Endogenous Expression of Adenosine A<Subscript>1</Subscript>, A<Subscript>2 </Subscript>and A<Subscript>3</Subscript> Receptors in Rat C6 Glioma Cells

Inhibitory and stimulatory adenosine receptors have been identified and characterized in both membranes and intact rat C6 glioma cells. In membranes, saturation experiment performed with [³H]DPCPX, selective A₁R antagonist, revealed a single binding site with a K _D = 9.4 ± 1.4 nM and B _max = 62.7 ± 8.6 fmol/mg protein. Binding of [³H]DPCPX in intact cell revealed a K _D = 17.7 ± 1.3 nM and B _max = 567.1 ± 26.5 fmol/mg protein. On the other hand, [³H]ZM241385 binding experiments revealed a single binding site population of receptors with K _D = 16.5 ± 1.3 nM and B _max = 358.9 ± 52.4 fmol/mg protein in intact cells, and K _D = 4.7 ± 0.6 nM and B _max = 74.3 ± 7.9 fmol/mg protein in plasma membranes, suggesting the presence of A_2A receptor in C6 cells. A₁, A_2A, A_2B and A₃ adenosine receptors were detected by Western-blotting and immunocytochemistry, and their mRNAs quantified by real time PCR assays. Giα and Gsα proteins were also detected by Western-blotting and RT-PCR assays. Furthermore, selective A₁R agonists inhibited forskolin- and GTP-stimulated adenylyl cyclase activity and CGS 21680 and NECA stimulated this enzymatic activity in C6 cells. These results suggest that C6 glioma cells endogenously express A₁ and A₂ receptors functionally coupled to adenylyl cyclase inhibition and stimulation, respectively, and suggest these cells as a model to study the role of adenosine receptors in tumoral cells.     

Enhancement of artemisinin content and biomass in <Emphasis Type="Italic">Artemisia annua</Emphasis> by exogenous GA<Subscript>3</Subscript> treatment

Artemisinin is a promising and potent antimalarial drug naturally produced by the plant Artemisia annua L. but in very low yield. Its artemisinin content is known to be greatly affected by both genotype and environmental factors. In this study, the production of artemisinin and leaf biomass in Artemisia annua L. was significantly increased by exogenous GA₃ treatment. The effect of GA₃ application on expression of proposed key enzymes involved in artemisinin yield was examined in both wild type (007) and FPS-overexpression (253-2) lines of A. annua. In the wild type (007) at 6 h post GA₃ application there was an abrupt rise in FPS, ADS and CYP71AV1 expression and at 24 h a temporary and significant peak in artemisinin (1.45-fold higher than the control). After GA₃ application in line 253-2, there was a dramatic rise in expression of FPS at 3 h, CYP71AV1 at 9 h and ADS at 72 h and accumulation of artemisinin after 7 days, which was a delay when compared with the wild type plant. Thus, increased artemisinin content from exogenous GA₃ treatment was associated with increased expression of key enzymes in the artemisinin biosynthesis pathway. Interestingly, exogenous GA₃ continuously enhanced artemisinin content from the vegetative stage to flower initiation in both plant lines and gave significantly higher leaf biomass than in control plants. Consequently, the artemisinin yield in GA₃-treated plants was much higher than in control plants. Although the maximum artemisinin content was found at the full blooming stage [2.1% dry weight (DW) in 007 and 2.4% DW in 253-2], the highest artemisinin yield in GA₃-treated plants was obtained during the flower initiation stage (2.4 mg/plant in 007 and 2.3 mg/plant in 235-2). This was 26.3 and 27.8% higher, respectively, than in non-treated plants 007 and 253-2. This study showed that exogenous GA₃ treatment enhanced artemisinin production in pot experiments and should be suitable for field application.     

