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     

The quantitative relationship between gibberellin A1 and internode growth in Pisum sativum L.

The metabolism and growth-promoting activity of gibberellin A₂₀ (GA₂₀) were compared in the internode-length genotypes of pea, na le and na Le. Gibberellin A₂₉ and GA₂₉-catabolite were the major metabolites of GA₂₀ in the genotype na le. However, low levels of GA₁, GA₈ and GA₈-catabolite were also identified as metabolites in this genotype, confirming that the le allele is a leaky mutation. Gibberellin A₂₀ was approximately 20 to 30 times as active in promoting internode growth of genotype na Le as of genotype na le. However, the levels of the 3-hydroxylated metabolite of GA₂₀, GA₈ (2-hydroxy GA₁), were similar for a given growth response in both genotypes. In each case a close linear relationship was observed between internode growth and the logarithm of GA₈ levels. A similar relationship was found on comparing GA₂₀ metabolism in the three genotypes le ^d, le and Le. The former mutation results in a more severe dwarf phenotype than the le allele (which has previously been shown to reduce the 3-hydroxylation of GA₂₀ to GA₁). These results indicate that GA₂₀ has negligible intrinsic activity and support the contention that GA₁ is the only GA active per se in promoting stem growth in pea.Abbreviations GA_n gibberellin A_n - GC-MS gas chromatography-mass spectrometry - HPLC high-pressure liquid chromatography     

Internode length in Pisum sativum L. The kinetics of growth and [3H]gibberellin A20 metabolism in genotype na Le

The relationship between shoot growth and [³H]gibberellin A₂₀ (GA₂₀) metabolism was investigated in the GA-deficient genotype of peas, na Le. [17-¹³C, ³H₂]gibberellin A₂₀ was applied to the shoot apex and its metabolic fate examined by gas chromatographic-mass spectrometric analysis of extracts of the shoot and root tissues. As reported before, [¹³C, ³H₂]GA₁, [¹³C, ³H₂]GA₈ and [¹³C, ³H₂]GA₂₉ constituted the major metabolites of [¹³C, ³H₂]GA₂₀ present in the shoot. None of these GAs showed any dilution by endogenous ¹²C-material. [¹³C, ³H₂]GA₂₉-catabolite was also a prominent metabolite in the shoot tissue but showed pronounced isotope dilution probably due to carry-over of endogenous [¹²C]GA₂₉-catabolite from the mature seed. In marked contrast to the shoot tissue, the two major metabolites present in the roots were identified as [¹³C, ³H₂]GA₈-catabolite and [¹³C, ³H₂]GA₂₉-catabolite. Both of these compounds showed strong dilution by endogenous ¹²C-material. Only low levels of [¹³C, ³H₂]GA₁, [¹³C, ³H₂]GA₈, [¹³C, ³H₂]GA₂₀ and [¹³C, ³H₂]GA₂₉ accumulated in the roots. It is suggested that compartmentation of GA-catabolism may occur in the root tissue in an analogous manner to that shown in the testa of developing seeds. Changes in the levels of [1,3-³H₂]GA₂₀ metabolites over 10 d following application of the substrate to the shoot apex of genotype na Le confirmed the accumulation of [³H]GA-catabolites in the root tissues. No evidence was obtained for catabolic loss of [³H]GA₂₀ by complete oxidation or conversion to a methanol-inextractable form. The results indicate that the root system may play an important role in the regulation of biologically active GA levels in the developing shoot of Na genotypes of peas.Abbreviations GA_n gibberellin A_n - GC-MS gas chromatography-mass spectrometry - HPLC high-pressure liquid chromatography     

Metabolism of [13C1]gibberellin A29 to [13C1]gibberellin-catabolite in maturing seeds of Pisum sativum cv. Progress No. 9

