Identification of gibberellins in leaf exudates of strawberry (Fragaria ×ananassaDuch.)

Leaf exudates from the short-day (SD) strawberry (Fragaria × ananassa Duch.) cv. Elsanta were analysedfor gibberellin (GA) content using combined gaschromatography – mass spectrometry (GC-MS). Comparison of full-scan mass spectra and Kovatsretention indices (KRIs) with those of standardsrevealed the presence of GA₁, GA₃, 3-epiGA₁ and GA₃-isolactone.     

The role of endogenous gibberellins in the formation of α-amylase by aleurone layers of germinating barley caryopses

Using sensitive and selective immunological assays we have shown that in germinating caryopses of Hordeum vulgare L. cv. Himalaya, the level of gibberellin A₄ (GA₄) rises approximately 18-to 20-fold shortly (2–4 h) before -amylase activity increases. Gibberellin A₄ is the predominant immunoreactive gibberelin during these developmental stages and reaches a peak amount of approximately 9 pmol per caryopsis about 48 h after imbibition. Isolated aleurone layers produce GA₄ in the presence of an exogenous gibberellin, such as GA₁, which is not a biosynthetic precursor for GA₄. Experiments with inhibitors of gibberellin biosynthesis indicate that gibberellin synthesis is required in this tissue for the induction of -amylase. The inductive effect of exogenously applied GA₁ is indirect and appears to be mediated by GA₄. Embryos form predominantly GA₁; however, very little of this material is released by isolated embryos into the incubation medium. The results presented make it unlikely that the role of the embryo in the process of -amylase induction in aleurone layers is to provide gibberellins or gibberellin precursors.Abbreviations ABA abscisic acid - GA gibberellin - GA₃ gibberellic acid - RIA radioimmunoassay - TLC thin-layer chromatography     

A highly-sensitive rice seedling bioassay for the detection of femtomole quantities of 3β-hydroxylated gibberellins

A very sensitive and specific bioassay using prohexadione calcium [BX-112, which blocks 2- and 3-hydroxylation of gibberellins (GAs)] with uniconazole (which blocks oxidation of ent-kaurene, ent-kaurenol and ent-kaurenal) in a microdrop assay was developed for several rice (Oryza sativa L.) varieties, including cv. Waito-C, which is already specific to 3-hydroxylated GAs. The sensitivity and specificity of cvs. Waito-C, Tan-ginbozu and Koshihikari to 3-hydroxylated GAs was greatly enhanced by treatment of the seeds with a combination of 40 mM prohexadione calcium and 80 M uniconazole. The minimum detectable doses of 3-hydroxylated GAs (GA₁, GA₃, GA₄ and GA₇) in the three cultivars treated with both chemicals were 1 to 10 fmol (i.e. ca. 350 fg to 3.5 pg) per plant. This is equal to 30-fold more sensitive than Waito-C treated with uniconazole alone, and 30 to 1000-fold more sensitive than Waito-C with no growth retardant soak. Minimum detectable doses of 3-nonhydroxylated GAs (GA₉, GA₁₉ GA₂₀) and GAs with very low biological activity (GA₈ and GA₁₇) were equal to or more than 1000 fmol per plant. This is about equal to the activity in Waito-C treated with uniconazole alone. Application of this assay to an extract from Raphanus sativus was compared with the data by gas chromatography/mass spectrometry (GC/MS), confirming the conclusions reached using authentic test GAs, namely that use of uniconazole plus BX-112 appreciably enhanced the detection sensitivity to fractions shown by GC/MS to contain GA₁ and GA₄, both 3-hydroxylated GAs.Abbreviations GA gibberellin - BX-112 prohexadione calcium     

Identification of gibberellins in leaf tissues of day-neutral strawberry (Fragaria × ananassa Duch.) cultivars

The endogenous gibberellins (GAs) in leaf tissues of two day-neutral cultivars (Rapella and Selva) of strawberry (Fragaria × ananassa Duch.) were analysed using combined gas chromatography -- mass spectrometry (GC-MS). Seven of the later members of the 13-hydroxylation GA biosynthetic pathway were identified, by comparison of Kovats retention indices and mass spectral data obtained for methyl ester trimethylsilyl ether derivatives, either with data obtained from authentic compounds or literature values. GA₁, GA₃, GA₈, GA₁₇, GA₁₉, GA₂₀ and GA₂₉ were detected in extracts of both cultivars.     

