Gibberellin metabolism in cell-free extracts from spinach leaves in relation to photoperiod

Cell-free extracts capable of converting [¹⁴C]-labeled gibberellins (GAs) were prepared from spinach (Spinacia oleracea L.) leaves. [¹⁴C]-labeled GAs, prepared enzymically from [¹⁴C]mevalonic acid, were incubated with these extracts, and products were identified by gas chromatography-mass spectrometry. The following pathway was found to operate in extracts from spinach leaves grown under long day (LD) conditions: GA₁₂ → GA₅₃ → GA₄₄ → GA₁₉ → GA₂₀. The pH optima for the enzymic conversions of [¹⁴C]GA₅₃, [¹⁴C]GA₄₄ and [¹⁴C]GA₁₉ were approximately 7.0, 8.0, and 6.5, respectively. These three enzyme activities required Fe²⁺, α-ketoglutarate and O₂ for activity, and ascorbate stimulated the conversion of [¹⁴C]GA₅₃ and [¹⁴C]GA₁₉. Extracts from plants given LD or short days (SD) were examined, and enzymic activities were measured as a function of exposure to LD, as well as to darkness following 8 LD. The results indicate that the activities of the enzymes oxidizing GA₅₃ and GA₁₉ are increased in LD and decreased in SD or darkness, but that the enzyme activity oxidizing GA₄₄ remains high irrespective of light or dark treatment. This photoperiodic control of enzyme activity is not due to the presence of an inhibitor in plants grown in SD. These observations offer an explanation for the higher GA₂₀ content of spinach plants in LD than in SD.     

Identification of Gibberellins in Spinach and Effects of Light and Darkness on their Levels

The endogenous gibberellin (GA) content of spinach (Spinacia oleracea) was reinvestigated by combined gas chromatography-mass spectrometry analysis. The 13-hydroxy GAs: GA₅₃, GA₄₄, GA₁₉, GA₁₇, GA₂₀, GA₅, GA₁, GA₂₉, and GA₈; the non-3, 13-hydroxy GAs: GA₁₂, GA₁₅, GA₉, and GA₅₁; and the 3β-hydroxy GAs: GA₄, GA₇, and GA₃₄, were identified in spinach extracts by comparing full-scan mass spectra and Kovats retention indices with those of reference GAs. In addition, spinach plants contained GA₇-isolactone, 16,17-dihydro-17-hydroxy-GA₅₃, GA₂₉-catabolite, 3-epi-GA₁, and 10 uncharacterized GAs with mass spectra indicative of mono- and dihydroxy-GA₁₂, monohydroxy-GA₂₅, dihydroxy-GA₂₄, and dihydroxy-GA_g. The effect of light-dark conditions on the GA levels of the 13-hydroxylation pathway was studied by using labeled internal standards in selected ion monitoring mode. In short day, the GA levels were higher at the end of the light period than at the end of the dark period. Levels of GAs at the end of each short day were relatively constant. During the first supplementary light period of long day treatment, GA₅₃ and GA₁₉ declined dramatically, GA₄₄ and GA₁ decreased slightly, and GA₂₀ increased. During the subsequent high-intensity light period, the GA₂₀ level decreased and the levels of GA₅₃, GA₄₄, GA₁₉, and GA₁ increased slightly. Within 7 days after the beginning of long day treatment, similar patterns for GA₅₃ and GA₁₉ occurred. Furthermore, when these plants were transferred to darkness, an increase in the levels of GA₅₃ and GA₁₉ was observed. These results are compatible with the idea that in spinach, the flow through the GA biosynthetic pathway is much enhanced during the high-intensity light period, although GA turnover occurs also during the supplementary period of long day, both effects being responsible for the increase of GA₂₀ and GA₁ in long day.     

