Apical localization of 1-aminocyclopropane-1-carboxylic acid and its conversion to ethylene in etiolated pea seedlings

The biosynthetic basis for the high rates of ethylene production by the apical region of etiolated pea (Pisum sativum L.) seedlings was investigated. The ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) was quantified in extracts of various regions of seedlings by measuring isotopic dilution of a ²H-labelled internal standard using selected-ion-monitoring gas chromatography/mass spectrometry. The ACC levels in the apical hook and leaves were much higher than in the expanded internodes of the epicotyl. The capacity of excised tissue sections to convert exogenous ACC to ethylene was also much greater in the apical region, reflecting the distribution of soluble protein in the epicotyl.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - FW fresh weight - GC/MS coupled gas chromatography/mass spectrometry - HPLC high-performance liquid chromatography     

Phytochrome-controlled ethylene biosynthesis of intact etiolated bean seedlings

Intact etiolated bean (Phaseolus vulgaris L. cv. Limburgse vroege) seedlings were illuminated with red light (10.5 W·m^-2) for 10 min. After different time intervals ethylene production, and contents of 1-aminocyclopropane-1-carboxylic acid (ACC) and 1-(malonylamino)cyclopropane-1-carboxylic acid were measured. The red-light-induced decrease of ethylene production in 8-d-old intact etiolated bean seedlings was fast, strong and long-lasting ad was mediated through the phytochrome system. This effect appeared to be strictly age-dependent, as it could not be detected in plants younger than 6 d or older than 11 d.The capacity for the conversion of ACC to ethylene was not affected by red light. The inhibitory effect of the light treatment on ethylene production could be related to a reduced free-ACC content. This reduction was a consequence of a temporary non-reversible increase of ACC malonylation and a long-lasting, for a certain time reversible, inhibition of ACC synthesis. The effect of a brief irradiation with red light on the decrease of ethylene production and free-ACC content was completed after about 2 h. Reversibility by far-red, however, persisted for at least 3 h, and was lost between 3 and 6 h.Abbrevation ACC 1-aminocyclopropane-1-carboxylic acid - M-ACC 1-(malonylamino)cyclopropane-1-carboxylic acid     

The role of ethylene in the regeneration of Helianthus annuus (sunflower) plants from callus

Hypocotyl-derived callus from the Helianthus annuus L. inbred line SS415B regenerated significantly more plants if the seedlings were grown in the light. The difference between light- and dark-grown seedlings was not correlated with differences in seedling ethylene production, but seemed to be due to a difference in sensitivity to ethylene at a specific time during seedling growth. Treating 3-day-old dark-grown seedlings with 10 μ M aminoethoxyvinylglycine (AVG) effectively inhibited ethylene production for at least 7 days. Hypocotyl callus derived from AVG-treated seedlings gave the same amount of regeneration as callus from light-grown seedlings. Promotion of regeneration by AVG was not seen unless the 3-day-old seedlings were grown for 4 additional days prior to culturing hypocotyl explants. The effects of AVG could be reversed by treatment with 1-aminocyclopropane-1-carboxylic acid (ACC) during these 4 days. After the 4 days, ACC was no longer effective.     

Effects of 1-aminocyclopropane-1-carboxylic acid and oxygen concentrations on in vivo and in vitro activity of ACC oxidase of sunflower hypocotyl segments

In vivo ethylene production by hypocotyl segments of sunflower seedlings and in vitro activity of 1-aminocyclopropane-1-carboxylic acid oxidase (formerly ethylene-forming enzyme) extacted from the same tissues increase with increasing concentrations of 1-aminocyclopropane-1-carboxylic acid (ACC) and oxygen. ACC oxidase activity follows Michaelis-Menten kinetics. The apparent Km values of the enzyme towards ACC, estimated in vivo and in vitro, are respectively 219 M and 20.6 M. Both Km values towards O₂ are similar, ca 10.6–11.4%. A decrease in concentration in one of the substrates (ACC or O₂) results in an increase in in vivo apparent Km of ACC oxidase for the other substrate. On the contrary, Km values of the enzyme towards ACC or O₂ estimated in vitro are not dependent upon the concentration of the other substrate (ACC or O₂).Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - EFE ethylene-forming enzyme - MACC malonylate 1-aminocyclopropane-1-carboxylic acid - SD standard deviation     

