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.     

Developing Inflorescences of Male and Female <Emphasis Type="Italic">Rumex acetosa</Emphasis> L. Show Differences in Gibberellin Content

Rumex acetosa L. (common sorrel) is a dioecious perennial in the family Polygonaceae. Gibberellins (GAs) of the early 13-hydroxylation pathway and the putative early 3, 13-hydroxylation pathway were previously identified in young R. acetosa inflorescences by GC-MS. In this investigation to examine the GA content of individual inflorescences ELISAs were used for quantitative analysis. Significant differences were revealed between the sexes in the GA content of young inflorescences, and GC-SRM was used to validate the observed trends. Males had higher levels of the 3, 13-hydroxylated C₂₀-GA GA₁₈ and the 2, 13-hydroxylated C₁₉-GA GA₂₉, whereas females had higher levels of the 13-hydroxylated C₂₀-GAs GA₅₃ and GA₁₉. It is suggested that the conversion from C₂₀-GAs to C₁₉-GAs is under tighter control in the inflorescences of females compared to male plants and therefore there is accumulation of the C₂₀-GAs in the females. Results from flowering bioassays using authentic GAs indicate that differences in GA content between the sexes are unlikely to be a consequence of sex determination.     

Gibberellin Signal Transduction in Rice

Rumex acetosa L. (common sorrel) is a dioecious perennial in the family Polygonaceae. Gibberellins (GAs) of the early 13-hydroxylation pathway and the putative early 3, 13-hydroxylation pathway were previously identified in young R. acetosa inflorescences by GC-MS. In this investigation to examine the GA content of individual inflorescences ELISAs were used for quantitative analysis. Significant differences were revealed between the sexes in the GA content of young inflorescences, and GC-SRM was used to validate the observed trends. Males had higher levels of the 3, 13-hydroxylated C₂₀-GA GA₁₈ and the 2, 13-hydroxylated C₁₉-GA GA₂₉, whereas females had higher levels of the 13-hydroxylated C₂₀-GAs GA₅₃ and GA₁₉. It is suggested that the conversion from C₂₀-GAs to C₁₉-GAs is under tighter control in the inflorescences of females compared to male plants and therefore there is accumulation of the C₂₀-GAs in the females. Results from flowering bioassays using authentic GAs indicate that differences in GA content between the sexes are unlikely to be a consequence of sex determination.     

OsGA2ox5, a Gibberellin Metabolism Enzyme,Is Involved in Plant Growth,the Root Gravity Response and Salt Stress

Gibberellin (GA) 2-oxidases play an important role in the GA catabolic pathway through 2β-hydroxylation. There are two classes of GA2oxs, i.e., a larger class of C₁₉-GA2oxs and a smaller class of C₂₀-GA2oxs. In this study, the gene encoding a GA 2-oxidase of rice, Oryza sativa GA 2-oxidase 5 (OsGA2ox5), was cloned and characterized. BLASTP analysis showed that OsGA2ox5 belongs to the C₂₀-GA2oxs subfamily, a subfamily of GA2oxs acting on C₂₀-GAs (GA₁₂, GA₅₃). Subcellular localization of OsGA2ox5-YFP in transiently transformed onion epidermal cells revealed the presence of this protein in both of the nucleus and cytoplasm. Real-time PCR analysis, along with GUS staining, revealed that OsGA2ox5 is expressed in the roots, culms, leaves, sheaths and panicles of rice. Rice plants overexpressing OsGA2ox5 exhibited dominant dwarf and GA-deficient phenotypes, with shorter stems and later development of reproductive organs than the wild type. The dwarfism phenotype was partially rescued by the application of exogenous GA₃ at a concentration of 10 µM. Ectopic expression of OsGA2ox5 cDNA in Arabidopsis resulted in a similar phenotype. Real-time PCR assays revealed that both GA synthesis-related genes and GA signaling genes were expressed at higher levels in transgenic rice plants than in wild-type rice; OsGA3ox1, which encodes a key enzyme in the last step of the bioactive GAs synthesis pathway, was highly expressed in transgenic rice. The roots of OsGA2ox5-ox plants exhibited increased starch granule accumulation and gravity responses, revealing a role for GA in root starch granule development and gravity responses. Furthermore, rice and Arabidopsis plants overexpressing OsGA2ox5 were more resistant to high-salinity stress than wild-type plants. These results suggest that OsGA2ox5 plays important roles in GAs homeostasis, development, gravity responses and stress tolerance in rice.     