Partial purification of gibberellin oxidases from spinach leaves

Four enzyme activities catalyzing the following oxidative steps in the gibberellin (GA) biosynthetic pathway have been extracted from spinach (Spinacia oleracea L.) leaves after exposure to 8 long days: GA₁₂ → GA₅₃ → GA₄₄ → GA₁₉ → GA₂₀. Two of these, GA₅₃ oxidase and GA₁₉ oxidase, were separable from the other two, GA₄₄ oxidase and GA₁₂ 13-hydroxylase, by anion exchange high performance liquid chromatography (HPLC). Apparent molecular weights of the four enzymes as determined by gel filtration HPLC are: GA₁₂ 13-hydroxylase, 28,400; GA₅₃ oxidase, 42,500; GA₄₄ oxidase, 38,100; GA₁₉ oxidase, 39,500. GA₄₄ oxidase was purified approximately 100-fold in 0.3% yield by a combination of ammonium sulfate fractionation, anion exchange HPLC, phenyl-Sepharose chromatography and gel filtration HPLC.     

Comparison of the levels of six endogenous gibberellins in roots and shoots of spinach in relation to photoperiod

This communication describes the distribution of gibberellins (GAs) in roots and shoots of spinach in relation to photoperiod. From previous work (Metzger, Zeevaart 1980 Plant Physiol 65: 623-626) shoots were known to contain GA₅₃, GA₄₄, GA₁₉, GA₁₇, GA₂₀, and GA₂₉. We now show by combined gas chromatography—mass spectrometry that roots contain GA₄₄, GA₁₉, and GA₂₉. Trace amounts of GA₅₃ were detected by combined gas chromatography—selected ion current monitoring. Neither GA₁₇ nor GA₂₀ were detected in root extracts. Analysis by the d-5 corn bioassay also showed no effect of photoperiodic treatment on the levels of GA-like substances in root extracts. Both phloem and xylem exudates had patterns of GA-like activity similar to those found in shoots and roots, respectively. Moreover, foliar application of [³H]GA₂₀ resulted in the transport of label from the shoot to the roots. Over half of the label in the roots represented unmetabolized [³H]GA₂₀, indicating that part of the GA₂₀ in the phloem is transported to the roots. Consequently, if GA₂₀ is made in, or transported to the roots, it is rapidly metabolized in that organ. This is a clear indication that regulation of GA metabolism is greatly different in roots and shoots.     

The effect of photoperiod on the levels of seven endogenous gibberellins in the long-day plant Agrostemma githago L.

The following seven gibberellins (GAs) have been identified by gas chromatography-mass spectrometry in shoots and leaves of the long-day plant Agrostemma githago: GA₅₃, GA₄₄, GA₁₉, GA₁₇, GA₂₀, GA₁, and 3-epi-GA₁. The levels of these compounds were measured, using selected ion monitoring, during photoperiodic induction. The levels of GA₄₄, GA₁₉, GA₁₇, and GA₂₀ all increased to a peak at eight long days (LD), followed by a decline, while the levels of GA₁ and 3-epi-GA₁ did not reach a peak until 12 LD. The level of GA₅₃ remained steady over the first 10–12 LD. Later in the LD treatment the levels of GA₅₃, GA₄₄, GA₁₉, and GA₁₇ increased again. The rate of metabolism of all GAs except GA₅₃ was higher after 12–16 LD than under short days. These data thus provide indirect evidence for an effect of photoperiodic induction on GA turnover in A. githago.Abbreviations AMO-1618 2-isopropyl-4-dimethylamino-5-methylphenyl-1-piperidine-carboxylate methyl chloride - GA(s) gibberellin(s) - GC-MS gas chromatography-mass spectrometry - HPLC high performance liquid chromatography - LD long day(s) - MeTMS trimethylsilylether of the methyl ester - SD short day(s) - SIM selected ion monitoring     

Comparison of the endogenous gibberellins in the shoots and roots of vernalized and non-vernalized chinese spring wheat seedlings