Changes in concentrations of soluble carbohydrates during germination of <Emphasis Type="Italic">Amaranthus caudatus</Emphasis> L. seeds in relation to ethylene,gibberellin A<Subscript>3</Subscript> and methyl jasmonate

Unimbibed Amaranthus caudatus seeds were found to contain stachyose, raffinose, verbascose, sucrose, galactinol, myo-inositol, glucose and fructose, while no galactose, maltose and maltotriose was detected. During imbibition, seed concentrations of verbascose, stachyose, raffinose, galactinol, myo-inositol (temporary) and fructose (transient) were observed to decrease; concentrations of galactose and maltose remained fairly constant, while those of sucrose, glucose and maltotriose increased, the increase in sucrose concentration was only temporary. Effects of gibberellin A₃ (GA₃) at 3 × 10⁻⁴ M and ethephon at 3 × 10⁻⁴ M alone or in the presence of methyl jasmonate (Me-JA) at 10⁻³ M on concentrations of soluble sugars during germination of A. caudatus seeds were examined. Me-JA was found to inhibit seed germination and fresh weight of the seeds, but did not affect sucrose, myo-inositol, galactose and maltose concentrations during imbibition for up to 20 h. The exogenously applied GA₃ was observed to enhance germination, stachyose breakdown and glucose concentration after 20 h of incubation. Ethephon stimulated seed germination as well as utilisation of stachyose, galactinol (both after 14 and 20 h) and raffinose (after 14 h of incubation). Although the stimulatory effect of either GA₃ or ethephon on seed germination was blocked by Me-JA; these stimulators increased mobilisation of raffinose and stachyose, but only ethephon enhanced both glucose and fructose after 14 and/or 20 h of incubation in the presence of Me-JA. The maltose concentration was increased by both GA₃ and ethephon alone and in the presence of Me-JA. Of the growth regulators studied, ethephon alone and/or in combination with Me-JA significantly increased the concentrations of glucose, fructose, galactose, maltose and maltotriose. The differences in sugar metabolism appear to be linked to ethylene or GA₃ applied simultaneously with Me-JA.     

Influence of gibberellin A<Subscript>3</Subscript> application,pH of the medium,photoperiod and temperature on the enhancement of in vitro flowering in <Emphasis Type="Italic">Vitex negundo</Emphasis> L.

Vitex negundo L. is an economically important small medicinal tree and an essential oil is extracted from its flowers. In nature, flowering is season specific and irregular, appearing only after a long vegetative phase. A very much improved protocol has been developed for the enhancement of flowering in vitro, one which increases both the number of flowers and the percentage (98.6%) of micro-plants flowering. The inclusion of gibberellin A₃ (GA₃) in the culture medium played the key role in controlling the in vitro flowering in V. negundo. The promotive effect of GA₃ was further improved by optimising pH, photoperiod and temperature. Our in vitro flower induction procedures provide an extremely effective method for further research on flowering regulation mechanisms in this important medicinal tree.     

Improved GA<Subscript>1</Subscript> production by<Emphasis Type="Italic"> Fusarium fujikuroi</Emphasis>

Recent studies into gibberellin A₁ (GA₁) showed it to be physiologically more active than GA₃ in plants of great agricultural interest, such as tomatoes, rice, peas, and sweet cherries. We describe here a simple procedure for obtaining large quantities of GA₁ (1,500 mg/l) by incubating the FKMC1995 strain of Fusarium fujikuroi in a standard complex medium (SCM). We also compare the GA production of this strain with that of two other wild-type strains of F. fujikuroi (IMI58289, m567) in SCM and low-nitrogen medium and discuss the possible biogenetic mechanisms involved in the over-accumulation of GA₁ by FKMC1995.     

Obligate methylotroph <Emphasis Type="Italic">Methylobacillus arboreus</Emphasis> Iva<Superscript>T</Superscript> synthesizes a plant hormone,gibberellic acid GA<Subscript>3</Subscript>

An obligate methylotroph Methylobacillus arboreus Iva^Т (VKM B-2590^Т, CCUG 59684^T, DSM 23628T) is the first known aerobic methylotrophic bacterium capable of synthesis of the bioactive gibberellic acid GA₃. Primary separation and identification of gibberellic acid from the culture liquid of methanol-grown culture were carried out using thin-layer chromatography and high-performance liquid chromatography. The concentration and structure of the gibberellic acid GA₃ were determined by liquid chromatography?mass spectrometry (LC/MS). Biological activity of the isolated compound was confirmed by tests on sprouts of lettuce (Laсtuca sativa L.).     