Gibberellins in suspensor,embryo and integument from very young seeds of Phaseolus coccineus L.

Endogenous gibberellins (GAs) were extracted from suspensor, embryo and integument of very young seeds of Phaseolus coccineus L. and detected by combined gas chromatography-mass spectrometry (GC-MS). Results show the presence of one C₂₀-GA, GA₄₄ and five C₁₉-GAs in the suspensor: GA₁, GA₄, GA₅, GA₆ and GA₈, and four C₁₉-GAs in the integument: GA₁, GA₅, GA₆ and GA₈. Only traces of GA₁ and GA₅ were identified in the embryo. A compound structurally related to GAs was identified as tetrahydroxy-Kauranoic acid in suspensor, integument and, only in trace amounts, in the embryo.     

Gibberellin Metabolism and Transport During Germination and Young Seedling Growth of Pea (<Emphasis Type="Italic">Pisum sativum</Emphasis> L.)

The role of gibberellins (GAs) during germination and early seedling growth is examined by following the metabolism and transport of radiolabeled GAs in cotyledon, shoot, and root tissues of pea (Pisum sativum L.) using an aseptic culture system. Mature pea seeds have significant endogenous GA₂₀ levels that fall during germination and early seedling growth, a period when the seedling develops the capacity to transport GA₂₀ from the cotyledon to the shoot and root of the seedling. Even though cotyledons at 0–2 days after imbibition have appreciable amounts of GA₂₀, the cotyledons retain the ability to metabolize labeled GA₁₉ to GA₂₀ and express significant levels of PsGA20ox2 message (which encodes a GA biosynthesis enzyme, GA 20-oxidase). The large pool of cotyledonary GA₂₀ likely provides substrate for GA₁ synthesis in the cotyledons during germination, as well as for shoots and roots during early seedling growth. The shoots and roots express GA metabolism genes (PsGA3ox genes which encode GA 3-oxidases for synthesis of bioactive GA₁, and PsGA2ox genes which encode GA 2-oxidases for deactivation of GAs to GA₂₉ and GA₈), and they develop the capacity to metabolize GAs as necessary for seedling establishment. Auxins also show an interesting pattern during early seedling growth, with higher levels of 4-chloro-indole-3-acetic acid (4-Cl-IAA) in mature seeds and higher levels of indole-3-acetic acid (IAA) in young root and shoot tissues. This suggests a changing role for auxins during early seedling development.     

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 C₁₉-GAs, GA₁, GA₄, GA₅, GA₆, and one C₂₀ GA, GA₄₄, were identified. From the embryo, five C₁₉-GAs GA₁, GA₄, GA₅, GA₆, GA₆₀ and two C₂₀ GAs, GA₁₉ and GA₄₄ were identified. The data, in relation to previous results, suggest a dependence of the embryo on the suspensor during early stages of development.     

Gibberellins in shoots of Hordeum vulgare. A comparison between cv. Triumph and two dwarf mutants which differ in their response to gibberellin

The gibberellin (GA) content of barley (Hordeum vulgare L.) cv. Triumph was analysed by full scan gas chromatography-mass spectrometry. Developing grain contained several di-, tri-, and tetra-hydroxylated GAs, with the most abundant ones being hydroxylated at C-2, C-3, C-12β, and/or C-18. In contrast, the only GAs to be detected in shoots of 9-day old dark- and light-grown seedlings of Triumph were 13-hydroxylated C₁₉-GAs, namely GA₁, GA₈, GA₂₀, and GA₂₉, (all of which are components of the early 13-hydroxylation GA biosynthetic pathway) and GA₃. Feeds of [¹³C.³H₂GA₂₀, confirmed that GA₂₀ is a precursor of GA₁, GA₈, and GA₂₉ in barley shoots. From these results it is suggested that stem growth of barley, in common with that of several other mono- and dicotyledons, is controlled by GA,. Homozygous gal and gal lines were obtained after backcrossing to Triumph. These were then compared to Triumph with respect to their GA content and response to applied GAs and GA precursors. Shoots of the homozygous gal gal plants contained ca 6-fold less GA₁, than Triumph. These plants responded to all ent-kaurenoids and 13-hydroxylated C₂₀- and C₁₉-GAs tested. It is concluded that the gal locus impairs the GA biosynthetic pathway prior to ent-kaurene, most probably at ent-kaurene synthetase. In contrast, shoots of homozygous gal gal line contained ca 10-fold higher levels of GA, than Triumph, but failed to respond to applied GA, or GA₃. The gal locus therefore confers insensitivity to both exogenous and endogenous GAs, possibly by perturbing the reception or transduction of the GA₁ signal.     