Endogenous gibberellins (GAs) in Chinese Spring wheat seedlings were isolated by high performance liquid chromatography (HPLC) and identified by combined capillary gas chromatography-selected ion monitoring (GC-SIM). Gibberellins A₁, A₃, A₁₉, A₂₀, A₄₄, and A₅₃ were identified in the shoots, A₁₉ and A₂₀ in the roots. The identification of these 13-hydroxylated GAs demonstrates the presence of the early-13-hydroxylation pathway in wheat seedlings. Based on peak area of total ion response of five characteristic ions by GC-SIM, the approximate levels of GAs in the shoots is GA₄₄ > GA₁₉ > GA₁ = GA₃ > GA₂₀ for the non-vernalized wheat seedlings, and GA₄₄ > GA₁₉ > GA₅₃ = GA₃ > GA₁ = GA₂₀ for the vernalized wheat seedlings. The C₂₀ GAs, GA₅₃, GA₄₄ and GA₁₉, are present in shoots of the vernalized (flowering) wheat seedlings at somewhat higher levels than that in the non-vernalized (rapidly growing) wheat seedlings. Approximate levels of the C₁₉ GAs, GA₂₀, GA₁ and GA₃ were lower in the shoots of the vernalized wheat seedlings than in the non-vernalized wheat seedlings. The conversion of GA₁₉ to GA₂₀ (C₂₀ to C₁₉ GAs) may be a rate-limiting step in the vernalized wheat seedlings.     

Endogenous Gibberellins in Aralia cordata

Eight gibberellins (GAs) were identified in extracts of buds of Aralia cordata by full scan GC/MS and by Kovats retention indices. These GAs comprised five GAs on the early-13-hydroxylation pathway [GA₁, GA₁₉, GA₂₀, GA₄₄, and GA₅₃] and three other GAs [GA₄, GA₁₅, and GA₃₇]. The major GAs were GA₁₉ and GA₄₄.     

Effect of Photoperiod on the Levels of Endogenous Gibberellins in Spinach as Measured by Combined Gas Chromatography-selected Ion Current Monitoring

The changes in the levels of five endogenous gibberellins (GAs) in spinach in relation to photoperiodic treatment have been examined by combined gas chromatography-selected ion current monitoring. Long-day treatment caused a 5-fold decline in the level of GA₁₉ while the levels of GA₂₀ and GA₂₉ increased dramatically during the same period. In absolute terms, the level of GA₂₀ increased from 0.8 microgram per 100 grams dry weight in short days to 5.5 micrograms per 100 grams dry weight after 14 long days. The levels of GA₁₇ and GA₄₄ did not change significantly with long-day treatment. These results are consistent with the hypothesis that GA₁₉ is converted to GA₂₀ and that this conversion is under photoperiodic control. Since stem growth in spinach is correlated with an increase in the level of GA₂₀, one major aspect of photoperiodic control of stem growth might be the availability of GA₂₀ through regulation of the conversion of GA₁₉ to GA₂₀.     

Accumulation of C19-gibberellins in the gibberellin-insensitive dwarf mutantgai ofArabidopsis thaliana (L.) Heynh

