Mobile signals in day length-regulated flowering: Gibberellins, flowering locus T, and sucrose

Despite low activity for stem growth, the gibberellins GA₅ and GA₆ act as long-day (LD) florigens in Lolium temulentum L. This claim is based on extensive evidence covering GA synthesis in LD in the induced leaf and their transport to the shoot apex where they act in a dose-dependent manner. GAs also act as a LD florigen in association with cold vernalization of L. perenne. In contrast, highly bioactive GA₄ and, possibly, GA₁ are important florigens in Arabidopsis thaliana (L.) Heynh. This species contrast reflects differences in GA deactivation, which is unimportant for Arabidopsis but dominant in L. temulentum. It is unclear if GAs participate in flowering responses of short-day (SD) species since it is LD, which up-regulate enzymes for GA biosynthesis. Sugars (sucrose) may also act directly as a florigen and, specifically, with increase in photosynthesis as in LD or when light intensity is increased in SD. In addition, in LD sucrose can indirectly cause flowering by up-regulating FT expression, the FT protein acting as a further leaf-to-apex transported florigen. Thus, there are not only multiple florigens but there can be complex interactions between the signaling pathways controlling production of these various florigens.     

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.     

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     

Assessing Gibberellins Oxidase Activity by Anion Exchange/Hydrophobic Polymer Monolithic Capillary Liquid Chromatography-Mass Spectrometry

Bioactive gibberellins (GAs) play a key regulatory role in plant growth and development. In the biosynthesis of GAs, GA3-oxidase catalyzes the final step to produce bioactive GAs. Thus, the evaluation of GA3-oxidase activity is critical for elucidating the regulation mechanism of plant growth controlled by GAs. However, assessing catalytic activity of endogenous GA3-oxidase remains challenging. In the current study, we developed a capillary liquid chromatography – mass spectrometry (cLC-MS) method for the sensitive assay of in-vitro recombinant or endogenous GA3-oxidase by analyzing the catalytic substrates and products of GA3-oxidase (GA₁, GA₄, GA₉, GA₂₀). An anion exchange/hydrophobic poly([2-(methacryloyloxy)ethyl]trimethylammonium-co-divinylbenzene-co-ethylene glycol dimethacrylate)(META-co-DVB-co-EDMA) monolithic column was successfully prepared for the separation of all target GAs. The limits of detection (LODs, Signal/Noise = 3) of GAs were in the range of 0.62–0.90 fmol. We determined the kinetic parameters (K _m) of recombinant GA3-oxidase in Escherichia coli (E. coli) cell lysates, which is consistent with previous reports. Furthermore, by using isotope labeled substrates, we successfully evaluated the activity of endogenous GA3-oxidase that converts GA₉ to GA₄ in four types of plant samples, which is, to the best of our knowledge, the first report for the quantification of the activity of endogenous GA3-oxidase in plant. Taken together, the method developed here provides a good solution for the evaluation of endogenous GA3-oxidase activity in plant, which may promote the in-depth study of the growth regulation mechanism governed by GAs in plant physiology.     

Identification of gibberellins in leaf tissues of day-neutral strawberry (Fragaria × ananassa Duch.) cultivars

The endogenous gibberellins (GAs) in leaf tissues of two day-neutral cultivars (Rapella and Selva) of strawberry (Fragaria × ananassa Duch.) were analysed using combined gas chromatography -- mass spectrometry (GC-MS). Seven of the later members of the 13-hydroxylation GA biosynthetic pathway were identified, by comparison of Kovats retention indices and mass spectral data obtained for methyl ester trimethylsilyl ether derivatives, either with data obtained from authentic compounds or literature values. GA₁, GA₃, GA₈, GA₁₇, GA₁₉, GA₂₀ and GA₂₉ were detected in extracts of both cultivars.     

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.     

Comparison of Biological Activities of Gibberellins and Gibberellin-Precursors Native to Thlaspi arvense L

Field pennycress (Thlaspi arvense L.) is a winter annual weed with a cold requirement for stem elongation and flowering. The relative abilities of several native gibberellins (GAs) and GA-precursors to elicit stem growth were compared. Of the eight compounds tested, gibberellin A₁, (GA₁), GA₉, and GA₂₀ caused stem growth in noninduced (no cold treatment) plants. No stem growth was observed in plants treated with ent-kaurene, ent-kaurenol, ent-kaurenoic acid, GA₅₃, or GA₈. Moreover, of the biologically active compounds, GA₉ was the most active followed closely by GA₁. In thermoinduced plants (4-week cold treatment at 6°C) that were continuously treated with 2-chlorocholine chloride to reduce endogenous GA production, GA₉ was the most biologically active compound. However, the three kaurenoid GA precursors also promoted stem growth in thermoinduced plants, and were almost as active as GA₂₀. No such increase in activity was observed for either GA_[unk] or GA₅₃. The results are discussed in relation to thermoinductive regulation of GA metabolism and its significance to the initiation of stem growth in field pennycress. It is proposed that thermoinduction results in increased conversion of ent-kaurenoic acid to GAs through the C-13 desoxy pathway and that GA₉ is the endogenous mediator of thermoinduced stem growth in field pennycress.     

