A simplified method for determining 1-aminocyclopropane-1-carboxylic acid (ACC) in plant tissues using a mass selective detector

A sensitive and specific method is described for the routine assay of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) in 100–200 mg fresh weight samples of green or etiolated tissue. The method involves high performance liquid chromatography (HPLC) and gas chromatography linked to mass spectrometry (GCMS) and uses ¹⁴C-labelled ACC as an internal standard, N-benzoyl n-propyl ACC as an easily prepared derivative for HPLC and GCMS, and N-benzoyl isobutyl ACC as an internal standard for GCMS. The procedure is faster and safer than an existing GCMS method and more specific and reliable than indirect assays widely in use. The method has been used to measure ACC in maize roots, young leaves of cucumber, and aerobic or anaerobic seedlings of rice.     

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.     

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     

The influence of oxygen deficiency on ethylene synthesis, 1-aminocyclopropane-1-carboxylic acid levels and aerenchyma formation in roots of Zea mays

The relationship between ethylene production, 1-aminocyclopropane-l-carboxylic acid (ACC) concentration and aerenchyma formation (ethylene-promoted cavitation of the cortex) was studied using nodal roots of maize (Zea mays L. cv. LG11) subjected to various O₂ treatments. Ethylene evolution was 7–8 fold faster in roots grown at 3 kPa O₂ than in those from aerated solution (21 kPa O₂), and transferring roots from aerated solution to 3 kPa O₂ enhanced ethylene synthesis within less than 2 h. Ethylene production and ACC accumulation were closely correlated in different zones of hypoxic roots, regardless of whether O₂ was furnished to the roots through aerenchyma or external solution. Both ethylene production and ACC concentrations (fresh weight basis) were more than 10-fold greater in the distal 0–10 mm than in the fully expanded zone of roots at 3 kPa O₂. Aerenchyma formation occurred in the apical 20 mm of these roots. Roots transferred from air to anoxia accumulated less than 0. 1 nmol ACC (mg protein)^-1 for the first 1.75 h; no ethylene was produced in this time. The subsequent rise in ACC levels shows that ACC can reach high concentrations even in the absence of O₂, presumably due to a de-repression of ACC synthase. The hypothesis was therefore tested that anoxia in the apical region of the root caused enhanced synthesis of ACC, which was transported to more mature regions (10–20 mm behind the apex), where ethylene could be produced and aerenchyma formation stimulated. Surprisingly, exposure of intact root tips to anoxia inhibited aerenchyma formation in the mature root axis. High osmotic pressures around the growing region or excision of apices had the same effect, demonstrating that a growing apex is required for high rates of aerenchyma formation in the adjacent tissue.     

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     

Transport of 1-aminocyclopropane-1-carboxylic acid into isolated maize mesophyll vacuoles

Intracellular transport of the ethylene precursor, I-aminocyclopropane-1-carboxylic acid (ACC) can change the ACC concentration in cell compartments and impact ethylene biosynthesis. Transport of ACC into isolated maize ( Zea mays L.) mesophyll vacuoles was studied by silicon layer flotation filtering. The transport of ACC across the tonoplast was stimulated 2. 4- to 8. 1-fold by 5 m M MgATP, showed saturation kinetics with an apparent K_m for ACC of 20 μ M , and was optimal at 25°C. Transport of ACC was sensitive to the pH of the medium, falling as external pH rose. Effectors known to inhibit proton-translocating ATPases (N, N-dicyclohexylcarbodiimide) and to collapse the electrical (thiocyanate, valinomycin) and chemical (carbonylcyanide m -chlorophenylhydrazone, gramicidin) potential gradients for protons across the tonoplast all reduced ACC transport. The nonhydrolyzable MgATP analog. Mg adenylyl-imidodiphosphate, stimulated ACC transport as effectively as MgATP. Other nucleotides (MgADP, MgCTP, MgUTP, MgGTP) and MgPP_i had little or no effect. These results suggest that ACC uptake into isolated maize mesophyll vacuoles is carrier mediated, is dependent upon an electrochemical potential gradient for protons and is specifically regulated, but not necessarily energized, by MgATP     

Determination of 1-aminocyclopropane-1-carboxylic acid (ACC) in leaf tissue and xylem sap using capillary column gas chromatography and a nitrogen/phosphorus detector

The Lizada and Yang method, commonly used for analyzing 1-aminocyclopropane-1-carboxylic acid (ACC), the immediate precursor of the plant hormone ethylene, is subject to interference and lacks internal standards. The use of combined gas chromatography-mass spectrometry (GC-MS) overcomes these shortcomings but the method is expensive and unavailable to many laboratories. We describe an alternative physico-chemical method using a capillary column gas chromatograph fitted with a standard nitrogen/phosphorus detector. After forming the N-benzoyl n-propyl derivative, measurements of ACC concentrations in extracts of leaves and in xylem sap of tomato plants using the nitrogen/phosphorus detector were within 10% of those obtained by GC-MS. Concentrations in plants grown in well-drained soil were approximately 0.16 nmol g^–1 fresh weight (leaves) and 0.04–0.01 mmol m^–3 (sap). Flooding the soil for 48–72 h increased these values approximately 9-fold.     

