Formation of GA20 glucosyl conjugates in seedlings ofVicia faba

[³H]GA₂₀ (1)¹, fed toVicia faba seedlings, was converted to [³H]GA₂₀ glucosyl ester (5) and [³H]GA₂₀-13-0-glucoside (6). The GA₂₀ glucosyl ester (5) was identified by HPLC-RC and by GC-MS of GA₂₀-Me formed by transesterification of (5). The [³H]GA₂₀-Me was crystallized to constant specific radioactivity with authentic GA₂₀-Me. On HPLC-RC the GA₂₀-13-0-glucoside (6) was shown to have the same retention time as an authentic sample. Subsequent enzymic hydrolysis gave a product with an HPLC retention time identical to that of authentic GA₂₀ (1).     

Phytohormone Profiling During Tuber Development of Chinese Yam by Ultra-high performance Liquid Chromatography–Triple Quadrupole Tandem Mass Spectrometry

Changes in agronomic characters and the profile of various endogenous phytohormones during tuber development were studied in Dioscorea opposite (Chinese yam) cv. Guihuai 16. Tuber development exhibited a sigmoidal growth pattern according to the changes in tuber agronomic characters. The growth cycle of yam tuber could be divided into three stages: initiation stage, enlargement stage, and maturation stage. Moreover, the enlargement stage could be separated into three phases—slow growth phase, rapid growth phase, and late growth phase. Endogenous changes in phytohormones were associated with developmental changes in the tubers. The pulses of bioactive gibberellins (such as GA₃ and GA₄) were measured in tubers. The highest contents of GA₃ and GA₄ were reached 90 days after field planting, corresponding to the beginning of the rapid growth phase of tuber enlargement. Changes in trans-zeatin (tZ), jasmonic acid (JA), indole-3-acetic acid (IAA), and abscisic acid (ABA) levels were also observed, and seemed to be related to tuber enlargement at different phases. Continuous increases in JA and tZ contents accompanied tuber enlargement. Transient pulses of both IAA and ABA contents were also observed at the start of tuber rapid growth. Additionally, a second peak level of IAA was detected at the tuber maturation stage. These results suggest GAs play a key role at the beginning of the tuber rapid growth stage, and there is a close relationship between whole tuber enlargement and the contents of JA and tZ. Moreover, it is suggested that IAA and ABA also may be linked to the beginning of tuber rapid growth, and IAA also seems to be correlated to late tuber maturation.     

Gibberellic Acid Activates Chromatin-bound DNA-dependent RNA Polymerase in Wounded Potato Tuber Tissue

Chromatin-bound DNA-dependent RNA polymerases react upon wounding of white potato tuber tissues with an increase in activity, which is additionally enhanced to 300% in the presence of 0.1 micromolar gibberellic acid (GA₃). 2,4-Dichlorophenoxyacetic acid is only weakly effective and indoleacetic acid not at all. Wounding and treatment with GA₃ affect template availability of chromatin only slightly. The hormone has no effect on chromatin-bound RNA polymerases, if added in vitro.     

Gibberellic acid-induced cell elongation in cotton suspension cultures

Gibberellic acid (GA₃) causes cell elongation in cotton suspension cultures derived from cotton ovule callus tissue of both auxin-dependent and-independent lines. Cell elongation was more pronounced in auxin-dependent cultures. Cells were cultured for a period of 14 days but differences in cell lengths could be detected after 6 days in culture. Cell elongation took place in cultures in which GA₃ was present throughout the culture period or only for the first 3 days. Auxins and cytokinin alone or in the presence of GA₃ did not promote cotton cell elongation above the value for the treatment with GA₃ alone.     