The metabolism of GA₂₉ during seed maturation in Pisum sativum cv. Progress No. 9 was further investigated. [17-¹³C₁]GA₂₉ was metabolised to a GA-catabolite (structure 3), with incorporation of the [¹³C] label from the GA₂₉ substrate into the GA-catabolite being demonstrated by GC-MS. Quantitation of the GA-catabolite using GC-MS was achieved by adding GA-catabolite, labelled with [¹⁸O], to seed extracts as an internal standard. At least 50% conversion of [¹³C₁]GA₂₉ to [¹³C₁]GA-catabolite was demonstrated with the build up of exogenous [¹³C₁]GA-catabolite strictly paralleling the accumulation of native GA-catabolite. These results strongly suggest that conversion of GA₂₉ to the GA-catabolite is a natural metabolic step occurring during the final stages of seed maturation. 25 g per seed of native GA-catabolite was recorded in 37 day old seeds. Some problems encountered in the analysis of extracts containing the GA-catabolite are discussed briefly.Abbreviations BSTFA bis(trifluoromethylsilyl)acetamide - GA_n gibberellin A_n - GC gas chromatography - GC-MS combined gas chromatography-mass spectrometry - Me methyl ester - SICM selected ion current monitoring - TMSi trimethylsilyl ether     

Identification,quantitation and distribution of gibberellins in fruits of Pisum sativum L. cv. Alaska during pod development

In addition to the previously-reported gibberellins: GA₁; GA₈, GA₂₀ and GA₂₉ (García-Martínez et al., 1987, Planta 170, 130–137), GA₃ and GA₁₉ were identified by combined gas chromatography-mass spectrometry in pods and ovules of 4-d-old pollinated pea (Pisum sativum cv. Alaska) ovaries. Pods contained additionally GA₁₇, GA₈₁ (2-hydroxy GA₂₀) and GA₂₉-catabolite. The concentrations of GA₁, GA₃, GA₈, GA₁₉, GA₂₀ and GA₂₉ were higher in the ovules than in the pod, although, with the exception of GA₃, the total content of these GAs in the pod exceeded that in the seeds. About 80% of the GA₃ content of the ovary was present in the seeds. The concentrations of GA₁₉ and GA₂₀ in pollinated ovaries remained fairly constant for the first 12 ds after an thesis, after which they increased sharply. In contrast, GA₁ and GA₃ concentrations were maximal at 7 d and 4–6 d, respectively, after anthesis, at about the time of maximum pod growth rate, and declined thereafter. Emasculated ovaries at anthesis contained GA₈, GA₁₉ and GA₂₀ at concentrations comparable with pollinated fruit, but they decreased rapidly. Gibberellins a₁ and A₃ were present in only trace amounts in emasculated ovaries at any stage. Parthenocarpic fruit, produced by decapitating plants immediately above an emasculated flower, or by treating such flowers with 2,4-dichlorophenoxyacetic acid or GA₇, contained GA₁₉ and GA₂₀ at similar concentrations to seeded fruit, but very low amounts of GA₁ and GA₃ Thus, it appears that the presence of fertilised ovules is necessary for the synthesis of these last two GAs. Mature leaves and leaf diffusates contained GA₁, GA₈, GA₁₉ and GA₂₀ as determined by combined gas chromatography-mass spectrometry using selected ion monitoring. This provides further evidence that vegetative tissues are a possible alternative source of GAs for fruit-set, particularly in decapitated plants.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - FW fresh weight - GA_n gibberellin A_n - GC-MS combined gas chromatography-mass spectrometry - HPLC high-performance liquid chromatography - KRI Kovats retention index - m/z mass to charge ratio We thank Mr M.J. Lewis for qualitative GC-MS analyses and Ms M.V. Cuthbert (LARS), R. Martinez Pardo and T. Sabater (IATA) for technical assistance. We are also grateful to Professor B.O. Phinney, University of California, Los Angeles, for gifts of [17-¹³C]GA₈ and -GA₂₉ and to Mr Paul Gaskin, University of Bristol, for the mass spectrum of GA₂₉-catabolite and for a sample of GA₈₁ The work in Spain was supported by Dirección General de Investigación Cientifica y Técnica (grant PB87-0402 to J.L.G.-M.). We also acknowledge the British Council and Ministerio de Educacion y Ciencia for travel grants through Accion Integrada Hispano-Britanica 56/142 (J.L.G.-M. and P.H.).     

