Accumulation of Gibberellin A1 and the Metabolism of Gibberellin A9 to Gibberellin A1 in a Phaeosphaeria sp. L487 Culture

Gibberellin A₁ (GA₁), which was identified as the major GA from the GA-producing fungus Phaeosphaeria sp. L487, was accumulated in the culture with a maltose-yeast extract medium, its amount in the culture filtrate being about 50 mg per liter after a 3-week culture. The new fungal biosynthetic pathway to GA₁ from GA₉ via GA₄ was elucidated by feeding experiments with synthetic [17-²H₂]GA₉ and [17-²H₂]GA₄.     

Characterization of novel mutants with an altered gibberellin spectrum in comparison to different wild-type strains of Fusarium fujikuroi

The rice pathogen Fusarium fujikuroi is known for producing a wide range of secondary metabolites such as pigments, mycotoxins, and a group of phytohormones, the gibberellic acids (GAs). Bioactive forms of these diterpenes are responsible for hyperelongation of rice stems, yellowish chlorotic leaves, and reduced grain formation during the bakanae disease leading to severely decreased crop yields. GAs are also successfully applied in agriculture and horticulture as plant growth regulators to enhance crop yields, fruit size, and to induce earlier flowering. In this study, six F. fujikuroi wild-type and mutant strains differing in GA yields and the spectrum of produced GAs were cultivated in high-quality lab fermenters for optimal temperature and pH control and compared regarding their growth, GA production, and GA gene expression levels. Comparative analysis of the six strains revealed that strain 6314/ΔDES/ΔPPT1, holding mutations in two GA biosynthetic genes and an additional deletion of the 4'-phosphopantetheinyl transferase gene PPT1, exhibits the highest total GA amount. Expression studies of two GA biosynthesis genes, CPS/KS and DES, showed a constantly high expression level for both genes under production conditions (nitrogen limitation) in all strains. By cultivating these genetically engineered mutant strains, we were able to produce not only mixtures of different bioactive GAs (GA₃, GA_4, and GA₇) but also pure GA₄ or GA₇. In addition, we show that the GA yields are not only determined by different production rates, but also by different decomposition rates of the end products GA₃, GA₄, and GA₇ explaining the varying GA levels of genetically almost identical mutant strains.     

<Emphasis Type="Italic">Chrysosporium pseudomerdarium</Emphasis> produces gibberellins and promotes plant growth

We isolated 10 endophytic fungi from the roots of drought stressed soybean cultivar Hwangkeumkong and bioassyed on waito-c rice and soybean seedlings, in order to identify plant growth-promoting fungi. The fungal isolate D-2-1 provided the best result for plant height and biomass promotion as compared to wild type Gibberella fujikuroi. The D-2-1 culture filtrate (CF) was analyzed for the presence of gibberellins (GAs) and it was observed that all physiologically active GAs, especially gibberellic acid, were present in higher amounts (GA₁, 0.24 ng/ml; GA₃, 8.99 ng/ml; GA₄, 2.58 ng/ml and GA₇, 1.39 ng/ml) in conjunction with physiologically inactive GA₅, GA₉, GA₁₅, GA₁₉, and GA₂₄. The fungal isolate D-2-1 was identified as a new strain of Chrysosporium pseudomerdarium through phylogenetic analysis of 18S rDNA sequence. Plant growth promotion and GAs production capacity of genus Chrysosporium have been reported for the first time in this study.     

Restoration of Gibberellin Production in Fusarium proliferatum by Functional Complementation of Enzymatic Blocks

