Promotion of Flowering on Mature Picea sitchensis by Gibberellin and Environmental Treatments. The Influence of Timing and Hormonal Concentration

Male and female flowering were significantly promoted by gibberellin A_4/7+ A₃ mixtures applied beneath bark flaps on branches of 11-year-grafted mature scions of Picea sitchensis (Bong.) Carr. growing in the open in Scotland; 4 mg/ml gave more flowering than higher or lower concentrations of gibberellins and application in June was more effective than in July. Gibberellins also increased male flowering on mature scions when applied directly to the buds in a polythene house. A gibberellin A_4/7+ A₃ mixture produced greater numbers of strobili when applied to 2-year-grafted plants in May and June than when applied in July and August. Flowering of 5-year-grafted plants increased with increasing concentrations of a gibberellin A_4/7+ A₉ mixture up to 19.7 mg/ml above which flowering decreased. Modifying the environment by keeping in a polythene house enhanced the combined male and female flowering of 5-year-grafted mature scions above the amount on control plants outside, the effect being more marked in June and July than in August and September. Larger numbers of gibberellin applications or longer total lengths of time in the polythene house gave significantly greater amounts of combined male and female strobili.     

Synergism between Gibberellin and Auxin in the Growth of Intact Tomato

Gibberellin A_4&7 was more effective than gibberellic acid in increasing shoot elongation when applied to the apex of intact Lycopersicum esculentum seedlings of Tiny Tim, a dwarf cultivar, and Winsall, a tall cultivar. After 14 days, gibberellic acid and gibberellin A_4&7 stimulated growth of the dwarf more than the tall tomato. In tall tomato the application of indole-3-acetic acid alone (6.1 μg/plant) showed an inhibitory growth effect, but when applied with 17.5 μg per plant of gibberellic acid, it had a synergistic effect at 7 days but not at 14 days. When the auxin concentration was reduced to 0.61 μg per plant a synergistic effect was observed on tall plants at 7 and 14 days between indole-3-acetic acid and gibberellic acid. Application of gibberellin A_4&7 with auxin did not give a synergistic response in tall or dwarf tomato.     

Comparative effects of gibberellins and an N-substituted phthalimide on seed germination and extension growth of celery (Apium graveolens L.)

The N-substituted phthalimide AC 94377 (1-(3-chlorophthalimido)-cyclohexanecarboxamide) was equally effective as a mixture of the gibberellins A₄ and A₇ (GA_4/7) in breaking dormancy and stimulating germination of celery seeds when either was used in combination with ethephon or daminozide as a seed soak. Whereas seedlings emerging from GA_4/7-treated seeds became etiolated in comparison with those from untreated seeds, those from AC 94377-treated seeds showed normal development. Preharvest sprays of gibberellic acid (GA₃) increased the height of mature plants in comparison with untreated controls by about 16 per cent whereas AC 94377 was ineffective. The yield from GA₃-treated plots was about 10 per cent greater than that from AC 94377-treated plots.     

The effect of seed drying and gibberellin treatment on the germination performance of developing carrot seed

In a succession of seed harvests of carrot, the highest percentage and most rapid germination was obtained from seed harvested 51 days after anthesis (DAA) when dried by conditioning at 25°C and 60% RH for one week and from seeeds harvested 65 or 79 DAA with or without conditioning treatment. Seed from these harvests had reached maximum weight when dried, had embryos of maximum length and were considered mature. The germinaton of seed from these treatments was unaffected by a mixture of the gibberellins A₄ and A₇ (GA_4/7) applied in the incubation medium. Seed harvested 37 DAA also gave maximum percentage germination when it was both conditioned and incubated in GA_4/7 solution. Seed harvested earlier than this germinated poorly. Germination times of both mature and immature seed were reduced after storage for 18 months but there was no response to GA_4/7.Abbreviations ABA abscisic acid - DAA days after anthesis - GA gibberellin     

Dormancy-release of celery seed by a growth retardant,N-dimethylaminosuccinamic acid (alar)

Summary Alar (N-dimethylaminosuccinamic acid) was more effective than the cytokinin benzyladenine (BA) in breaking dormancy of light-sensitive celery seed treated with a mixture of gibberellins A₄ and A₇ (GA_4/7).     

Allosteric modulators of human A2B adenosine receptor

Background

Among adenosine receptors (ARs) the A_2B subtype exhibits low affinity for the endogenous agonist compared with the A₁, A_2A, and A₃ subtypes and is therefore activated when concentrations of adenosine increase to a large extent following tissue damages (e.g. ischemia, inflammation). For this reason, A_2B AR represents an important pharmacological target.