The endogenous gibberellins (GAs) from shoots of the GA-insensitive mutant,gai, ofArabidopsis thaliana were analyzed and compared with the GAs from the Landsberg erecta (Ler) line. Twenty GAs were identified in Ler plants by full-scan gas chromatography-mass spectrometry (GC-MS) and Kovats retention indices (KRI's). These GAs are members of the early-13-hydroxylation pathway (GA₅₃, GA₄₄, GA₁₉, GA₁₇, GA₂₀, GA₁, GA₂₉, and GA₈), the non-3,13-hydroxylation pathway (GA₁₂, GA₁₅, GA₂₄, GA₂₅, GA₉, and GA₅₁), and the early-3-hydroxylation pathway (GA₃₇, GA₂₇, GA₃₆, GA₁₃, GA₄, and GA₃₄). The same GAs, except GA₅₃, GA₄₄, GA₃₇, and GA₂₉ were detected in thegai mutant by the same methods. In addition, extracts fromgai plants contained GA₄₁ and GA₇₁. Both lines also contained several unknown GAs. In Ler plants these were mainly hydroxy-GA₁₂ derivatives, whereas in thegai mutant hydroxy-GA₂₄, hydroxy-GA₂₅, and hydroxy-GA₉ compounds were detected. Quantification of seven GAs by GC-selected ion monitoring (SIM), using internal standards, and comparisons of the ion intensities in the SIM chromatograms of the other thirteen GAs, demonstrated that thegai mutant had reduced levels of all C₂₀-dicarboxylic acids (GA₅₃, GA₄₄, GA₁₉, GA₁₂, GA₁₅, GA₂₄, GA₃₇, GA₂₇, and GA₃₆). In contrast,gai plants had increased levels of C₂₀-tricarboxylic acid GAs (GA₁₇, GA₂₅, and GA₄₁) and of all C₁₉-GAs (GA₂₀, GA₁, GA₈, GA₉, GA₅₁, GA₄, GA₃₄, and GA₇₁) except GA₂₉. The 3β-hydroxylated GAs, GA₁ and GA₄, and their respective 2β-hydroxylated derivatives, GA₈ and GA₃₄, were the most abundant GAs found in shoots of thegai mutant. Thus, thegai mutation inArabidopsis results in a phenotype that resembles GA-deficient mutants, is insensitive to both applied and endogenous GAs, and contains low levels of C₂₀-dicarboxylic acid GAs and high levels of C₁₉-GAs. This indicates that theGAI gene controls a step beyond the synthesis of an active GA. Thegai mutant is presumably a GA-receptor mutant or a mutant with a block in the transduction pathway between the receptor and stem elongation. We thank Dr. L.N. Mander, Australian National University, Canberra, for providing [²H]gibberellins, Dr. B.O. Phinney, University of California, Los Angeles, USA for [¹³C]GA₈, and Dr. D.A. Gage, MSU-NIH Mass Spectrometry Facility (grant No. DRR00480), for advice with mass spectrometry. This work was supported by a fellowship from the Spanish Ministry of Agriculture (I.N.I.A.) to M.T., by the U.S. Department of Energy under Contract DE-ACO2-76ERO-1338, and by U.S. Department of Agriculture grant No. 88-37261-3434 to J.A.D.Z.     

Gibberellin concentration and transport in genetic lines of pea : effects of grafting

Effects of the Na and Le loci on gibberellin (GA) content and transport in pea (Pisum sativum L.) shoots were studied. GA₁, GA₈, GA₁₇, GA₁₉, GA₂₀, GA₂₉, GA₄₄, GA₈ catabolite, and GA₂₉ catabolite were identified by full-scan gas chromatography-mass spectrometry in extracts of expanding and fully expanded tissues of line C79-338 (Na Le). Quantification of GAs by gas chromatography-single-ion monitoring using deuterated internal standards in lines differing at the Na and Le alleles showed that na reduced the contents of GA₁₉, GA₂₀, and GA₂₉ on average to <3% and of GA₁ and GA₈ to <30% of those in corresponding Na lines. In expanding tissues from Na le lines, GA₁ and GA₈ concentrations were reduced to approximately 10 and 2%, respectively, and GA₂₉ content increased 2- to 3-fold compared with those in Na Le plants. There was a close correlation between stem length and the concentrations of GA₁ or GA₈ in shoot apices in all six genotypes investigated. In na/Na grafts, internode length and GA₁ concentration of nana scions were normalized, the GA₂₀ content increased slightly, but GA₁₉ levels were unaffected. Movement of labeled GAs applied to leaves on Na rootstocks indicated that GA₁₉ was transported poorly to apices of na scions compared with GA₂₀ and GA₁. Our evidence suggests that GA₂₀ is the major transported GA in peas.     