Gibberellin-induced inhibition and promotion of sprouting in aerial tubers of Begonia evansiana Andr. in relation to photoperiodic treatment and tuber stage

Gibberellic-acid (GA₃) treatment, when applied within a period ranging from the start of short-day (SD) treatment until about 10 SD, GA₃ strongly inhibited formation of aerial tubers in response to SD and brought about sprouting of developing aerial tubers. In contrast, when applied after about 10 SD or more, GA₃ hastened the completion of the dormant state in the tubers and prolonged their dormancy. The dormancy-promoting effect of GA₃ on detached tubers increased with their degree of maturation. Application of growth retardants N-dimethylaminosuccinamic acid (B-9), 2-isopropyl-4-dimethylamino-5-methylphenyl-1-piperidine carboxylate methyl chloride (AMO-1618) and 2-chloroethyltrimethylammonium chloride (CCC) to the cuttings delayed the onset of dormancy in the aerial tuber. When the retardants were applied to detached aerial tubers, however, such a delay of dormancy was not observed, and GA₃ application did not inhibit sprouting in aerial tubers detached from CCC-treated cuttings.Abbreviations GA gibberellin - GA₃ gibberellic acid - SD short day(s) - LD long day(s) - SDP short-day plant - LDP long-day plant - CCC 2-chloroethyltrimethylammonium chloride - B-9 N-dimethylaminosuccinamic acid - AMO-1618 2-isopropyl-4-dimethyl-amino-5-methylphenyl-1-piperidine carboxylate methyl chloride     

Regulation of Bud Rest in Tubers of Potato, Solanum tuberosum L: VI. Biochemical Changes Induced in Excised Potato Buds by Gibberellic Acid

The rest period of the potato tuber was studied in relation to certain biochemical changes that are induced by gibberellic acid (GA₃). The concentration of reducing sugars in excised plugs with buds treated with 10⁻⁴m GA₃ decreased in the first 4 hours after treatment and then rapidly increased up to 70 hours. The pattern in control buds was similar, but the changes occurred more slowly. The response to GA₃ is temperature-dependent and is not limited to any particular tissue of the tuber. The concentration of reducing sugars in excised buds increased proportionally to the log of the concentration of GA₃ in a range from 10⁻⁸ to 10⁻⁴m. At 10⁻³m, GA₃ slightly inhibited production of reducing sugars. Malonate inhibits the initial decrease and the subsequent increase in reducing sugars in control buds, but not the increase induced by GA₃.     

Changes in jasmonate and gibberellin levels during development of potato plants (Solanum tuberosum)

Among the multiple environmental signals and hormonal factors regulatingpotato plant morphogenesis and controlling tuber induction, jasmonates (JAs)andgibberellins (GAs) are important components of the signalling pathways in theseprocesses. In the present study, with Solanum tuberosum L.cv. Spunta, we followed the endogenous changes of JAs and GAs during thedevelopmental stages of soil-grown potato plants. Foliage at initial growthshowed the highest jasmonic acid (JA) concentration, while in roots the highestcontent was observed in the stage of tuber set. In stolons at the developmentalstage of tuber set an important increase of JA was found; however, in tubersthere was no change in this compound during tuber set and subsequent growth.Methyl jasmonate (Me-JA) in foliage did not show the same pattern as JA; Me-JAdecreased during the developmental stages in which it was monitored, meanwhileJA increased during those stages. The highest total amount of JAs expressed asJA+Me-JA was found at tuber set. A very important peak ofJA in roots was coincident with that observed in stolons at tuber set. Also, aprogressive increase of this compound in roots was shown during the transitionof stolons to tubers. Of the two GAs monitored, gibberellic acid(GA₃) was the most abundant in all the organs. While GA₁and GA₃ were also found in stolons at the time of tuber set, noothermeasurements of GAs were obtained for stolons at previous stages of plantdevelopment. Our results indicate that high levels of JA and GAs are found indifferent tissues, especially during stolon growth and tuber set.     