Rhythmicity in ethylene production in cotton seedlings

Cotyledons of cotton (Gossypium hirsutum L.) seedlings grown under a photoperiod of 12 hour darkness and 12 hour light showed daily oscillations in ethylene evolution. The rate of ethylene evolution began to increase toward the end of the dark period and reached a maximum rate during the first third of the light period, then it declined and remained low until shortly before the end of the dark period. The oscillations in ethylene evolution occurred in young, mature, and old cotyledons (7 to 21 day old). These oscillations in ethylene evolution seemed to be endogenously controlled since they continued even when the photoperiod was inverted. Moreover, in continuous light the oscillations in ethylene evolution persisted, but with shorter intervals between the maximal points of ethylene evolution. In continuous darkness the oscillations in ethylene evolution disappeared. The conversion of [3,4-¹⁴C]methionine into [¹⁴C] ethylene followed the oscillations in ethylene evolution in the regular as well as the inverted photoperiod. On the other hand, the conversion of applied 1-aminocyclopropane-1-carboxylic acid into ethylene did not follow the oscillations in ethylene evolution, but was affected directly by the light conditions. Always, light decreased and darkness increased the conversion of applied 1-aminocyclopropane-1-carboxylic acid into ethylene. It is concluded that in the biosynthetic pathway of ethylene the conversion of 1-aminocyclopropane-1-carboxylic acid into ethylene is directly affected by light while an earlier step is controlled by an endogenous rhythm.     

The quantification of 1-(malonylamino)cyclopropane-1-carboxylic acid in plant tissues by quantitative mass spectrometry

A method for the quantitation of 1-(malonylamino)cyclopropane-1-carboxylic acid (MACC), a conjugated form of 1-aminocyclopropane-1-carboxylic acid (ACC), in plants is described. [2,2,3,3-²H₄]MACC has been used as an internal standard for selected ion monitoring/isotope dilution quantitation of MACC in wheat seedlings and in tomato leaves. This method is compared with a widely-used two step indirect assay for MACC, which is based upon hydrolysis of MACC to ACC and conversion of ACC by hypochlorite reagent to ethylene which is subsequently quantified by gas chromatography.     

Uniconazole inhibits stress-induced ethylene in wheat and soybean seedlings

Previous studies have shown that uniconazole inhibits ethylene synthesis and protects plants from various stresses. The present research was conducted to delineate the mechanism of ethylene inhibition by uniconazole [(E)-(p-chlorophenyl)-4,4-dimethyl-2-(1,2,4-triazol-1-yl)-1-penten-3-ol]. Following heat stress of 48°C for 3 h, the shoots of the control wheat seedlings became desiccated, and the seedlings lost 23% of their fresh mass 8 h after stress. The control soybean seedlings had epinastic unifoliate leaves 5 h after foliar application (4.4 g.a.i./ha) of the herbicide triclopyr [(3,5,6-trichloro-2-pyridinyl)oxyacetic acid]. Soil drench applications of uniconazole, a potent member of the triazole family, reduced these symptoms associated with heat and herbicide stress in wheat (5.0 mg/L) and soybean (0.4 mg/L) seedlings, respectively. Basal ethylene production was inhibited 32 and 48% by uniconazole in the wheat and acotyledonous soybean seedlings, respectively. Following a 48°C heat stress, 1-aminocyclopropane-1-carboxylic acid (ACC) levels increased 40% in both the control and uniconazole-treated wheat seedlings. After triclopyr application, ACC levels increased 400% in both the control and uniconazoletreated soybean seedlings. The increased ACC levels, following stress, were accompanied by increased ethylene production from the control, but not from the uniconazole-treated wheat and acotyledonous soybean seedlings. Uniconazole treatment did not significantly change the basal or stress-induced N-malonyl-1-aminocyclopropane-1-carboxylic acid (MACC) levels compared to controls. These results suggest that uniconazole inhibits ethylene synthesis by interfering with the conversion of ACC to ethylene in wheat and acotyledonous soybean seedlings. Ethylene production and ACC conversion were not inhibited by uniconazole in excised soybean cotyledons. These results indicate that different ethylene-forming enzyme (EFE) systems operate in the soybean acotyledonous seedling and cotyledon, and the system in the former is inhibited by uniconazole.     