Ethylene-Mediated Regulation of Gibberellin Content and Growth in Helianthus annuus L

Elongation of hypocotyls of sunflower can be promoted by gibberellins (GAs) and inhibited by ethylene. The role of these hormones in regulating elongation was investigated by measuring changes in both endogenous GAs and in the metabolism of exogenous [³H]- and [²H₂]GA₂₀ in the hypocotyis of sunflower (Helianthus annuus L. cv Delgren 131) seedlings exposed to ethylene. The major biologically active GAs identified by gas chromatography-mass spectrometry were GA₁, GA₁₉, GA₂₀, and GA₄₄. In hypocotyls of seedlings exposed to ethylene, the concentration of GA₁, known to be directly active in regulating shoot elongation in a number of species, was reduced. Ethylene treatment reduced the metabolism of [³H]GA₂₀ and less [²H₂]GA₁ was found in the hypocotyls of those seedlings exposed to the higher ethylene concentrations. However, it is not known if the effect of ethylene on GA₂₀ metabolism was direct or indirect. In seedlings treated with exogenous GA₁ or GA₃, the hypocotyls elongated faster than those of controls, but the GA treatment only partially overcame the inhibitory effect of ethylene on elongation. We conclude that GA content is a factor which may limit elongation in hypocotyls of sunflower, and that while exposure to ethylene results in reduced concentration of GA₁ this is not sufficient per se to account for the inhibition of elongation caused by ethylene.     

Endogenous Gibberellins of Pine Pollen: II. Changes during Germination of Pinus attenuata, P. coulteri, and P. ponderosa Pollen

The endogenous gibberellins (GAs) of pollen of Pinus attenuata, P. coulteri, and P. ponderosa were bioassayed at hour 0, 3, 15, 24, 48 and 72 of germination. Dormant pollen showed relatively high GA activity throughout the elution spectrum (i.e. ranging from relatively nonpolar to highly polar). The maximum GA activity was obtained at hour 15 in more polar regions and especially in the zone corresponding to GA₃ (for P. attenuata estimated as 250 micrograms of GA₃/kilogram pollen). It is probable that the “nonpolar” GAs present in high quantities in dormant pollen and in early stages of germination were converted to “more polar” GAs as germination progressed. The amount of all GAs decreased after hour 15 of germination and by hour 72 no GAs could be detected. Among the species tested P. attenuata showed the highest over-all GA activity.     

Gibberellins in Relation to Growth and Flowering in Pharbitis nil Chois

Flowering can be modified by gibberellins (GAs) in Pharbitis nil Chois. in a complex fashion depending on GA type, dosage, and the timing of treatment relative to a single inductive dark period. Promotion of flowering occurs when GAs are applied 11 to 17 hours before a single inductive dark period. When applied 24 hours later the same GA dosage is inhibitory. Thus, depending on their activity and the timing of application there is an optimum dose for promotion of flowering by any GA, with an excessive dose resulting in inhibition. Those GAs highly promotory for flowering at low doses are also most effective for stem elongation (2,2-dimethyl GA₄ GA₃₂ > GA₃ > GA₅ > GA₇ > GA₄). However, the effect of GAs on stem elongation contrasts markedly with that on flowering. A 10- to 50-fold greater dose is required for maximum promotion of stem elongation, and the response is not influenced by time of application relative to the inductive dark period. These differing responses of flowering and stem elongation raise questions about the use of relatively stable, highly bioactive GAs such as GA₃ to probe the flowering response. It is proposed that the `ideal' GAs for promoting flowering may be highly bioactive but with only a short lifetime in the plant and, hence, will have little or no effect on stem elongation.     