Gibberellic-acid-regulated expression of α-amylase and six other genes in wheat aleurone layers

The effect of gibberellic acid (GA₃) on gene expression in wheat aleurone cells has been characterised. In-vitro translation of polyadenylated RNA indicated that α-amylase and other messenger-RNA (mRNA) species increase in relative concentration in GA₃-treated tissue. At least one mRNA species declines in relative level in response to GA₃. There is also a GA₃-dependent, four-fold increase in the level of polyadenylated RNA. This effect is largely the result of increased levels of many mRNA species which are also present in untreated tissue. Seven GA₃-induced polyadenylated RNA species including the Amyl α-amylase gene product have been cloned as complementary DNA in the plasmid pBR322. These cloned DNAs have been used as hybridisation probes to show that the GA₃-induced increase in α-amylase mRNA is more prolonged than the accumulation of the other GA₃-regulated mRNA species. A polyadenylated-RNA sequence showing reduced concentration in GA₃-treated tissue has also been cloned.     

Regulation of potato tuber protein accumulation by gibberellic Acid

Many studies have shown that gibberellic acid (GA₃) inhibits tuberization in potato (Solanum tuberosum L.). In this study, we have utilized the 40 kilodalton glycoprotein, patatin, as a marker for biochemical events associated with the process of tuberization. To determine the effects of exogenous applications of GA₃ on the induction of the accumulation of this major tuber protein, we measured patatin levels in tubers from treated whole plants, petioles from a single-node cutting system with GA₃ applied in a lanolin paste, and stolon tips cultured in vitro on an inductive medium supplemented with GA₃. In all three systems, GA₃ inhibited the accumulation of patatin and the major 15 and 22 kilodalton tuber proteins. This effect appeared to be selective since most of the other proteins were not affected and, in tubers, at least one protein was stimulated by GA₃. These results suggest that GA₃ can reverse biochemical events of tuberization in tubers as well as prevent the accumulation of the major tuber proteins in other inducible tissues.     

Enzymic glucosylation of gibberellins

Starting from the well-known conversion of exogenously applied free gibberellic acid (GA₃) to its 3(O)-glucoside by intact immature fruits of runner beans (Phaseolus coccineus L.), a protein fraction has been prepared from this plant material possessing glucosylating activity towards GAs. This glucosyltransferase is located in the pericarp only and utilizes preferably UDP-glucose as a sugar donor. The product formed enzymically from GA₃ and UDP-glucose could be identified by derivatization and comparison with the authentic compound to be GA₃-3(O)-glucoside. Among 15 native or chemically modified GAs, the enzyme glucosylates only GA₃ and to a lower extent GA₇ and GA₃₀, indicating a high enzyme specificity with regard to the A ring of gibberellins. The physiological significance of the enzymic GA₃-3(O)-glucoside formation inPhaseolus coccineus is not clear, since this glucoside is not known to be endogenous in this plant. The enzyme preparation did not glucosylate substances of phenolic structure, such as hydroquinone, aesculetin, and quercetin. Glucosylation of GA₃ was achieved also by enzyme preparations fromVigna sinensis and from cell suspension cultures ofDigitalis purpurea. A number of other plant materials showed no activity.     

Endogenous gibberellins and kauranoids identified from developing and germinating barley grain

Several gibberellins (GAs) and kauranoids were identified in extracts of barley (Hordeum vulgare) by combined capillary gas chromatography-mass spectrometry (GC-MS). A partially purified acidic ethyl acetate extract from 21-day postanthesis developing barley grain (cv. Proctor) contained GA₁ (trace), GA₄ (trace), GA₈ (trace), GA₁₂, GA₁₇, GA₂₀ (tentative) (trace), GA₂₅, GA₃₄, GA₄₈, 18-hydroxy-GA₄, 12β-hydroxy-GA₉, and 18-hydroxy-GA₃₄ (tentative). A hydrolyzed butanol extract contained GA₁₇, GA₂₀, GA₄₈, and 18-hydroxy-GA₃₄ (tentative). An acidic ethyl acetate extract from 3-day-old germinating barley grain (cv. Maris Otter) contained GA₁, GA₃ (possibly a contaminant), GA₁₇, GA₁₉, GA₂₀, GA₃₄, GA₄₈, and 18-hydroxy-GA₃₄ (tentative). A hydrolyzed butanol extract contained GA₃₄, GA₄₈, and 18-hydroxy-GA₃₄ (tentative). In germinating grain, levels of all GAs were very low. Two hydroxylated kauranoic acids and a number of other kauranoids were also detected in the above extracts. 1β-Hydroxylated GAs previously found in wheat were not found in barley in this study.     