The physiological role of lipoxygenase in ethylene formation from 1-aminocyclopropane-1-carboxylic acid in oat leaves

In order to understand the physiological significance of the in-vitro lipoxygenase (EC 1.13.11.12)-mediated ethylene-forming system (J.F. Bousquet and K.V. Thimann 1984, Proc. Natl. Acad. Sci. USA 81, 1724–1727), its characteristics were compared to those of an in-vivo ethylene-forming system. While oat (Avena sativa L.) leaves, as other plant tissues, preferentially converted only one of the 1-amino-2-ethylcyclopropane-1-carboxylic acid (AEC) isomers to 1-butene, the lipoxygenase system converted all four AEC isomers to 1-butene with nearly equal efficiencies. While the in-vivo ethylene-forming system of oat leaves was saturable with ACC with a K_m of 16 M, the lipoxygenase system was not saturated with ACC even at 10 mM. In contrast to the in-vivo results, only 10% of the ACC consumed in the lipoxygenase system was converted to ethylene, indicating that the reaction is not specific for ethylene formation. Increased ACC-dependent ethylene production in oat leaves following pretreatment with linoleic acid has been inferred as evidence of the involvement of lipoxygenase in ethylene production. We found that pretreating oat leaves with linoleic acid resulted in increased ACC uptake and thereby increased ethylene production. A similar effect was observed with oleic acid, which is not a substrate of lipoxygenase. Since linoleic acid hydroperoxide can substitute for lipoxygenase and linoleic acid in this system, it is assumed that the alkoxy radicals generated during the decomposion of linoleic acid hydroperoxide are responsible for the degradation of ACC to ethylene. Our results collectively indicate that the reported lipoxygenase system is not the in-vivo ethylene-forming enzyme.Abbreviations ACC 1-Aminocyclopropane-1-carboxylic acid - AEC 1-amino-2-ethylcyclopropane-1-carboxylic acid - Epps N-(2-hydroxyethyl)-piperazine-N-3-propanesulfonic acid - LH linoleic acid - LOOH linoleic acid hydroperoxide - pyridoxal-P pyridoxal-phosphate This work was presented at the 12th International Conference on Plant Growth Substances, Heidelberg, FRG, August 1985 (Abstract No. PO 5-52)     

Ethylene formation from 1-aminocyclopropane-1-carboxylic acid in homogenates of etiolated pea seedlings

Homogenates of etiolated pea (Pisum sativum L.) shoots formed ethylene upon incubation with 1-aminocyclopropane-1-carboxylic acid (ACC). In-vitro ethylene formation was not dependent upon prior treatment of the tissue with indole-3-acetic acid. When homogenates were passed through a Sephadex column, the excluded, high-molecular-weight fraction lost much of its ethylene-synthesizing capacity. This activity was largely restored when a heat-stable, low-molecular-weight factor, which was retarded on the Sephadex column, was added back to the high-molecular-weight fraction. The ethylene-synthesizing system appeared to be associated, at least in part, with the particulate fraction of the pea homogenate. Like ethylene synthesis in vivo, cell-free ethylene formation from ACC was oxygen dependent and inhibited by ethylenediamine tetraacetic acid, n-propyl gallate, cyanide, azide, CoCl₃, and incubation at 40°C. It was also inhibited by catalase. In-vitro ethylene synthesis could only be saturated at very high ACC concentrations, if at all. Ethylene production in pea homogenates, and perhaps also in intact tissue, may be the result of the action of an enzyme that needs a heat-stable cofactor and has a very low affinity for its substrate, ACC, or it may be the result of a chemical reaction between ACC and the product of an enzyme reaction. Homogenates of etiolated pea shoots also formed ethylene with 2-keto-4-mercaptomethyl butyrate (KMB) as substrate. However, the mechanism by which KMB is converted to ethylene appears to be different from that by which ACC is converted.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - IAA indole-3-acetic acid - KMB 2-keto-4-mercaptomethyl butyrate - SAM S-adenosylmethionine     

Role of IAA-oxidase in the formation of ethylene from 1-aminocyclopropane-1-carboxylic acid

The IAA-oxidase system of olive tree (Olea europea) in the presence of its substrate, IAA, and cofactors, DCP and Mn², forms ethylene from 1-aminocyclopropane-l-carboxylic acid (ACC) bound as a Schiffs base to pyridoxal phosphate. Similarly, olive leaf discs upon incubation with ACC liberate considerable amounts of ethylene. The results suggest that this IAA-oxidase system may be the one active in the last step in the biosynthesis of ethylene from methionine.     

