Cloning and Characterization of a Gibberellin-Induced RNase Expressed in Barley Aleurone Cells1

We cloned a cDNA for a gibberellin-induced ribonuclease (RNase) expressed in barley (Hordeum vulgare) aleurone and the gene for a second barley RNase expressed in leaf tissue. The protein encoded by the cDNA is unique among RNases described to date in that it contains a novel 23-amino acid insert between the C2 and C3 conserved sequences. Expression of the recombinant protein in tobacco (Nicotiana tabacum) suspension-cultured protoplasts gave an active RNase of the expected size, confirming the enzymatic activity of the protein. Analyses of hormone regulation of expression of mRNA for the aleurone RNase revealed that, like the pattern for α-amylase, mRNA levels increased in the presence of gibberellic acid, and its antagonist abscisic acid prevented this effect. Quantitative studies at early times demonstrated that cycloheximide treatment of aleurone layers increased mRNA levels 4-fold, whereas a combination of gibberellin plus cycloheximide treatment was required to increase α-amylase mRNA levels to the same extent. These results are consistent with loss of repression as an initial effect of gibberellic acid on transcription of those genes, although the regulatory pathways for the two genes may differ.

Arabidopsis ent-Kaurene Oxidase Catalyzes Three Steps of Gibberellin Biosynthesis

The Arabidopsis GA3 cDNA was expressed in yeast (Saccharomyces cerevisiae) and the ability of the transformed yeast cells to metabolize ent-kaurene was tested. We show by full-scan gas chromatography-mass spectrometry that the transformed cells produce ent-kaurenoic acid, and demonstrate that the single enzyme GA3 (ent-kaurene oxidase) catalyzes the three steps of gibberellin biosynthesis from ent-kaurene to ent-kaurenoic acid.

Feedback Control and Diurnal Regulation of Gibberellin 20-Oxidase Transcript Levels in Potato1

Tuber formation in potato (Solanum tuberosum) is promoted by short photoperiods and is inhibited by gibberellins (GAs). Endogenous levels of GA1 were shown to decrease in stolons and leaves of potato plants induced to tuberize, which suggests that photoperiodic regulation of GA biosynthesis may play a role in tuber induction. We report the isolation of three potato cDNA clones (StGA20ox1–3) encoding GA 20-oxidase, a key regulatory enzyme in the GA-biosynthetic pathway. Using northern analysis, we detected a differential pattern of tissue-specific expression of the mRNAs corresponding to these clones. StGA20ox mRNAs were also very abundant in leaves of the potato ga1 mutant, which is blocked in the 13-hydroxylation step, and were strongly down-regulated by gibberellic acid, suggesting a feedback regulation of these genes. In plants grown in short-day (inductive) conditions, levels of the StGA20ox transcripts in leaves fluctuated during a 24-h period, with a peak of accumulation observed about 4 h after the lights were turned off. Interruption of the night with a 30-min “night break” of light (noninductive conditions) did not have a marked effect on the levels of accumulation of the three GA 20-oxidase mRNAs during the day, but it induced a second peak of expression of StGA20ox1 and StGA20ox3 transcripts late in the night. This observation, together with the finding that StGA20ox1 mRNA is expressed at high levels in leaves, suggests that night-break induction of this gene might play a role in the control of tuberization by regulating endogenous levels of GAs in response to daylength conditions.

The P450–1 gene of Gibberella fujikuroi encodes a multifunctional enzyme in gibberellin biosynthesis

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-[14C]kaurenoic acid efficiently into [14C]GA14, indicating that P450–1 catalyzes four sequential steps in the GA-biosynthetic pathway: 7β-hydroxylation, contraction of ring B by oxidation at C-6, 3β-hydroxylation, and oxidation at C-7. The GA precursors ent-7α-hydroxy[14C]kaurenoic acid, [14C]GA12-aldehyde, and [14C]GA12 were also converted to [14C]GA14. 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.

The First Step of Gibberellin Biosynthesis in Pumpkin Is Catalyzed by at Least Two Copalyl Diphosphate Synthases Encoded by Differentially Regulated Genes

The first step in gibberellin biosynthesis is catalyzed by copalyl diphosphate synthase (CPS) and ent-kaurene synthase. We have cloned from pumpkin (Cucurbita maxima L.) two cDNAs, CmCPS1 and CmCPS2, that each encode a CPS. Both recombinant fusion CmCPS proteins were active in vitro. CPS are translocated into plastids and processed by cleavage of transit peptides. For CmCPS1 and CmCPS2, the putative transit peptides cannot exceed the first 99 and 107 amino acids, respectively, because longer N-terminal deletions abolished activity. Levels of both CmCPS transcripts were strictly regulated in an organ-specific and developmental manner. Both transcripts were almost undetectable in leaves and were abundant in petioles. CmCPS1 transcript levels were high in young cotyledons and low in roots. In contrast, CmCPS2 transcripts were undetectable in cotyledons but present at significant levels in roots. In hypocotyls, apices, and petioles, CmCPS1 transcript levels decreased with age much more rapidly than those of CmCPS2. We speculate that CmCPS1 expression is correlated with the early stages of organ development, whereas CmCPS2 expression is correlated with subsequent growth. In contrast, C. maxima ent-kaurene synthase transcripts were detected in every organ at almost constant levels. Thus, ent-kaurene biosynthesis may be regulated through control of CPS expression.