The action of exogenous gibberellic acid on polysome formation and translation of mRNA in germinating castor-bean seeds

The amount of protein synthesis in germinating castor-bean seeds has been estimated by the quantitative and qualitative exmainatin of polysomes from the seeds in the presence and absence of gibberellic acid (GA₃). Careful optimisation of polysome extraction procedures was required to minimise the ribonuclease activity in the extracts. Ribonuclease activity in seed extracts increased fourfold over the first 5 d of germination. Gibberellic acid stimulated polysome formation about twofold during the first 4 d of germination. It also stimulated the amount of mRNA associated with polysomes by about twofold during the first 3 d of germination. Between days 1 and 5 of germination, polysome formation was primarily limited by mRNA availability. During the period 0–24 h, polysome formation was independent of mRNA levles. The increase in enzyme activities stimulated by GA₃ was probably the result of an increase in the amount of cellular mRNA. No evidence was obtained for an action of GA₃ on translation other than on the increased production of RNA. Examination of the recruitment of isocitrate-lyase mRNA into polysomes showed that GA₃ did not specifically stimulate production of this enzyme.     

Genetic manipulation of 6-phosphofructokinase in potato tubers

The aim of this work was to discover whether genetic manipulation of 6-phosphofructokinase [EC 2.7.1.11; PFK(ATP)] influenced the rate of respiration of tuber tissue of Solanum tuberosum L. Transgenic plants were produced that contained the coding sequence of the Escherichia coli pfkA gene linked to a patatin promoter. Expression of this chimaeric gene in tubers resulted in a 14to 21-fold increase in the maximum catalytic activity of PFK(ATP) without affecting the activities of the other glycolytic enzymes. Tubers, and aged disks of tuber tissue, from transformed plants showed no more than a 30% fall in the content of hexose 6-monophosphates; the other intermediates of glycolysis increased threeto eightfold. Fructose-2,6-bisphosphate was barely detectable in aged disks of transformed tubers. The relative rates of ¹⁴CO₂ production from [1-¹⁴C]-and [6-¹⁴C]-glucose supplied to disks of transformed and control tubers were similar. Oxygen uptake and CO₂ production by aged disks of transformed tubers did not differ significantly from those from control tubers. The same was true of CO₂ production, in air, and in nitrogen, for tuber tissue. It is concluded that PFK(ATP) does not dominate the control of respiration in potato tubers.Abbreviations Fru2,6bisP fructose-2,6-bisphosphate - FW freshweight - GUS -glucuronidase - PFK(ATP) 6-phosphofructokinase - PFK(PPi) pyrophosphate: fructose-6-phosphate 1-phosphotransferase     

Effect of 2-chloroethyltrimethyl ammonium chloride on tuberization and endogenous GA3 in roots of potato cuttings