Gibberellins in immature seeds and dark-grown shoots of Pisum sativum

Gibberellins (GAs) A₁₇, A₁₉, A₂₀, A₂₉, A₄₄, 2OH-GA₄₄ (tentative) and GA₂₉-catabolite were identified in 21-day-old seeds of Pisum sativum cv. Alaska (tall). These GAs are qualitatively similar to those in the dwarf cultivar Progress No. 9 with the exception of GA₁₉ which does not accumulate in Progress seeds. There was no evidence for the presence of 3-hydroxylated GAs in 21 day-old Alaska seeds. Dark-grown shoots of the cultivar Alaska contein GA₁, GA₈, GA₂₀, GA₂₉, GA₈-catabolite and GA₂₉-catabolite. Dark-grown shoots of the cultivar Progress No.9 contain GA₈, GA₂₀, GA₂₉ and GA₂₉-catabolite, and the presence of GA₁ was strongly indicated. Quantitation using GAs labelled with stable isotope showed the level of GA₁ in dark-grown shoots of the two cultivars to be almost identical, whilst the levels of GA₂₀, GA₂₉ and GA₂₉-catabolite were significantly lower in Alaska than in Progress No. 9. The levels of these GAs in dark-grown shoots were 10²- to 10³-fold less than the levels in developing seeds. The 2-epimer of GA₂₉ is present in dark-grown-shoot extracts of both cultivars and is not thought to be an artefact.Abbreviations cv cultivar - GA_n gibberellin A_n - GC gas chromatography - GC-MS combined gas chromatographymass spectrometry - HPLC high-pressure liquid chromatography - KRI Kovats retention index - MeTMSi methyl ester trimethylsilyl ether     

Identification and quantitative analysis of gibberellins inCitrus

Nine gibberellins (GAs) have been identified from tissues of Valencia orange (Citrus sinensis Osbeck) using gas chromatography—mass spectrometry and gas chromatography-selected ion monitoring of high-performance liquid chromatography (HPLC)-fractionated extracts. These GAs are GA₁, GA₃, GA₈, GA₁₉, GA₂₀, GA₂₉, 3-epi-GA₁, 2-epi-GA₂₉, and iso-GA₃. Selected-ion monitoring and stable-isotope dilution assays have been used to estimate levels of some of these GAs in vegetative and reproductive tissues. GA₂₉ was found to be the most abundant GA measured. GA₁ was found in all samples examined, and there was always less 3-epi-GA₁ than GA₁. GA₂₀ was present in most extracts. Leaves of developing inflorescence shoots contained six times more GA₂₉ than did leaves of comparable vegetative shoots. Levels of GA₂₉ increased during the early stages of fruit development. GA₂₀ may be more abundant in growing fruitlets than in those about to abscise; however, there were no consistent differences in the relative amounts of the other GAs. No major differences were found between tissues of immature seeded and seedless fruit, and developing seeds did not contain high levels of any of the GAs measured. It is concluded that seed-produced GAs are not essential for normal fruit development in Valencia orange.     

Metabolism of Gibberellin A(12) and A(12)-Aldehyde in Developing Seeds of Pisum sativum L

Metabolism of [¹⁴C]gibberellin (GA) A₁₂ (GA₁₂) and [¹⁴C]gibberellin A₁₂-aldehyde (GA₁₂-aldehyde) was examined in cotyledons and seed coats from developing seeds of pea (Pisum sativum L.). Both were metabolized to only 13-hydroxylated GAs in cotyledons but to 13-hydroxylated and non-13-hydroxylated GAs in seed coats. The metabolism of [¹⁴C]GA₁₂ was slower in seed coats than in cotyledons. [¹⁴C]GA₁₂-aldehyde was also metabolized to conjugates in seed coats. Seed coat [¹⁴C]-metabolites produced from [¹⁴C]GA₁₂-aldehyde were isolated by high-performance liquid chromatography (HPLC). Conjugates were base hydrolyzed and the free GAs reisolated by HPLC and identified by gas chromatography-mass spectrometry. [¹⁴C]GA₅₃-aldehyde, [¹⁴C]GA₁₂-aldehyde conjugate, and [¹⁴C]GA₅₃-aldehyde conjugate were major metabolites produced from [¹⁴C]GA₁₂-aldehyde by seed coats aged 20-22 days or older. The dilution of ¹⁴C in these compounds by ¹²C, as compared to the supplied [¹⁴C]GA₁₂-aldehyde, indicated that they are endogenous. Feeding [¹⁴C]GA₅₃-aldehyde led to the production of [¹⁴C]GA₅₃-aldehyde conjugate in seed coats and shoots and also to 13-hydroxylated GAs in shoots. Labeled GAs, recovered from plant tissue incubated with either [¹⁴C]GA₁₂, [¹⁴C]GA₁₂-aldehyde, or [³H]GA₉, were used as appropriate markers for the recovery of endogenous GAs from seed coats or cotyledons. These GAs were purified by HPLC and identified and quantified by gas chromatography-mass spectrometry. GA₁₅, GA₂₄, GA₉, GA₅₁, GA₅₁-catabolite, GA₂₀, GA₂₉, and GA₂₉-catabolite were detected in seed coats, whereas GA₉, GA₅₃, GA₄₄, GA₁₉, GA₂₀, and GA₂₉ were found in cotyledons. The highest GA levels were for GA₂₀ and GA₂₉ in cotyledons (783 and 912 nanograms per gram fresh weight, respectively) and for GA₂₉ and GA₂₉-catabolite in seed coats (1940 and > 1940 nanograms per gram fresh weight, respectively).     