Decreased GA1 Content Caused by the Overexpression of OSH1 Is Accompanied by Suppression of GA 20-Oxidase Gene Expression

We previously reported that overexpression of the rice homeobox gene OSH1 led to altered morphology and hormone levels in transgenic tobacco (Nicotiana tabacum L.) plants. Among the hormones whose levels were changed, GA₁ was dramatically reduced. Here we report the results of our analysis on the regulatory mechanism(s) of OSH1 on GA metabolism. GA₅₃ and GA₂₀, precursors of GA₁, were applied separately to transgenic tobacco plants exhibiting severely changed morphology due to overexpression of OSH1. Only treatment with the end product of GA 20-oxidase, GA₂₀, resulted in a striking promotion of stem elongation in transgenic tobacco plants. The internal GA₁ and GA₂₀ contents in OSH1-transformed tobacco were dramatically reduced compared with those of wild-type plants, whereas the level of GA₁₉, a mid-product of GA 20-oxidase, was 25% of the wild-type level. We have isolated a cDNA encoding a putative tobacco GA 20-oxidase, which is mainly expressed in vegetative stem tissue. RNA-blot analysis revealed that GA 20-oxidase gene expression was suppressed in stem tissue of OSH1-transformed tobacco plants. Based on these results, we conclude that overexpression of OSH1 causes a reduction of the level of GA₁ by suppressing GA 20-oxidase expression.     

The loss of carbon-20 in C19-gibberellin biosynthesis in a cell-free system from Pisum sativum L.

The fate of the carbon-20 atom in gibberellin (GA) biosynthesis was studied in a cell-free system from Pisum sativum. This carbon atom is lost at the aldehyde stage of oxidation when C₂₀-GAs are converted to C₁₉-GAs. Gibberellin A₁₂ labeled with ¹⁴C at C-20 was prepared from [3-¹⁴C]mevalonic acid with a cell-free system from Cucurbita maxima and incubated with the pea system. Analysis of the gas and aqueous phases showed that ¹⁴CO₂ was formed at the same rate and in nearly equivalent amounts as ¹⁴C-labeled C₁₉-GAs whereas [¹⁴C]formic acid and [¹⁴C]formaldehyde were not detectable. The possibility that C-20 had been lost as formic acid which had then been converted to CO₂ was investigated by control incubations with [¹⁴C]formic acid. The rate of release of ¹⁴CO₂ from [¹⁴C]formic acid was only one fiftieth of the rate of ¹⁴CO₂ release from [¹⁴C]GA₁₂ as the substrate. We conclude that in the formation of C₁₉-GAs from C₂₀-GAs, the C-20 is removed directly as CO₂.Abbreviations GA_n Gibberellin A_n     

Engineering gibberellin metabolism in <Emphasis Type="Italic">Solanum nigrum</Emphasis> L. by ectopic expression of gibberellin oxidase genes