The relative significance for stem elongation and flowering in Lolium temulentum of 3β-hydroxylation of gibberellins

In previous experiments with many gibberellins (GAs) and GA derivatives applied to Lolium temulentum L., quite different structural requirements were evident for stem elongation on the one hand and for the promotion of flowering on the other. Whereas hydroxylation at carbons 12, 13 and 15 enhanced flowering relative to stem growth, the reverse was the case at carbon 3 (L.T. Evans et al. 1990, Planta 182, 97–106). The significance of hydroxylation at carbon 3 is examined in this paper. The application of inhibitors of 3β-hydroxylation, including C/D-ring-rearranged GAs, reduced stem growth but, in the case of the two acylcyclohexanediones, increased the flowering response when applied on the inductive long day. Later applications of the acylcyclohexanediones, made after floral initiation had occurred, were inhibitory to flowering, suggesting that subsequent inflorescence development requires 3β-hydroxylated GAs. Applications of the 3α-hydroxy epimers of GA₁, GA₃ and GA₄ gave slightly less promotion of flowering in comparison with the 3β-hydroxy GAs, but far less promotion of stem elongation, except in the case of 3-epi-GA₄, which was comparable to GA₄. The 3α-hydroxy epimer of 2,2-dimethyl GA₄ gave less promotion of flowering than its 3β-hydroxy epimer but almost no promotion of stem elongation. The 3α-hydroxy epimers of GA₃ and 2,2-dimethyl GA₄ did not act as competitive inhibitors of the stem elongation elicited by GA₃ and 2,2-dimethyl GA₄, respectively. These results extend the differences in GA structure which favour flowering as opposed to stem elongation, and indicate that 3-hydroxylation and its epimeric configuration are of much greater importance to stem elongation than to flower initiation in Lolium.     

Identification of gibberellins in leaf tissues of strawberry (Fragaria × ananassa Duch.) grown under different photoperiods

In studies of the effect of long or short-day photoperiod treatments on the qualitative gibberellin (GA) content of mature leaves of a facultative short-day (SD) strawberry cultivar (Fragaria × ananassa Duch. cv. Elsanta), GA₁, GA₈, GA₁₇, GA₁₉, GA₂₀, GA₂₉ and GA₄₄ were identified by full-scan gas chromatography - mass spectrometry (GC-MS) in extracts from plants grown under long-day (LD) conditions, and GA₁, GA₅, GA₈, GA₁₉, GA₂₀ and GA₂₉ in similar extracts from plants subjected to eight SD cycles after growth under LD conditions. The early 13-hydroxylation GA biosynthetic pathway thus appeared to predominate, with the apparent absence of GA₅ in LD and of GA₁₇ and GA₄₄ in SD extracts providing evidence of modulation of this pathway by photoperiod. A search, including GC-MS with selected ion monitoring, failed to detect GA₃, or the polyhydroxylated GA₈₅, GA₈₆, GA₈₇ or GA₃₂ for which some extracts were specifically purified.This paper is respectfully dedicated to the memory of Gordon Browning, who died suddenly on the 1st July, 1993. He will be sorely missed, both as a friend and colleague.     