Gibberellins in developing fruits of Pisum sativum cv. Alaska: Studies on their role in pod growth and seed development

Gibberellins A₁, A₈, A₂₀ and A₂₉ were identified by capillary gas chromatography-mass spectrometry in the pods and seeds from 5-d-old pollinated ovaries of pea (Pisum sativum cv. Alaska). These gibberellins were also identified in 4-d-old non-developing, parthenocarpic and pollinated ovaries. The level of gibberellin A₁ within these ovary types was correlated with pod size. Gibberellin A₁, applied to emasculated ovaries cultured in vitro, was three to five times more active than gibberellin A₂₀. Using pollinated ovary explants cultured in vitro, the effects of inhibitors of gibberellin biosynthesis on pod growth and seed development were examined. The inhibitors retarded pod growth during the first 7 d after anthesis, and this inhibition was reversed by simultaneous application of gibberellin A₃. In contrast, the inhibitors, when supplied to 4-d-old pollinated ovaries for 16 d, had little effect on seed fresh weight although they reduced the levels of endogenous gibberellins A₂₀ and A₂₉ in the enlarging seeds to almost zero. Paclobutrazol, which was one of the inhibitors used, is xylem-mobile and it efficiently reduced the level of seed gibberellins without being taken up into the seed. In intact fruits the pod may therefore be a source of precursors for gibberellin biosynthesis in the seed. Overall, the results indicate that gibberellin A₁, present in parthenocarpic and pollinated fruits early in development, regulates pod growth. In contrast the high levels of gibberellins A₂₀ and A₂₉, which accumulate during seed enlargement, appear to be unnecessary for normal seed development or for subsequent germination.Abbreviations GA_(a) gibberellin A_n - GC-MS combined gas chromatography-mass spectrometry - HPLC high-performance liquid chromatography - PFK perfluorokerosene - PVP polyvinylpyrrolidone     

Ethylene metabolism in Pisum sativum L.

The possible role of C₂H₄ metabolism in mediating the responses of plants to C₂H₄ is re-examined. It is demonstrated that (i) the effects of inhibitors upon C₂H₄ action do not correspond with their effects on metabolism, (ii) elicitors of C₂H₄ effects do not have appropriate effects on C₂H₄ metabolism, (iii) inhibitors of C₂H₄ metabolism do not affect the response of plants to C₂H₄. It is concluded that metabolism of C₂H₄ is not linked to the mode of action of the growth regulator.Abbreviations DTC sodium diethyldithiocarbamate - FW fresh weight     

Internode length in Pisum

The influence of the Na and Le genes in peas on gibberellin (GA) levels and metabolism were examined by gas chromatographic-mass spectrometric analysis of extracts from a range of stem-length genotypes fed with [¹³C, ³H]GA₂₀. The substrate was metabolised to [¹³C, ³H]GA₁, [¹³C, ³H]GA₈ and [¹³C, ³H]GA₂₉ in the immature, expanding apical tissue of all genotypes carrying Le. In contrast, [¹³C, ³H]GA₂₉ and, in one line, [¹³C, ³H]GA₂₉-catabolite, were the only products detected in plants homozygous for the le gene. These results confirm that the Le gene in peas controls the 3-hydroxylation of GA₂₀ to GA₁. Qualitatively the same results were obtained irrespective of the genotype at the Na locus. In all Na lines the [¹³C, ³H]GA₂₀ metabolites were considerably diluted by endogenous [¹²C]GAs, implying that the metabolism of [¹³C, ³H]GA₂₀ mirrored that of endogenous [¹²C]GA₂₀. In contrast, the [¹³C, ³H]GA₂₀ metabolites in na lines showed no dilution with [¹²C]GAs, confirming that the na mutation prevents the production of C₁₉-GAs. Estimates of the levels of endogenous GAs in the apical tissues of Na lines, made from the ¹²C:¹³C isotope ratios and the radioactivity recovered in respective metabolites, varied between 7 and 40 ng of each GA per plant in the tissue expanded during the 5 d between treatment with [¹³C, ³H]GA₂₀ and extraction. No [¹²C]GA₁ and only traces of [¹²C]GA₈ (in one line) were detected in the two Na le lines examined. These results are discussed in relation to recent observations on dwarfism in rice and maize.Abbreviations GA_n gibberellin A_n - GC-MS gas chromatography-mass spectrometry - HPLC high-pressure liquid chromatography     

