Further identification of endogenous gibberellins in the shoots of pea, line g2

To interpret the metabolism of radiolabeled gibberellins A₁₂-aldehyde and A₁₂ in shoots of pea (Pisum sativum L.), the identity of the radiolabeled peaks has to be determined and the endogenous presence of the gibberellins demonstrated. High specific activity [¹⁴C]GA₁₂ and [¹⁴C]GA₁₂-aldehyde were synthesized using a pumpkin endosperm enzyme preparation, and purified by high performance liquid chromatography (HPLC). [¹⁴C]GA₁₂ was supplied to upper shoots of pea, line G2, to produce radiolabeled metabolites on the 13-OH pathway. Endogenous compounds copurifying with the [¹⁴C]GAs on HPLC were analyzed by gas chromatography-mass spectrometry. The endogenous presence of GA₅₃, GA₄₄, GA₁₉ and GA₂₀ was demonstrated and their HPLC peak identity ascertained. The ¹⁴C was progressively diluted in GAs further down the pathway, proportional to the levels found in the tissue and inversely proportional to the speed of metabolism, ranging from 63% in GA₅₃ to 4% in GA₂₀. Calculated levels of GA₂₀, GA₁₉, GA₄₄, and GA₅₃ were 42, 8, 10, and 0.5 nanograms/gram, respectively.     

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.     

The endogenous gibberellins of dwarf mutants of lettuce

The gibberellin (GA) content of E-1, a tall genotype of early flowering lettuce (Lactuca sativa L.), and of three selected GA-responsive dwarfs, dwf1, dwf2, and dwf2¹, has been determined using ¹³C-labeled internal standards and gas chromatographymass spectrometry (GC-MS). In the shoots of the E-1 parent, GA₁, 3-epi-GA₁, GA₃, GA₅, GA₈, GA₁₉, GA₂₀, GA₂₉, and GA₅₃ were identified by full scan GC-MS and Kovats retention indices. Purification by immunoaffinity chromatography selective for 13-hydroxy GAs, was necessary for GA identification. Relative to the parent E-1, the concentrations of GA₁, GA₈, GA₂₀, and GA₂₉ in the shoots of dwf2 plants were reduced to about 10% and in shoots of dwf2¹ plants to less than 50%. In dwf1 the levels of GA₁, GA₈, and GA₂₉ were also reduced to less than 50% of the parent E-1, but the level of GA₂₀ was fivefold higher than in E-1. Plant height was correlated with the endogenous levels of GA₁ and GA₈.     

Biosynthetic Origin of Gibberellins A(3) and A(7) in Cell-Free Preparations from Seeds of Marah macrocarpus and Malus domestica

Cell-free preparations from seeds of Marah macrocarpus L. and Malus domestica L. catalyzed the conversion of gibberellin A₉ (GA₉) and 2,3-dehydroGA₉ to GA₇; GA₉ was also metabolized to GA₄ in a branch pathway. The preparation from Marah seeds also metabolized GA₅ to GA₃ in high yield; GA₆ was a minor product and was not metabolized to GA₃. Using substrates stereospecifically labeled with deuterium, it was shown that the metabolism of GA₅ to GA₃ and of 2,3-dehydroGA₉ to GA₇ occurs with the loss of the 1β-hydrogen. In cultures of Gibberella fujikuroi, mutant B1-41a, [1β,2β-²H₂]GA₄, was metabolized to [1,2-²H₂]GA₃ with the loss of the 1α- and 2α-hydrogens. These results provide further evidence that the biosynthetic origin of GA₃ and GA₇ in higher plants is different from that in the fungus Gibberella fujikuroi.     