<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.     

Interactive effects of gibberellin A<Subscript>3</Subscript> and ascorbic acid on lipid peroxidation and antioxidant enzyme activities in <Emphasis Type="Italic">Glycine max</Emphasis> seedlings under nickel stress

The effects of nickel in combination with ascorbic acid (AsA) and gibberellin on 7-day-old soybean seedlings were examined. Exposure to 0.25 mM NiCl₂ × 6H₂O for 5 days resulted in toxicity symptoms, such as formation of reddish-brown mottled spots on the leaf blade. Addition of 0.05 mM GA₃ or 1 mM AsA reduced toxic effects of nickel. After their simultaneous application, these symptoms did not appear. Ni decreased dry weights of roots and shoots and reduced chlorophyll content in leaves. An enhanced level of lipoxygenase activity and malondialdehyde, and changes in the activities of the antioxidant enzymes, catalase, guaiacol peroxidase and ascorbate peroxidase, in both roots and leaves indicated that Ni caused an oxidative stress in soybean plants. The Ni-stressed seedlings exposed to AsA or GA₃, especially to GA₃ plus AsA, exhibited an improved growth as compared to Ni-treated plants. GA₃ decreased Ni uptake by roots, while ascorbic acid considerably reduced root-to-shoot translocation of Ni. Interaction of AsA plus GA₃ prevented the decrease in chlorophyll content and lipid peroxidation as well as increased the activities of the antioxidant enzymes. These results suggest that GA₃ plus AsA treatment counteracts the negative effects of Ni on soybean seedlings. Published in Russian in Fiziologiya Rastenii, 2007, Vol. 54, No. 1, pp. 85–91. The text was submitted by the authors in English.     

A new strain of <Emphasis Type="Italic">Arthrinium phaeospermum</Emphasis> isolated from <Emphasis Type="Italic">Carex kobomugi</Emphasis> Ohwi is capable of gibberellin production

Plant growth-promoting endophytic fungi with gibberellin-producing ability were isolated from the roots of Carex kobomugi Ohwi, a common sand-dune plant, and bioassayed for plant growth-promotion. A new strain, Arthrinium phaeospermum KACC43901, promoted growth of waito-c rice and Atriplex gemelinii. Analysis of its culture filtrate showed the presence of bioactive GA₁ (0.5 ng/ml), GA₃ (8.8 ng/ml), GA₄ (4.7 ng/ml) and GA₇ (2.2 ng/ml) along with physiologically inactive GA₅ (0.4 ng/ml), GA₉ (0.6 ng/ml), GA₁₂ (0.4 ng/ml), GA₁₅ (0.4 ng/ml), GA₁₉ (0.9 ng/ml) and GA₂₄ (1.8 ng/ml). The fungal isolate was identified through sequence homology and phylogenetic analysis of 18S rDNA (internal transcribed region). Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Growth promotion of cucumber by pure cultures of gibberellin-producing <Emphasis Type="Italic">Phoma</Emphasis> sp. GAH7

The beneficial effects of plant growth promoting fungi (PGPF) on plant growth and development are well documented. However, limited information is available on gibberellin (GA) production capacity of PGPF of endophytic origin. In current study, 11 fungal endophytes were isolated from cucumber roots and then screened on Waito-C rice, in order to identify plant growth promoting fungal strains. The fungal isolate GAH7 provided the maximum shoot length (11.3 cm) in comparison to control treatment (7.8 cm). In a separate experiment, bioassay of GAH7 significantly promoted growth attributes of cucumber. The GAH7 culture filtrate (CF) was found to contain physiologically active gibberellins in higher concentrations (GA₁, 0.81 ng/ml; GA₃, 4.34 ng/ml and GA₄, 9.31 ng/ml) in conjunction with physiologically inactive GA₉ (0.74 ng/ml), GA₁₅ (0.97 ng/ml), GA₁₉ (1.67 ng/ml) and GA₂₀ (0.46 ng/ml). Isolate GAH7 produced higher amounts of GA₃, GA₄, GA₉ and GA₁₉ than wild type Fusarium fujikuroi, which was used as control for GA production. Gibberellins were analyzed through gas chromatograph/mass spectrometer (GC/MS) with selected ion monitoring (SIM). The fungal isolate GAH7 was later identified as a new strain of Phoma on the basis of sequence homology (99%) and phylogenetic analysis of 18S rDNA sequence.     

