The CYP88A cytochrome P450, ent-kaurenoic acid oxidase, catalyzes three steps of the gibberellin biosynthesis pathway.
(41/793)
We have shown that ent-kaurenoic acid oxidase, a member of the CYP88A subfamily of cytochrome P450 enzymes, catalyzes the three steps of the gibberellin biosynthetic pathway from ent-kaurenoic acid to GA(12). A gibberellin-responsive barley mutant, grd5, accumulates ent-kaurenoic acid in developing grains. Three independent grd5 mutants contain mutations in a gene encoding a member of the CYP88A subfamily of cytochrome P450 enzymes, defined by the maize Dwarf3 protein. Mutation of the Dwarf3 gene gives rise to a gibberellin-responsive dwarf phenotype, but the lesion in the gibberellin biosynthesis pathway has not been identified. Arabidopsis thaliana has two CYP88A genes, both of which are expressed. Yeast strains expressing cDNAs encoding each of the two Arabidopsis and the barley CYP88A enzymes catalyze the three steps of the GA biosynthesis pathway from ent-kaurenoic acid to GA(12). Sequence comparison suggests that the maize Dwarf3 locus also encodes ent-kaurenoic acid oxidase. (+info)
KNOX homeodomain protein directly suppresses the expression of a gibberellin biosynthetic gene in the tobacco shoot apical meristem.
(42/793)
To identify genes targeted by the tobacco KNOX homeodomain protein, Nicotiana tabacum homeobox 15 (NTH15), we have generated an inducible system using the human glucocorticoid receptor. In this system, steroid treatment strictly induced NTH15 function and immediately suppressed the expression of a gibberellin (GA) biosynthetic gene encoding GA 20-oxidase (Ntc12) and also resulted in a decrease in bioactive GA levels. The repression of Ntc12 was observed even when indirect effects were blocked by cycloheximide. NTH15 mRNA was present in corpus cells of the shoot apical meristem (SAM), whereas Ntc12 mRNA was observed in leaf primordia and rib meristem but not in the corpus. Recombinant NTH15 protein strongly bound to a 5-bp dyadsymmetric sequence, GTGAC, in the first intron of Ntc12 in vitro. Mutation of this sequence in the Ntc12 gene abolished the NTH15-dependent suppression of Ntc12 in the corpus of the SAM. Our results indicate that NTH15 directly represses Ntc12 expression in the corpus of the wild-type SAM to maintain the indeterminate state of corpus cells. The suppression of NTH15 within cells at the flanks of the SAM permits GA biosynthesis, which promotes organized cell proliferation and consequently induces the determination of cell fate. (+info)
Gibberellin/abscisic acid antagonism in barley aleurone cells: site of action of the protein kinase PKABA1 in relation to gibberellin signaling molecules.
(43/793)
The antagonism between gibberellins (GA) and abscisic acid (ABA) is an important factor regulating the developmental transition from embryogenesis to seed germination. In barley aleurone layers, the expression of genes encoding alpha-amylases and proteases is induced by GA but suppressed by ABA. It has been shown that an ABA-induced protein kinase, PKABA1, mediates the ABA suppression of alpha-amylase expression. Using a barley aleurone transient expression system, we have now localized the site of action of PKABA1 relative to other signal transduction components governing the expression of alpha-amylase. The expression of alpha-amylase can be transactivated by the transcription factor GAMyb, which is itself induced by GA. A truncated GAMyb containing the DNA binding domain but lacking the transactivation domain prevents the GA induction of alpha-amylase, further supporting the notion that GAMyb mediates the GA induction of alpha-amylase expression. Although ABA and PKABA1 strongly inhibit the GA induction of alpha-amylase, they have no effect on GAMyb-transactivated alpha-amylase expression. Using a GAMyb promoter--beta-glucuronidase construct, we also show that both ABA and PKABA1 repress the GA induction of GAMyb. In the slender mutant, GAMyb and alpha-amylase are highly expressed, even in the absence of GA. However, this constitutive expression can still be inhibited by ABA, PKABA1, or an inhibitor of cGMP synthesis. On the basis of these observations, we suggest that PKABA1 acts upstream from the formation of functional GAMyb but downstream from the site of action of the Slender gene product. Because PKABA1 inhibits the GA induction of the GAMyb promoter--beta-glucuronidase construct, it appears that at least part of the action of PKABA1 is to downregulate GAMyb at the transcriptional level. (+info)
Isolation and expression analysis of gibberellin 20-oxidase homologous gene in apple.
(44/793)
To characterize the gibberellin (GA) 20-oxidase gene in apple, the genomic and cDNA clone from "Fuji" apple (accession no. AB037114) was isolated. The deduced amino acid sequence of this cDNA showed 71% and 66% identity to those of GA 20-oxidase cloned from French bean and Arabidopsis, respectively. The transcript of this gene was detected mainly in immature seeds between 1-3 months after full bloom. These results suggested that this apple GA 20-oxidase gene might be involved in GA biosynthesis in developing apple seed. (+info)
Release of reactive oxygen intermediates (superoxide radicals, hydrogen peroxide, and hydroxyl radicals) and peroxidase in germinating radish seeds controlled by light, gibberellin, and abscisic acid.