Qualitative and Quantitative Analyses of Gibberellins in Vegetative Shoots of Normal, dwarf-1, dwarf-2, dwarf-3, and dwarf-5 Seedlings of Zea mays L

Gibberellins A₁₂ (GA₁₂), GA₅₃, GA₄₄, GA₁₉, GA₁₇, GA₂₀, GA₂₉, GA₁, and GA₈ have been identified from extracts of vegetative shoots of normal (wild type) maize using full scan capillary gas chromatography-mass spectrometry and Kovats retention indices. Seven of these gibberellins (GAs) have been quantified by capillary gas chromatography-selected ion monitoring using internal standards of [¹⁴C₄]GA₅₃, [¹⁴C₄]GA₄₄, [²H₂] GA₁₉, [¹³C₁]GA₂₀, [¹³C₁]GA₂₉, [¹³C₁]GA₁, and [¹³C₁]GA₈. Quantitative data from extracts of normal, dwarf-1, dwarf-2, dwarf-3, and dwarf-5 seedlings support the operation of the early 13-hydroxylation pathway in vegetative shoots of Zea mays. These data support the positions in the pathway blocked by the mutants, previously assigned by bioassay data and metabolic studies. The GA levels in dwarf-2, dwarf-3, and dwarf-5 were equal to, or less than, 2.0 nanograms per 100 grams fresh weight, showing that these mutants are blocked for steps early in the pathway. In dwarf-1, the level of GA₁ was very low (0.23 nanograms per 100 grams fresh weight) and less than 2% of that in normal shoots, while GA₂₀ and GA₂₉ accumulated to levels over 10 times those in normals; these results confirm that the dwarf-1 mutant blocks the conversion of GA₂₀ to GA₁. Since the level of GAs beyond the blocked step for each mutant is greater than zero, each mutated gene probably codes for an altered gene product, thus leading to impaired enzyme activities.     

Gibberellin biosynthesis in cell-free extracts from developing Cucurbita maxima embryos and the identification of new endogenous gibberellins

Gibberellin (GA) biosynthetic pathways from GA₁₂-aldehyde, GA₁₂ and GA₅₃ were investigated in cell-free systems from developing embryos of Cucurbita maxima L. Gibberellin A₁₂-aldehyde and GA₁₂ were converted to GA₂₅, putative 12α-hydroxyGA₂₅, GA₁₃ and GA₃₉ as main products. Minor products were GA₄, GA₃₄ and, when GA₁₂ was the substrate, putative 12α-hydroxyGA₁₂. The intermediates GA₁₅ and GA₂₄ accumulated at low protein concentrations. The influence of various factors on GA₁₂ metabolism was examined. At low 2-oxoglutarate and ascorbate concentrations, or at acid pH, 3β-hydroxylated products predominated, whereas with increasing 2-oxoglutarate and ascorbate concentrations, or at neutral pH, the yield of 12α-hydroxylated GAs increased. Gibberellin A₅₃ was metabolised mainly to the C₂₀-GAs GA₄₄, GA₁₉, GA₁₇, GA₂₃ and GA₂₈, with the C₁₉-GAs GA₂₀, GA₁ and GA₈ as minor products. Only C₁₉-GAs were 2β-hydroxylated, which is a main characteristic of the embryo systems. In addition to GA₁₃, GA₂₅, GA₃₉, GA₄₃, GA₄₉, GA₅₈, GA₇₄, 12α-hydroxyGA₂₅ and GA₃₉ 3-isovalerate, which were known previously from embryos of C. maxima, GA₁, GA₄, GA₁₇, GA₂₈, GA₃₇, GA₃₈, GA₄₈, GA₈₅, 12α-hydroxyGA₃₇ and putative 12α-hydroxyGA₄₃ were identified as endogenous components by full-scan capillary gas chromatography-mass spectrometry and Kovats retention indices. Evidence for putative 2β-hydroxyGA₂₈ and GA₂₃ was also obtained but it was less conclusive because of contamination.     

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).     

Gibberellins in Suspensors of Phaseolus coccineus L. Seeds

Analysis of extracts from 6300 (1.2 grams fresh weight) Phaseolus coccineus suspensors by combined gas chromatography-mass spectrometry has demonstrated the presence of five C₁₉-gibberellins, GA₁, GA₄, GA₅, GA₆, GA₈, and one C₂₀-GA, GA₄₄. The major GAs present were GA₁ and GA₈. Data are discussed in relation to previous results obtained in P. coccineus seed as well as in the embryo-suspensor system.     