Biochemical characteristics of immune-associated phospholipase A<Subscript>2</Subscript> and its inhibition by an entomopathogenic bacterium, <Emphasis Type="Italic">Xenorhabdus nematophila</Emphasis>

An entomopathogenic bacterium, Xenorhabdus nematophila, induces an immunosuppression of target insects by inhibiting phospholipase A₂ (PLA₂) activity. Recently, an immune-associated PLA₂ gene was identified from the red flour beetle, Tribolium castaneum. This study cloned this PLA₂ gene in a bacterial expression vector to produce a recombinant enzyme. The recombinant T. castaneum PLA₂ (TcPLA₂) exhibited its characteristic enzyme activity with substrate concentration, pH, and ambient temperature. Its biochemical characteristics matched to a secretory type of PLA₂ (sPLA₂) because its activity was inhibited by dithiothreitol (a reducing agent of disulfide bond) and bromophenacyl bromide (a specific sPLA₂ inhibitor) but not by methylarachidonyl fluorophosphonate (a specific cytosolic type of PLA₂). The X. nematophila culture broth contained PLA₂ inhibitory factor(s), which was most abundant in the media obtained at a stationary bacterial growth phase. The PLA₂ inhibitory factor(s) was heat-resistant and extracted in both aqueous and organic fractions. Effect of a PLA₂-inhibitory fraction on the immunosuppression of T. castaneum was equally comparable with that resulted from inhibition of the TcPLA₂ gene expression by RNA interference.     

<Emphasis Type="Italic">Burkholderia</Emphasis> sp. KCTC 11096BP as a newly isolated gibberellin producing bacterium

We isolated 864 bacteria from 553 soil samples and bioassayed them on cucumber and crown daisy for plant growth promotion. A new bacterial strain, Burkholderia sp. KCTC 11096BP gave maximum growth promotion and was selected for further investigations. The culture filtrate of this bacterium was thus analyzed for the presence of gibberellins and we found physiologically active gibberellins were found (GA₁, 0.23 ng/100 ml; GA₃, 5.11 ng/100 ml and GA₄, 2.65 ng/100 ml) along with physiologically inactive GA₉, GA₁₂, GA₁₅, GA₂₀, and GA₂₄. The bacterial isolate also solubilised tricalcium phosphate and lowered the pH of the medium during the process. The isolate was identified as a new strain of Burkholderia through phylogenetic analysis of 16S rDNA sequence. Gibberellin production capacity of genus Burkholderia is reported for the first time in current study. These authors contributed equally to the work     

Gibberellin production by pure cultures of a new strain of <Emphasis Type="Italic">Aspergillus fumigatus</Emphasis>

Fungal endophytes produce a variety of favorable metabolites for plant growth and survival, but there is limited information on their gibberellin (GA) production capacity. In the current study, we isolated eight endophytic fungi from the roots of a drought stressed soybean cultivar Hwangkeumkong, and screened them on waito-c rice for plant growth promotion. Seven fungal isolates promoted plant growth, while one inhibited it. The culture filtrate (CF) of fungal isolate HK-5-2 provided the best results for growth promotion and was thus bioassayed on soybean. HK-5-2 CF enhanced plant length, plant fresh and dry weight and endogenous bioactive GA₁ and GA₄ contents of soybean as compared to control. The GA analysis of HK-5-2 CF showed the presence of bioactive GA₃ (8.38 ng/ml), GA₄ (2.16 ng/ml) and GA₇ (1.56 ng/ml) in conjunction with physiologically inactive GA₅, GA₁₉ and GA₂₄. Gibberella fujikuroi was used as positive control during this experiment. The fungal isolate HK-5-2 was identified as a new strain of Aspergillus fumigatus through molecular and phylogenetic analysis of 18S and 28S rDNA sequences.     

