Fractionation of gibberellins in plant extracts by reverse phase high performance liquid chromatography

In studies on endogenous plant gibberellins (GAs), reverse phase (Bondapak C₁₈) high performance liquid chromatography (HPLC) has proved to be a useful method for the fractionation of plant extracts. The behavior of 18 authentic GAs in such a chromatographic system is described. The main factors determining chromatographic behavior are the degree and the position of hydroxylation of the GA. Generally, dihydroxylated GAs elute before monohydroxylated GAs, whereas 13-hydroxylated GAs elute before 3-hydroxylated GAs. The number of carboxyl groups and the degree of saturation of the A-ring have little effect. For 20-carbon GAs, the oxidation state at C-20 is only relevant insofar as GAs having a methyl group at this position elute later than those with other groups (lactone, aldehyde, or carboxyl).     

Gibberellins and <Emphasis Type="Italic">N</Emphasis>-(2-chloro-4-pyridyl)-<Emphasis Type="Italic">N′</Emphasis>-phenylurea improve retention force and reduce water core in pre-mature fruit of Japanese pear cv. Housui

A study of the effect of a gibberellin A₃ + A₄ mixture (GAs) on pre-harvest fruit drop of Japanese pear cv. Housui is reported. The GAs was applied alone or in combination with N-(2-Chloro-4-pyridyl)-N′-phenylurea (CPPU) in a lanolin paste to the abscission zone at the spur-end of the pedicel. The results showed that the GAs and CPPU combination treatment increased the pedicel–spur retention force relative to that of the untreated controls. Histological studies showed an accelerated rate of cambium division and the development of secondary xylem in the abscission zone near the spur-end of pedicels treated with the GAs plus CPPU paste, which delayed the formation of the abscission zone. Fruit quality (sugar, acid, firmness, color) was not adversely affected by the application of GAs plus CPPU, although the application of GAs alone promoted ripening. In contrast, the addition of CPPU to the two GAs delayed fruit ripening, which was measured as ethylene efflux. All treatments were without adverse effects on return bloom, measured as bud size. The CPPU plus GAs treatment also suppressed the incidence of water core, whereas the application of the GAs alone accelerated water core in this water core susceptible pear variety.     

One‐pot sample preparation approach for profiling spatial distribution of gibberellins in a single shoot of germinating cereal seeds

Sample preparation remains a bottleneck in the rapid and reliable quantification of gibberellins (GAs) for obtaining an insight into the physiological processes mediated by GAs. The challenges arise from not only the extremely low content of GAs in complex plant matrices, but the poor detectability of GAs by mass spectrometry (MS) in negative ion mode. In an effort to solve these urgent difficulties, we present a spatial‐resolved analysis method to investigate the distribution of GAs in tiny plant tissues based on a simplified one‐pot sample preparation approach coupled with ultrahigh‐performance liquid chromatography‐tandem MS. By integrating extraction and derivatization into one step, target GAs were effectively extracted from plant materials and simultaneously reacted with N‐(3‐dimethylaminopropyl)‐N′‐ethylcarbodiimide, the sample preparation time was largely shortened, the probability of sample loss was minimized and the detection sensitivity of MS was also greatly improved compared with underivatized GAs. Under optimal conditions, the method was validated from the quantification linearity, limits of detection and limits of quantification in the presence of plant matrices, recoveries, and precision. With the proposed method, 15 endogenous GAs were detected and, among these, 11 GAs could be quantified in 0.50 mg fresh weight (FW) wheat shoot samples, and five GAs were quantified in only 0.15 mg FW developing seed samples of Arabidopsis thaliana. The distribution patterns of GAs along both the non‐13‐hydroxylation pathway and the early 13‐hydroxylation pathway in a single shoot of germinating wheat, rice and maize seeds were finally profiled with a spatial resolution down to approximately 1 mm².     