Nine biological species, or mating populations (MPs), denoted by letters A to I, and at least 29 anamorphic Fusarium species have been identified within the Gibberella fujikuroi species complex. Members of this species complex are the only species of the genus Fusarium that contain the gibberellin (GA) biosynthetic gene cluster or at least parts of it. However, the ability of fusaria to produce GAs is so far restricted to Fusarium fujikuroi, although at least six other MPs contain all the genes of the GA biosynthetic gene cluster. Members of Fusarium proliferatum, the closest related species, have lost the ability to produce GAs as a result of the accumulation of several mutations in the coding and 5′ noncoding regions of genes P450-4 and P450-1, both encoding cytochrome P450 monooxygenases, resulting in metabolic blocks at the early stages of GA biosynthesis. In this study, we have determined additional enzymatic blocks at the first specific steps in the GA biosynthesis pathway of F. proliferatum: the synthesis of geranylgeranyl diphosphate and the synthesis of ent-kaurene. Complementation of these enzymatic blocks by transferring the corresponding genes from GA-producing F. fujikuroi to F. proliferatum resulted in the restoration of GA production. We discuss the reasons for Fusarium species outside the G. fujikuroi species complex having no GA biosynthetic genes, whereas species distantly related to Fusarium, e.g., Sphaceloma spp. and Phaeosphaeria spp., produce GAs.     

Qualitative and Quantitative Analysis of Endogenous Gibberellins in Onion Plants and Their Effects on Bulb Development

Evidence has been reported that bulb development in onion plants (Allium cepa L.) is controlled by endogenous bulbing and anti-bulbing hormones, and that gibberellin (GA) is a candidate for anti-bulbing hormone (ABH). In this study, we identified a series of C-13-H GAs (GA₁₂, GA₁₅, GA₂₄, GA₉, GA₄, GA₃₄, and 3-epi-GA₄) and a series of C-13-OH GAs (GA₄₄, GA₂₀, GA₁ and GA₈) from the leaf sheaths including the lower part of leaf blades of onion plants (cv. Senshu-Chuko). These results suggested that two independent GA biosynthetic pathways, the early-non-hydroxylation pathway to GA₄ (active GA) and early-13-hydroxylation pathway to GA₁ (active GA), exist in onion plants. It was also suggested that GA₄ and GA₁ have almost the same ability to inhibit bulb development in onion plants induced by treatment with an inhibitor of GA biosynthesis, uniconazole-P. The endogenous levels of GA₁ and GA₄, and their direct precursors, GA₂₀ and GA₉, in leaf blades, leaf sheaths, and roots of 4-week-old bulbing and non-bulbing onion plants were measured by gas chromatography/selected ion monitoring with the corresponding [²H]labeled GAs as internal standards. In most cases, the GA levels in long-day (LD)-grown bulbing onion plants were higher than those of short-day (SD)-grown non-bulbing onion plants, but the GA₁ level in leaf blades of SD-grown onion plants was rather higher than that of LD-grown onion plants. Relationship between the endogenous GAs and bulb development in onion plants is discussed.     

Immunoaffinity techniques applied to the purification of gibberellins from plant extracts

The use of immunoaffinity columns containing anti-gibberellin (GA) antibodies for the selective purification of GAs in plant extracts is described. GA₁, GA₃, GA₄, GA₅, GA₇, and GA₉ conjugates to bovine serum albumin were synthesized and used to elicit anti-GA polyclonal antibodies (Abs) in rabbits. Protein A purified rabbit serum, containing a mixture of anti-GA Abs, was immobilized on matrices of Affi-gel 10 or Fast-Flow Sepharose 4B. Columns of these immunosorbents retained a wide range of C-19 GA methyl esters, but no C-20 GA methyl esters. Quantitative recovery of C-19 GA methyl esters was achieved from the columns, which, after reequilibration in buffer, could be reused up to 500 times. The immunosorbents were tested by examination of extracts from immature soybean and pea seeds. GAs were initially purified by passing the extracts through DEAE-cellulose and concentrating them on octadecylsilica. The extracts were methylated and further purified on the mixed anti-GA immunoaffinity columns. GAs were detected and quantified as methyl esters or methyl ester trimethylsilyl ethers by gas chromatography-mass spectrometry-selected ion monitoring. GA₇ was found in soybean seeds, 17 days after anthesis, at low levels (8.8 nanograms per gram fresh weight). C-19 GAs were examined in cotyledons, embryonic axes, and testae of G2 pea seeds harvested 20 days after anthesis. High levels of GA₂₀ and GA₂₉ were found in cotyledons (3580 and 310 nanograms per gram fresh weight, respectively) and embryonic axes (5375 and 1430 nanograms per gram) fresh weight, respectively). Lower levels of GA₉ were found in cotyledons and embryonic axes (147 and 161 nanograms per gram fresh weight, respectively). GA₉ was the major GA of testae at levels of 195 nanograms per gram fresh weight. Trace quantities of GA₂₀ and GA₅₁ were also observed in testae.     