Uniconazole inhibits stress-induced ethylene in wheat and soybean seedlings

Previous studies have shown that uniconazole inhibits ethylene synthesis and protects plants from various stresses. The present research was conducted to delineate the mechanism of ethylene inhibition by uniconazole [(E)-(p-chlorophenyl)-4,4-dimethyl-2-(1,2,4-triazol-1-yl)-1-penten-3-ol]. Following heat stress of 48°C for 3 h, the shoots of the control wheat seedlings became desiccated, and the seedlings lost 23% of their fresh mass 8 h after stress. The control soybean seedlings had epinastic unifoliate leaves 5 h after foliar application (4.4 g.a.i./ha) of the herbicide triclopyr [(3,5,6-trichloro-2-pyridinyl)oxyacetic acid]. Soil drench applications of uniconazole, a potent member of the triazole family, reduced these symptoms associated with heat and herbicide stress in wheat (5.0 mg/L) and soybean (0.4 mg/L) seedlings, respectively.Basal ethylene production was inhibited 32 and 48% by uniconazole in the wheat and acotyledonous soybean seedlings, respectively. Following a 48°C heat stress, 1-aminocyclopropane-1-carboxylic acid (ACC) levels increased 40% in both the control and uniconazole-treated wheat seedlings. After triclopyr application, ACC levels increased 400% in both the control and uniconazoletreated soybean seedlings. The increased ACC levels, following stress, were accompanied by increased ethylene production from the control, but not from the uniconazole-treated wheat and acotyledonous soybean seedlings. Uniconazole treatment did not significantly change the basal or stress-induced N-malonyl-1-aminocyclopropane-1-carboxylic acid (MACC) levels compared to controls. These results suggest that uniconazole inhibits ethylene synthesis by interfering with the conversion of ACC to ethylene in wheat and acotyledonous soybean seedlings. Ethylene production and ACC conversion were not inhibited by uniconazole in excised soybean cotyledons. These results indicate that different ethylene-forming enzyme (EFE) systems operate in the soybean acotyledonous seedling and cotyledon, and the system in the former is inhibited by uniconazole.     

ACC conversion to ethylene by sunflower seeds in relation to maturation,germination and thermodormancy

Conversion of exogenous 1-aminocyclopropane-1-carboxylic acid (ACC) to ethylene was studied in sunflower (Helianthus annuus L., cv. Mirasol) seeds in relation to germinability. Ethylene production from ACC decreased during seed maturation, and non-dormant mature seeds were practically unable to synthesize ethylene until germination and growth occurred, indicating that ethylene forming enzyme (EFE) activity developed during tissue imbibition and growth. ACC conversion to ethylene was reduced by the presence of pericarp, and in young seedlings it was less in cotyledons than in growing axes.ACC conversion to ethylene by cotyledons from young seedlings was optimal at c. 30°C, and was strongly inhibited at 45°C. Pretreatment of imbibed seeds at high temperature (45°C) induced a thermodormancy and a progressive decrease in EFE activity.Abscisic acid and methyl-jasmonate, two growth regulators which inhibit seed germination and seedling growth, and cycloheximide were also shown to inhibit ACC conversion to ethylene by cotyledons of 3-day-old seedlings and by inbibed seeds.Abbreviations ABA abscisic acid - ACC 1-aminocyclopropane-1-carboxylic acid - CH cycloheximide - EFE ethylene forming enzyme - IAA indole-3-acetic acid - Me-Ja methyl-jasmonate     

Biochemical Decomposition of the Herbicide N-(3,4-Dichlorophenyl)-2-Methylpentanamide and Related Compounds

Organisms capable of decomposing N-(3,4-dichlorophenyl)-2-methylpentanamide (Karsil) were isolated, identified, and tested for their ability to hydrolyze this herbicide. Primary products of Karsil decomposition by cells and cell-free extracts of a Penicillium sp. were identified as 2-methyl-valeric acid and 3,4-dichloroaniline. The Karsil acylamidase (EC 3.5.1.a aryl acylamine amidohydrolase) was an induced enzyme. It was partially purified and tested for its ability to hydrolyze 25 related compounds. Some relations between the structures of these compounds and their susceptibility to enzymatic hydrolysis were discerned.     

