The ectopic overexpression of a citrus gibberellin 20-oxidase enhances the non-13-hydroxylation pathway of gibberellin biosynthesis and induces an extremely elongated phenotype in tobacco

Transgenic plants of Nicotiana tabacum overexpressing a gibberellin (GA) 20-oxidase cDNA ( CcGA20ox1 ) from citrus, under the control of the 35S promoter, were taller (up to twice) and had larger inflorescences and longer flower peduncles than those of control plants. Hypocotyls of transgenic seedlings were also longer (up to 4 times), and neither the seedlings nor the growing plants elongated further after application of GA₃. Hypocotyl and stem lengths were reduced by application of paclobutrazol, and this inhibition was reversed by exogenous GA₃. The ectopic overexpression of CcGA20ox1 enhanced the non-13-hydroxylation pathway of GA biosynthesis leading to GA₄, apparently at the expense of the early-13-hydroxylation pathway. The level of GA₄ (the active GA from the non-13-hydroxylation pathway) in the shoot of transgenic plants was 3–4 times higher than in control plants, whereas that of GA₁, formed via the early-13-hydroxylation pathway (the main GA biosynthesis pathway in tobacco), decreased or was not affected. GA₄ applied to the culture medium or to the expanding leaves was found to be at least equally active as GA₁ on stimulating hypocotyl and stem elongation of tobacco plants. The results suggest that the tall phenotype of tobacco transgenic plants was due to their higher content of GA₄, and that the GA response was saturated by the presence of the transgene.     

Internode length in Pisum. Estimation of GA1 levels in genotypes Le, le and led

The levels of GA₁, 3-epiGA₁ and GA₈ in genotypes Le, le and le^d of Pisum sativum L. were determined by gas chromatography-selected ion monitoring (GC-SIM) after feeds of [³H, ¹³C]-GA₂₀ to each genotype. The levels of endogenous and [¹³C]-labelled metabolites were determined by reverse isotope dilution with unlabelled GA₁, 3-epiGA₁ and GA₈. The results demonstrate a quantitative relationship between the level of GA₁ and the extent of elongation both on a per plant and a per g fresh weight basis. These results are consistent with previous findings in peas and other species possessing a predominant early 13-hydroxylation pathway for GA biosynthesis.
The levels of 3-epiGA₁ also decreased in the genotypic sequence Le, le, le^d although not as rapidly as for the level of GA₁. This may suggest that the alleles at the le locus also influence the formation of 3-epiGA₁.     

An acylcyclohexadione retardant inhibits gibberellin A1 metabolism, thereby nullifying phytochrome-modulation of cowpea epicotyl explants

The regulation by phytochrome of stem elongation in light-grown plants depends on gibberellins (GAs). To investigate whether this is mediated by a change in GA metabolism, the effect of the GA biosynthesis inhibitor LAB 198 999 (an acylcyclohexadione derivative) on the end-of-day far-red (FR) response in cowpea ( Vigna sinensis L.) epicotyl explants has been investigated. Growth of epicotyl explants of light-grown seedlings was enhanced when treated with far-red light before incubation in the dark (end-of-day FR effect). Low doses of LAB 198 999 (0.05 and 0.5 μg explant⁻¹) reduced the effect of FR, whereas 5 to 50 μg explant⁻¹ stimulated elongation of both red light (R)- and FR-treated epicotyl explants while nullifying the differences between R and FR treatments. In paclobutrazol-treated epicotyl explants, FR enhanced the response to applied GA₁ and GA₂₀, whereas LAB 198 999 increased the activity of GA₁ and decreased that of GA₂₀, [³H]Gibberellin A₁, injected into the basal part of the epicotyl, was transported and metabolized mainly to [³H]GA₈ in the apical 20 mm of the epicotyl. The conversion of [³H]GA₁ to [³H]GA₈ was dramatically reduced by both end-of-day FR treatments and LAB 198 999 applications. In addition, both treatments enhanced epicotyl elongation. It is proposed that the regulation of cowpea epicotyl growth by phytocrome is mediated, at least partially, by modifying GA₁ degradation.     