Gibberellins (GAs) control many aspects of plant development, including seed germination, shoot growth, flower induction and growth and fruit expansion. Leaf explants of Solanum nigrum (Black Nightshade; Solanaceae) were used for Agrobacterium-mediated delivery of GA-biosynthetic genes to determine the influence of their encoded enzymes on the production of bioactive GAs and plant stature in this species. Constructs were prepared containing the neomycin phosphotransferase (nptII) gene for kanamycin resistance as a selectable marker, and the GA-biosynthetic genes, their expression under the control of the CaMV 35S promoter. The GA-biosynthetic genes comprised AtGA20ox1, isolated from Arabidopsis thaliana, the product from which catalyses the formation of C₁₉-GAs, and MmGA3ox1 and MmGA3ox2, isolated from Marah macrocarpus, which encode functionally different GA 3-oxidases that convert C₁₉-GAs to biologically active forms. Increase in stature was observed in plants transformed with AtGA20ox1, MmGA3ox2 and MmGA3ox1 + MmGA3ox2, their presence and expression being confirmed by PCR and RT-PCR, respectively, accompanied by an increase in GA₁ content. Interestingly, MmGA3ox1 alone did not induce a sustained increase in plant height, probably because of only a marginal increase in bioactive GA₁ content in the transformed plants. The results are discussed in the context of regulating plant stature, since this strategy would decrease the use of chemicals to promote plant growth.     

Over-expression of a gibberellin 2-oxidase gene from Phaseolus coccineus L. enhances gibberellin inactivation and induces dwarfism in Solanum species

Gibberellins (GAs) are endogenous hormones that play a predominant role in regulating plant stature by increasing cell division and elongation in stem internodes. The product of the GA 2-oxidase gene from Phaseolus coccineus (PcGA2ox1) inactivates C₁₉-GAs, including the bioactive GAs GA₁ and GA₄, by 2β-hydroxylation, reducing the availability of these GAs in plants. The PcGA2ox1 gene was introduced into Solanum melanocerasum and S. nigrum (Solanaceae) by Agrobacterium-mediated transformation with the aim of decreasing the amounts of bioactive GA in these plants and thereby reducing their stature. The transgenic plants exhibited a range of dwarf phenotypes associated with a severe reduction in the concentrations of the biologically active GA₁ and GA₄. Flowering and fruit development were unaffected. The transgenic plants contained greater concentrations of chlorophyll b (by 88%) and total chlorophyll (11%), although chlorophyll a and carotenoid contents were reduced by 8 and 50%, respectively. This approach may provide an alternative to the application of chemical growth retardants for reducing the stature of plants, particularly ornamentals, in view of concerns over the potential environmental and health hazards of such compounds. C. Dijkstra, E. Adams, A. Bhattacharya and A. F. Page contributed equally to this paper.     

The transport and metabolism of gibberellins A1 and A5 in excised segments from internodes of Phaseolus coccineus

Jacobs, W. P., Beall, F. D. and Pharis, R. P. 1988. The transport and metabolism of gibberellins A₁ and A₅ in excised segments from internodes of Phaseolus coccineus. -Physiol. Plant. 72: 529–534. The transport and metabolism of gibberellins (GAs) ([³H]-GA, and [³H]-GA₅) of high specific radioactivity were investigated in excised segments from young internodes of Phaseolus coccineus L. Both GA₁ and GA₅ are native to this species and present in shoot tissue. The segments, 5.1 mm long, were incubated for 6 h in the horizontal position with agar donor blocks containing the [³H]-GA on the morphological apical or basal ends and with plain agar receiver blocks on the opposite end. At the end of incubation, the individual agar blocks were analyzed immediately for total radioactivity, or both blocks and intervening tissue were frozen and freeze-dried for later chromatographic analysis. The movement of both [³H]-GA, and [³H]-GA₅ was found to be consistently without polarity. However, approximately 5-fold more [³H]-GA, than [³H]-GA₅ was transported through the Phaseolus segments into receivers when equal amounts were in the donors. The extractable radioactivity from receiver blocks was primarily that of the donor GA. No putative GA conjugates were found in any class of receivers, but more GA metabolites were found in the free acid fraction from acropetal than basipetal receivers. Chromatographic analysis by reversed phase C₁₈ high performance liquid chromatography of the tissue segments showed that [³H]-GA, was metabolized more than [³H]-GA₅. Tissue adjacent to receiver blocks contained not only the precursor GA from the donor, but also polar ‘free GA metabolites’ and putative GA glucosyl conjugates. These results provide evidence that GA., which is the known ‘effector’ GA for elongation in shoot tissue of several species, is more effectively transported than GA₅ (a known precursor of GA₁) or than GA₁s more polar metabolites.     

Genome-Wide Identification,Evolution and Expression Analyses of <i>GA2ox</i> Gene Family in <i>Brassica napus</i> L.