Identification of Gibberellins A(1), A(5), A(29), and A(32) from Immature Seeds of Apricot (Prunus armeniaca L.)

Gibberellins (GAs) A₁, A₅, and A₂₉ were identified, and also GA₃₂ was confirmed, as endogenous GAs of immature seeds (3-4 weeks after anthesis, 0.25-0.5 gram fresh weight) of apricot (Prunus armeniaca L.) based on capillary gas chromatography (GC), retention time (Rt), and selected ion monitoring (SIM), in comparison with authentic standards. Fractions subjected to GC-SIM were purified and separated using sequential solvent partitioning → paper chromatography → reverse phase C₁₈ high performance liquid chromatography (HPLC) → bioassay on dwarf rice cv Tan-ginbozu. Two other peaks of free GA-like bioactivity (microdrop and immersion dwarf rice assays) were eluted from C₁₈ HPLC at Rts where GA_4/7 and GA₈ (or other GAs with similar structures) would elute. Also, three unidentified GA glucoside-like compounds (based on bioactivity on the immersion assay, and no bioactivity on the microdrop assay) were noted. There were very high amounts of GA₃₂ (112 ng of GA₃ equivalents per gram fresh weight), and minor amounts (0.5 ng of GA₃ equivalents) for each of GA₁ and GA₅, respectively, based on the microdrop assay.     

Gibberellin A(3) Is Biosynthesized from Gibberellin A(20) via Gibberellin A(5) in Shoots of Zea mays L

[17-¹³C,³H]-Labeled gibberellin A₂₀ (GA₂₀), GA₅, and GA₁ were fed to homozygous normal (+/+), heterozygous dominant dwarf (D8/+), and homozygous dominant dwarf (D8/D8) seedlings of Zea mays L. (maize). ¹³C-Labeled GA₂₉, GA₈, GA₅, GA₁, and 3-epi-GA₁, as well as unmetabolized [¹³C]GA₂₀, were identified by gas chromatography-selected ion monitoring (GC-SIM) from feeds of [17-¹³C, ³H]GA₂₀ to all three genotypes. ¹³C-Labeled GA₈ and 3-epi-G₁, as well as unmetabolized [¹³C]GA₁, were identified by GC-SIM from feeds of [17-¹³C, ³H]GA₁ to all three genotypes. From feeds of [17-¹³C, ³H]GA₅, ¹³C-labeled GA₃ and the GA₃-isolactone, as well as unmetabolized [¹³C]GA₅, were identified by GC-SIM from +/+ and D8/D8, and by full scan GC-MS from D8/+. No evidence was found for the metabolism of [17-¹³C, ³H]GA₅ to [¹³C]GA₁, either by full scan GC-mass spectrometry or by GC-SIM. The results demonstrate the presence in maize seedlings of three separate branches from GA₂₀, as follows: (a) GA₂₀ → GA₁ → GA₈; (b) GA₂₀ → GA₅ → GA₃; and (c) GA₂₀ → GA₂₉. The in vivo biogenesis of GA₃ from GA₅, as well as the origin of GA₅ from GA₂₀, are conclusively established for the first time in a higher plant (maize shoots).     