Phytochrome Regulates Gibberellin Biosynthesis during Germination of Photoblastic Lettuce Seeds1

Germination of lettuce (Lactuca sativa L.) seed is regulated by phytochrome. The requirement for red light is circumvented by the application of gibberellin (GA). We have previously shown that the endogenous content of GA1, the main bioactive GA in lettuce seeds, increases after red-light treatment. To clarify which step of GA1 synthesis is regulated by phytochrome, cDNAs encoding GA 20-oxidases (Ls20ox1 and Ls20ox2, for L. sativa GA 20-oxidase) and 3β-hydroxylases (Ls3h1 and Ls3h2 for L. sativa GA 3β-hydroxylase) were isolated from lettuce seeds by reverse-transcription polymerase chain reaction. Functional analysis of recombinant proteins expressed in Escherichia coli confirmed that the Ls20ox and Ls3h encode GA 20-oxidases and 3β-hydroxylases, respectively. Northern-blot analysis showed that Ls3h1 expression was dramatically induced by red-light treatment within 2 h, and that this effect was canceled by a subsequent far-red-light treatment. Ls3h2 mRNA was not detected in seeds that had been allowed to imbibe under any light conditions. Expression of the two Ls20ox genes was induced by initial imbibition alone in the dark. The level of Ls20ox2 mRNA decreased after the red-light treatment, whereas that of Ls20ox1 was unaffected by light. These results suggest that red light promotes GA1 synthesis in lettuce seeds by inducing Ls3h1 expression via phytochrome action.

Overexpression of 20-Oxidase Confers a Gibberellin-Overproduction Phenotype in Arabidopsis

In the gibberellin (GA) biosynthesis pathway, 20-oxidase catalyzes the oxidation and elimination of carbon-20 to give rise to C19-GAs. All bioactive GAs are C19-GAs. We have overexpressed a cDNA encoding 20-oxidase isolated from Arabidopsis seedlings in transgenic Arabidopsis plants. These transgenic plants display a phenotype that may be attributed to the overproduction of GA. The phenotype includes a longer hypocotyl, lighter-green leaves, increased stem elongation, earlier flowering, and decreased seed dormancy. However, the fertility of the transgenic plants is not affected. Increased levels of endogenous GA1, GA9, and GA20 were detected in seedlings of the transgenic line examined. GA4, which is thought to be the predominantly active GA in Arabidopsis, was not present at increased levels in this line. These results suggest that the overexpression of this 20-oxidase increases the levels of some endogenous GAs in transgenic seedlings, which causes the GA-overproduction phenotype.

Gibberellin Dose-Response Regulation of GA4 Gene Transcript Levels in Arabidopsis1

The gibberellins (GAs) are a complex family of diterpenoid compounds, some of which are potent endogenous regulators of plant growth. As part of a feedback control of endogenous GA levels, active GAs negatively regulate the abundance of mRNA transcripts encoding GA biosynthesis enzymes. For example, Arabidopsis GA4 gene transcripts encode GA 3β-hydroxylase, an enzyme that catalyzes the conversion of inactive to active GAs. Here we show that active GAs regulate GA4 transcript abundance in a dose-dependent manner, and that down-regulation of GA4 transcript abundance is effected by GA4 (the product of 3β-hydroxylation) but not by its immediate precursor GA9 (the substrate). Comparison of several different GA structures showed that GAs active in promoting hypocotyl elongation were also active in regulating GA4 transcript abundance, suggesting that similar GA:receptor and subsequent signal transduction processes control these two responses. It is interesting that these activities were not restricted to 3β-hydroxylated GAs, being also exhibited by structures that were not 3β-hydroxylated but that had another electronegative group at C-3. We also show that GA-mediated control of GA4 transcript abundance is disrupted in the GA-response mutants gai and spy-5. These observations define a sensitive homeostatic mechanism whereby plants may regulate their endogenous GA levels.