Endophytic Cephalotheca sulfurea AGH07 reprograms soybean to higher growth

Abstract

Gibberellins (GAs) are well known for plant growth promotion. GAs production by fungi has received little attention, although substantial work has been carried out on other aspects of plant growth-promoting fungi (PGPF). We investigated GAs production and plant growth-promoting capacity of an endophytic fungus isolated from the roots of soil grown soybean plants. The endophytic fungus is reported as GAs producer and as PGPF for the first time in this study. Nine endophytic isolates were collected from the roots of soybean, and culture filtrates (CFs) obtained from their pure cultures were screened on Waito-C, a dwarf rice cultivar, for the presence of GAs. Of these, seven fungal isolates promoted shoot length as compared to control (distilled water), while one inhibited it. Three fungal isolates were selected on the basis of higher shoot elongation as compared to wild type Gibberella fujikuroi, which was used as positive control. The growth-prompting capacity of selected fungal isolates SB5-1, SB3-2, and SB3-3 was bio-assayed on soybean cv. Hwangkeumkong. Fungal isolate SB5-1 provided maximum plant height (31.6 cm), shoot length (21.1 cm), whole plant fresh biomass (2.41 g), shoot fresh biomass (1.99 g), and leaf area (24.37 cm²). The CF of isolate SB5-1 was analyzed for the presence of GAs, and it was found that all physiologically active GAs were present (GA₁, 0.15 ng/ml, GA₃, 1.2 ng/ml, GA₄, 7.37 ng/ml, and GA₇, 3.18 ng/ml) in conjunction with physiologically inactive GA₅, GA₉, GA₁₅, GA₁₉, GA₂₀, and GA₂₄. The fungal isolate SB5-1 was identified as a new strain of Cephalotheca sulfurea through molecular and phylogenetic approaches.     

Gibberellins in Leaves of Alstroemeria hybrida: Identification and Quantification in Relation to Leaf Age

In alstroemeria (Alstroemeria hybrida), leaf senescence is retarded effectively by the application of gibberellins (GAs). To study the role of endogenous GAs in leaf senescence, the GA content was analyzed by combined gas chromatography and mass spectrometry. Five 13-hydroxy GAs (GA₁₉, GA₂₀, GA₁, GA₈, and GA₂₉) and three non-13-hydroxy GAs (GA₉ and GA₄) were identified in leaf extracts by comparing Kováts retention indices (KRIs) and full scan mass sprectra with those of reference GAs. In addition, GA₁₅, GA₄₄, GA₂₄, and GA₃₄ were tentatively identified by comparing selected ion monitoring results and KRIs with those of reference GAs. A number of GAs were detected in conjugated form as well. Concentrations of GAs in alstroemeria changed with the development of leaves. The proportion of biologically active GA₁ and GA₄ decreased with progressive senescence and the fraction of conjugated GAs increased. Received May 26, 1997; accepted August 12, 1997     

Identification of endogenous gibberellins, and metabolism of tritiated and deuterated GA4, GA9 and GA20, in Scots pine (Pinus sylvestris) shoots