Endogenous gibberellins in flushing buds of three deciduous trees: Alder,aspen, and birch

Endogenous gibberellins (GAs) were extracted from flushing (expanding) vegetative buds of river alder (Alnus tenuifolia), European white birch (Betula pendula), and aspen (Populus tremuloides) and identified by gas chromatography-mass spectrometry with full scans and/or selected ion monitoring. Five 13-hydroxylated GAs were detected from the three trees: GA_{1, 8}, and ₂₀ from alder, GA_{1, 8, 19} and ₂₀ from aspen and GA_{1, 8, 19, 20}, and ₂₉ from birch. Thirteen other GAs previously detected in Salix or common in other plants were specifically investigated but not detected. The presence of GA₁, its probable precursors GA₁₉ and GA₂₀, and its probable metabolite, GA₈, suggests that the early 13-hydroxylated GA biosynthetic pathway is dominant in vegetative buds of these trees. Abundant endogenous GAs of these trees are similar to the principal GAs of willows (various Salix spp.) and poplars (various Populus spp.). This suggests similarities in the GA physiology and is consistent with a common role of GA₁ as a regulator of shoot growth in woody angiosperms.     

The biosynthesis of gibberellic acids by the transformants of orchid-associated <Emphasis Type="Italic">Fusarium oxysporum</Emphasis>

The genus Fusarium, including multiple strains in the Gibberella fujikuroi species complex (GFC), is well known for its production of diverse secondary metabolites. F. fujikuroi, associated with the “bakanae” disease of rice, is an active producer of gibberellins (GAs), a wide class of plant hormones. In addition to some members of the GFC, the GA biosynthetic gene cluster, or parts of it, occurs also in some isolates of the closely related species of F. oxysporum, which does not belong to the GFC. However, production of GAs has never been observed in any F. oxysporum strain. In this study, we report on the GA biosynthetic activity in an orchid-associated F. oxysporum strain by transforming a cosmid with the entire F. fujikuroi GA gene cluster. Southern and Northern blot analyses confirmed not only the integration of the entire gene cluster into the genome but also the active expression of the seven GA biosynthetic genes under nitrogen-limiting conditions. The transformants produced GAs at levels similar to those of F. fujikuroi. These data show that the regulatory network for expression of GA genes is fully active in the F. oxysporum background.     

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.     

Delay of early fruitlet abscission by branch girdling in citrus coincides with previous increases in carbohydrate and gibberellin concentrations

Current evidence in citrus indicates that gibberellins (GAs) are main determinants of early fruit set while subsequent growth of developing fruits is mostly dependent upon carbohydrate availability. In this work, branch girdling performed at anthesis in Satsuma mandarin (Citrus unshiu (Mak.) Marc.) cv. Okitsu transitorily reduced early abscission rates (12–32 days after anthesis, DAA) delaying initially the process of natural fruitlet drop. The effects of girdling on growth, gibberellin (GA) and carbohydrate concentrations in developing ovaries and fruitlets were assessed during this initial growth stage (0–69 DAA). In girdled branches, abscission rate reduction was preceded by elevated concentrations of carbohydrate and GA in developing ovaries and fruitlets. Girdling at anthesis stimulated higher hexose (21 DAA) and starch (6–20 DAA) concentrations and also higher GA₁ (6 DAA), GA₁₉ (13–20 DAA) and GA₂₀ (6–20 DAA). The results established a relationship between the reduction of early abscission rates and higher concentrations of carbohydrates and GAs induced by girdling in developing fruitlets. These findings revealed that girdling certainly increased GA concentration and strongly suggested that its effect on early fruitlet abscission delay is likely mediated by both GA and carbohydrates.     

Gibberellin and auxin signals control scape cell elongation and proliferation in <Emphasis Type="Italic">Agapanthus praecox</Emphasis> ssp. <Emphasis Type="Italic">orientalis</Emphasis>

Plant height is determined by the processes of cell proliferation and elongation. Plant hormones play key roles in a species-dependent manner in these processes. We used paclobutrazol (PAC) at 400 mg·L^-1 in this study to spray Agapanthus praecox ssp. orientalis plants in order to induce dwarf scape (inflorescence stem). Morphological examination showed that PAC reduced scape height by inhibiting the cell elongation by 54.56% and reducing cell proliferation by 10.45% compared to the control. Quantification and immunolocalization of endogenous gibberellins (GAs) and indole-3-acetic acid (IAA) showed that the GA₁, GA₃, and GA₄ levels and the IAA gradient were reduced. Among these hormones, GA4 was the key component of GAs, which decreased 59.51-92.01% compared to the control in scape. The expression of cell wall synthesis related genes cellulose synthase (CESA) and UDP-glucuronic acid decarboxylase (UXS) were upregulated significantly, whereas cell wall loosening gene xyloglucan endotransglucosylase 2 (XET2) was downregulated by 99.99% surprisingly. Correlation analysis suggested GA regulated cell elongation and auxin modulated cell proliferation in Agapanthus scape. Additionally, the accumulation of sugars played roles in cell wall synthesis and cell expansion. These results indicated GA and IAA signals triggered a downstream signaling cascade, controlled cell expansion and proliferation during scape elongation.     