Gibberellin 2-oxidases (GA2ox) are important enzymes that maintain the balance of bioactive GAs in plants. GA2ox genes have been identified and characterized in many plants, but these genes were not investigated in Brassica napus. Here, we identified 31 GA2ox genes in B. napus and 15 of these BnaGA2ox genes were distributed in the A and C subgenomes. Subcellular localization predictions suggested that all BnaGA2ox proteins were localized in the cytoplasm, and gene structure analysis showed that the BnaGA2ox genes contained 2–4 exons. Phylogenetic analysis indicated that BnGA2ox family proteins in monocotyledons and dicotyledons can be divided into four groups, including two C₁₉-GA2ox and two C₂₀-GA2ox clades. Group 4 is a C₂₀-GA2ox Class discovered recently. Most BnaGA2ox genes had a syntenic relationship with AtGA2ox genes. BnaGA2ox genes in the C subgenome had experienced stronger selection pressure than genes in the A subgenome. BnaGA2ox genes were highly expressed in specific tissues such as those involved in growth and development, and most of them were mainly involved in abiotic responses, regulation of phytohormones and growth and development. Our study provided a valuable evolutionary analysis of GA2ox genes in monocotyledons and dicotyledons, as well as an insight into the biological functions of GA2ox family genes in B. napus.     

Daylength and spatial expression of a gibberellin 20-oxidase isolated from hybrid aspen (Populus tremula L. × P. tremuloides Michx.)

Physiologically active gibberellins (GAs) are key regulators of shoot growth in trees. To investigate this mechanism of GA-controlled growth in hybrid aspen, we cloned cDNAs encoding gibberellin 20-oxidase (GA 20-oxidase), a key, highly regulated enzyme in the biosynthesis of GAs. Clones were isolated from leaf and cambium cDNA libraries using probes generated by polymerase chain reaction, based on conserved domains of GA 20-oxidases. Upon expression in Escherichia coli, the GST-fusion protein was shown to oxidise GA12 as well as oxidising the 13-hydroxylated substrate GA53, successively to GA9 and GA20, respectively. The gene PttGA20ox1 was expressed in meristematic cells and growing tissues such as expanding internodes, leaves and roots. The expression was negatively regulated by both GA4 and overexpression of phytochrome A. RNA analysis also showed that the expression was down-regulated in late-expanding leaf tissue in response to short days (SDs). Actively growing tissues such as early elongating internodes, petioles and leaf blades had the highest levels of C19-GAs. Upon transfer to SDs an accumulation of GA19 was observed in early elongating internodes and leaf blades. The levels of C19-GAs were also to some extent changed upon transfer to SDs. The levels of GA20 were down-regulated in internodes, and those of GA1 were significantly reduced in early expanding leaf blades. In roots the metabolites GA19 and GA8 decreased upon shifts to SDs, while GA20 accumulated slightly. The down-regulation of GA 20-oxidase activity in response to SDs was further indicated by studies of [14C]GA12 metabolism in shoots, demonstrating that the substrate for GA 20-oxidase, [14C]GA53, accumulates in SDs.     

Function and Expression Analysis of Gibberellin Oxidases in Apple

Three cDNAs, encoding gibberellin (GA) 20-oxidase (MdGA20ox1, identical to AB037114), 3-oxidase (MdGA3ox1), and 2-oxidase (MdGA2ox1), were isolated from apple cv. Fuji (Malus x domestica). Southern blot analysis indicated that each of these genes belongs to a gene family. Standard enzyme assays show that the MdGA20ox1-MBP fusion protein can sequentially oxidize three times at C-20 position of GA₁₂ and GA₅₃ and generate GA₉ and GA₂₀; the MdGA3ox1-MBP fusion protein converts GA₂₀ and GA₉ to GA₄ and GA₁, and the MdGA2ox1-MBP fusion protein converts GA₄ and GA₁ to GA₃₄ and GA₈, respectively. In addition, we confirmed that MdGA20ox1 is strongly expressed in immature seeds and scarcely detected in other tissues, whereas MdGA3ox1 and MdGA2ox1 are mainly expressed in flowers. Therefore, all the three cDNAs are localized in reproductive tissues. Functional and expression analysis of the three GA oxidases would provide fundamental molecular information to analyze GA metabolic regulation in apple.