Cuttings of potato shoots treated with the plant growth retardant 2-chloroethyltrimethyl ammonium chloride (CCC) form tubers earlier and have less biologically-active gibberellin (GA)-like substances in the roots than control cuttings. The major GA-like substance in roots of potato cuttings was identified as GA₃ by gas-chromatography-mass spectrometry (GC-MS). The content of GA₃ in roots of control cuttings, estimated by GC-MS-selected ion monitoring (SIM) using [17, 17-²H]GA₃ as a quantitative internal standard, was 38.8 ng per g fresh weight (fw), and in roots of CCC-treated cuttings, in which tuberization was promoted, was 0.6 ng per g fw. Gibberellin A₁, GA₈ and GA₂₀ were also indicated as minor components of roots from both control and CCC-treated cuttings. The comparatively high GA₃ content in roots of control cuttings might be the root factor responsible for delaying tuberization in potato.Abbreviations CCC 2-chloroethyltrimethyl ammonium chloride - dw dry weight - EtOAc ethyl acetate - GA gibberellin - GC-MS-SIM gas chromatography-mass spectrometry-selected ion monitoring - HPLC high performance liquid chromatography - IAA indole-3-acetic acid - KRI Kovats' retention index - MeOH methanol - MeTMSi methyl ester trimethylsilyl ether - NAA naphthalene acetic acid - SD short day(s) - 2,4-D 2,4-dichlorophenoxy acetic acid     

Influence of CuSO4 spectral filters,daminozide, and exogenous gibberellic acid on growth ofDendranthema Xgrandiflorum (Ramat.) Kitamura ‘bright golden anne’

The response of chrysanthemum plants to gibberellic acid (GA₃) and daminozide, grown under 6% CuSO₄ and water (control) spectral filters, was evaluated to determine the involvement of gibberellins in regulation of plant height under CuSO₄ filters. The CuSO₄ filter increased the red (R)/farred (FR), and blue (B)/R ratio (R=600–700 nm; FR =700–800 nm; B=400–500 nm) of transmitted light. PPF under 6% CuSO₄ filter was reduced by about 34% compared to PPF under water filter which averaged about 750 μM·m^?2·s^?1. Control plants were shaded with Saran Wrap to ensure equal PPF as in the CuSO₄ chamber. GA₃ application increased plant height under both the control and CuSO₄ filter, but the height increase under the CuSO₄ filter was about 20% greater than that under the control filter. Daminozide treatment reduced plant height under the control and CuSO₄ filter, but the height reduction in control plants was slightly greater than under the CuSO₄ filter. The height reduction caused by daminozide was prevented by GA₃ application in plants grown under the control or CuSO₄ filter. The results suggest that GA₃ may be partially involved in height reduction under CuSO₄ filters.     

Identification of gibberellins from sugarcane plants

Five GAs, GA₁, GA₃, GA₁₉, GA₂₀, and GA₂₉, were identified in extracts from mature leaf and shoot apical meristem of flowering and non-flowering sugarcane (Saccharum spp. hybrids) by combined GC/MS. The presence of ABA was also confirmed.     

Factors affecting shoot initiation from tuber discs of potato (Solanum tuberosum)

Discs of cortical and perimedullary tissue from tubers of potato (Solanum tuberosum L. cv. Superior) formed adventitious shoots when cultured on a medium containing Murashige and Skoog's salts, myo-inositol, 100 mg/l; folic acid, 0.5 mg/l; D-biotin, 0.05 mg/l; gibberellic acid (GA₃), 0.5 mg/l; thiamine ˙ HCl, 0.1 mg/l; glycine, 2.0 mg/l; pyridoxine ˙ HCl, 0.5 mg/l; nicotinic acid, 0.5 mg/l; sucrose, 25 g/l; casein hydrolysate, 1 g/l; Bacto agar, 9.0 g/l; and a cytokinin [N⁶-benzylaminopurine (BAP), N⁶-γ,γ-dimethylallylaminopurine (2iP), or N⁶-furfurylaminopurine (kinetin)]. Discs of pith tissue either failed to survive or produced callus but did not undergo morphogenesis. Cytokinin was essential for explant survival, while BAP at 3.0 mg/l was most efficacious in promoting shoot initiation. Auxin was not essential for shoot initiation or development; however, a low concentration (0.03 mg/l) of α-naphthaleneacetic acid (NAA) stimulated both explant survival and the number of shoots produced per disc. Indole-3-butyric acid (IBA) and indole-3-acetic acid (IAA) did not stimulate shoot initiation. GA₃ was essential for both shoot initiation and subsequent shoot development. The highest incidence of morphogenesis (over 100 shoots in 12 weeks) occurred from tuber discs cultured at 18°C constant and under a photon flux density of 60 μE m^-2s^-1. No difference in regenerative ability was found in explants taken from source tubers stored for 0 to 20 weeks at 4°C. A histological examination indicated that shoots developed from small clusters of meristematic cells which were initiated from within small protuberances on the surface of the tuber disc 3 weeks after transfer.     