Gibberellin biosynthesis in fungi: genes, enzymes, evolution, and impact on biotechnology

Gibberellins (GAs) constitute a large family of tetracyclic diterpenoid carboxylic acids, some members of which function as growth hormones in higher plants. As well as being phytohormones, GAs are also present in some fungi and bacteria. In recent years, GA biosynthetic genes from Fusarium fujikuroi and Arabidopsis thaliana have been cloned and well characterised. Although higher plants and the fungus both produce structurally identical GAs, there are important differences indicating that GA biosynthetic pathways have evolved independently in higher plants and fungi. The fact that horizontal gene transfer of GA genes from the plant to the fungus can be excluded, and that GA genes are obviously missing in closely related Fusarium species, raises the question of the origin of fungal GA biosynthetic genes. Besides characterisation of F. fujikuroi GA pathway genes, much progress has been made in the molecular analysis of regulatory mechanisms, especially the nitrogen metabolite repression controlling fungal GA biosynthesis. Basic research in this field has been shown to have an impact on biotechnology. Cloning of genes, construction of knock-out mutants, gene amplification, and regulation studies at the molecular level are powerful tools for improvement of production strains. Besides increased yields of the final product, GA₃, it is now possible to produce intermediates of the GA biosynthetic pathway, such as ent-kaurene, ent-kaurenoic acid, and GA₁₄, in high amounts using different knock-out mutants. This review concentrates mainly on the fungal biosynthetic pathway, the genes and enzymes involved, the regulation network, the biotechnological relevance of recent studies, and on evolutionary aspects of GA biosynthetic genes.     

The relative significance for stem elongation and flowering in Lolium temulentum of 3β-hydroxylation of gibberellins

In previous experiments with many gibberellins (GAs) and GA derivatives applied to Lolium temulentum L., quite different structural requirements were evident for stem elongation on the one hand and for the promotion of flowering on the other. Whereas hydroxylation at carbons 12, 13 and 15 enhanced flowering relative to stem growth, the reverse was the case at carbon 3 (L.T. Evans et al. 1990, Planta 182, 97–106). The significance of hydroxylation at carbon 3 is examined in this paper. The application of inhibitors of 3β-hydroxylation, including C/D-ring-rearranged GAs, reduced stem growth but, in the case of the two acylcyclohexanediones, increased the flowering response when applied on the inductive long day. Later applications of the acylcyclohexanediones, made after floral initiation had occurred, were inhibitory to flowering, suggesting that subsequent inflorescence development requires 3β-hydroxylated GAs. Applications of the 3α-hydroxy epimers of GA₁, GA₃ and GA₄ gave slightly less promotion of flowering in comparison with the 3β-hydroxy GAs, but far less promotion of stem elongation, except in the case of 3-epi-GA₄, which was comparable to GA₄. The 3α-hydroxy epimer of 2,2-dimethyl GA₄ gave less promotion of flowering than its 3β-hydroxy epimer but almost no promotion of stem elongation. The 3α-hydroxy epimers of GA₃ and 2,2-dimethyl GA₄ did not act as competitive inhibitors of the stem elongation elicited by GA₃ and 2,2-dimethyl GA₄, respectively. These results extend the differences in GA structure which favour flowering as opposed to stem elongation, and indicate that 3-hydroxylation and its epimeric configuration are of much greater importance to stem elongation than to flower initiation in Lolium.     

Diversity,regulation, and evolution of the gibberellin biosynthetic pathway in fungi compared to plants and bacteria

Bioactive gibberellins (GAs) are diterpene plant hormones that are biosynthesized through complex pathways and control diverse aspects of growth and development. GAs were first isolated as metabolites of a fungal rice pathogen, Gibberella fujikuroi, since renamed Fusarium fujikuroi. Although higher plants and the fungus produce structurally identical GAs, significant differences in their GA pathways, enzymes involved and gene regulation became apparent with the identification of GA biosynthetic genes in Arabidopsis thaliana and F. fujikuroi. Recent identifications of GA biosynthetic gene clusters in two other fungi, Phaeosphaeria spp. and Sphaceloma manihoticola, and the high conservation of GA cluster organization in these distantly related fungal species indicate that fungi evolved GA and other diterpene biosynthetic pathways independently from plants. Furthermore, the occurrence of GAs and recent identification of the first GA biosynthetic genes in the bacterium Bradyrhizobium japonicum make it possible to study evolution of GA pathways in general.In this review, we summarize our current understanding of the GA biosynthesis pathway, specifically the genes and enzymes involved as well as gene regulation and localization in the genomes of different fungi and compare it with that in higher and lower plants and bacteria.