Metabolism of Tritiated Gibberellin A(20) in Immature Seeds of Dwarf Pea, cv. Meteor

Tritium-labeled gibberellin A₂₀ ([³H]GA₂₀) was applied via the pedicel to immature pods and seeds of dwarf peas and three harvests were made at days 5, 10, and 23 (mature) after application. Of the five metabolites of [³H]GA₂₀, the three in highest yield were GA₂₉, an α,β-unsaturated ketone, and a compound (B), whose structure was only tentatively assigned. The metabolic sequence GA₂₀ → GA₂₉ → compound B → the ketone was indicated. The amount of [³H]GA₂₉ in both seeds and pods was highest at day 5 and declined to its lowest level at maturity. The amount of the [³H]ketone in the seed increased with time to its highest level at maturity. It is suggested that compound B and the ketone represent the major pathway of catabolism of GA₂₉, a 2β-hydroxylated GA of low biological activity, and that the ketone is not metabolized, or only slowly metabolized, during seed maturation.     

Gibberellins and Stem Growth as Related to Photoperiod in Silene armeria L

Stem growth and flowering in the long-day plant Silene armeria L. are induced by exposure to a minimum of 3 to 6 long days (LD). Stem growth continues in subsequent short days (SD), albeit at a reduced rate. The growth retardant tetcyclacis inhibited stem elongation induced by LD, but had no effect on flowering. This indicates that photoperiodic control of stem growth in Silene is mediated by gibberellins (GA). The objective of this study was to analyze the effects of photoperiod on the levels and distribution of endogenous GAs in Silene and to determine the nature of the photoperiodic after-effect on stem growth in this plant. The GAs identified in extracts from Silene by full-scan combined gas chromatography-mass spectrometry (GC-MS), GA₁₂, GA₅₃, GA₄₄, GA₁₇, GA₁₉, GA₂₀, GA₁, GA₂₉, and GA₈, are members of the early 13-hydroxylation pathway. All of these GAs were present in plants under SD as well as under LD conditions. The GA₅₃ level was highest in plants in SD, and decreased in plants transferred to LD conditions. By contrast, GA₁₉, GA₂₀, and GA₁ initially increased in plants transferred to LD, and then declined. Likewise, when Silene plants were returned from LD to SD, there was an increase in GA₅₃, and a decrease in GA₁₉, GA₂₀, and GA₁ which ultimately reached levels similar to those found in plants kept in SD. Thus, measurements of GA levels in whole shoots of Silene as well as in individual parts of the plant suggest that the photoperiod modulates GA metabolism mainly through the rate of conversion of GA₅₃. As a result of LD induction, GA₁ accumulates at its highest level in shoot tips which, in turn, results in stem elongation. In addition, LD also appear to increase the sensitivity of the tissue to GA, and this effect is presumably responsible for the photoperiodic after-effect on stem elongation in Silene.     

Metabolism of gibberellins in a cell-free system from immature seeds of Phaseolus vulgaris L.

The biosynthetic steps from gibberellin A₁₂-aldehyde (GA₁₂-aldehyde) to C₁₉-GAs were studied by means of a cell-free system from the embryos of immature Phaseolus vulgaris seeds. Stable-isotope-labeled GAs were used as substrates and the products were identified by gas chromatography-mass spectrometry. Gibberellin A₁₂-aldehyde was converted to GA₄ via non-hydroxylated intermediates and to GA₁ via 13-hydroxylated intermediates. 13-Hydroxylation took place at the beginning of the pathway by the conversion of GA₁₂-aldehyde to GA₅₃-aldehyde. The conversion of GA₂₀ to GA₅ and GA₆ was also shown but no 2-hydroxylating activity was found. Endogenous GAs from embryos and testas of 17-dold seeds were re-examined by gas chromatography-selected ion monitoring using stable-isotopelabeled GAs as internal standards. Gibberellins A₉, A₁₂, A₁₅, A₁₉, A₂₃, A₂₄, and A₅₃ were identified for the first time in P. vulgaris, in addition to GA₁, GA₄, GA₅, GA₆, GA₈, GA₁₇, GA₂₀, GA₂₉, GA₃₇, GA₃₈ and GA₄₄, which were previously known to occur in this species. The levels of all GAs, except the 2-hydroxylated ones, were greater in the embryos than in the testas. Conversely, the contents of GA₈ and GA₂₉, both 2-hydroxylated, were much higher in the testas than in the embryos.Abbreviations GA_n gibberellin A_n - GC-MS gas chromatography-mass spectrometry - GC-SIM gas chromatography-selected ion monitoring - HPLC high-performance liquid chromatography - TLC thin-layer chromatography - m/z ion of mass     