Gibberellin biosynthetic pathway and the physiologically active gibberellin in the shoot ofCucumis sativus L.

[²H₂]Gibberellin A₂₄ (GA₂₄) and [²H₄]-GA₉ were applied to the apices of normal-type cucumber (Cucumis sativus L. cv. Yomaki) seedlings treated with uniconazole, an inhibitor of GA biosynthesis. The metabolites from these feeds were identified by full-scan gas chromatography-mass spectrometry (GC-MS) to confirm the conversions of [²H₂]GA₂₄ to [²H₂]GA₉ and of [²H₄]GA₉ to [²H₄]GA₄. The results show that GA₄ is biosynthesized from GA₂₄ via GA₉. In a cucumber hypocotyl elongation bioassay using cv. Yomaki, prohexadione (DOCHC), an inhibitor of 2-oxoglutaratedependent dioxygenase, inhibited the hypocotyl elongation caused by application of GA₉, while DOCHC enhanced the elongation caused by application of GA₄. These results indicate that GA₄ is a physiologically active GA and that the activity of GA₉ is due to its conversion to GA₄ in cucumber shoots.     

The control of bud dormancy in potato tubers. Measurement of the seasonal pattern of changing concentrations of zeatin-cytokinins

A radioimmunoassay, combined with high-performance liquid chromatography, has been used to analyse the zeatin-type cytokinins of potato (Solanum tuberosum L. cv. Majestic) tubers and tuber buds throughout growth and storage. During tuber growth, zeatin riboside was the predominant cytokinin detected in all tissues. Immediately after harvest, the total cytokinin concentration fell dramatically in the storage tissue, largely as a consequence of the disappearance of zeatin riboside. During storage, levels of cytokinins in the storage tissue remained relatively constant, but increased in the tuber buds. In the buds of tubers stored at 2°C there was a 20-to 50-fold increase in total cytokinin over six weeks, coinciding with the natural break of innate dormancy. At 10°C the rise in the level of bud cytokinins was slower, correlating with the longer duration of innate dormancy. Injecting unlabelled cytokinins into tubers in amounts known to induce sprouting gave rise to increases in cytokinin concentrations in the buds of the same order as the increase associated with the natural break of dormancy. Metabolism of injected cytokinins was greater in non-dormant than in dormant tubers. The roles of cytokinin concentration and the sensitivity of the buds to cytokinin in the control of dormancy are discussed.Abbreviations CK cytokinin - FW fresh weight - HPLC high-performance liquid chromatography - RIA radioimmunoassay - tio⁶ade 6-(4-hydroxy-3-methylbut-trans-2-enylamino)-purine=zeatin - tio⁶adeglc⁹ 6-(4-hydroxy-3-methylbut-trans-2-enylamino)-9--D-glucopyranosyl purine=zeatin-9-glucoside - tio⁶ado 6-(4-hydroxy-3-methylbut-trans-2-enylamino)-9--D-ribofuranosyl purine=zeatin riboside - tio⁶ado-[³H]-diol a radioactive derivative of zeatin riboside, synthesised by periodate-oxidation followed by [³H]NaBH₄-reduction - tio⁶AMP 6-(4-hydroxy-3-methylbut-trans-2-enylamino)-9--D-5-phosphoribofuranosyl purine=zeatin riboside 5-monophosphate - t(ioglc⁴)⁶ade 6-(4-O--D-glucopyranosyl-3-methylbut-trans-2-enylamino)-purine=zeatin-O-glucoside