Pre-harvest foliar application of Prohexadione-Ca and gibberellins modify canopy source-sink relations and improve quality and shelf-life of ‘Bing’ sweet cherry

This research evaluated the potential of gibberellins (GA), and Prohexadione-Ca (PCa) to affect sweet cherry (Prunus avium) fruit size and quality. The results demonstrate the ability of ostensibly counter-acting plant growth regulators to significantly improve sweet cherry cv ‘Bing’ fruit size, fruit quality and postharvest characteristics compared to the current commercial application of GA₃ alone. In 2008, we found that the combination of GA₃ or GA_4/7 (30 mg l⁻¹) with PCa (150 mg l⁻¹) applied to entire 3-year-old limbs 30 days after anthesis increased fruit size and improved fruit quality in ‘Bing’. In 2009, we investigated the effect of application timing in larger-scale field trials, comparing treatments made at 30 or 37 days after anthesis, on fruit quality, storability and sensory attributes after storage. Treatment with PCa + GA₃ or PCa + GA_4/7 delayed fruit maturity by about 7 days compared to the untreated control. Both the first and second applications of PCa + GA_4/7 resulted in 35–40% fruit being ≥10 g, compared with only 20% in the control. PCa + GA₃ treatment also showed greater potential for improving fruit storability by maintaining fruit firmness, sweetness, and consumer appeal than PCa + GA_4/7. PCa alone or in combination with GAs inhibited current shoot growth and delayed fruit coloring development. After 30 days of 4°C storage, fewer than 5% fruit from untreated trees were rated as healthy and marketable, compared to 50 and 30% fruit from PCa + GA₃ treatment applied at 30 or 37 days after anthesis, respectively. In conclusion, preharvest foliar application of PCa + GA₃ at the onset of Stage II of fruit development shows potential to affect canopy source-sink relations and improved quality and shelf life of ‘Bing’ sweet cherries.     

Effect of gibberellins A3, A4+7 and A13 and of (−)-kaurene on flowering and extension growth of Impatiens balsamina under different photoperiods

Summary Gibberellins A₃, A₄₊₇ and A₁₃ and (–)-kaurene delay floral-bud initiation and flowering and decrease the number of floral-buds and flowers in Impatiens balsamina under 4-hr photoperiods. They do not have any marked effect under 8-hr photoperiods. Under 16- and 24-hr photoperiods they hasten floral-bud initiation and flowering and increase the number of flowers, the effect being greater under 16- than under 24-hr days and the order of effectiveness being GA₄₊₇>GA₃>GA₁₃>(–)-kaurene.While GA₃ and GA₄₊₇ promote extension growth, the effect being greater with the former, GA₁₃ and (–)-kaurene do not promote it under any photoperiod. The magnitude of stem elongation in different treatments prior to floral-bud initiation increases from 4- to 8-hr photoperiods but decreases under 16- and 24-hr periods, the effect being more under 24-hr although both 16-and 24-hr photoperiods are noninductive for flowering.     

The effect of gibberellins A4+7 on fruit set and seed set in unpollinated and pollinated ‘Sonia’ roses

The effect of GA₄₊₇ (GA) on fruit and seed set of Sonia roses unpollinated and pollinated with Ilona, was studied. GA at 0, 10, 50, 250 or 1250 ppm in water with 25% iso-propyl alcohol was applied 0, 7 or 14 days after emasculation. In general, GA improved fruit and seed set, and induced heavier, earlier ripening fruits. In the unpollinated and pollinated categories fruit weight and the number of achenes per fruit in comparable treatments were about the same. In the unpollinated category all achenes were parthenocarpic. In the pollinated category, 10 and 50 ppm GA after 14 days more than doubled the number of achenes per pollinated flower. Achenes with or without an embryo are a probable condition for fruit set. Effects of GA are discussed.     

Effects of gibberellins, darkness and osmotica on endosperm rupture and class I β-1,3-glucanase induction in tobacco seed germination