Methods

We evaluated seven 1-benzyl-3-ketoindole derivatives (7–9) for their ability to act as positive or negative allosteric modulators of human A_2B AR through binding and functional assays using CHO cells expressing human A₁, A_2A, A_2B, and A₃ ARs.

Results

The investigated compounds behaved as specific positive or negative allosteric modulators of human A_2B AR depending on small differences in their structures. The positive allosteric modulators 7a,b and 8a increased agonist efficacy without any effect on agonist potency. The negative allosteric modulators 8b,c and 9a,b reduced agonist potency and efficacy.

Conclusions

A number of 1-benzyl-3-ketoindole derivatives were pharmacologically characterized as selective positive (7a,b) or negative (8c, 9a,b) allosteric modulators of human A_2B AR.

General significance

The 1-benzyl-3-ketoindole derivatives 7–9 acting as positive or negative allosteric modulators of human A_2B AR represent new pharmacological tools useful for the development of therapeutic agents to treat pathological conditions related to an altered functionality of A_2B AR.     

Promotion of Flowering in the Pinaceae by Gibberellins

Flowering was significantly promoted in 4-year-old grafts of mature coastal Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco) clones by exogenous gibberellins (GAs) A₄ and A₇ (as a mixture) applied alone and in combination with A₅ and A₉. Biweekly applications of 400 μg GA_4/7 per branch between late March and late June gave a 5-fold increase in ovulate and 3-fold increase in staminate strobilus production over untreated controls. ⁶N-benzyladenine and 2,3,5-triiodobenzoic acid applied in combination with GAs had no consistent effect on strobilus production. Non-destructive branch girdling, ineffective by itself as a cultural treatment, enhanced the GA benefit to flowering. Exogenous application of GA_4/7 is effective and appears to be a practical method for promotion of early and enhanced flowering in grafted Douglas-fir seed orchards.     

Dormancy break of celery (Apium graveolens L.) seeds by plant derived smoke extract

Seed dormancy of a highly-dormant cultivar of celery (Apium graveolens L.) was broken by combinations of plant-derived smoke extract or N⁶-benzyladenine (BA) and gibberellins A_4/7 (GA_4/7) in the dark at temperatures between 18 and 26°C. A less dormant cultivar which responded to GA_4/7 alone showed no additional response to smoke extract or BA. Neither smoke extract nor BA affected either cultivar in the dark in the absence of GA_4/7. The partial dormancy-breaking effect of short exposures to red-light was also enhanced by smoke extracts in this highly-dormant cultivar. The results suggest that smoke extracts act in a similar way to cytokinins, by enhancing gibberellin activity in the celery seed system.Abbreviations BA N⁶-benzyladenine - GA_4/7 A₄ and A₇ gibberellin mixture     

Thermodormancy in Celery Seeds and its Removal by Cytokinins and Gibberellins

Imbibition of celery (Apium graveolens L.) seeds at 32°C for up to 96 h lowered the upper temperature limit for germination. If this high temperature treatment was given in the light, these seeds germinated slightly earlier than those treated in the dark although the final percentage germination was similar for both treatments. The inhibitory effect of the high temperature treatment was completely removed by allowing the seeds to imbibe in a mixture of the gibberellins A₄ and A₇ (GA_4/7) and partially removed by the cytokinin N⁶-benzylaminopurine (BA). GA_4/7 was less effective when added before rather than after the high temperature treatment, whereas the opposite was true of BA. At constant temperatures more GA_4/7 was required to promote germination as the temperature was raised but addition of BA reduced the concentration of GA_4/7 required. A model is proposed for the control of celery seed germination by light and temperature through the action of endogenous cytokinins and gibberellins.     

Interactions of Abscisic Acid, Cytokinin and Gibberellin in the Control of Betacyanin Synthesis in Seedlings of Amaranthus caudatus

In de-rooted seedlings of Amaranthus caudatus L., betacyanin synthesis induced by white light or cytokinin was inhibited by abscisic acid (ABA) or a mixture of gibberellins A₄ and A₇ (GA_4/7). The GA_4/7 and ABA effects were additive. Thus ABA inhibited the cytokinin action but had no effect on the gibberellin response.     