Function and Substrate Specificity of the Gibberellin 3β-Hydroxylase Encoded by the Arabidopsis GA4 Gene1

cDNA corresponding to the GA4 gene of Arabidopsis thaliana L. (Heynh.) was expressed in Escherichia coli, from which cell lysates converted [14C]gibberellin (GA)9 and [14C]GA20 to radiolabeled GA4 and GA1, respectively, thereby confirming that GA4 encodes a GA 3β-hydroxylase. GA9 was the preferred substrate, with a Michaelis value of 1 μm compared with 15 μm for GA20. Hydroxylation of these GAs was regiospecific, with no indication of 2β-hydroxylation or 2,3-desaturation. The capacity of the recombinant enzyme to hydroxylate a range of other GA substrates was investigated. In general, the preferred substrates contained a polar bridge between C-4 and C-10, and 13-deoxy GAs were preferred to their 13-hydroxylated analogs. Therefore, no activity was detected using GA12-aldehyde, GA12, GA19, GA25, GA53, or GA44 as the open lactone (20-hydroxy-GA53), whereas GA15, GA24, and GA44 were hydroxylated to GA37, GA36, and GA38, respectively. The open lactone of GA15 (20-hydroxy-GA12) was hydroxylated but less efficiently than GA15. In contrast to the free acid, GA25 19,20-anhydride was 3β-hydroxylated to give GA13. 2,3-Didehydro-GA9 and GA5 were converted by recombinant GA4 to the corresponding epoxides 2,3-oxido-GA9 and GA6.

The Role of Gibberellin, Abscisic Acid, and Sucrose in the Regulation of Potato Tuber Formation in Vitro1

The effects of plant hormones and sucrose (Suc) on potato (Solanum tuberosum L.) tuberization were studied using in vitro cultured single-node cuttings. Tuber-inducing (high Suc) and -noninducing (low Suc or high Suc plus gibberellin [GA]) media were tested. Tuberization frequencies, tuber widths, and stolon lengths were measured during successive stages of development. Endogenous GAs and abscisic acid (ABA) were identified and quantified by high-performance liquid chromatography and gas chromatography-mass spectrometry. Exogenous GA4/7 promoted stolon elongation and inhibited tuber formation, whereas exogenous ABA stimulated tuberization and reduced stolon length. Indoleacetic acid-containing media severely inhibited elongation of stolons and smaller sessile tubers were formed. Exogenous cytokinins did not affect stolon elongation and tuber formation. Endogenous GA1 level was high during stolon elongation and decreased when stolon tips started to swell under inducing conditions, whereas it remained high under noninducing conditions. GA1 levels were negatively correlated with Suc concentration in the medium. We conclude that GA1 is likely to be the active GA during tuber formation. Endogenous ABA levels decreased during stolon and tuber development, and ABA levels were similar under inducing and noninducing conditions. Our results indicate that GA is a dominant regulator in tuber formation: ABA stimulates tuberization by counteracting GA, and Suc regulates tuber formation by influencing GA levels.

The GA2 Locus of Arabidopsis thaliana Encodes ent-Kaurene Synthase of Gibberellin Biosynthesis

The ga2 mutant of Arabidopsis thaliana is a gibberellin-deficient dwarf. Previous biochemical studies have suggested that the ga2 mutant is impaired in the conversion of copalyl diphosphate to ent-kaurene, which is catalyzed by ent-kaurene synthase (KS). Overexpression of the previously isolated KS cDNA from pumpkin (Cucurbita maxima) (CmKS) in the ga2 mutant was able to complement the mutant phenotype. A genomic clone coding for KS, AtKS, was isolated from A. thaliana using CmKS cDNA as a heterologous probe. The corresponding A. thaliana cDNA was isolated and expressed in Escherichia coli as a fusion protein. The fusion protein showed enzymatic activity that converted [3H]copalyl diphosphate to [3H]ent-kaurene. The recombinant AtKS protein derived from the ga2–1 mutant is truncated by 14 kD at the C-terminal end and does not contain significant KS activity in vitro. Sequence analysis revealed that a C-2099 to T base substitution, which converts Gln-678 codon to a stop codon, is present in the AtKS cDNA from the ga2–1 mutant. Taken together, our results show that the GA2 locus encodes KS.