The “Havana 425” cultivar of Nicotiana tabacum L. is photodormant. Gibberellins (e.g. 10^?5 M GA₄ or GA₇) can substitute for light in releasing dormancy. Measurements of β-1,3-glucanase activity, mRNA accumulation and the activity of the class I β-1,3-glucanase B promoter indicated that class I β-1,3-glucanases are induced by GA₄ in the dark in association with germination. As in the light, this induction occurred prior to endosperm rupture and was localized exclusively in the micropylar region of the endosperm where the radicle will penetrate. Abscisic acid (ABA, 10^?5 M) did not appreciably affect GA-induced release of photodormancy or seed-coat rupture, but it delayed endosperm rupture and inhibited the rate of class I β-1,3-glucanase accumulation. Seeds imbibed in the light in the presence of osmotica, e.g. 0.04 M polyethylene glycol 6000, showed delayed seed-coat and endosperm rupture, delayed onset of β-1,3-glucanase induction, and decreased rates of β-1,3-glucanase accumulation. These delays were shortened by GA₄ treatment. Our results suggest that GAs and ABA act at two distinct sites during germination and that expansive growth of the embryo acts in two ways by triggering β-1,3-glucanase induction and by providing force for endosperm penetration. This provides further support for our working hypothesis that class I β-1,3-glucanases promote endosperm weakening and facilitate radicle penetration.     

Growth-active gibberellins overcome the very slow shoot growth of Hancornia speciosa, an important fruit tree from the Brazilian “Cerrado”

Shoot elongation of Hancornia speciosa, an endangered tree from the Brazilian savannah “Cerrado”, is very slow, thus limiting nursery production of plants. Gibberellins (GAs) A₁, A₃, and A₅, and two inhibitors of GA biosynthesis, trinexapac-ethyl and ancymidol were applied to shoots of Hancornia seedlings. GA₁ and GA₃ significantly stimulated shoot elongation, while GA₅ had no significant effect. Trinexapac-ethyl and ancymidol, both at 100 μg per seedling, inhibited shoot elongation up to 45 days after treatment, though the effect was statistically significant only for ancymidol. Somewhat surprisingly, exogenous GA₃ more effectively stimulated shoot elongation in SD-grown plants, than in LD-grown plants. The results from exogenous application of GAs and inhibitors of GA biosynthesis imply that Hancornia shoot growth is controlled by GAs, and that level of endogenous growth-active GAs is likely to be the limiting factor for shoot elongation in Hancornia. Application of GAs thus offer a practical method for nursery production of Hancornia seedlings for outplanting into the field.     

Inhibition of growth of microcultured <Emphasis Type="Italic">Hancornia speciosa</Emphasis> shoots by 3β-hydroxylated gibberellins and one of their C-3 deoxy precursors

Gibberellins (GAs) A(1), A(3), A(4) and A(7), all 3beta-hydroxylated, growth-active GAs, significantly inhibited shoot elongation and the formation of nodes in in vitro-grown Hancornia speciosa, as did GA(20), a 3-deoxy precursor of GA(1). Ancymidol, an early-stage inhibitor of GA biosynthesis, significantly retarded shoot elongation without affecting the formation of nodes. Co-application of ancymidol and GA(1 )did not overcome the ancymidol-induced growth retardation. Trinexapac-ethyl, which can inhibit 3beta-hydroxylation (GA activation) and 2beta-hydroxylation (GA inactivation), gave no significant response on either shoot elongation or node formation, while two isomers of 16,17-dihydro GA(5), also inhibitors of GA 3beta-hydroxylation, significantly inhibited both shoot growth and the formation of nodes. These unusual results may indicate a unique metabolism for GAs in microcultured shoots of H. speciosa.     

Influence of photoperiod on seed development in the genetic line of peas G2 and its relation to changes in endogenous gibberellins measured by combined gas chromatography — Mass spectrometry

When apical senescence in the genetic line of peas G2 was prevented by short days fruit development was also found to be retarded. The levels of GA₂₀ and GA₂₉ in cotyledons and pods grown under long or short days were measured by gas chromatography — mass spectrometry multiple ion monitoring using extracts derivatised with deuterated trimethylsilyl groups as internal standards. The levels of GA₂₀ but not GA₂₉, were increased by short days. Conventional gas chromatography — mass spectrometry showed that relative to GA₂₉ the levels of GA₁₉, the other GA identified in G2 cotyledons, were also increased in short days. The levels of GA₂₀ in the pods were highest during the main phase of pod growth early in fruit development.Abbreviations GA_n gibberellin A_n - GC/MS gas chromatography — mass spectrometry - MIM multiple ion monitoring - Me methyl ester - SIM single ion monitoring - TIC total ion current - TMS trimethylsilyl ether - TLC thin layer chromatography - TTLC instant thin layer chromatography     