4-Substituted-7-N-alkyl-N-acetyl 2-aminobenzothiazole amides: Drug-like and non-xanthine based A2B adenosine receptor antagonists

7-N-Acetamide-4-methoxy-2-aminobenzothiazole 4-fluorobenzamide (compound 1) was chosen as a drug-like and non-xanthine based starting point for the discovery of A_2B receptor antagonists because of its slight selectivity against A₁ and A_2A receptors and modest A_2B potency. SAR exploration of compound 1 described herein included modifications to the 7-N-acetamide group, substitution of the 4-methoxy group by halogens as well as replacement of the p-flouro-benzamide side chain. This work culminated in the identification of compound 37 with excellent A_2B potency, modest selectivity versus A_2A and A₁ receptors, and good rodent PK properties.     

3-hydroxylation of gibberelin A,12-aldehyde in Gibberella fujikuroi strain rec-193A

When grown on PDL medium for 11 days the strain REC-193A of Gibberella fujikuroi produces the usual range of gibberellins and ent-kaurenoid metabolites. After 3–5 days under the same conditions of culture, this slow growing strain produces virtually none of these metabolites. These short term cultures were found to convert gibberellin A₁₂-aldehyde into gibberellins A₁₂ (8.3 % A₁₄ (45%), A₄ (ca. 17%) and A₇ (ca. 6%). Under identical conditions of culture gibberellin A₁₂ was largely unmetabolised. These results show that 3-hydroxylation is the first step in the conversion of gibberellin A₁₂-aldehyde into gibberellins A₁₄, A₄ and A₇.     

MD-2-mediated Ionic Interactions between Lipid A and TLR4 Are Essential for Receptor Activation

Lipopolysaccharide (LPS) activates innate immune responses through TLR4·MD-2. LPS binds to the MD-2 hydrophobic pocket and bridges the dimerization of two TLR4·MD-2 complexes to activate intracellular signaling. However, exactly how lipid A, the endotoxic moiety of LPS, activates myeloid lineage cells remains unknown. Lipid IV_A, a tetra-acylated lipid A precursor, has been used widely as a model for lipid A activation. For unknown reasons, lipid IV_A activates proinflammatory responses in rodent cells but inhibits the activity of LPS in human cells. Using stable TLR4-expressing cell lines and purified monomeric MD-2, as well as MD-2-deficient bone marrow-derived macrophages, we found that both mouse TLR4 and mouse MD-2 are required for lipid IV_A activation. Computational studies suggested that unique ionic interactions exist between lipid IV_A and TLR4 at the dimerization interface in the mouse complex only. The negatively charged 4′-phosphate on lipid IV_A interacts with two positively charged residues on the opposing mouse, but not human, TLR4 (Lys³⁶⁷ and Arg⁴³⁴) at the dimerization interface. When replaced with their negatively charged human counterparts Glu³⁶⁹ and Gln⁴³⁶, mouse TLR4 was no longer responsive to lipid IV_A. In contrast, human TLR4 gained lipid IV_A responsiveness when ionic interactions were enabled by charge reversal at the dimerization interface, defining the basis of lipid IV_A species specificity. Thus, using lipid IV_A as a selective lipid A agonist, we successfully decoupled and coupled two sequential events required for intracellular signaling: receptor engagement and dimerization, underscoring the functional role of ionic interactions in receptor activation.     

Structural modification of gibberellins by in-vitro hydroxylating systems

Summary The behaviour of gibberellins A₄, A₅ and A₇ (GA₄, GA₅ and GA₇) has been studied in various in vitro hydroxylating systems. Incubation of GA₄ and GA₇ in peroxidase/dihydroxyfumarate or tyrosinase/ascorbate formulations resulted in the production of additional biologically-active zones on thin-layer chromatograms of the reaction mixtures. The product from GA₇/peroxidase incubations was conclusively identified as GA₇ norketone by gas/liquid chromatography-mass spectrometry and nuclear magnetic resonance spectrometry. A similar reaction is inferred for GA₄ in the same system and for both GA₄ and GA₇ with tyrosinase/ascorbate. By analogy, the product derived from GA₅ in the non-enzymatic ascorbate/ferrous iron system may be GA₅ norketone. The reaction kinetics and the biological activity of the products are discussed.     

Evidence for the accumulation of a germination inhibitor during progressive thermoinhibition of seeds of celery (Apium graveolens L.)