Stimulation of shoot elongation in Salix pentandra by gibberellin A9; activity appears to be dependent upon hydroxylation to GAl via GA20

Cessation of shoot elongation in seedlings of Salix pentandra L. is induced by short photoperiod. Gibbereliin A₉ (GA₉) applied either to the apical bud or injected into a mature leaf, induced shoot elongation under a short photoperiod of 12 h, and GA₉ could completely substitute for a transfer to a long photoperiod. When [³H]GA₉ or [²H₂]GA₉ was injected into a leaf, no [³H]GA₉ was detected in the elongating apex and only traces of [³H]GA₉ were found in the shoot above the treated leaf. By the use of gas chromatography-mass spectrometry (GC-MS), [²H₂]GA₂₀ was identified as the main metabolite of [²H₂]GA₉ in both the shoot and the treated leaf. In addition, [²H₂]GA₁ and [²H₂]GA₂₉ were also identified as metabolites of [²H₂]GA₉. These results are consistent with the hypothesis that exogenous GA, promotes shoot elongation in Salix through its metabolism to GA₂₀ and GA,.     

Biological activity, identification and quantification of gibberellins in seedlings of Norway spruce (Picea abies) grown under different photoperiods

Short photoperiod induces growth cessation in seedlings of Norway spruce ( Picea abies (L.] Karst.). Application of different gibberellins (GAS) to seedlings growing under a short photoperiod show that GA₉ and GA₂₀ can not induce growth. In contrast application of GA, and GA₄ induced shoot elongation. The results indicate that 3β-hydroxylation of GA₉ to GA₄ and of GA₂₀ to GA₁ is under photoperiodic control. To confirm that conclusion, both qualitative and quantitative analyses of endogenous GAs were performed. GA₁, GA₃, GA₄, GA₇, GA₉, GA₁₂, GA₁₅, GA₁₅, GA₂₀, GA₂₉, GA₃₄ and GA₅₁ were identified by combined gas chromatography-mass spectrometry in shoots of Norway spruce seedlings. The effect of photoperiod on GA levels was determined by using deuterated and ¹⁴C-labelled GAs as intermal standards. In short days, the amounts of GA₉, GA₄ and GA₁ are less than in plants grown in continuous light. There is no significant difference in the amounts of GA₃, GA₁₂, and GA₂₀ between the different photoperiods. The lack of accumulation of GA₉ and GA₂₀ under short days is discussed.     

Immunomodulation of bioactive gibberellin confers gibberellin-deficient phenotypes in plants

Immunomodulation is a means to modulate an organism's function by antibody production to capture either endogenous or exogenous antigens. This method was applied to plants to repress the function of gibberellins (GAs), a class of phytohormones responsible for plant elongation, by anti-bioactive GA antibodies. Two different antibodies were produced in Arabidopsis as single-chain variable fragment (scFv) fused to green fluorescent protein (GFP) with four different subcellular localizations: endoplasmic reticulum (ER), cytosol, apoplastic space or the outer surface of the plasma membrane. When targeting scFv-GFP to ER, plants showed the highest accumulation of scFv-GFP, with binding activity, strong GFP fluorescence in ER-derived compartments and mild but clear GA-deficient phenotypes, including a smaller leaf size, delayed bolting, shorter inflorescence length and decreased germination. Plants expressing scFv-GFP in ER responded to exogenous GA₄ and contained 15–40 times greater endogenous GA₄ than wild-type plants. They also showed increased gene expression for GA3ox1 , GA20ox1 and GA20ox2 , but decreased expression for GA2ox1 , which are feedback and feedforward regulated by GA signalling, respectively. These results suggest that the level of free functional GA₄ decreased when trapped in the ER with scFv to the extent that mild GA-deficient phenotypes were created. A dramatic increase in the total sum of GA₄ (free plus scFv-GFP bound) was detected as a result of the up-regulation of GA biosynthesis (feedback regulated), and a decrease in GA₄ catabolism as a result of protection by scFv-GFP binding. This study demonstrates that the use of immunomodulation to inhibit the action of bioactive GAs is an effective method of creating GA-deficient plants.     

The end-of-day far-red irradiation increases gibberellin A1 content in cowpea (Vigna sinensis) epicotyls by reducing its inactivation

It has been shown previously that gibberellins (GAs) mediate the phytochrome (Phy) control of cowpea ( Vigna sinensis L.) epicotyl elongation induced by end-of-day (EOD)-far-red light (FR). In the present work, the EOD-FR effect on GA metabolism and GA levels in cowpea has been investigated. GA₁, GA₈, GA₁₉ and GA₂₀ were identified in epicotyls, and GA₁, GA₁₉, GA₂₀ and GA₂₉-catabolite in leaves of 6-day-old cowpea seedlings. The content of GA₁ in the epicotyl paralleled the decrease of its growth rate, supporting the hypothesis that this is the GA bioactive in controlling cowpea epicotyl elongation. FR enhanced both the amount of [3H]GA₁ in the epicotyl produced from applied [3H]GA₂₀, and that of applied [3H]GA₁ that remained unmetabolized in epicotyl explants, suggesting that Phy may regulate the inactivation of GA₁. In agreement with this effect of light on GA₁ metabolism, the contents of GA₁ in the epicotyl remained higher in FR-treated than in R-treated explants. Moreover, in intact seedlings EOD-FR treatment increased both epicotyl elongation and GA₁ content in the responsive epicotyl, whereas it was not altered in the leaves. These results show, for the first time, that photostable Phys modulate the stem elongation in light-grown plants by locally controlling the GA₁ levels through regulation of its inactivation.     