Exogenous GA<Subscript>3</Subscript> and flowering induce the conversion of artemisinic acid to artemisinin in <Emphasis Type="Italic">Artemisia annua</Emphasis> plants

The contents of artemisinin and artemisinic acid were monitored in the Artemisia annua plants treated with GA₃ at vegetative and flowering initiation stages. The highest artemisinin content was observed at full bloom. The decrease in artemisinic acid content occurred during the transition from the vegetative stage to the beginning of flowering. Endogenous GA₃ content in the leaves peaked at full bloom. At the vegetative stage, in plants treated with various concentrations of GA₃ , the content of artemisinin increased while that of artemisinic acid decreased. Apparently, the rate-limiting step in artemisinin biosynthesis was from artemisinic acid to artemisinin. The bottleneck of artemisinin biosynthesis was probably unlocked during the flowering or in the vegetative plants treated with GA₃ , which triggered off the conversion of artemisinic acid to artemisinin.From Fiziologiya Rastenii, Vol. 52, No. 1, 2005, pp. 68–73.Original English Text Copyright © 2005 by Zhang, Ye, Liu, Wang, Li.This article was presented by the authors in English.     

Gibberellin production and plant growth promotion by a newly isolated strain of <Emphasis Type="Italic">Gliomastix murorum</Emphasis>

Endophytic fungi are known to play a vital role in the growth and development of their host plants. We isolated eleven endophytic fungi from the roots of sand-dune plant Elymus mollis and their growth-promoting ability was studied on waito-c rice and Atriplex gemelinii. We found that eight fungal isolates promoted growth of both plants. Fungal isolate EM-7-1 induced maximum growth promotion in waito-c rice (9.25 cm) and Atriplex gemelinii (3.1 cm), which was higher than wild-type Gibberella fujikuroi. Gibberellin analysis of EM-7-1 culture filtrate showed the presence of bioactive gibberellins GA₁ (0.32 ng/ml), GA₃ (5.76 ng/ml), GA₄ (0.82 ng/ml) and GA₇: (0.1 ng/ml) along with physiologically inactive GA₅ (0.59 ng/ml), GA₉ (5.38 ng/ml), GA₂₀ (0.25 ng/ml) and GA₂₄ (2.03 ng/ml). The fungal isolate EM-7-1 was identified as new strain of Gliomastix murorum (G. murorum KACC43902) with 99% sequence homology. This study reports the plant growth-promoting ability of genus Gliomastix and the presence of GA₅ in the culture filtrate of fungi for the first time. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

F-box Protein Arabidillo-1 Promotes Lateral Root Development by Depressing the Functioning of GA<Subscript>3</Subscript> in <Emphasis Type="Italic">Arabidopsis</Emphasis>

In Arabidopsis, Arabidillo-1 and Arabidillo-2 have great sequence homology to Dictyostelium and metazoan β–catenin/Armadillo, which are important to animal and Dictyostelium development. Arabidillo-1 and Arabidillo-2 promote lateral root formation redundantly in Arabidopsis. Here, we showed that gibberellins (GA₃) has a greater inhibitory effect on lateral root growth from the null mutant arabidillo-1 than from the wild type, suggesting that the mechanism for Arabidillo-1-regulated modulation of lateral root proliferation is associated with GA₃-metabolic or signaling pathways. Our yeast two-hybrid analysis demonstrated that Arabidillo-1 interacts with ASK2 and ASK11, and that ASK2 can bind with the F-box domain of Arabidillo-1. Therefore, Arabidillo-1 is involved in the ubiquitin/26S proteasome-mediated proteolytic pathway. Based on these results, we conclude that Arabidillo-1 can degrade some positive regulator of the GA₃ signaling pathway through selective protein degradation of ubiquitin/26S. Moreover, that process is believed to be the mechanism for Arabidillo-1 promotion of lateral root development in Arabidopsis.     