Photoperiod Control of Gibberellin Levels and Flowering in Sorghum1

Regulation of rhythmic peaks in levels of endogenous gibberellins (GAs) by photoperiod was studied in the short-day monocot sorghum (Sorghum bicolor [L.] Moench). Comparisons were made between three maturity (Ma) genotypes: 58M (Ma1Ma1, Ma2Ma2, phyB-1phyB-1, and Ma4Ma4 [a phytochrome B null mutant]); 90M (Ma1Ma1, Ma2Ma2, phyB-2phyB-2, and Ma4Ma4); and 100M (Ma1Ma1, Ma2Ma2, PHYBPHYB, and Ma4Ma4). Plants were grown for 14 d under 10-, 14-, 16-, 18-, and 20-h photoperiods, and GA levels were assayed by gas chromatography-mass spectrometry every 3 h for 24 h. Under inductive 10-h photoperiods, the peak of GA20 and GA1 levels in 90M and 100M was shifted from midday, observed earlier with 12-h photoperiods, to an early morning peak, and flowering was hastened. In addition, the early morning peaks in levels of GA20 and GA1 in 58M under conditions allowing early flowering (10-, 12-, and 14-h photoperiods) were shifted to midday by noninductive (18- and 20-h) photoperiods, and flowering was delayed. These results are consistent with the possibility that the diurnal rhythm of GA levels plays a role in floral initiation and may be one way by which the absence of phytochrome B causes early flowering in 58M under most photoperiods.

Cloning and functional analysis of two gibberellin 3β-hydroxylase genes that are differently expressed during the growth of rice

We have cloned two gibberellin (GA) 3β-hydroxylase genes, OsGA3ox1 and OsGA3ox2, from rice by screening a genomic library with a DNA fragment obtained by PCR using degenerate primers. We have used full-scan GC-MS and Kovats retention indices to show function for the two encoded recombinant fusion proteins. Both proteins show 3β-hydroxylase activity for the steps GA20 to GA1, GA5 to GA3, GA44 to GA38, and GA9 to GA4. In addition, indirect evidence suggests that the OsGA3ox1 protein also has 2,3-desaturase activity, which catalyzes the steps GA9 to 2,3-dehydro-GA9 and GA20 to GA5 (2,3-dehydro GA20), and 2β-hydroxylase activity, which catalyzes the steps GA1 to GA8 and GA4 to GA34. Molecular and linkage analysis maps the OsGA3ox1 gene to the distal end of the short arm of chromosome 5; the OsGA3ox2 gene maps to the distal end of the short arm of chromosome 1 that corresponds to the D18 locus. The association of the OsGA3ox2 gene with the d18 locus is confirmed by sequence and complementation analysis of three d18 alleles. Complementation of the d18-AD allele with the OxGA3ox2 gene results in transgenic plants with a normal phenotype. Although both genes show transient expression, the highest level for OsGA3ox1 is from unopened flower. The highest level for OsGA3ox2 is from elongating leaves.

The dwarf-1 (dt) Mutant of Zea mays blocks three steps in the gibberellin-biosynthetic pathway.

In plants, gibberellin (GA)-responding mutants have been used as tools to identify the genes that control specific steps in the GA-biosynthetic pathway. They have also been used to determine which native GAs are active per se, i.e., further metabolism is not necessary for bioactivity. We present metabolic evidence that the D1 gene of maize (Zea mays L.) controls the three biosynthetic steps: GA20 to GA1, Ga20 to GA5, and GA5 to GA3. We also present evidence that three gibberellins, GA1, GA5, and GA3, have per se activity in stimulating shoot elongation in maize. The metabolic evidence comes from the injection of [17-13C,3H]GA20 and [17-13C,3H]GA5 into seedlings of d1 and controls (normal and d5), followed by isolation and identification of the 13C-labeled metabolites by full-scan GC-MS and Kovats retention index. For the controls, GA20 was metabolized to GA1,GA3, and GA5; GA5 was metabolized to GA3. For the d1 mutant, GA20 was not metabolized to GA1, GA3, or to GA5, and GA5 was not metabolized to GA3. The bioassay evidence is based on dosage response curves using d1 seedlings for assay. GA1, GA3, and GA5 had similar bioactivities, and they were 10-times more active than GA20.

SPINDLY, a tetratricopeptide repeat protein involved in gibberellin signal transduction in Arabidopsis.

Gibberellins (GAs) are a major class of plant hormones that control many developmental processes, including seed development and germination, flower and fruit development, and flowering time. Genetic studies with Arabidopsis thaliana have identified two genes involved in GA perception or signal transduction. A semidominant mutation at the GIBBERELLIN INSENSITIVE (GAI) locus results in plants resembling GA-deficient mutants but exhibiting reduced sensitivity to GA. Recessive mutations at the SPINDLY (SPY) locus cause a phenotype that is consistent with constitutive activation of GA signal transduction. Here we show that a strong allele of spy is completely epistatic to gai, indicating that SPY acts downstream of GAI. We have cloned the SPY gene and shown that it encodes a new type of signal transduction protein, which contains a tetratricopeptide repeat region, likely serving as a protein interaction domain, and a novel C-terminal region. Mutations in both domains increase GA signal transduction. The presence of a similar gene in Caenorhabditis elegans suggests that SPY represents a class of signal transduction proteins that is present throughout the eukaryotes.