Growth-promoting activity of gibberellins on shoot elongation in Salix pentandra is reduced by 16,17-dihydro derivatisation

The metabolism of GA₁₀ is thought to be under photoperiodic control in the woody plant Salix pentandra . However, in a recent study using 16,17-[³H₂]GA₁₉ as a mimic of Ga₁₀, no effect of photoperiod was found on its metabolism to 16,17-dihydro-GA₂₀ and 16,17-dihydro-GA₁. To investigate if this was due to differential action of exogenous 16,17-dihydro-GAs and GAs, the effects of the 16,17-dihydro-derivatives of the gibberellins GA₁₉, GA₁, and GA₁ as compared with their parent GAs, on shoot elongation in seedlings of S. pentandra were studied. 16,17-Dihydro-GA₁₉, and -GA₂₀ were both almost inactive, while 16,17-dihydro-GA₁ induced some shoot elongation in seedlings treated with ancymidol as well as under short days. GA₁₉, GA₂₀ and GA₁ were all able to counteract the inhibitory effect of ancymidol under continuous light, while inhibition induced by a 12-h photoperiod was antagonised only by GA₂₀ and GA₁. Thus, the growth-stimulating activity of the tested GAs is significantly reduced by 16,17-dihydro derivatisation, but the derivatives do not inhibit stem elongation in S, pentandra , as has been found in monocotyledons.     

Effects of gibberellic acid on patterns of carbohydrate distribution and acid invertase activity in Phaseolus vulgaris

The application of gibberellic acid (GA₃,10 μ M ) as a root drench to 16-day-old plants of Phaseolus vulgaris L. cv. Masterpiece stimulated growth of the stem internodes and reduced root growth. GA₃ treatment did not affect the export of ¹⁴C from a primary leaf to which [¹⁴C]-sucrose was applied, but greatly increased upward translocation to the elongation region of the stem at the expense of transport to the hypocotyl and root system. The observed changes in the patterns of growth and [¹⁴C]-labelled assimilate distribution were correlated with an increase in the specific activity of acid invertase in the elongating stem internodes and a decrease in invertase activity in the hypocotyl and root. Sucrose concentration in the elongating internodes fell substantially after treatment with GA₃ while the concentration of hexose sugars increased. We suggest that by stimulating acid invertase synthesis in the elongating internodes, GA₃ acts to establish a more favourable sucrose gradient between these sinks and source leaves. Under source-limiting conditions this, in turn, will lead to a reduced rate of assimilate translocation to competing sinks in the root system.     

The plant-growth-promoting rhizobacteria Bacillus pumilus and Bacillus licheniformis produce high amounts of physiologically active gibberellins

The plant-growth-promoting rhizobacteria (PGPR), Bacillus pumilus and Bacillus licheniformis, isolated from the rhizosphere of alder ( Alnus glutinosa [L.] Gaertn.) have a strong growth-promoting activity. Bioassay data showed that the dwarf phenotype induced in alder seedlings by paclobutrazol (an inhibitor of gibberellin [GA] biosynthesis) was effectively reversed by applications of extracts from media incubated with both bacteria and also by exogenous GA₃. Full-scan gas chromatography-mass spectrometry analyses on extracts of these media showed the presence of GA₁, GA₃, GA₄and GA₂₀, in addition to the isomers 3- epi -GA₁ and iso -GA₃. Isotope dilution analysis indicated that epi -GA₁ was an artefact. Likewise, iso -GA₃ is also probably an artifact spontaneously formed during extraction and/or analysis. In both culture media, GA₁ was present in higher concentrations (130–150 ng ml⁻¹) than GA₃ (50–60 ng ml⁻¹), GA₄ (8–12 ng ml⁻¹) and GA₂₀ (2–3 ng ml⁻¹). The data indicated that culture of both bacteria accumulate bioactive C19-gibberellins in relative high amounts and that these GAs appear to be physiologically active in the host plant. The evidence suggests that the promotion of stem elongation induced by the PGPR could be mediated by bacterial GAs.     