Metabolism of gibberellin A29 in seeds of Pisum sativum cv. Progress No. 9; Use of [2H] and [3H]GAs,and the identification of a new GA catabolite

The metabolism of GA₂₉ in maturing seeds of Pisum sativum cv. Progress No. 9 was further investigated, and the utility of ²H-labelled GAs in conjuction with GC-MS is illustrated. Using [2-²H₁]GA₂₉ as an internal standard, endogenous GA₂₉ was shown to reach a maximal level (ca. 10 g/seed) 27 days from anthesis, and to decline to ca. 1.6 g/seed in mature seeds. In a time-course feed the metabolism of [2-²H₁] [2-³H₁]GA₂₉ applied to 27 day old seeds, and of endogenous GA₂₉, was compared from the ¹H:²H ratios in the recovered GA₂₉. Although both [2-²H₁] [2-³H₁]GA₂₉ and endogenous GA₂₉ were metabolised to the same limited extent to a putative conjugate, in the main metabolic process endogenous GA₂₉ was preferentially converted to an untraceable (i.e. unlabelled) metabolite. In contrast, endogenous GA₂₉ and [1,3-²H₂] [1,3-³H₂]GA₂₉, derived from [1,3-²H₂] [1,3-³H₂]GA₂₀ in a time-course feed, were metabolised in an identical manner. In the latter case isotope loss precluded identification of the metabolite. The structure (8) has been assigned to a GA catabolite present in maturing seeds and seedlings of pea. The isotope data are consistent with this compound being the hitherto untraced metabolite of GA₂₉ in pea.Abbreviations GA_n gibberellin A_n - GC gas chromatography - GC-MS combined gas chromatography-mass spectrometry - GC-RC combined gas chromatography-radio counting - M⁺ molecular ion - Me methyl ester - R_T retention time - SICM selected ion current monitoring - TLC thin layer chromatography - TMS trimethylsilyl ether     

Metabolism of gibberellins by immature barley grain

Gibberellins A₁, A₄, A₉, A₁₂-aldehyde, A₂₀ and A₅₁, each labelled with both a radioactive and stable isotope were fed to immature barley grain by injection into the endosperm. After 7 d, extensive metabolism of all substrates had occurred, and metabolites were identified by combined capillary gas chromatography-mass spectrometry. A proposed scheme of gibberellin metabolism in immature barley grain is presented.Abbreviations GA_n gibberellin A_n - GC-MS combined gas chromatography-mass spectrometry - HPLC high-performance liquid chromatography     

Gibberellin translocation in Pisum sativum L.

The physiological and biochemical consequences of treating Le (tall) and le (dwarf) pea seedlings with varying quantities of the gibberellins [³H]GA₂₀ and GA₁ have been investigated. Although the percentage uptake of these compounds from the site of application on the 3 stipules was low and most of the applied GA remained unmetabolised in situ, the quantitative relationship between GA translocation and GA dosage was found to be linear for GA₁ but saturating for GA₂₀. The movement of the GAs and their subsequently produced metabolites was mainly acropetal. They accumulated in greatest quantity in the apical extremities of the shoot. Overall, the extent to which GA₂₀ was metabolished in le seedlings was considerably less than in Le pea seedlings. Although all le tissues contained significantly less [³H]GA₁ than their Le counterparts, phenotypic effects of the le mutation were apparent only on internode and tendril development. Increased tissue growth, consequent upon GA treatment, was also apparent only in the internodes and tendrils of le plants. For internodes, GA₁ content determined the mid-logarithmic-phase growth rate and, consequently, final length. For tendrils, GA₂₀ rather than GA₁ may be the primary stimulatory agent.Abbreviations GA gibberellin - HPLC high-performance liquid chromatography - 1–6 consecutive developmental numbering system for plant tissues/organs as shown in Fig. 1 The author gratefully acknowledges financial support from Imperial Chemical Industries, Plant Protection, Jealott's Hill, Bracknell, Berks., UK and the Science and Engineering Research Council.     