The application of gibberellin A_4/7 (GA_4/7) to the stem of previous-year (1-year-old) terminal shoots of Scots pine (Pinus sylvestris) seedlings has been observed to stimulate cambial growth locally, as well as at a distance in the distal current-year terminal shoot, but the distribution and metabolic fate of the applied GA_4/7, as well as the pathway of endogenous GA biosynthesis in this species, has not been investigated. As a first step, we analysed for endogenous GAs and monitored the transport and metabolism of labelled GAs 4, 9 and 20. Endogenous GAs from the elongating current-year terminal shoot of 2-year-old seedlings were purified by column chromatography and high-performance liquid chromatography and analysed by combined gas chromatography-mass spectrometry (GC-MS). GAs 1, 3, 4, 9, 12 and 20 were identified in the stem, and GAs 1, 3 and 4 in the needles, by full-scan mass spectrometry (GAs 1, 3, 4, 9 and 12) or selected-ion monitoring (GA₂₀) and Kovats retention index. Tritiated and deuterated GA₄, GA₉ or GA₂₀ were applied around the circumference at the midpoint of the previous-year terminal shoot, and metabolites were extracted from the elongating current-year terminal shoot, the application point, and the 1-year-old needles and the cambial region above and below the application point. After purification, detection by liquid scintillation spectrometry and analysis by GC-MS, it was evident that, for each applied GA, unmetabolised [²H₂]GA and [³H]radioactivity were present in every seedling part analysed. Most of the radioactivity was retained at the application point when [³H]GA₉ and [³H]GA₂₀ were applied, whereas the largest percentage of radioactivity derived from [³H]GA₄ was recovered in the current-year terminal shoot. It was also found that [²H₂]GA₉ was converted to [²H₂]GA₂₀ and to both [²H₂]GA₄ and [²H₂]GA₁, [²H₂]GA₄ was metabolised to [²H₂]GA₁, and [²H₂]GA₂₀ was converted to [²H₂]GA₂₉. The data indicate that for Pinus sylvestris shoots (1) GAs applied laterally to the outside of the vascular system of previous-year shoots not only are absorbed and translocated extensively throughout the previous-year and current-year shoots, but also are readily metabolised, (2) the GA metabolic pathways found are closely related to the endogenous GAs identified, and (3) GA₉ metabolism follows two distinctly different routes: in one, GA₉ is converted to GA₁ through GA₄, and in the other it is converted to GA₂₀, which is then metabolised to GA₂₉. The results suggest that the late 13-hydroxylation pathway is an important route for GA biosynthesis in shoots of Pinus sylvestris, and that the stimulation of cambial growth in Scots pine by exogenous GA_4/7 may be due to its conversion to GA₁, rather than to it being active per se.     

The role of gibberellin biosynthesis in the control of growth and flowering in Matthiola incana

Recently, it was found that stem elongation and flowering of stock Matthiola incana (L.) R. Br. are promoted by exogenous gibberellins (GAs), including GA₄, and also by acylcyclohexanedione inhibitors of GA biosynthesis, such as prohexadione‐calcium (PCa) and trinexapac‐ethyl (TNE). Here, because it was unclear how GA biosynthetic inhibitors could promote stem elongation and flowering, their effect on GA biosynthesis has been examined by quantifying endogenous GA levels; also, the sensitivity of stem elongation and flowering to various GAs in combination with the inhibitors was examined. Stem elongation and flowering were most effectively promoted by GA₄ when combined with PCa and, next in order, by 2,2‐dimethyl‐GA₄, PCa, GA₄+TNE, TNE, GA₉+PCa and by GA₄. There was little or no promotion by GA₁, GA₃, GA₉, GA₁₃, GA₂₀ and 3‐epi‐2,2‐dimethyl‐GA₄. Both the promotive effects of the acylcyclohexanediones on stem elongation and flowering, particularly when applied with GA₄, and the fact that TNE caused a build‐up of endogenous GA₄ imply that one effect of TNE at the lower dose involved an inhibition of 2β‐hydroxylation of GA₄ rather than an inhibition of 20‐oxidation and 3β‐hydroxylation of GAs which were precursors of GA₄. Overall, these results indicate that: (1) GAs with 3β‐OH and without 13‐OH groups (e.g. GA₄) are the most important for stem elongation and flowering in M. incana; (2) growth promotion rather than inhibition can result if an acylcyclohexanedione acts predominantly to slow 2β‐hydroxylation and so slows inactivation of active gibbberellins, including GA₄. It follows that a low dose of an acylcyclohexanedione can be a ‘growth enhancer’ for any applied GA that is liable to inactivation by 2β‐hydroxylation.     