Abscisic acid,gibberellins and brassinosteroids in Kelpak®, a commercial seaweed extract made from Ecklonia maxima

The seaweed extract Kelpak^® made from the kelp Ecklonia maxima is registered as a biostimulant for use in agriculture. It elicits many beneficial responses including improved root and shoot growth, higher yields and greater resistance to abiotic and biotic stresses. Previously, cytokinins, auxins and polyamines were identified in Kelpak^®. The aim of the present study was to quantify other groups of plant growth regulators (PGRs)—abscisic acid (ABA), gibberellins (GAs) and brassinosteroids—that may be present in E. maxima and Kelpak^®. Kelpak^® samples harvested between 2008 and 2010 and stored for up to 26 months were analysed using ultra performance liquid chromatography tandem mass spectrometry. ABA levels were below the limits of detection in E. maxima but were detected in low concentrations in Kelpak^®, ranging from 0.31 to 20.70 pg mL^?1 Kelpak^®. Eighteen GAs were found in E. maxima and Kelpak^® with concentrations from 187.54 to 565.96 pg mL^?1 Kelpak^®. The biologically active GAs (GA₁, GA₃, GA₄, GA₅, GA₆ and GA₇) comprised less than 3 % in Kelpak^®. Although GA₁₃ (a final product in the metabolic pathway) was present in low concentrations in E. maxima, very high concentrations were present in Kelpak^®. The brassinosteroids brassinolide (BL) and castasterone (CS) were identified in E. maxima and Kelpak^®. Concentrations varied with harvest and storage time, ranging from 384.72 to 793.23 pg BL mL^?1 Kelpak^® and 62.84 to 567.51 pg CS mL^?1 Kelpak^®. It is likely that this cocktail of natural PGRs present in Kelpak^® may act individually or in concert and thus contribute to the numerous favourable physiological responses elicited by Kelpak^® application to plants.     

Distribution of endogenous gibberellins in dwarf and giant off-types banana (Musa AAA,cv.‘Grand nain’) plants from in vitro propagation

GA₃ and GA₂₀ were quantified in leaf extracts from true-to-type and somaclonal variants (dwarf and giant) of Musa AAA cv. Grand nain by GC-MS-SIM after purification on reverse- and normal-phase HPLC and detection by ELISA with GA₃ antibodies and by a dwarf rice bioassay. GA₃ concentration in dwarf plants was 811 ng g^–1 dry weight. For normal and giant plants, the endogeneous GA₃ levels were respectively 3.6 and 4.6 times higher. The GA₂₀ concentration in the giant plant was 68 ng g^–1 of dry weight. This concentration was, respectively, 4.6 and 7.3 times higher than those of normal and dwarf plants. These results suggest that the somaclonal variations affecting banana plant height are associated with modifications in GA metabolism.Abbreviations HPLC High Performance Liquid Chromatography - GC-MS Gas Chromatography-Mass Spectrometry - SIM Selected Ion Monitoring - GA Gibberellin - BSA Bovine Serum Albumin - PB Phosphate Buffer     

Interactions of daminozide, 2, 2′-dipyridyl,and gibberellins A3, A9 and A20, in stem extension inChrysanthemum morifolium Ramat: an indication that daminozide may not inhibit the responses to GA9 or GA20 by blocking gibberellin hydroxylation

Interactions between the growth retardant daminozide (a substituted succinamic acid) and a subsequent application (1 or 10 g) of either gibberellin A₃, A₉ or A₂₀, on stem extension inChrysanthemum morifolium cv. Bright Golden Anne, indicated that pre-treatment of plants with daminozide largely prevented the response to GA₂₀ as well as to GA₉. The daminozide-GA₃ interaction on total stem length was dependent upon the dose of GA₃ such that, by flowering time, 1 g of GA₃ had virtually eliminated the retardant effect, while 10 g of GA₃ increased stem length to a value similar to that achieved by control shoots receiving 10 g of GA₃. In contrast, prior application of 2, 2-dipyridyl (an inhibitor of hydroxylation in some plant and animal systems) had no significant influence on the time courses of response to any of the GAs. In the absence of daminozide (and 2, 2-dipyridyl) all three GAs were very active in promoting internode extension soon after their application. If 2, 2-dipyridyl can block hydroxylation reactions in chrysanthemum tissues, the results do not support the hypothesis that daminozide restricts GA₉- (or GA₂₀-) induced stem elongation by preventing the hydroxylation of GA₉.     