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     

Phloem translocation of gibberellins in three species of higher plants

Phloem sap was collected from white lupin (Lupinus albus L.), cowpea (Vigna unguiculata L.) and castor bean (Ricinus communis L.) and analysed for gibberellins (GAs) using gas chromatography-mass spectrometry (GC-MS). A large number of GAs were found in the phloem exudate of all three species, particularly where the sap was collected from pods (white lupin and cowpea) and in these legumes GAs representing both the early C-13-hydroxylation and non-hydroxylation pathways of biosynthesis were identified. In the sap collected from the vegetative tissues of castor bean the number of GAs identified was fewer than that in the other species, representing mainly the non-hydroxylation pathway. Data from sap collected from the pedicel and stylar ends of pods and by making feeds of radiolabelled GAs to seeds in situ in white lupin indicate that the GAs present in the phloem are derived mainly from the vegetative tissues of the plant. No evidence for metabolism of GAs in the phloem could be found.     

Endogenous Gibberellins in Aralia cordata

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

Glycoantigen expression is regulated both temporally and spatially during development in the cellular slime molds Dictyostelium discoideum and D. mucoroides

Six monoclonal antibodies were isolated which react with common antigens shared by multiple glycoconjugate species in the cellular slime mold Dictyostelium discoideum. Based on competition of antibody binding by glycopeptides and simple sugars, and inhibition of antibody binding by antigen pretreatment with Na periodate, it is argued that at least five of the six antibodies recognize epitopes which contain carbohydrate. These epitopes are consequently referred to as glycoantigens (GAs).Three of the GAs are expressed during growth and throughout the developmental cycle, but are eventually enriched in prestalk and stalk cells. The remaining three are expressed only during and/or after aggregation and are exclusively expressed or highly enriched in prespore cells and spores. These conclusions are derived from Western blot immunoanalysis of purified cell types, immunofluorescence, and EM immunocytochemistry.The two GAs found only in prespore cells appear to be exclusively enclosed within prespore vesicles. The third GA of this type, which is only enriched in prespore cells compared to prestalk cells, is also found in other vesicle types as well as on the cell surface.Two of the GAs enriched in prestalk cells are initially found in all cells of the slug. They are undetectable in spores and prominent in stalk cells. The third GA, though found in the interiors of both prestalk and prespore cells, is enriched on the cell surface of prestalk cells.The chief characteristics of expression of four of these GAs are conserved in the related species D. mucoroides. This species is characterized by continuous trans differentiation of prespore cells into prestalk cells. This shows that the prespore cells maintain specific mechanisms for turning over their cell type specific GAs and that prestalk cells express a specific mechanism for inducing at least one of their cell-type specific GAs.These observations identify specific carbohydrate structures (as GAs) whose synthesis, subsequent localization and turnover are developmentally regulated. The exclusive association of two GAs with prespore vesicles identifies these GAs as markers for this organelle and raises questions regarding the functional significance of this association. The restricted cell surface localization of the other four GAs, together with data from cell adhesion studies, suggest the possibility of a potential role for these GAs in intercellular recognition leading to cell sorting.This paper is dedicated to the memory of the late Daniel McMahon.     

Enhanced recovery of four antitumor ganoderic acids from Ganoderma lucidum mycelia by a novel process of simultaneous extraction and hydrolysis

Ganoderic acids (GAs) Mk, T, S and R exhibit promising anti-tumor effect, but they are difficult to purify from Ganoderma lucidum mycelia due to the presence of numerous analogs. In this work, a novel and efficient extraction/hydrolysis method was developed for the recovery of these four GAs from the mycelia of G. lucidum. By using a 50% aqueous ethanol solution containing 50 mmol/l HCl as extractant, extraction of GAs from mycelia and conversion of analogs impurities into the products of interest could be achieved in one step. This one-pot extraction/hydrolysis process increased the yield of GA-Mk, -T, -S and -R to 242%, 389%, 189% and 420%, respectively, compared to a raw sample without hydrolysis. Simultaneous purification of these four GAs was readily achieved in a single RP-HPLC run due to the conversion of analog impurities into corresponding desired GAs, and the purity and recovery of these four GAs were over 97% and 90%, respectively. The results demonstrated that the simultaneous extraction and hydrolysis process is simple and efficient and thus can act as a useful approach for enhanced recovery of those four GAs from G. lucidum mycelia.     