Gibberellins in seeds of Arabidopsis thaliana: biological activities,identification and effects of light and chilling on endogenous levels

The activities of several gibberellins in stimulating germination of wild-type and GA-deficient gal seeds of Arabidopsis thaliana were compared. Of the six compounds tested GA₄ and GA₇-isolactone had the highest activity and GA₇ and GA₉ the lowest; activities of GA₁ and GA₃ were intermediate. Combined application of pure GAs presented no indications that more than one GA receptor is involved. Four GAs were identified in extracts from wild-type and GA-insensitive gai seeds by combined gas chromatography mass spectrometry: GA₁, GA₃, GA₄ and GA₉. Effects of light and chilling on levels of GA₁, GA₄ and GA₉ were studied using deuterated standards. Light increased both GA levels and germination in unchilled wild-type and gai seeds. As a result of irradiation GA levels in gai seeds were 7–10 times as high as in wild-type seeds. In the dark germination was 0%, in the light 14% of gai seeds and 95% of wild-type seeds germinated. A chilling pre-treatment of 7 days at 2°C was required to enhance further the germination of gai seeds in the light. Light did not increase GA levels of chilled seeds of either genotype; levels of GA₄ and GA₉ of chilled gai seeds, in the light were respectively 7 and 12 times lower than in non-chilled seeds, whereas the latter seeds germinated better. Slightly elevated levels of GA₄ were detected in darkness after chilling, but germination capacity was still 0%. These results strengthened the conclusion that GAs are required for germination of A. thaliana seeds, whereby GA₄ has intrinsic biological activity. However, it is unlikely that light and chilling stimulate germination primarily by increasing levels of GA. Instead GA sensitivity is a possible alternative.     

Exophiala sp. LHL08 reprograms Cucumis sativus to higher growth under abiotic stresses

Endophytic fungi are potential sources of secondary metabolites; however, they are little known for phytohormones secretion and amelioration of plant growth under abiotic stresses. We isolated a novel endophyte from the roots of Cucumis sativus and identified it as a strain of Exophiala sp. by sequencing internal transcribed spacer/large subunit rDNA and phylogenetic analysis. Prior to identification, culture filtrate (CF) of Exophiala sp. has shown significant growth promotion of Waito‐C [a gibberellins (GAs)‐deficient mutant cultivar] and Dongjin‐byeo (normal GAs biosynthesis cultivar) rice seedlings. CF analysis of Exophiala sp. showed the presence of physiologically active GAs (GA₁, GA₃, GA₄ and GA₇) and inactive GAs (GA₅, GA₈, GA₉, GA₁₂ and GA₂₀). Exophiala sp. had higher GAs in its CF than wild‐type strain of Gibberella fujikuroi except GA₃. Influence of Exophiala sp. was assessed on cucumber plant's growth and endogenous abscisic acid (ABA), salicylic acid (SA) and bioactive GAs under salinity and drought stresses. Exophiala sp.‐treated plants have shown significantly higher growth and rescued the host plants from stress promulgated water deficit, osmotic and cellular damage. The altered levels of stress‐responsive ABA showed low level of stress confined to endophyte‐applied plants than control. Elevated levels of SA and bioactive GAs (GA₃ and GA₄) in endophyte‐associated plants suggest stress‐modulating response toward salinity and drought. In conclusion, symbiotic relations between Exophiala and cucumber have reprogrammed the host plant growth under abiotic stresses, thus indicating a possible threshold role of endophytic fungi in stress alleviation. This study could be extended for improving agricultural productivity under extreme environmental conditions.     

Microbial Production of Plant Gibberellins and Related Compounds from ent-Kaurene Derivatives in Gibberella fujikuroi

ent-15α-Hydroxykaurenoic acid (8) was synthesized and fed to a mycelium suspension of Gibberella fujikuroi in the presence of 1-n-decylimidazole, a gibberellin biosynthesis inhibitor. The metabolites included 15β-hydroxy GA₂₄, GA₄₅ (GA of Pyrus communis), 15β-hydroxy GA₁₅ and 15β-hydroxy GA₂₅. Microbial production of 12α-hydroxy GAs from ent-12β-hydroxykaurene is also described.     