The Effect of the Ethylene Action Inhibitor 1-Cyclopropenylmethyl Butyl Ether on Early Plant Growth

Increased levels of ethylene in plants are responsible for many deleterious effects such as early senescence, fruit deterioration and inhibition of root elongation. Several cyclopropene derivatives have previously been studied as inhibitors of ethylene action in plants. This study focuses on one such compound, 1-cyclopropenylmethyl butyl ether and its effect on the growth of roots and shoots of canola plants as well as rooting of mung bean seedlings 1-cyclopropenylmethyl butyl ether increased root length in canola plants, but had no significant effect on shoot length. In rooting studies, mung bean seedlings treated with 1-cyclopropenylmethyl butyl ether prior to root excision had fewer numbers of roots than control plants that were not treated with the ethylene action inhibitor. The same rooting study, when repeated in the presence of 1-aminocyclopropane-1-carboxylic acid (ACC), demonstrated an overall increase in the number of roots of inibitor-treated and non-treated plants, however, the inhibitor was still effective in decreasing the number of roots, compared to its non-treated conterpart. Online publication: 7 April 2005     

Ethylene precursors and antagonists increase embryogenesis of Hordeum vulgare L. anther culture

The role of ethylene in microspore embryogenesis and regeneration was analyzed by studying the effects of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) and the ethylene antagonists silver nitrate and silver thiosulphate on the androgenic response of in vitro cultured anthers of seven genotypes of barley. Incorporation of either ACC or silver salts in the culture medium lead to a significant increase in callus induction for five of the seven genotypes tested. The treatment that increased callus induction depended upon genotype. Only anthers cultured on 1 mg l^–1 silver thiosulphate gave rise to fertile plants in all seven genotypes tested.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole acetic acid - PAA phenyl acetic acid - STS silver thiosulphate - ACC 1-aminocyclopropane-1-carboxylic acid     

Characterization and kinetic parameters of ethylene-forming enzyme from avocado fruit.

Biosynthesis of the phytohormone ethylene in higher plants proceeds via the following pathway: S-adenosylmethionine----1-aminocyclopropane-1-carboxylic acid (ACC)----ethylene. Ethylene-forming enzyme (EFE), the enzyme responsible for the oxidation of ACC to ethylene, has been only partially characterized in vitro. We have obtained authentic EFE activity in vitro from extracts of avocado fruit (Persea americana Mill. cv Hass). Ammonium sulfate fractionation revealed the presence of two EFE activities, which we designate as EFE1 and EFE2. EFE1 activity utilizes ACC and O2 as substrates and requires Fe(II) and ascorbate as cofactors. The enzyme has a relatively low Km (32 microM) for ACC, discriminates diastereomers of 1-amino-2-ethyl-cyclopropane-1-carboxylic acid, and is inhibited competitively by 2-aminoisobutyric acid, thus confirming its identity with authentic EFE. Activity is retained in a 100,000 x g supernatant and has a pH optimum of 7.5-8.0, suggesting a cytosolic localization.     

Ethylene formation in Pisum sativum and Vicia faba protoplasts

Protoplasts isolated from leaves of peas (Pisum sativum L.) and of Vicia faba L. produced 1-aminocyclopropane-1-carboxylic acid (ACC) from endogenous substrate. Synthesis of ACC and conversion of ACC to ethylene was promoted by light and inhibited by 3-(3,4-dichlorophenyl)-1,1-dimethylurea and carbonyl cyanide m-chlorophenylhydrazone. Aminoethoxyvinylglycine inhibited ethylene synthesis to a minor extent when given during incubation of the protoplasts but was very effective when added both to the medium in which the protoplasts were isolated and to the incubation medium as well. Radioactivity from [U-¹⁴C]methionine was incorporated into ACC and ethylene. However, the specific radioactivity of the C-2 and C-3 atoms of ACC, from which ethylene is formed, increased much faster than the specific radioactivity of ethylene. It appears that ACC and ethylene are synthesized in different compartments of the cell and that protoplasts constitute a suitable system to study this compartmentation.Abbreviations ACC 1-Aminocyclopropane-1-carboxylic acid - AVG aminoethoxyvinylglycine - CCCP carbonyl cyanide m-chlorophenylhydrazone - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea     