Gibberellin A(1) Biosynthesis in Pisum sativum L. : II. Biological and Biochemical Consequences of the le Mutation

A comparative study of the metabolism of radiolabeled gibberellin (GA) 1, 19, and 20 in isolated vegetative tissues of isogenic Le and le pea (Pisum sativum) plants incubated in vitro with the appropriate GA substrate is described. The results of this study provide evidence that the enzymes involved in the latter stages of GA biosynthesis are spatially separated within the growing pea plant. Apical buds were not apparently involved in the production of bioactive GA₁ or its immediate precursors. The primary site of synthesis of GA₂₀ from GA₁₉ was immature leaflets and tendrils, and the synthesis of bioactive GA₁ and its inactive catabolite GA₈ occurred predominantly in stem tissue. GA₂₉, the inactive catabolite of GA₂₀, was produced to varying extents in all the tissues examined. Little or no difference was observed in the ability of corresponding Le and le tissues to metabolize radiolabeled GA₁, GA₁₉, or even GA₂₀. During a fixed period of 24 hours, stems of plants carrying the le mutation produced slightly more [³H]GA₁ (and [³H]GA₂₉) than those of Le plants. It has been concluded that the le mutation does not lie within the gene encoding the GA₂₀ 3β-hydroxylase protein.     

GC/MS analysis of the plant hormones in seeds of Cucurbita maxima

The GC/MS detection is reported of over 30 compounds, in extracts of the endosperm and embryos from seeds of Cucurbita maxima. The compounds which were identified from reference spectra include: cis,trans-ABA; trans,trans-ABA; dihydrophaseic acid; IAA; GA₄; GA₁₂; GA₁₃; GA₂₅; GA₃₉; GA₄₃; GA₄₉; ent-13-hydroxy-, ent-6α,7α-and ent-7α,13-dihydroxy-, and ent-6α,7α,13-trihydroxykaur-16-en-19-oic acids; ent-7α,16,17-trihydroxy- and ent-6α,7α,16,17-tetrahydroxy-kauran-19-oic acids, ent-6,7-seco-7-oxokauren-6,19-dioic acid and/or ent-6,7-secokauren-6,7,19-trioic acid, and 7β,12α-dihydroxykaurenolide. New compounds, the structures of which were deduced from GC/MS data, include: the 12α-hydroxy-derivatives of GA₁₂, GA₁₄, GA₃₇ and GA₄, and the 12β-hydroxy-derivatives of ent-7α-hydroxy- and ent-6α,7α-dihydroxykaurenoic acids.     

Identification of Endogenous Gibberellins in Dormant Bulbils of Chinese Yam, Dioscorea opposita

It is known that dormancy of the genus Dioscorea is induced by application of gibberellin (GA) A₃. To understand the role of GAs in dormancy induction, endogenous GAs have been identified by Kovats retention indices and full mass spectra from capillary gas chromatography-mass spectrometry analysis of purified extract from dormant bulbils of Dioscorea opposita Thunb. These include GA₄, GA₉, GA₁₂, GA₁₉, GA₂₀, GA₂₄, GA₃₆, and GA₅₃; their presence suggests the occurrence of two biosynthetic pathways in D. opposita bulbils, the early 13-hydroxylation pathway and the non-13-hydroxylation pathway.     

Identification of Endogenous Gibberellins in the Winter Annual Weed Thlaspi arvense L

Eleven endogenous gibberellins (GAs) were identified by combined gas chromatography-mass spectrometry in purified extracts from shoots of field pennycress (Thlaspi arvense L.): GA_{1,9,12,15,19,20,24,29,44,51,53}. Traces of GA₈ and GA₂₅ were tentatively indicated by combined gas chromatography-mass spectrometry-selected ion monitoring. Comparison of the total ion current traces indicated that GA₁₉ and GA₄₄ were most abundant, while GA_{12,15,20,24,29,53} occurred in lesser amounts. Only small amounts of GA_1,9,51 were present. The levels of GA₈ and GA₂₅ were barely detectable. Consideration of hydroxylation patterns of the ent-gibberellane ring structure indicates two families of GAs: one with a C-13 hydroxyl group (GA_{1,8,19,20,29,44,53}) and another whose members are either nonhydroxylated (GA_{9,12,15,24,25}) or lack a C-13 hydroxyl group (GA₅₁). This suggests that in field pennycress there are two parallel pathways for GA metabolism with an early branch point from GA₁₂: an early C-13 hydroxylation pathway, leading ultimately to GA₁ and GA₈ and a C-13 deoxy pathway culminating in the formation of GA₉ and GA₅₁.     