Gibberellins and the photoperiodic control of leaf growth in Poa pratensis

The role of gibberellins (GAs) in photoperiodic control of leaf elongation in Poa pratensis was studied by both application of exogenous GAs and analysis of endogenous GAs. Leaf elongation was strongly increased under long day (LD, 24 h) conditions at both 9 and 21°C, leaf length at 9°C LD being similar to that in plants grown in short days (SD, 8 h) at 21°C. However, even at 21°C leaf elongation was enhanced by LD. Exogenous GA₁ could completely compensate for LD at both 9 and 21°C. Gibberellins A₂₀, A₁₉ and A₄₄ could also partly replace LD, but they were significantly less active than GA₁, GA₅₃ was inactive when applied to plants grown at 9°C in SD and exhibited only marginal activity at 9°C LD and 21°C SD. The total level of GAs of the early 13-hydroxylation pathway (A₅₃, A₄₄, A₁₉, A₂₀ and A₁) increased rapidly when plants were transferred from SD to LD at 9°C. After transfer from 9 to 21°C, there was an increase in GA levels at both LD and SD, followed by a decrease under LD conditions. In all cases, GA₁₉ was the predominant GA, accounting for 60 to 80% of the analysed GAs. Levels of the bioactive GA₁ were low and increased transiently by LD four days after transfer from SD to LD. At both temperatures, the ratio GA₁₉ to GA₂₀ and GA₂₀ to GA₁ at 9°C was enhanced by LD compared with SD. Taken together, these results support the hypothesis that photoperiodic regulation of leaf elongation in Poa pratensis is GA-mediated, and they indicate a photoperiodic control of oxidation of GA₅₃ to GA₄₄ and GA₁₉ to GA₂₀, and perhaps also of 3β-hydroxylation of GA₂₀ to GA₁.     

Implication of Gibberellins in Head Smut (Sporisorium reilianum) of Sorghum bicolor

The head smut fungus, Sporisorium reilianum ([Kuhn] Landon and Fullerton), was shown to reduce plant height in infected Sorghum bicolor ([L.] Moench) plants. The major reductions occurred in the internodes nearest the panicle and were more severe in naturally infected than in inoculated plants. Less affected plants developed reproductively sterile panicles, and eventually smutted panicles developed phyllodied growths which progressed into leafy shoots. Extracts of smutted, sterile, and healthy (control) panicles of field-grown plants exhibited gibberellin (GA)-like activity in the dwarf rice bioassay. When extracts were purified and assayed with deuterium-labeled GA standards by gas chromatography-mass spectrometry-selected ion monitoring (GC-MS-SIM), GA₁, GA₃, GA₁₉, GA₂₀, and GA₅₃ were detected based on coelution with the standards, identical Kovats retention index values, and matching ion masses and relative abundances for three major ions. In addition, based on published Kovats retention index values, ion masses, and relative abundance values, GA₄, GA₇, GA₈, GA₁₄, GA₂₉, and GA₄₄ were tentatively identified. Quantitative analysis revealed that panicles of healthy control plants contained from 60 to 100% higher total concentrations of GAs than panicles of smutted plants. These comparisons were most striking for the early 13-hydroxylation pathway precursors GA₅₃, GA₄₄, and GA₁₉ but not for GA₂₀. Extracts of S. reilianum sporidia and culture medium exhibited GA-like bioactivity, and GA₁ and GA₃ were detected based on GC-MS-SIM assay with ²H-labeled internal standards. Quantitative analysis of these GAs showed increasing concentrations from 4 to 7 to 10 days of culture and a decline at 20 days. This is the first GC-MS-SIM detection of GAs in a non-Ascomycete fungus, and the disease symptoms and quantitative data suggested that fungal infection may interfere with biosynthesis of GAs by the host plant.