The germination of seeds of celery (Apium graveolens L.) becomes progressively thermoinhibited on incubation in the dark at high temperatures, the inhibitory temperature being dependent on the cultivar used. In two high-dormancy cultivars of celery, the production of germination inhibitors in seeds incubated in the dark at 26°C gradually increased over a 7-day period. Inhibitor production was measured by incubating seeds of the low-dormancy cultivar Florida 683 in homogenates of the thermoinhibited seeds of the high-dormancy cultivars and recording germination either in the light or with the gibberellins A₄ and A₇ (GA_4/7) in the dark. Most Florida 683 seeds which failed to germinate in the homogenates after 15 days were induced to germinate by addition of N⁶-benzyladenine (BA). The presence of BA in addition to GA_4/7 throughout incubation in the dark completely overcame the inhibitory effects of homogenates. This indicates that thermoinhibition of celery seeds is associated with the accumulation of a germination inhibitor which interacts with cytokinins. This does not appear to be abscisic acid (ABA) since ABA levels in thermoinhibited seeds were lower than in untreated seeds and did not increase with duration of high temperature treatment.Abbreviations ABA Abscisic acid - BA N⁶-benzyladenine - GA_4/7 a mixture of the gibberellins A₄ and A₇ - HTP high-temperature pretreatment     

QUANTITATIVE AND QUALITATIVE DIFFERENCES IN PLANT RESPONSE TO THE GIBBERELLINS

Wittwer , S. H., and M. J. Bukovac . (Michigan State U., E. Lansing.) Quantitative and qualitative differences in plant response to the gibberellins. Amer. Jour. Bot. 49(5): 524–529. Illus. 1962.—The comparative biological activities of gibberellins A₁ through A₉ were evaluated, over a wide concentration range and in several test systems. All gibberellins were effective in promoting stem elongation of dwarf peas (Pisum sativum), and, with the exception of A₈, epicotyl growth in Phaseolus vulgaris. Elongation of Cucumis sativus seedlings was strikingly greater with A₄, A₇, and A₉ than with the other gibberellins. With mutant dwarfs of Zea mays, A₅ and A₉ were the most active gibberellins for d₃ and d₅, and relatively ineffective compared to A₃ on d₁. Gibberellins A₂, A₇, and A₈ were less effective than A₃ on all dwarfs. Qualitative and quantitative differences among the gibberellins were noted on seedstalk elongation and flowering of Lactuca sativa, with A₃ the most active followed by A₁, A₇, A₄, and A₉. No flowering or seedstalk elongation occurred with A₂, A₆ or A₈. Parthenocarpic fruit growth in Lycopersicon esculentum was a function of dosage with all gibberellins. At the lowest levels, A₅ and A₇ were the most active, while at the highest levels all gibberellins with the exception of A₈ were equally effective. The results suggest a high degree of species and response specificity among the known fungal and higher plant gibberellins, and demonstrate the importance of utilizing a wide spectrum of plant responses and dosage levels in the biological assay of plant extracts for native gibberellins.     

Photosynthetic performance at low temperature of Bouteloua gracilis Lag., a high-altitude C4 grass from the Rocky Mountains, USA

The mechanisms controlling the photosynthetic performance of C₄ plants at low temperature were investigated using ecotypes of Bouteloua gracilis Lag. from high (3000 m) and low (1500 m) elevation sites in the Rocky Mountains of Colorado. Plants were grown in controlled‐environment cabinets at a photon flux density of 700 μ mol m^?2 s^?1 and day/night temperatures of 26/16 °C or 14/7 °C. The thermal response of the net CO₂ assimilation rate (A) was evaluated using leaf gas‐exchange analysis and activity assays of ribulose‐1,5‐bisphosphate carboxylase/oxygenase (Rubisco), phosphoenolpyruvate carboxylase (PEPCase) and pyruvate,orthophosphate dikinase (PPDK). In both ecotypes, a reduction in measurement temperature caused the CO₂‐saturated rate of photosynthesis to decline to a greater degree than the initial slope of A versus the intercellular CO₂ response, thereby reducing the photosynthetic CO₂ saturation point. As a consequence, A in normal air was CO₂‐saturated at sub‐optimal temperatures. Ecotypic variation was low when grown at 26/16 °C, with the major difference between the ecotypes being that the low‐elevation plants had higher A; however, the ecotypes responded differently when grown at cool temperature. At temperatures below the thermal optimum, A in high‐elevation plants grown at 14/7 °C was enhanced relative to plants grown at 26/16 °C, while A in low‐elevation plants grown at 14/7 °C was reduced compared to 26/16 °C‐grown plants. Photoinhibition at low growth temperature was minor in both ecotypes as indicated by small reductions in dark‐adapted F_v/F_m. In both ecotypes, the activity of Rubisco was equivalent to A below 17 °C but well in excess of A above 25 °C. Activities of PEPCase and PPDK responded to temperature in a similar proportion relative to Rubisco, and showed no evidence for dissociation that would cause them to become principal limitations at low temperature. Because of the similar temperature response of Rubisco and A, we propose that Rubisco is a major limitation on C₄ photosynthesis in B. gracilis below 17 °C. Based on these results and for theoretical reasons associated with how C₄ plants use Rubisco, we further suggest that Rubisco capacity may be a widespread limitation upon C₄ photosynthesis at low temperature.     