Effects of exogenous gibberellins and paclobutrazol on floral stalk growth of tulip sprouts isolated from cooled and non-cooled tulip bulbs

The involvement of gibberellins (GAs) in the regulation of floral stalk elongation and flower development has been studied in tulip. The biological activity of GA₄ and GA₉, both endogenous in tulip bulb sprouts, and GA₁, was tested in vitro on sprouts of cooled and non-cooled tulip bulbs ( Tulipa gesneriana L. cv. Apeldoorn), in the presence or absence of the GA biosynthesis inhibitor paclobutrazol. At early starting dates of incubation, floral stalks from both cooled and non-cooled bulbs hardly showed any elongation in the absence of exogenous GA. Paclobutrazol had no effect on floral stalk elongation, and the response to GAs of sprouts from cooled bulbs was greater than that of sprouts from non-cooled bulbs. At later starts of incubation, considerable floral stalk elongation occurred without GA application. Paclobutrazol inhibited this floral stalk elongation, and the growth of sprouts from both cooled and non-cooled bulbs was stimulated by GA application. The effect of paclobutrazol was reversed by simultaneous application of GA₄ or GA₉. Application of GA with and without paclobutrazol resulted in the same elongation of the floral stalk, indicating the absence of substantial side effects of the inhibitor. The isolated sprouts did not develop a full-grown flower without the addition of GA. GA₄ was more effective than GA₉ in stimulating this flower development. The results demonstrate that both sprouts from cooled and non-cooled bulbs are responsive to exogenous GAs in vitro, and may be a site of GA biosynthesis.     

Gibberellin-biosynthesis and -sensitivity mediated stimulation of seed germination of Sisymbrium officinale by red light and nitrate

Light stimulated seed germination of Sisymbrium officinale (L.) Scop, (hedge mustard) by means of two different mechanisms. Light effect I was absolutely dependent on the simultaneous presence of nitrate. Without nitrate, red (R) irradiated seeds did not escape from the antagonizing action of far-red (FR) irradiation. The data indicated that nitrate acted as a cofactor at the level of the FR absorbing form of phy-tochrome (P_fr). The combined action of R and nitrate could be replaced by addition of the gibberellins 4 and 7 (GA,₄₊₇). This action could be inhibited by the growth re-tardant tetcyclacis, an inhibitor to GA biosynthesis in cell free systems and intact plants. The action of tetcyclacis was fully neutralized by GA₄₊₇. It is concluded that the combination of R and nitrate stimulated GA biosynthesis. Omission of nitrate from the incubation medium enabled the study of light effects apart from G A biosynthesis. In such conditions R stimulated the sensitivity to GA₄₊₇, (light effect II). The two light effects could also be distinguished by their different reactions to the temperature of a pre-treatment in water and darkness. The sensitivity to R and nitrate was subject to breaking and induction of dormancy. Both processes were stimulated at rising temperatures. Due to a different optimum, breaking of dormancy prevailed at lower temperatures and induction of secondary dormancy at more elevated temperatures. The sensitivity to GA₄₊₇ rose and fell in a comparable way during dark incubation at a broad range of temperatures. The capacity of light to stimulate GA₄₊₇, action did not diminish at higher temperatures, it even tended to rise. The study indicated that seed germination is regulated by an increase in both the levels of GAs and the sensitivity to GAs.     

Identification and quantification of endogenous gibberellins in apical buds and the cambial region of Eucalyptus

Endogenous gibberellins (GAs) were extracted and purified from apical buds of Eucalyptus nitens (Deane and Maid.) Maid. and the cambial region of E. globulus (Labill.). then analysed by capillary gas chromatography-mass spectrometry. GA₁ GA₁₉ GA₂₀ and GA₂₉ were identified by full scan mass spectra. Kovats retention indices and high resolution selected ion monitoring. Using deuterated internal standards. GA₁. GA₁₉. GA₂₀ and putative GA₂₉ and GA₅₃ were quantified in the apical buds, while GA₄. GA₈. GA₉ and GA₄₄ were shown to be either absent or present at very low levels. From the cambial region. GA₁ and GA₂₀ were quantified at levels of 0.30 ng (g fresh weight)-¹ and 8.8 ng (g fresh weight)-¹ respectively. These data suggest that the early 13-hydroxylation pathway is the dominant pathway for GA biosynthesis in Eucalyptus .     