A comparative analysis of karyotypes of three species of Macleaya—producers of a complex of isoquinoline alkaloids

Using a set of methods (C-banding, DAPI-staining, fluorescence hybridization in situ (FISH) with probes of 26S and 5S rDNA, and analysis of meiosis), the first comparative cytogenetic study of three species of Macleaya, producers of complex isoquinoline alkaloids, cordate Macleaya cordata (Willd.) R. Br. (2n = 20), small-fruited Macleaya microcarpa (Maxim.) Fedde (2n = 20) and Macleaya kewensis Turrill (2n = 20), was first carried out. On the basis of morphometric analysis, formulas of karyotypes were made for each species. Species ideograms for M. cordata, M. microcarpa, and M. kewensis were constructed taking into account the polymorphic variants of the C-banding patterns and indicating the location of 26S and 5S rDNA sites. A comparative study revealed that the karyotypes of M. microcarpa and M. kewensis have more in common with each other than with M. cordata. Analysis of meiotic chromosomes suggests of genetic stability of Macleaya genomes. The results of chromosome analysis were used to confirm the close relationship of Macleaya and to clarify their phylogenetic relationships.     

Effect of γ-Irradiation on Development of Lachrymator of Onion

A thermosensitive uracil requiring mutant of Bacillus subtilis Marburg 168 thy trp₂ ts42 was examined as to the colony forming ability at the permissive and nonpermissive temperatures. The viability of the mutant cells decreased rapidly at the restrictive temperature in the modified Woese’s (MW) medium. However, the cells retained viability when sodium succinate or potassium chloride was added to the medium at that temperature although uracil deficiency was unchanged. A little but significant incorporation of adenine-8-¹⁴C into RNA still continued even after the incorporation of N-acetyl-³H-d-glucosamine into acid insoluble fraction of the cells terminated in the MW medium at 48°C. Both incorporations as well as increase of absorbance were slowed down in the presence of sodium succinate at 48°C. This mutant, ts42, was more sensitive to deoxycholate (DOC) than the parent strain. The restoration of colony forming ability after the temperature shift back from 48 to 37°C was suppressed by the addition of DOC to the medium. However, the cell became resistant to DOC when uracil was added to the medium prior to the temperature shift.     

Mechanisms of action of the α-amylase of Micromonospora melanosporea

The -amylase of Micromonospora melanosporea was produced extracellularly during batch fermentation in a 5.0-1 fermentor. The absence of an organic nitrogen source in its growth medium facilitated subsequent purification of the enzyme by ammonium sulphate fractionation and two consecutive Superose-12 gel-filtration steps. The enzyme exhibited maxima for activity at pH 7.0 and 55° C and was 72% stable at pH 6.0–12.0 for 30 min at 40° C. It had a relative molecular mass of 45 000 and an isoelectric point at pH 7.6. The enzyme catalyses the conversion of starch to maltose (53%, w/w) as the predominant final end-product. Initial hydrolysis of this substrate, however, gave rise to the formation of maltooligosaccharides in the range maltotriose to maltohexaose. Maximum yields of these intermediate sugars accumulated to between 31 and 42% (w/w) as the reaction proceeded. The action of the M. melanosporea amylase on high concentrations of saccharides larger than maltotriose resulted in the formation of mainly maltose and maltotriose without concomitant glucose production. A combination of hydrolytic and transfer events is postulated to be responsible for this phenomenon and for the high maltose levels achieved. Correspondence to: C. T. Kelly