Further studies on the metabolism of gibberellins (GAs) A9, A20 and A29 in immature seeds of Pisum sativum cv. progress No. 9

Seed maturation of Pisum sativum cv. Progress No. 9 proceeds more slowly in winter than in summer even when the parent plants are grown in greenhouse conditions with light-and heat-supplementation. For parent plants grown under summer and winter conditions the metabolism of [³H]GA₉ in cultured seeds is qualitatively different in seeds of equivalent age and qualitatively the same in seeds of equivalent weight. 13-Hydroxylation of [³H]GA₉[³H]GA₂₀ is restricted to early stages of seed development. 2-Hydroxylation of [³H]GA₉2-OH-[³H]GA₉ has only been observed at a stage of development after endogenous GA₉ has accumulated. 2-OH-GA₉ has been shown to be endogenous to pea and is named GA₅₁. H₂-GA₃₁ and its conjugate have not been shown to be present in pea and may be induced metabolites of [³H]GA₉. The metabolism of GA₂₀GA₂₉ is used to illustrate a technique of feeding [²H][³H]GAs in order to distinguish a metabolite from the same endogenous compound. The in vitro conversion of [³H]GA₂₀[³H]GA₂₉, and the virtual non-metabolism of [³H]GA₂₉ have been confirmed for seeds in intact fruits. These results are discussed in relation to the apparent absence of conjugated GAs in mature pea seeds.Abbreviations GA_n gibberellin A_n - GC gas chromatography - GC-MS combined gas chromatography-mass spectrometry - GC-RC combined gas chromatography-radio counting - Me methyl ester - R_T etention time - SICM selected ion current monitoring - TLC thin layer chromatography - TMS trimethyl silyl ether The author is née Frydman     

Endomitosis and the effect of gibberellic acid in different Pisum sativum L. cultivars

The evolution of the total amount of DNA in epicotyls and of the amount of DNA per cell nucleus in epicotyl cortex cells during germination was followed in two closely related pea varieties, Pisum sativum cv. Finale and Pisum sativum cv. Rondo. Under etiolating conditions, growth of the cv. Rondo occurs only by cell elongation which is preceded by endomitotic DNA synthesis, while in the cv. Finale growth is the result of cell elongation accompanied by endomitotic DNA synthesis and cell division. The maximum C-level attained in both cultivars under etiolating conditions is 8 C (C=haploid amount of DNA in a gamete cell). Both the maximum C-level reached and the percentage of cells reaching this C-level seem to be under strict genetic control. In both cultivars, light inhibits the endomitotic DNA replication.Neither gibberellic acid (GA₃), nor AMO 1618 alter the maximum C-level or the percentage distribution of the C-classes. Both growth regulators are effective, although in an opposite way, only in tissues where cell division occurs or where endomitotic DNA synthesis is blocked, as in light-grown pea epicotyls.     

Enzymes of serine and glycine metabolism in leaves and non-photosynthetic tissues of Pisum sativum L.