Gibberellin Metabolism and Transport During Germination and Young Seedling Growth of Pea (<Emphasis Type="Italic">Pisum sativum</Emphasis> L.)

The role of gibberellins (GAs) during germination and early seedling growth is examined by following the metabolism and transport of radiolabeled GAs in cotyledon, shoot, and root tissues of pea (Pisum sativum L.) using an aseptic culture system. Mature pea seeds have significant endogenous GA₂₀ levels that fall during germination and early seedling growth, a period when the seedling develops the capacity to transport GA₂₀ from the cotyledon to the shoot and root of the seedling. Even though cotyledons at 0–2 days after imbibition have appreciable amounts of GA₂₀, the cotyledons retain the ability to metabolize labeled GA₁₉ to GA₂₀ and express significant levels of PsGA20ox2 message (which encodes a GA biosynthesis enzyme, GA 20-oxidase). The large pool of cotyledonary GA₂₀ likely provides substrate for GA₁ synthesis in the cotyledons during germination, as well as for shoots and roots during early seedling growth. The shoots and roots express GA metabolism genes (PsGA3ox genes which encode GA 3-oxidases for synthesis of bioactive GA₁, and PsGA2ox genes which encode GA 2-oxidases for deactivation of GAs to GA₂₉ and GA₈), and they develop the capacity to metabolize GAs as necessary for seedling establishment. Auxins also show an interesting pattern during early seedling growth, with higher levels of 4-chloro-indole-3-acetic acid (4-Cl-IAA) in mature seeds and higher levels of indole-3-acetic acid (IAA) in young root and shoot tissues. This suggests a changing role for auxins during early seedling development.     

Changes in levels of gibberellins,their putative conjugates,and ABA in zygotic embryos during late maturation and dry seed stages of <Emphasis Type="Italic">Brassica napus</Emphasis> cv Topaz

Two late stages [days 35 and 40 after pollination (DAP)] in zygotic embryo (ZE) development of Brassica napus were utilized to quantify, by the stable isotope-labeled dilution method, levels of “free” and “aglycone” gibberellins (GAs), as well as abscisic acid (ABA), during the programmed dehydration of the seed. GAs from both the early 13 hydroxylation and early non-hydroxylation pathways were present in these ZEs of B napus. Between 35 and 40 DAP endogenous ABA dropped precipitously (almost 30-fold) and this drop in ABA was accompanied by a significant reduction in levels of GA₁ and even in levels of the inactive GA catabolites, GA₈ and GA₂₉. Levels of GA₄ and putative GA₈₅ also dropped appreciably, though not significantly. In contrast, the levels of GA₂₀ and GA₉ (the immediate precursors of GA₁ and GA₄, respectively) did not change in the ZEs during this transition. A fungal-derived cellulase was used to hydrolyze the highly water-soluble fraction, which will contain GA conjugates. Relatively high levels of several GAs (GA₉, GA₂₀) were thus quantified after hydrolysis as the aglycones, e.g., 56 and 25 ng/g DW of GA₂₀ and 23 and 5 ng/g DW, of GA₉, respectively at DAP 35 and DAP 40. Other GAs found after hydrolysis of the highly water-soluble fraction remained relatively constant between 35 and 40 DAP. An exception was the putative GA₈₅ aglycone, which increased sixfold (free GA₈₅ decreased by ca. half). The transition to the dry seed stage for ZEs of B. napus is thus accompanied not only by the expected reduction in ABA, but also by reduced levels of many “free” GAs, especially the bioactive, 3β-hydroxylated GAs. In contrast, levels of 3-deoxy GAs remain relatively high, implying a partial block in the 3β-hydroxylation “activation” step of GA biosynthesis.     