Internode length in Lathyrus odoratus. Effects of mutants l and lb on gibberellin metabolism and levels

After the application of [¹³C³H]-gibberellin A₂₀ to wild-type (tall) sweet peas ( Lathyrus odoratus L.) labelled gibberellin A₁ (GA₁), GA₈, GA₂₉ and 2-epiGA₂₉ were identified as major metabolities by gas chromatography-mass spectrometry after high performance liquid chromatography. By contrast in genetically comparable dwarf ( II ) plants only labelled GA₂₉ and 2-epiGA₂₉ were produced in significant amounts, although evidence was obtained for trace amounts of labelled GA₁ and GA₈. The apical portions of dwarf plants contained 8–10 times less GA₁ than those of tall plants but at least as much GA₂₀ (measured using di-deuterated internal standards). In conjunction with previous data these results strongly indicate that in genotype ll internode length is reduced and leaf growth altered by a reduction in GA1 levels attributable to a partial block in the 3β-hydroxylation of GA₂₀ to GA₁.
In contrast to dwarf plants, semidwarf plants (genotype lblb ) contained more GA₁ in the apical portion than wild-type counterparts. This is consistent with the suggestion that lb alters some aspect of GA sensitivity.     

Isoprenoid biosynthesis in bacteria: Two different pathways?

Abstract The biosynthesis of isopentenylpyrophosphate, a central intermediate of isoprenoid formation, was investigated in six different bacterial organisms. Cell-free extracts of Myxococcus fulvus, Staphylococcus carnosus, Lactobacillus plantarum and Halobacterium cutirubrum converted [¹⁴C]acetyl-CoA or [¹⁴C]hydroxymethylglutaryl-CoA to [¹⁴C]mevalonic acid. Furthermore, [¹⁴C]mevalonic acid, [¹⁴C]mevalonate-5-phosphate and [¹⁴C]mevalonate-5-pyrophosphate were metabolized to [¹⁴C]isopentenylpyrophosphate in bacteria. In contrast, no intermediates of this reaction sequence could be detected using cell-free extracts of Zymomonas mobilis and Escherichia coli . These results indicate that at least two different pathways for the biosynthesis of isopentenylpyrophosphate are present in bacteria.     

Hormone directed sucrose transport during fruit set induced by gibberellins in Pisum sativum

A new system has been developed to study hormone-directed transport in intact plants during parthenocarpic fruit set induced by gibberellins. Gibberellic acid (GA₃) and gibberellin A₁ (GA₁) applied to unpollinated ovaries of pea ( Pisum sativum L. cv. Alaska) promoted sucrose transport from the leaf to the site of hormone application. In vivo experiments showed an early (30 min) accumulation of [¹⁴C]-sucrose in ovaries of pea stimulated by gibberellins. This activation of sucrose transport appears to be mediated by gibberellins (GA₁, GA₃), increasing both loading of phloem with sucrose in the leaf (source) and sucrose unloading in the ovary (sink). The ability of pea tissue segments to take up sucrose in vitro was not affected by the hormonal treatment.     

Effects of the triazole plant growth retardant BAS 111¨W on gibberellin levels in oilseed rape, Brassica napus

Three-week-old shoots of the spring oilseed rape cv. Petranova ( Brassica napus L. ssp. napus ) were found by combined gas chromatography-mass spectrometry to contain GA₁, GA₈, GA₁₅, GA₁₇, GA₁₉, GA₂₀, GA₂₄, GA₂₉, 3-epi-GA₁ and a previously uncharacterised C₁₉ dicarboxylic acid that is probably structurally related to GA₂₄. Shoots of the winter cultivar Belinda, harvested at the early flowering stage, contained the same GAs with the exception of the C₁₉ dicarboxylic acid and, in addition, GA₃₄ and GA₅₁ were identified. All material contained higher levels of GA₂₀ than of GA₁; the ratio of GA₁ to GA₂₀ was highest in shoots containing the largest proportion of young immature tissues. Soil treatment of cv. Petranova seedlings with the growth retardant BAS 111¨W [1-phenoxy-5,5-dimethyl-3-(1,2,4-triazol-1-yl)-hexan-4-ol] caused 80% reduction in height 18 days after treatment and the levels of all GAs were 20% or less that of control plants. Foliar treatment at the same dosage reduced height by 50% and caused an 85% or greater reduction in the concentrations of the GA₁ precursors GA₂₀, GA₁₉ and GA₄₄. However, the levels of GA₁, GA₈ and GA₂₉ were affected to a much smaller extent. Foliar application of BAS 111¨W to cv. Belinda 1 month after sowing resulted in only a 20% height reduction at flowering, but no uniform decrease in the concentrations of endogenous GAs at this stage.