Ethylene perception generates gravicompetence in gravi-incompetent leaves of rye seedlings

The elongating leaves of young rye seedlings do not show a gravitropic response when placed horizontally. However, after treatment with ethylene, either supplied exogenously via ethephon or by application of its precursor 1-aminocyclopropane-1-carboxylic acid (ACC), gravicompetence is seen. The inhibition of ethylene perception by 1-methylcyclopropene (MCP) prevents gravicompetence. Young rye leaves provide a useful model system in which to identify the components of the gravity sensing or response systems, the presence of which govern gravicompetence.     

High-performance liquid chromatography analysis of 1-aminocyclopropane-1-carboxylic acid using o-phthaldialdehyde precolumn derivatization

A simple, rapid, direct method for the HPLC analysis of 1-aminocyclopropane-1-carboxylic acid (ACC) as its o-phthaldialdehyde derivative is described. The method is sensitive to about 1 pmol and can be used on plant tissue extracts with no cleanup. It will prove valuable in plant extracts where the chemical conversion of ACC in the tissue extracts to ethylene is variable, or when analyzing the specific radioactivity of ACC produced from radiolabeled precursors.     

Effects of Cold Acclimation and Salicylic Acid on Changes in ACC and MACC Contents in Maize during Chilling

The effect of 0.5 mM salicylic acid (SA) pretreatment and of growing at hardening temperatures on chilling-induced changes in 1-aminocyclopropane-1-carboxylic acid (ACC) and malonyl 1-aminocyclopropane-1-carboxylic acid (MACC) was investigated in young maize (Zea mays L.) plants grown in hydroponic solution at 22/20 °C. Chilling at 5 °C caused an increase in ACC content;however, this increase was less pronounced in plants cold acclimated at 13/11 °C 4 d before the chilling treatment, and in those which were pretreated with SA for 1 d before the cold stress. Changes in MACC at low temperature showed no correlation with chilling tolerance in maize.     

Isolation of a glucosyltransferase from Arabidopsis thaliana active in the metabolism of the persistent pollutant 3,4-dichloroaniline

The pollutant 3,4-dichloroaniline (DCA) was rapidly detoxified by glucosylation in Arabidopsis thaliana root cultures, with the N-beta-d-glucopyranosyl-DCA exported into the medium. The N-glucosyltransferase (N-GT) responsible for this activity was purified from Arabidopsis suspension cultures and the resulting 50 kDa polypeptide analysed by matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) following tryptic digestion. The protein was identified as GT72B1. The GT was cloned and the purified recombinant enzyme shown to be highly active in conjugating DCA and 2,4,5-trichlorophenol, as well as several other chlorinated phenols and anilines, demonstrating both N-GT and O-GT activity. GT72B1 showed little activity towards natural products with the exception of the tyrosine catabolite 4-hydroxyphenylpyruvic acid. Both O-GT and N-GT activities were enhanced in both plants and cultures treated with herbicide safeners, demonstrating the chemical inducibility of this detoxification system in Arabidopsis.     

Relationship between the malonylation of 1-aminocyclopropane-1-carboxylic acid and D-amino acids in mung-bean hypocotyls

In sections from hypocotyls of dark-grown mung-bean (Vigna radiata L.) seedlings, D-phenylalanine and D-methionine (D-met) inhibited the formation of 1-(malonylamino)cyclopropane-1-carboxylic acid from exogenously administered 1-aminocyclopropane-1-carboxylic acid (ACC), resulting in an increase in free ACC content and stimulation of ethylene production, whereas their L-enantiomers had little or no such effect. When the hypocotyls were administered D-Met, it was mainly metabolized to N-malonylmethionine and N-malonylmethionine sulfoxide, and this malonylation process was inhibited to a greater extent by ACC and D-amino acids (phenylalanine and serine) than by L-amino acids. These results indicate that malonylation of D-amino acids and of ACC are intimately interrelated.