A comparative study is presented of the activities of enzymes of glycine and serine metabolism in leaves, germinated cotyledons and root apices of pea (Pisum sativum L.). Data are given for aminotransferase activities with glyoxylate, hydroxypyruvate and pyruvate, for enzymes associated with serine synthesis from 3-phosphoglycerate and for glycine decarboxylase and serine hydroxymethyltransferase. Aminotransferase activities differ between the tissues in that, firstly, appreciable transamination of serine, hydroxypyruvate and asparagine occurs only in leaf extracts and, secondly, glyoxylate is transaminated more actively than pyruvate in leaf extracts, whereas the converse is true of extracts of cotyledons and root apices. Alanine is the most active amino-group donor to both glyoxylate and hydroxypyruvate. 3-Phosphoglycerate dehydrogenase and glutamate: O-phosphohydroxypyruvate aminotransferase have comparable activities in all three tissues, except germinated cotyledons, in which the aminotransferase appears to be undetectable. Glycollate oxidase is virtually undetectable in the non-photosynthetic tissues and in these tissues the activity of glycerate dehydrogenase is much lower than that of 3-phosphoglycerate dehydrogenase. Glycine decarboxylase activity in leaves, measured in the presence of oxaloacetate, is equal to about 30–40% of the measured rate of CO₂ fixation and is therefore adequate to account for the expected rate of photorespiration. The activity of glycine decarboxylase in the non-photosynthetic tissues is calculated to be about 2–5% of the activity in leaves and has the characteristics of a pyridoxal-and tetrahydrofolate-dependent mitochondrial reaction; it is stimulated by oxaloacetate, although not by ADP. In leaves, the measured activity of serine hydroxymethyltransferase is somewhat lower than that of glycine decarboxylase, whereas in root apices it is substantially higher. Differential centrifugation of extracts of root apices suggests that an appreciable proportion of serine hydroxymethyltransferase activity is associated with the plastids.Abbreviation GOGAT l-Glutamine:2-oxoglutarate aminotransferase     

Carnitine acyltransferases in chloroplasts of Pisum sativum L.

Carnitine-acetyltransferase (EC 2.3.1.7) and carnitine-palmitoyltransferase (EC 2.3.1.21) activities were shown to be present in chloroplasts of green pea leaves and possibly to occur in leaf mitochondrial and peroxisomal fractions. A role for the enzymes in the transfer of acyl groups across membranes is suggested.     

The partial purification and characterization of a gibberellin C-20 hydroxylase from immature Pisum sativum L. seeds

A gibberellin (GA) C-20 hydroxylase that catalyses the conversion of GA₅₃ to GA₄₄ was purified from developing pea embryos by ammonium-sulfate precipitation, gel filtration and anion-exchange column chromatography. The purification was about 270-fold and 15% of the enzymic activity was recovered. The relative molecular mass was 44000 by Sephadex G-200 gel filtration. The apparent Michaelis constant was 0.7 M and the isoelectric point was 5.6–5.9. The enzymic activity was optimal at pH 7.0 2-Oxoglutarate and ascorbate were required for activity. Low concentrations of Fe²⁺ stimulated the reaction, but externally added Fe²⁺ was not essential, even in the most purified preparation. Catalase and bovine serum albumin also stimulated. Dithiothreitol preserved the activity during purification but was not needed during incubation. In fact, the simultaneous presence of dithiothreitol and Fe²⁺ in the incubation mixture was inhibitory to the purified enzyme. The cofactor requirements are typical for those of 2-oxoglutarate-dependent dioxygenases.When the incubation time was long enough, GA₅₃ was converted to both GA₄₄ and GA₁₉. The proportions of these two products remained constant throughout the purification, but this does not necessarily mean that their formations is catalysed by a single enzyme. Sodium dodecyl sulfatepolyacrylamide gel electrophoresis showed that the final preparation contained several proteins. Although the most prominent protein band was located within the range expected for the enzyme on the grounds of its molecular weight, this band did not represent the enzyme, since it separated from the GA C-20 hydroxylase activity on ultrathin-layer isoeletric focusing.Abbreviation BSA bovine serum albumin - DEAE diethylaminoethyl - DTT dithiothreitol - EDTA ethylenediamine-tetraacetic acid - GA_n gibberellin A_n - HPLC high-performance liquid chromatography - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - Tris 2-amino-2-(hydroxymethyl)-1,3-propanediol     