2种石蒜生长发育期鳞茎内源激素的动态变化

应用酶联免疫吸附测定法（ELISA）研究了石蒜与中国石蒜不同生长发育期鳞茎内源激素质量摩尔浓度的动态变化,分析了内源激素与2种石蒜生长发育的关系。结果表明,花芽分化前期2种石蒜鳞茎IAA浓度较低,而iPAs、ABA与GAs浓度较高;临近抽葶IAA浓度均有所增加,而iPAs与Zrs、ABA与GAs浓度均降到极值。花期ABA、GAs与ZRs浓度均有所回升,说明高浓度的ABA与GAs对2种石蒜的开花可能起正向调控的作用,但对叶生长的协同规律不明显。内源激素比例分析表明,ZRs/IAA、GAs/IAA、ABA/GAs三者比例高有利于2种石蒜的花原基分化,三者比例低则有助于2种石蒜的叶生长。     

Endogenous gibberellin and abscisic Acid content as related to senescence of detached lettuce leaves

Levels of gibberillins (GAs) and of abscisic acid (ABA) in attached leaves of romaine lettuce (Lactuca sativa L.) declined as the leaf became older. The time course of changes in hormone levels, determined in detached lettuce leaves kept in darkness, revealed that a sharp decline in GAs accompanied by a moderate rise in ABA occurred before the onset of chlorophyll degradation. As senescence advanced, no GAs could be detected and a considerable rise of ABA was observed. A similar sequence of hormonal modifications, but more pronounced, was observed in the course of accelerated senescence induced by either Ethephon or water stress. When kinetin or GA₃ was applied to detached leaves, the loss of chlorophyll and the rise in ABA were reduced. Bound GAs were detected in senescent leaves. They were not found in the kinetin-treated leaves, which contained a relatively high level of free GAs. The results suggest that senescence in detached romaine lettuce leaves is connected with a depletion of free GAs and cytokinins, which is thereafter followed by a great surge in ABA.     

Biotechnological production and application of ganoderic acids

Ganoderic acids (GAs), a kind of highly oxygenated lanostane-type triterpenoids, are important bioactive constituents of the famous medicinal mushroom Ganoderma lucidum. They have received wide attention in recent years due to extraordinarily pharmacological functions. Submerged fermentation of G. lucidum is viewed as a promising technology for production of GAs, and substantial efforts have been devoted to process development for enhancing GA production in the last decade. This article reviews recent publication about fermentative production of GAs and their potential applications, especially the progresses toward manipulation of fermentation conditions and bioprocessing strategies are summarized. The biosynthetic pathway of GAs is also outlined.     

Effects of light and temperature on seed dormancy and gibberellin-stimulated germination in Arabidopsis thaliana: studies with gibberellin-deficient and -insensitive mutants.

Effects of light and temperature on gibberellin (GA)-induced seed germination were studied in Arabidopsis thaliana (L.) Heynh. with the use of GA-deficient ( gal ) mutants, mutants with a strongly reduced sensitivity to GA ( gai ) and with the recombinant gai/gal . Seeds of the gal mutant did not germinate in the absence of exogenous GAs, neither in darkness, nor in light, indicating that GAs are absolutely required for germination of this species. Wild-type and gai seeds did not always require applied GAs in light. The conclusion that light stimulates GA biosynthesis was strengthened by the antagonistic action of tetcyclacis, an inhibitor of GA biosynthesis. In wild-type, gal and gai/gal seeds light lowered the GA requirement, which can be interpreted as an increase in sensitivity to GAs. In gai and gai/gal seeds light became effective only after dormancy was broken by either a chilling treatment of one week or a dry after-ripening period at 2°C during some months. The present genetic and physiological evidence strongly suggests that temperature regulates the responsiveness to light in A. thaliana seeds. The responsiveness increases during dormancy breaking, whereas the opposite occurs during induction of dormancy (8 days at 15°C pre-incubation). Since light stimulates the synthesis of GAs as well as the responsiveness to GAs, temperature-induced changes in dormancy may indirectly change the capacities to synthesize GAs and to respond to GAs. GA sensitivity is also directly controlled by temperature. It is concluded that both GA biosynthesis and sensitivity to GAs are not the primary controlling factors in dormancy, but are essential for germination.     

