GA secretion. The gene is expressed in mycelium grown under GA-production conditions, but not when the GA biosynthesis is repressed by high amounts of ammonium. To investigate the function of SMT, gene replacement experiments were performed. The smt-mutants did not show any reduction in the GA yield; and gibberellic acid or its precursors did not influence the gene expression. However, sugar alcohols, such as myo-inositol, sorbitol and mannitol, induced the expression of smt. The results demonstrate that the smt gene does not play an essential role in biosynthesis and secretion of GAs in G. fujikuroi, despite the location adjacent to the GA gene cluster.

DNA, Fungal↗

PubMed2003

Thermoinduction of genes encoding the enzymes of gibberellin biosynthesis and a putative negative regulator of gibberellin signal transduction in Eustoma grandiflorum.

Eustoma grandiflorum Shinn requires vernalization for the induction of stem elongation and flowering. To investigate the role of gibberellins (GAs) in vernalization, the expression levels of genes encoding enzymes of GA biosynthesis, copalyl diphosphate synthetase, GA 20-oxidase and GA 3 beta-hydroxylase, were examined using two culitvars that show different responses to vernalization. The three genes were induced in a vernalization- and a cultivar-dependent manner. EgSPY, a putative negative regulator of GA signal transduction, was also induced during the vernalization period. The results suggest that the expression of the genes encoding GAs biosynthesis is regulated by vernalization. We postulate that EgSPY functions as a negative regulator of GA signal transduction during vernalization, inhibiting adventitious shoot elongation during vernalization.

Alkyl and Aryl Transferases↗

PubMed2003

Regulation of gibberellin 20-oxidase gene expression and gibberellin content in citrus by temperature and citrus exocortis viroid.

A cDNA clone coding for a gibberellin (GA) 20-oxidase ( CcGA20ox1), an enzyme of GA biosynthesis, which when expressed in vitro catalyzed the conversion of GA(12) to GA(9) and of GA(53) to GA(20), was isolated from the citrus hybrid Carrizo citrange (C itrus sinensis x Poncirus trifoliata). Transcripts of CcGA20ox1 were abundant in the apex and leaves and much less abundant in internodes, nodes and roots. Seedlings of Carrizo citrange cultured under a 32 degrees C/27 degrees C (day/night) regime elongated more than seedlings growing under 17 degrees C/12 degrees C conditions. The effect of higher temperature was associated with more CcGA20ox1 transcripts and with higher content of GA(1), the main active GA in citrus, in the shoot. The infection of Etrog citron ( Citrus medica) plants with citrus exocortis viroid (CEVd), which produces a stunted phenotype, reduced the levels of transcripts in the apical shoot hybridizing to the gene CcGA20ox1 of Carrizo citrange and the content of GA(1). Thus GA(1) content correlated with CcGA20ox1 transcript levels. In contrast, results for gibberellic acid (GA(3)) and paclobutrazol applications to Carrizo citrange showed that CcGA20ox1 expression was subject to feed-back regulation. These observations indicate that the feed-back regulation of GA20ox operates mostly when the levels of active GAs have been dramatically altered. The results also show that the growth reduction induced by environmental (temperature) and biotic (CEVd) factors may be partially due to the modulation of the expression of GA20ox genes.

Citrus↗

PubMed2003

Expression of novel rice gibberellin 2-oxidase gene is under homeostatic regulation by biologically active gibberellins.

We have cloned two genes for gibberellin (GA) 2-oxidase from rice ( Oryza sativa L.). Expression of OsGA2ox2 was not observed. The other gene, OsGA2ox3, was expressed in every tissue examined and was enhanced by the application of biologically active GA. Recombinant OsGA2ox3 protein catalyzed the metabolism of GA(1) to GA(8) and GA(20) to GA(29)-catabolite. These results indicate that OsGA2ox3 is involved in the homeostatic regulation of the endogenous level of biologically active GA in rice.

Amino Acid Sequence↗

PubMed2005

Distribution of gibberellin biosynthetic genes and gibberellin production in the Gibberella fujikuroi species complex.

Gibberella fujikuroi is a species-rich monophyletic complex of at least nine sexually fertile biological species (mating populations, MP-A to MP-I) and more than 30 anamorphs in the genus Fusarium. They produce a variety of secondary metabolites, such as fumonisins, fusaproliferin, moniliformin, beauvericin, fusaric acid, and gibberellins (GAs), a group of plant hormones. In this study, we examined for the first time all nine sexually fertile species (MPs) and additional anamorphs within and outside the G. fujikuroi species complex for the presence of GA biosynthetic genes. So far, the ability to produce GAs was described only for Fusarium fujikuroi (G. fujikuroi MP-C), which contains seven clustered genes in the genome all participating in GA biosynthesis. We show that six other MPs (MPs B, D, E, F, G, and I) and most of the anamorphs within the species complex also contain the entire gene cluster, except for F. verticillioides (MP-A), and F. circinatum (MP-H), containing only parts of it. Despite the presence of the entire gene cluster in most of the species within the G. fujikuroi species complex, expression of GA biosynthetic genes and GA production were detected only in F. fujikuroi (MP-C) and one isolate of F. konzum (MP-I). We used two new molecular marker genes, P450-4 from the GA gene cluster, and cpr, encoding the highly conserved NADPH cytochrome P450 reductase to study phylogenetic relationships within the G. fujikuroi species complex. The molecular phylogenetic studies for both genes have revealed good agreement with phylogenetic trees inferred from other genes. Furthermore, we discuss the role and evolutionary origin of the GA biosynthetic gene cluster.

Gene Expression↗

PubMed1999

Isolation and molecular characterization of gibberellin-regulated H1 and H2B histone cDNAs in the leaf of the gibberellin-deficient tomato.

After differential screening we isolated cDNA clones encoding a histone H1 (leH1) and three variants of histone H2B (leH2B-1, -2 and -3) from the gibberellin (GA)-deficient mutant of tomato (gib-1). The deduced polypeptide of leH1 is 271 amino acids long and exhibits the typical tripartite structure of histones H1. The full-length cDNA clone leH2B-1 encodes for a protein of 142 amino residues and shows the tripartite organization of histones H2B. The histones leH1 and leH2B, which show no tissue specificity, are developmentally expressed in the leaf. The mRNA accumulation was higher in organs which contain meristematic tissue and/or which have a high proportion of actively cycling cells. In the leaf of the gib-1 mutant we demonstrated GA-enhanced histone leH1 and leH2B expression which was not observed in the wild type. GAs of the early-13-hydroxylated pathway (GA1 and GA3) caused most enhanced transcription compared to GAs of the early-non-hydroxylation pathway (GA4 and GA9). Application of GA to the mutant increased histone expression that could correlate with enhanced DNA replication in leaf tissue. Increased chromosome replication may indicate that there is a higher rate of cell division and/or increase of endopolyploidy which both may be dependent on cell elongation induced by GAs.

Amino Acid Sequence↗