Gibberellin biosynthesis and gibberellin oxidase activities in Fusarium sacchari,Fusarium konzum and Fusarium subglutinans strains

Several isolates of three Fusarium species associated with the Gibberella fujikuroi species complex were characterized for their ability to synthesize gibberellins (GAs): Fusarium sacchari (mating population B), Fusarium konzum (mating population I) and Fusarium subglutinans (mating population E). Of these, F. sacchari is phylogenetically related to Fusarium fujikuroi and is grouped in the Asian clade of the complex, while F. konzum and F. subglutinans are only distantly related to Fusarium fujikuroi and belong to the American clade. Variability was found between the different F. sacchari strains tested. Five isolates (B-12756; B-1732, B-7610, B-1721 and B-1797) were active in GA biosynthesis and accumulated GA₃ in the culture fluid (2.76–28.4 μg/mL), while two others (B-3828 and B-1725) were inactive. GA₃ levels in strain B-12756 increased by 2.9 times upon complementation with ggs2 and cps-ks genes from F. fujikuroi. Of six F. konzum isolates tested, three (I-10653; I-11616; I-11893) synthesized GAs, mainly GA₁, at a low level (less than 0.1 μg/mL). Non-producing F. konzum strains contained no GA oxidase activities as found for the two F. subglutinans strains tested. These results indicate that the ability to produce GAs is present in other species of the G. fujikuroi complex beside F. fujikuroi, but might differ significantly in different isolates of the same species.     

Gibberellins in shoots of Hordeum vulgare. A comparison between cv. Triumph and two dwarf mutants which differ in their response to gibberellin

The gibberellin (GA) content of barley (Hordeum vulgare L.) cv. Triumph was analysed by full scan gas chromatography-mass spectrometry. Developing grain contained several di-, tri-, and tetra-hydroxylated GAs, with the most abundant ones being hydroxylated at C-2, C-3, C-12β, and/or C-18. In contrast, the only GAs to be detected in shoots of 9-day old dark- and light-grown seedlings of Triumph were 13-hydroxylated C₁₉-GAs, namely GA₁, GA₈, GA₂₀, and GA₂₉, (all of which are components of the early 13-hydroxylation GA biosynthetic pathway) and GA₃. Feeds of [¹³C.³H₂GA₂₀, confirmed that GA₂₀ is a precursor of GA₁, GA₈, and GA₂₉ in barley shoots. From these results it is suggested that stem growth of barley, in common with that of several other mono- and dicotyledons, is controlled by GA,. Homozygous gal and gal lines were obtained after backcrossing to Triumph. These were then compared to Triumph with respect to their GA content and response to applied GAs and GA precursors. Shoots of the homozygous gal gal plants contained ca 6-fold less GA₁, than Triumph. These plants responded to all ent-kaurenoids and 13-hydroxylated C₂₀- and C₁₉-GAs tested. It is concluded that the gal locus impairs the GA biosynthetic pathway prior to ent-kaurene, most probably at ent-kaurene synthetase. In contrast, shoots of homozygous gal gal line contained ca 10-fold higher levels of GA, than Triumph, but failed to respond to applied GA, or GA₃. The gal locus therefore confers insensitivity to both exogenous and endogenous GAs, possibly by perturbing the reception or transduction of the GA₁ signal.     

Gibberellin content of immature apple seeds from paclobutrazol-treated trees over three seasons

Seeds from heavily fruiting (on-year), mature untreated, and paclobutrazol-treated apple trees (Malus domestica Borkh. cv. Spartan) were sampled in mid-June 1987, mid-July 1987, and mid-July 1990. After seeds were freeze-dried, gibberellins (GAs) were extracted, purified, and fractionated via C₁₈ reversed-phase high-performance liquid chromatography (HPLC). Nine GAs (GA₁, GA₃, GA₄, GA₇, GA₈, GA₉, GA₁₉, GA₂₀, and GA₅₃) were quantified by the use of deuterated GA internal standards. Paclobutrazol trunk drench treatments reduced vegetative shoot elongation in the seasons that seeds were sampled by 55% or more. Between June 17, 1987 and July 15, 1987, the dry weight of seeds from both untreated and treated trees increased about 2.5 times and there were reductions, on a per seed basis, of GA₄ in seeds from both untreated and treated trees, of GA₇ in seeds from treated trees, and of GA₉ in seeds from untreated trees. However, GA₉ increased in seeds from treated trees. Changes in levels of some of the early-13-hydroxylation pathway GAs (GA₁₅ GA₃, GA₈, GA₁₉, GA₂₀, and GA₅₃) also occurred during the month. For mid-July harvested seeds, the pattern, with some exceptions, was that 2 years after paclobutrazol treatment (1987), levels of early-13-hydroxylation pathway GAs in seeds from treated trees were lower compared to controls but after 5 years (1990) their levels tended to increase. For the non-13-hydroxylated GAs (GA₄, GA₇, and GA₉), 2 years after paclobutrazol treatment, GA₄ levels were equal in seeds from untreated and treated trees, GA₇ decreased in seeds from treated trees compared with controls, but GA₉ levels increased. Levels of these three GAs were higher in seeds from treated trees 5 years after treatment (1990) but levels of GA₄, GA₇, and GA₉ dramatically increased in seeds from treated trees 4 years after treatment (1989), as we previously reported.     