An aminotransferase from bacterium ATCC 55552 deaminates hydrolyzed fumonisin B<Subscript>1</Subscript>

Previous research identified several microorganisms and pathways capable of degrading the mycotoxin fumonisin B₁ (FB₁). Degradation of FB₁ by microorganisms seems to comprise two essential steps: hydrolysis to hydrolyzed fumonisin B₁ (HFB₁) and deamination of the hydrolysis product. One of the previously studied microorganisms was the Gram negative bacterium ATCC 55552. The gene corresponding to the first step of FB₁ degradation in this bacterium was identified, but the genetic basis for deamination of the hydrolyzed intermediate remained unexplained (Duvick et al. 2000, PCT patent application WO200004158). Here we report the sequence and HFB₁-deaminating activity of a novel aminotransferase encoded by the bacterium ATCC 55552. The corresponding gene was identified, sequenced, and over-expressed in Escherichia coli. Cell lysates of the recombinant E. coli strain showed distinct HFB₁-deaminating activity in the presence of pyridoxal phosphate and pyruvate, as was demonstrated by liquid chromatography–mass spectrometry. Thus, we suggest the novel enzyme to be part of the fumonisin catabolic pathway of the bacterium ATCC 55552.     

Interisland Mutation of a Novel Phospholipase A<Subscript>2</Subscript> from <Emphasis Type="Italic">Trimeresurus flavoviridis</Emphasis> Venom and Evolution of Crotalinae Group II Phospholipases A<Subscript>2</Subscript>

Trimeresurus flavoviridis (Crotalinae) snakes inhabit the southwestern islands of Japan: Amami-Oshima, Tokunoshima, and Okinawa. Affinity and conventional chromatographies of Amami-Oshima T. flavoviridis venom led to isolation of a novel phospholipase A₂ (PLA₂). This protein was highly homologous (91%) in sequence to trimucrotoxin, a neurotoxic PLA₂, which had been isolated from T. mucrosquamatus (Taiwan) venom, and exhibited weak neurotoxicity. This protein was named PLA-N. Its LD₅₀ for mice was 1.34 µg/g, which is comparable to that of trimucrotoxin. The cDNA encoding PLA-N was isolated from both the Amami-Oshima and the Tokunoshima T. flavoviridis venom-gland cDNA libraries. Screening of the Okinawa T. flavoviridis venom-gland cDNA library with PLA-N cDNA led to isolation of the cDNA encoding one amino acid-substituted PLA-N homologue, named PLA-N(O), suggesting that interisland mutation occurred and that Okinawa island was separated from a former island prior to dissociation of Amami-Oshima and Tokunoshima islands. Construction of a phylogenetic tree of Crotalinae venom group II PLA₂s based on the amino acid sequences revealed that neurotoxic PLA₂s including PLA-N and PLA-N(O) form an independent cluster which is distant from other PLA₂ groups such as PLA₂ type, basic [Asp⁴⁹]PLA₂ type, and [Lys⁴⁹]PLA₂ type. Comparison of the nucleotide sequence of PLA-N cDNA with those of the cDNAs encoding other T. flavoviridis venom PLA₂s showed that they have evolved in an accelerated manner. However, when comparison was made within the cDNAs encoding Crotalinae venom neurotoxic PLA₂s, their evolutionary rates appear to be reduced to a level between accelerated evolution and neutral evolution. It is likely that ancestral genes of neurotoxic PLA₂s evolved in an accelerated manner until they had acquired neurotoxic function and since then they have evolved with less frequent mutation, possibly for functional conservation. The nucleotide sequences reported in this paper are available from the GenBank/EMBL/DDBJ databases under accession numbers AB102728 and AB102729.     

The level of endogenous 1-aminocyclopropane-1-carboxylic acid in gametophytes of <Emphasis Type="Italic">Anemia phyllitidis</Emphasis> is increased during GA<Subscript>3</Subscript>-induced antheridia formation

Capillary electrophoresis revealed that the endogenous level of ACC (1-aminocyclopropane-1-carboxylic acid) in the gametophytes of Anemia phyllitidis was elevated during GA₃-induced male determination, whereas AOA (aminooxyacetic acid, specific inhibitor of ACC synthase) in untreated as well as in the GA₃-treated gametophytes decreased concentration of ACC. The mechanism of ethylene involvement in controlling antheridiogenesis reflected at the level of ACC, which is supposed to mediate interactions between ethylene and gibberellins, is proposed.