Gene expression of WNTs, β-catenin and E-cadherin during the periimplantation period of pregnancy in pigs--involvement of steroid hormones

WNTs (wingless-type MMTV integration site family, member) are morphogenes considered as important factors taking part in uterus developmental processes and implantation. β-catenin is a downstream effector of WNTs action within the cell as well as, through E-cadherin, affecting epithelial organization and function. This study was conducted to investigate WNT4, WNT5A, WNT7A, β-catenin (CTNNB1) and E-cadherin (CDH1) gene expression and protein localization in the endometrium during the periimplantation period. Furthermore, the effect of 17β-estradiol (E₂) and progesterone (P₄) on WNTs, CTNNB1 and CDH1 gene expression in the porcine endometrium in vitro was examined. WNT4 protein was localized in the luminal and glandular epithelium as well as in the basal lamina of the uterine mucosa. WNT5A protein was detected only in the luminal epithelium. WNT7A, β-catenin and E-cadherin protein were identified both in the luminal and glandular epithelial cells, however, WNT7A protein immunoreactivity varied during respective days of estrous cycle and/or pregnancy. Despite unchanged expression of WNT4 mRNA in the endometrium of cyclic and early pregnant pigs, the negative influence of E₂ on WNT4 gene during in vitro experiment was observed. WNT4 and CDH1 gene expression was negatively correlated with blood plasma E₂ and P₄ level in uterine luminal flushings (ULFs) on Day 12 of pregnancy. Expression of WNT5A gene was up-regulated in the endometrium on Day 9 of pregnancy when compared to the respective day of the estrous cycle. A significant decrease of WNT7A gene expression and increase of CDH1 mRNA amount was detected on Day 12 of pregnancy. Overall, the results show the spatial localization of WNT4, WNT5A, WNT7A, β-catenin and E-cadherin proteins in porcine endometrium during periimplantation period of pregnancy and indicate significant changes of WNT5A, WNT7A and CDH1 gene expression before implantation in the pig.     

Gibberellins in mature apple seeds — contaminants?

Evidence is presented that gibberellins A₄ and A₇, previously identified (Sinska, I., Lewak, St., Gaskin, P., MacMillan, J., Planta 114, 359–364, 1973) in extracts of mature apple (Pyrus malus L.) seeds, were present primarily as contaminants. The facts supporting this conclusion are: a) the ratio of GA₄ to GA₇ was similar to that of a standard mixture; b) the concentrations were extremely high; and c) the ratio of GA₄ to GA₇ did not change during stratification, as had been reported when extracts were bioassayed (Sinska, I., and Lewak, St., Physiol. Vég. 8, 661–667, 1970).Michigan Agricultural Experiment Station Journal Article No. 9036     

Responses of florence fennel (Foeniculum vulgare azoricum) seeds to light,temperature and gibberellin A4/7

The percentage and the rate of germination of seeds of three varieties of Florence fennel was higher in the dark than in the light, the high temperature cut-off points being between 27.2 and 29.4°C. The optimum temperature for germination was between 20 and 25°C. Seeds of all three varieties responded to incubation in solutions containing gibberellin A_4/7 mixture (GA_4/7; Regulex), giving higher germination in the light at temperatures from 20 to 30°C. Seeds steeped for 4 h at 25°C or for 24 h at 5°C in GA_4/7 solutions gave a higher percentage and increased rate of emergence as compared with untreated dry seeds, when sown in compost at 25°C; steeping in water alone was also beneficial. In general, drying the treated seeds before sowing reduced the rate but sometimes increased the percentage of germination as compared with seeds sown when still moist. Seeds harvested from secondary umbels of var. Zefa fino germinated better both in the light and dark than those taken from primary umbels.Abbreviations GA_4/7 a commercial mixture of the gibberellins A₄ and A₇ (trade name Regulex)