Ganoderma lucidum: A potential pleiotropic approach of ganoderic acids in health reinforcement and factors influencing their production

Ganoderma lucidum (G. lucidum) main attractive pharmacological characteristics are antitumor and immunomodulatory activities which are chiefly associated with its two principal bioactive compounds, those are polysaccharides and triterpenoids. Ganoderic acids (GAs) are one of the most discovered triterpenoids of G. lucidum among various triterpenoids. The prominent medicinal mushroom G. lucidum possesses GAs as essential bioactive constituents that are highly oxygenated lanostane-type triterpenoids. GAs exhibit diverse potential action against numerous diseases such as anticancer, antioxidant, anti-inflammatory, anti-HIV, cardioprotective, antiallergic, antihepatotoxic, neuroprotective and antinociceptive properties. GAs act through different mechanisms that include cytotoxic, apoptosis, inducing cell cycle arrest, inhibition of topoisomerases, antiproliferation, anti-invasion, inhibition of NF-kB AP1/uPA, farnesyl protein transferase and JAK-STAT3 pathway. The miraculous effects of GAs fascinate the researchers for their production. Various environmental factors such as biochemical signals, nutritional and physical that influence the biosynthesis of GA. However, the scarcities of pure compounds or accurately characterized extracts are the main problem of clinical studies. Substantial steps are required for characterized extracts of active compounds. This review contributes a thorough insight into the mode of actions of GAs and their possible reinforcements to overcome various diseases.     

Analysis of native gibberellins in the internode, nodes, leaves, and inflorescence of developing Avena plants

The native gibberellins (GAs) of various organs of the Avena plant were analyzed by bioassay and gas chromatography-mass spectrometry (GC-MS) after silicic acid partition column chromatography. The major GA of the inflorescence was identified as GA₃ by GC-MS, and this GA also forms the major component of the nodes, p-1 internode, and roots as determined by GLC or chromatography/bioassay. The inflorescence and nodes are the major sources of native GAs, the last two leaves, internode, and roots having significantly lower amounts of GA-like substances. In the internode, less polar GAs predominated at the lag stage of development, whereas by the log and plateau stages, the more polar GAs increased significantly.     

Effects of microbial volatile organic compounds on Ganoderma lucidum growth and ganoderic acids production in Co-v-cultures (volatile co-cultures)

Abstract

Co-v-culture (co-cultivations of physically separated microbes that only interact through the air) systems were designed to investigate the effects of microbial volatile organic compounds (mVOCs) from about 20 different microbes, on a medicinal fungus, Ganoderma lucidum. For more accuracy in co-cultivations, a novel synchronized cultivation approach was tested for culturing G. lucidum. The hyphal growth of G. lucidum and the content of its ganoderic acids (GAs) were measured. In almost all of the co-v-cultures, there was an inhibiting effect on hyphal growth and a promoting effect on GAs contents. In inducing GAs production, Bacillus cereus PTCC 1247 and Pseudomonas aeruginosa UTMC 1404 were the most effective ones, as, compared to control cultures, GAs content increased 2.8 fold. Comparing different co-v-cultivations demonstrated that the concentrations of mVOCs, oxygen, and carbon dioxide were the main players in co-v-cultures. No correlation was found between hyphal growth and GAs production. Strains of the same species imposed totally different effects on hyphal growth or GAs production. This study has investigated the effects of mVOCs on G. lucidum for the first time. Moreover, it suggests that co-v-cultivation may be a promising biotechnological approach to improve the production in G. lucidum.     