Gibberellin structure-activity effects on flower initiation in mature trees and on shoot growth in mature and juvenile Prunus avium

When applied to spurs of mature Prunus avium before floral initiation, gibberellins GA₁, GA₄ and GA₃ inhibited floral initiation by 9–17%, GA₇ by 43%, GA₃ by 65–71% and 2,2-dimethyl GA₄ by 78%. GA₉ and GA₂₀ were inactive. Thus activity only of the GAs with a C-3 hydroxyl was increased markedly by a double bond in the C-1,2 or C-2,3 position, and activity increased with increasing hydroxylation. None of the GAs affected the total number of buds (vegetative and floral) surviving in the spur. Measured by the threshold dose required for activity, seedling shoot growth responses to GA₃, GA₇, GA₁ or GA₄ resembled those of floral initiation, but di-methylation of GA₄ at C-2 had no effect, and GA₉ was as active as GA₇. Mature shoots, including those on rooted cuttings, were less responsive to GA treatment than were juvenile shoots, with terminal shoots on mature trees more responsive than spur shoots. Spur shoot growth on mature trees responded to GA₃ and to a lesser extent GA₇, but not to GA₁ or GA₄. However, all these GAs promoted the growth of terminal shoots on mature trees to similar extents, whereas 2,2-dimethyl GA₄ was less active than GA₄ The differences between juvenile and mature shoot growth in sensitivity to a C-1,2 or C-2,3 double bond, and between mature shoot growth and floral initiation in GA-structure requirements, indicate that phase change alters the GA complement and/or GA receptor/transduction mechanisms of P. avium. The difference in sensitivity to 2,2-dimethyl GA₄ indicates that floral initiation and growth have different requirements for GA transport and/or action.     

Gibberellin physiology of safflower: endogenous gibberellins and response to gibberellic acid

Endogenous gibberellins (GAs) were extracted from safflower (Carthamus tinctorius L.) stems and detected by capillary gas chromatography-mass spectrometry from which GA₁, GA₃, GA₁₉,, GA₂₀, GA₂₉, and probably, GA₄₄ were detected. The detection of these GAs suggests that the early 13-OH biosynthetic pathway is prevalent in safflower shoots. Deuterated GAs were used as internal standards and GA concentrations were determined in stems harvested at weekly intervals. GA₁ and GA₁₉ levels per stem increased but concentrations per gram dry weight decreased over time. GA₂₀ was only detected in young stem tissue.Gibberellic acid (GA₃) was also applied in field trials and both GA₃ and the GA biosynthetic inhibitor, paclobutrazol, were applied in growth chamber tests. GA₃ increased epidermal cell size, internode length, and increased internode cell number causing stem elongation. Conversely, paclobutrazol reduced stem height, internode and cell size, cell number and overall shoot weight. In field tests, GA₃ increased total stem weight, but decreased leaf weight, flower bud number and seed yield. Thus, GA₃ promoted vegetative growth at the expense of reproductive commitment. These studies collectively indicate a promotory role of GAs in the control of shoot growth in safflower, and are generally consistent with gibberellin studies of related crop plants. Author for correspondence