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     

Quantitation of gibberellins A1, A3, A4, A9 and an A9-conjugate in good- and poor-flowering clones of Sitka spruce (Picea sitchensis) during the period of flower-bud differentiation

The levels of endogenous gibberellin A₁ (GA₁), GA₃, GA₄, GA₉ and a cellulase-hydrolysable GA₉-conjugate in needles and shoot stems of Sitka spruce [Picea sitchensis (Bong.) Carr.] grafts with different coning or flowering histories were estimated by combined gas chromatography-mass spectrometry selected ion monitoring using deuterated GA₃, GA₄ and GA₉ as internal standards. The samples were taken at the approximate time of the start of flower-bud differentiation, i.e. when the shoots had elongated approx. 95% of the final length. The needles of the good-flowering clones contained 11–12 ng per g fresh weight (FW) and 15–28 ng· (g FW) ^–1 of GA₉-conjugate and GA₉, respectively. The shoot stems of the same material contained no detectable amounts of GA₉-conjugate and 11–15 ng-(g FW)^–1 of GA₉. The amounts of GA₉-conjugate and GA₉ were apparently lower in the poor-flowering clones, the needles containing 4–9 ng-(g FW)^–1 and 7–17 ng·(g FW)^–1, respectively. Also in this material the shoot stems contained no detectable amounts of GA₉-conjugate. The amounts of GA₄ were very small in both materials, ranging from 1–1.6 ng-(g FW)^–1. The good-flowering clones contained no detectable amounts of the more polar gibberellins, GA₁ and GA₃. The poor-flowering clones, on the other hand, contained high levels of GA₁₅ 17–19ng·(gFW)^–1 in the needles and 10–13 ng·(g FW) ^–1 in the shoot stems, and also smaller amounts of GA₃, 2–3 ng·(g FW)^–1 in the needles and approx. 1 ng·(g FW)^–1 in the shoot stems. The results demonstrate differences in GA-metabolism between the poor- and the good-flowering clones. The higher amounts of GA₉-conjugate and GA₉ might indicate a higher capacity for synthesizing GA₄ in the good-flowering material. This synthesis does not, however, result in a build-up of the GA₄-pool, maybe because of a high rate of turnover. Gibberellin A₄ was apparently neither hydroxylated to GA₁ nor converted to GA₃ in the goodflowering material, as was the case in the poor-flowering material. This might indicate that gibberellin metabolism in the poor-flowering material is directed towards GA₁ and GA₃, GAs preferentially used in vegetative growth.Abbreviations FW fresh weight - GA_n gibberellin A_n - HPLC high-performance liquid chromatography     

Paraquat-resistant lines in Pisum sativum cv. Alaska: biochemical and phenotypic characterization

In plants, the oxygen generated by photosynthesis can be excited to form reactive oxygen species (ROS) under excessive sunlight. Excess ROS including singlet oxygen (¹O₂) inhibit the growth, development and photosynthesis of plants. To isolate ROS-resistant crop plants, we used paraquat (PQ), a generator of O₂ ^·− as a source of screening and mutagen, and obtained two PQ-resistant lines in Pisum sativum, namely R3-1 and R3-2. Both lines showed greater resistance to PQ than their wild type (WT) siblings with respect to germination, root growth, and shoot growth. Biochemical analysis showed differences in these lines, in which ROS-scavenging enzymes undergo changes with a distinguishable increase in Mn-SOD. We further observed that the cytosolic catalases (CATs) in leaves in both lines were shifted in a native-PAGE analysis compared with that of the WT, indicating that the release of bound ¹O₂ was enhanced. Phenotypic analysis revealed distinguishable differences in leaf development, and in flowering time and position. In addition, R3-1 and R3-2 showed shorter individual internode lengths, dwarf plant height, and stronger branching compared with the WT. These results suggested that PQ-induced ROS-resistant Pisum have the potential pleiotropic effects on flowering time and stem branching, and that ROS including ¹O₂ plays not only important roles in plant growth and development as a signal transducer, but also appears as a strong inhibitor for crop yield. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.