Distribution and morphology of growth anomalies in Acropora from the Indo-Pacific

We assessed the distribution and prevalence of growth anomalies (GAs) in Acropora from French Frigate Shoals (Hawaii, USA), Johnston Atoll and Tutuila (American Samoa), developed a nomenclature for gross morphology, characterized GAs at the cellular level and obtained preliminary indices of their spatial patterns and progression within coral colonies. Acropora GAs were found in all 3 regions, but the distribution, variety and prevalence of Acropora GAs was highest in American Samoa. GAs were grouped into 7 gross morphologies (exophytic, bosselated, crateriform, nodular, vermiform, fimbriate or annular). On histology, GAs consisted of hyperplastic basal body wall (calicodermis, mesoglea and gastrodermis apposed to skeleton) with 3 distinct patterns of necrosis. There was no evidence of anaplasia or mitotic figures (common but not necessarily required morphologic indicators of neoplasia). Compared to normal tissues, GAs had significantly fewer polyps, zooxanthellae within the gastrodermis of the coenenchyme, mesenterial filaments and gonads but significantly more necrosis. On 2 colonies with GAs monitored at 2 points over 11 mo, numbers of GAs per colony increased from 0.9 to 3 times the original number seen, and significant clustering of GAs occurred within colonies. The evidence of GAs being true neoplasias (tumors) is mixed, so a cautionary approach is urged in use of morphologic terminology.     

Gibberellin Production in Cultured Cells of Nicotiana tabacum

Endogenous gibberellins (GAs) in several kinds of crown gallcells and cultured cells derived from normal tissue of Nicotianatabacum were systematically analyzed by gas chromatography-selectedion current monitoring (GC-SICM) after chromatographic purifications,and GA₁, GA₉, GA₁₉ and GA₂₀ were identified. Agrobacterium tumefaciens,a pathogen of crown gall, was confirmed not to produce GAs inits culture. We also investigated endogenous GAs of mother plant,tobacco, and found the same kinds of GAs as in cultured cells. ³ Present address: College of Agriculture, Chonnam NationalUniversity, Kwangju 500, Korea. (Received May 19, 1982; Accepted July 22, 1983)     

Synthesis and structure–activity relationship of 16,17-modified gibberellin derivatives

Gibberellins (GAs) are a group of diterpenoid plant hormones that control plant growth and development at various stages. Biologically active GAs share the common structures of a 3β-hydroxy group, a carboxy group at C-6, and a γ-lactone between C-4 and C-10. Hydroxylation at C-2β is a major deactivation step in many plant species, and hydroxylation at C-13 has been shown to weaken the binding affinity of GAs to their receptor proteins. In rice, bioactive GA₄ has also been shown to be deactivated through 16α,17-epoxidation. Moreover, 16,17-dihydro-16α,17-dihydroxy GA₄ has been identified as an aglycon of its glucoside from rice. However, our knowledge on the biological activity of 16,17-epoxidized GAs is currently limited to 16,17-dihydro-16α,17-epoxy GA₄. Moreover, the bioactivity of 16,17-dihydro-16α,17-dihydroxy GA₄ remains unknown. Here, we synthesized 16,17-epoxidized or dihydroxylated GA derivatives and performed a structure–activity relationship study using rice seedlings. 16,17-Epoxidation of bioactive GA₁ and GA₄ reduced their activity to promote elongation of rice leaf sheaths. Moreover, 16,17-dihydroxylation significantly decreased the activities of 16,17-dihydro-16α,17-epoxy GAs. These results suggest that GAs are deactivated in a stepwise manner via 16,17-epoxidation and hydrolysis of these epoxy groups.