Identification of Ten Gibberellins from Sporophytes of the Tree fern, Cyathea australis

Ten gibberellins (GAs) have been identified by Kovats retention indices and full mass spectra from GC-MS analysis of purified extracts of sporophytes of the tree-fern, Cyathea australis. These include the known GA₁, GA₄, GA₉, GA₁₅, GA₂₄, GA₃₅, and GA₅₈ and three new GAs, 12β-hydroxyGA₉ (GA₆₉), 12α-hydroxyGA₉ (GA₇₀) and 12β-hydroxyGA₄ (GA₇₁). The structure of GA₇₁ was established by the preparation and characterization of its methyl ester (as a metabolite of GA₄ methyl ester in a culture of prothallia of Lygodium japonicum).     

Shoot elongation in Lathyrus odoratus L.: Gibberellin levels in light- and dark-grown tall and dwarf seedlings

The levels of gibberellin A₁ (GA₁), GA₂₀, GA₁₉, GA₈, GA₂₉ and GA₈₁ (2-epiGA₂₉) were measured in tall (L-) and dwarf (ll) sweet-pea plants grown in darkness and in light. In both environments the apical portions of dwarf plants contained less GA₁; GA₈ and GA₁₉, but more GA₂₀, GA₂₉, and GA₈₁ than did those of tall plants. It is concluded that the partial block in 3β-hydroxylation of GA₂₀ to GA₁ is imposed by allele l in darkness as well as in the light. Furthermore, darkness does not appear to enhance elongation in sweet pea by increasing GA₁ levels. The reduction of the pool size of GA₁₉ in dwarf plants supports recent theories on the regulation of GA biosynthesis, formulated on the basis of observations in monocotyledonous species. Darkness results in decreased GA₂₀, GA₂₉, and GA₈₁ levels in the apical portions of tall and dwarf plants and possible reasons for this are discussed.     

Endogenous Gibberellins from Sporophytes of Two Tree Ferns, Cibotium glaucum and Dicksonia antarctica

Gibberellin A₁ (GA₁), 3-epi-GA₁, GA₄, GA₉, 11α-hydroxyGA₁₂, 12α-hydroxyGA₁₂, GA₁₅, GA₁₇, GA₁₉, GA₂₀, GA₂₅, GA₃₇, GA₄₀, GA₅₈, GA₆₉, GA₇₀, and GA₇₁ have been identified from Kovats retention indices and full scan mass spectra by capillary GC-MS analyses of purified extracts from sporophytes of the tree fern, Cibotium glaucum. Abscisic acid, dihydrophaseic acid, an epimer of 4′-dihydrophaseic acid, and the epimeric ent-6α, 7α, 16α, 17-(OH)₄ and ent-6α, 7α, 16β, 17-(OH)₄ derivatives of ent16, 17-dihydrokaurenoic acid, in addition to the epimeric 16α, 17- and 16β, 17-dihydroxy-16, 17-dihydro derivatives of GA₁₂, were also identified in extracts of C. glaucum. An oxodihydrophaseic acid and a hydroxydihydrophaseic acid were also detected. In extracts of sporophytes of Dicksonia antarctica, GA₄, GA₉, 12α- and 12β-hydroxyGA₁₂, GA₁₅, GA₂₅, and GA₃₇ were identified by the same criteria, as well as abscisic acid, phaseic acid, 8′-hydroxymethylabscisic acid and dihydrophaseic acid. This is the first time that GA₄₀ has been identified in a higher plant; it is also the first report of the natural occurrence of the two gibberellins, 11α- and 12β-hydroxyGA₁₂. The total gibberellin (GA) content in C. glaucum (tall) was at least one order of magnitude greater than that of D. antarctica (dwarf) based on total ion current response in GC-MS and bioassay data. Abscisic acid was a major component of D. antarctica and the oxodihydrophaseic acid was a major component of C. glaucum.     