(45/793)
Germination of radish (Raphanus sativus cv Eterna) seeds can be inhibited by far-red light (high-irradiance reaction of phytochrome) or abscisic acid (ABA). Gibberellic acid (GA3) restores full germination under far-red light. This experimental system was used to investigate the release of reactive oxygen intermediates (ROI) by seed coats and embryos during germination, utilizing the apoplastic oxidation of 2',7'-dichlorofluorescin to fluorescent 2',7'-dichlorofluorescein as an in vivo assay. Germination in darkness is accompanied by a steep rise in ROI release originating from the seed coat (living aleurone layer) as well as the embryo. At the same time as the inhibition of germination, far-red light and ABA inhibit ROI release in both seed parts and GA3 reverses this inhibition when initiating germination under far-red light. During the later stage of germination the seed coat also releases peroxidase with a time course affected by far-red light, ABA, and GA3. The participation of superoxide radicals, hydrogen peroxide, and hydroxyl radicals in ROI metabolism was demonstrated with specific in vivo assays. ROI production by germinating seeds represents an active, developmentally controlled physiological function, presumably for protecting the emerging seedling against attack by pathogens. (+info)
Azospirillum brasilense and Azospirillum lipoferum hydrolyze conjugates of GA20 and metabolize the resultant aglycones to GA1 in seedlings of rice dwarf mutants.
(46/793)
Azospirillum species are plant growth-promotive bacteria whose beneficial effects have been postulated to be partially due to production of phytohormones, including gibberellins (GAs). In this work, Azospirillum brasilense strain Cd and Azospirillum lipoferum strain USA 5b promoted sheath elongation growth of two single gene GA-deficient dwarf rice (Oryza sativa) mutants, dy and dx, when the inoculated seedlings were supplied with [17,17-2H2]GA20-glucosyl ester or [17,17- 2H2]GA20-glucosyl ether. Results of capillary gas chromatography-mass spectrometry analysis show that this growth was due primarily to release of the aglycone [17,17-2H2]GA20 and its subsequent 3beta-hydroxylation to [17,17-2H2]GA1 by the microorganism for the dy mutant, and by both the rice plant and microorganism for the dx mutant. (+info)
The P450-1 gene of Gibberella fujikuroi encodes a multifunctional enzyme in gibberellin biosynthesis.
(47/793)
Recent studies have shown that the genes of the gibberellin (GA) biosynthesis pathway in the fungus Gibberella fujikuroi are organized in a cluster of at least seven genes. P450-1 is one of four cytochrome P450 monooxygenase genes in this cluster. Disruption of the P450-1 gene in the GA-producing wild-type strain IMI 58289 led to total loss of GA production. Analysis of the P450-1-disrupted mutants indicated that GA biosynthesis was blocked immediately after ent-kaurenoic acid. The function of the P450-1 gene product was investigated further by inserting the gene into mutants of G. fujikuroi that lack the entire GA gene cluster; the gene was highly expressed under GA production conditions in the absence of the other GA-biosynthesis genes. Cultures of transformants containing P450-1 converted ent-[(14)C]kaurenoic acid efficiently into [(14)C]GA(14), indicating that P450-1 catalyzes four sequential steps in the GA-biosynthetic pathway: 7beta-hydroxylation, contraction of ring B by oxidation at C-6, 3beta-hydroxylation, and oxidation at C-7. The GA precursors ent-7alpha-hydroxy[(14)C]kaurenoic acid, [(14)C]GA(12)-aldehyde, and [(14)C]GA(12) were also converted to [(14)C]GA(14). In addition, there is an indication that P450-1 may also be involved in the formation of the kaurenolides and fujenoic acids, which are by-products of GA biosynthesis in G. fujikuroi. Thus, P450-1 displays remarkable multifunctionality and may be responsible for the formation of 12 products. (+info)
slender rice, a constitutive gibberellin response mutant, is caused by a null mutation of the SLR1 gene, an ortholog of the height-regulating gene GAI/RGA/RHT/D8.
(48/793)
The rice slender mutant (slr1-1) is caused by a single recessive mutation and results in a constitutive gibberellin (GA) response phenotype. The mutant elongates as if saturated with GAs. In this mutant, (1) elongation was unaffected by an inhibitor of GA biosynthesis, (2) GA-inducible alpha-amylase was produced by the aleurone layers without gibberellic acid application, and (3) endogenous GA content was lower than in the wild-type plant. These results indicate that the product of the SLR1 gene is an intermediate of the GA signal transduction pathway. SLR1 maps to OsGAI in rice and has significant homology with height-regulating genes, such as RHT-1Da in wheat, D8 in maize, and GAI and RGA in Arabidopsis. The GAI gene family is likely to encode transcriptional factors belonging to the GRAS gene superfamily. DNA sequence analysis revealed that the slr1-1 mutation is a single basepair deletion of the nuclear localization signal domain, resulting in a frameshift mutation that abolishes protein production. Furthermore, introduction of a 6-kb genomic DNA fragment containing the wild-type SLR1 gene into the slr1-1 mutant restored GA sensitivity to normal. These results indicate that the slr1-1 mutant is caused by a loss-of-function mutation of the SLR1 gene, which is an ortholog of GAI, RGA, RHT, and D8. We also succeeded in producing GA-insensitive dwarf rice by transforming wild-type rice with a modified SLR1 gene construct that has a 17-amino acid deletion affecting the DELLA region. Thus, we demonstrate opposite GA response phenotypes depending on the type of mutations in SLR1. (+info)