HPLC-based methods for the identification of gibberellin conjugates: Metabolism of [3H]gibberellin A4 in seedlings of Phaseolus coccineus

A series of chromatographic and derivatization techniques has been developed for the identification of radiolabelled gibberellin (GA) conjugates. The methods are based on reversed-phase HPLC, gel permeation chromatography, anion-exchange chromatography, enzymatic hydrolysis and transesterification of conjugates, and derivatization of free GAs to methoxycoumaryl esters. The procedures have been used to identify GA₄-glucosyl ester, GA₄-3-O-glucoside, a GA₃₄-O-glucoside and GA₈-2-O-glucoside, in addition to GA₁ and GA₈, as products of [1,2-³H]GA₄ metabolism in shoots of light-grown Phaseolus coccineus seedlings.     

A Gibberellin-Deficient Brassica Mutant-rosette

A single-gene mutant (rosette [ros/ros]) in which shoot growth and development are inhibited was identified from a rapid cycling line of Brassica rapa (syn campestris). Relative to normal plants, the mutant germinated slowly, had delayed or incomplete floral development, and reduced leaf, petiole, and internode growth. The exogenous application of GA₃ by foliar spray or directly to the shoot tip of rosette resulted in rapid flowering, bolting (shoot elongation), and viable seed production. Shoots of rosette contained endogenous levels of total gibberellin (GA)-like substances (`Tan-ginbozu' dwarf rice assay) of about one-tenth of that of the normal rapid-cycling line of B. rapa which consisted almost entirely of a very nonpolar, GA-like substance which yielded GA₁ and GA₃ upon mild acid hydrolysis. In a normal rapid-cycling B. rapa line, the nonpolar putative GA₁ and GA₃ conjugates were present, but additionally, free GA₁ and GA₃ were abundant and identified by gas chromatography-mass spectrometry-selected ion monitoring. The quantities of free GA₁ and GA₃ in the normal line and in rosette were quantified by GC-MS-SIM using [²H₂]GA₁ as an internal standard. Fourteen-day-old rosette and normal seedlings contained 5.3 and 23.2 ng GA₁ per plant, respectively. At day 21 the rosette plants contained 7.7 and 26.1 nanograms per plant of GA₁ and GA₃, while normal plants contained 31.1 and 251.5 nanograms per plant, respectively. Thus, normal plants contained from four to ten times higher levels of total GA-like substances, GA₁, or GA₃, than rosette. The ros allele results in reduced GA level, yielding the rosette phenotype whose delayed germination and flowering, and reduced shoot growth responses indicate a probable role for endogenous GA₁ and GA₃ in the regulation of these processes in Brassica.     

Use of an Acylcyclohexanedione Growth Retardant, LAB 198 999, to Determine Whether Gibberellin A(20) Has Biological Activity per se in Dark-Grown Dwarf (le) Seedlings of Pisum sativum

Dwarf (le⁵⁸³⁹) seedlings of Pisum sativum respond to gibberellin A₂₀ (GA₂₀) in the dark, although the same dosage of GA₂₀ applied to light-grown le⁵⁸³⁹ seedlings elicits no growth response. The acylcyclohexanedione growth retardant, LAB 198 999, which is known to inhibit gibberellin oxidation and in particular 3β-hydroxylation such as the conversion of GA₂₀ to GA₁, also inhibits the growth response of dark-grown dwarf (le⁵⁸³⁹) seedlings to GA₂₀. Thus, the biological activity of GA₂₀ in the dark appears to be a consequence of its conversion to GA₁, even though it is known from studies with light-grown seedlings that the le mutation reduces the conversion of GA₂₀ to GA₁.     

Biosynthesis of gibberellins A12, A15, A24, A36, and A37 by a cell-free system from Cucurbita maxima

GA₁₂-aldehyde obtained from mevalonate via ent-kaurene, ent-kaurenol, ent-kaurenoic acid and ent-7α-hydroxykaurenoic acid in a cell-free system from immature seeds of Cucurbita maxima was converted to GA₁₂ by the same system. When Mn²⁺ was omitted from the system GA₁₂-aldehyde and GA₁₂ were converted further to several products. Among these GA₁₅, GA₂₄, GA₃₆ and GA₃₇ were conclusively identified by GC-MS. With the exception of GA₃₇ these GAs have not previously been found in higher plants. Another biosynthetic pathway led from ent-7α-hydroxykaurenoic acid to very polar products via what was tentatively identified as ent-6α, 7α-dihydroxykaurenoic acid. An unidentified component with an MS resembling that of a dihydroxykaurenolide was also obtained from incubations with mevalonate.