Identification and characterization of three putative genes for 1-aminocyclopropane-1-carboxylate synthase from etiolated mung bean hypocotyl segments

The polymerase chain reaction (PCR) was used to produce 3 putative clones for ACC synthase from etiolated mung bean (Vigna radiata Rwilcz cv. Berken) hypocotyls. This was accomplished by utilizing genomic DNA from mung bean and degenerate primers made from information derived from highly conserved regions of ACC synthase from different plant tissues. The total length of pMAC-1, pMAC-2 and pMAC-3 are 308, 321, and 326 bp, respectively, all of which code for 68 amino acids. The introns for pMAC-1, pMAC-2 and pMAC-3 are 92, 105, and 110 bp, respectively. The degrees of homology at the DNA level for each of these clones is ca. 80% in their coding region and ca. 50% in their respective introns. This is the first report providing evidence that there are at least 3 genes for ACC synthase in etiolated mung bean.     

Isolation and characterization of an unusual 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase gene from Enterobacter cloacae UW4

A genomic library of the 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase-containing plant growth-promoting bacterium Enterobacter cloacae UW4 in pUC19 in Escherichia coli was screened for the ability to utilize ACC as a sole source of nitrogen. One of the clones that was isolated contained a plasmid with an insert of approximately 0.8 kb that conferred ACC deaminase activity. Sequence analysis revealed that this DNA fragment contains an open-reading frame of 696 nucleotides predicted to encode a protein of 232 amino acids, a member of the amidohydrolase protein superfamily, i.e., a deaminase that contains a mononuclear or binuclear metal center as compared to the canonical ACC deaminase which contains pyridoxal phosphate as a co-factor.     

The effect of light on the production of ethylene from 1-aminocyclopropane-1-carboxylic acid by leaves

White light inhibits the conversion of 1-amino-cyclopropane-1-carboxylic acid (ACC) in discs of green leaves of tobacco (Nicotiana tabacum L.) and segments of oat (Avena sativa L.) leaves by from 60 to 90%. Etiolated oat leaves do not show this effect. The general nature of the effect is shown by its presence in both a mono- and a dicotyledon. Since the leaves have been grown and pre-incubated in light, yet can produce from 2 to 9 times as much ethylene in the dark as in the light, it follows that the light inhibition is fully reversible. The inhibition by light is about equal to that exerted in the dark by CoCl₂; it can be partly reversed by dithiothreitol and completely by mercaptoethanol. Thus the light is probably acting, via the photosynthetic system, on the SH group(s) of the enzyme system converting ACC to ethylene.Abbreviation ACC 1-aminocyclopropane-1-carboxylic acid     

Iron: an essential cofactor for the conversion of 1-aminocyclopropane-1-carboxylic acid to ethylene

The activity of the ethylene-forming enzyme (EFE) in suspension-cultured tomato (Lycopersicon esculentum Mill.) cells was almost completely abolished within 10 min by 0.4 mM of the metal-chelating agent 1,10-phenanthroline. Subsequent addition of 0.4 mM FeSO₄ immediately reversed this inhibition. A partial reversion was also obtained with 0.6 mM CuSO₄ and ZnSO₄, probably as a consequence of the release of iron ions from the 1,10-phenanthroline complex. The inhibition was not reversed by Mn²⁺ or Mg²⁺. Tomato cells starved of iron exhibited a very low EFE activity. Addition of Fe²⁺ to these cells caused a rapid recovery of EFE while Cu²⁺, Zn²⁺ and other bivalent cations were ineffective. The recovery of EFE activity in iron-starved cells was insensitive to cycloheximide and therefore does not appear to require synthesis of new protein. The EFE activity in tomato cells was induced by an elicitor derived from yeast extract. Throughout the course of induction, EFE activity was blocked within 10–20 min by 1,10-phenanthroline, and the induced level was equally rapidly restored after addition of iron. We conclude that iron is an essential cofactor for the conversion of 1-aminocyclopropane-1-carboxylic acid to ethylene in vivo.     

Synthesis and Degradation of 1-Aminocyclopropane-1-carboxylic Acid by Penicillium citrinum

1-Aminocyclopropane-1-carboxylic acid (ACC), which is a precursor of ethylene in plants, has never been known to occur in microorganisms. We describe the synthesis of ACC by Penicillium citrinum, purification of ACC synthase [EC 4.4.1.14] and ACC deaminase [EC 4.1.99.4], and their properties. Analyses of P. citrinum culture showed occurrence of ACC in the culture broth and in the cell extract. ACC synthase was purified from cells grown in a medium containing 0.05% L-methionine and ACC deaminase was done from cells incubated in a medium containing 1% 2-aminoisobutyrate. The purified ACC synthase, with a specific activity of 327 milliunit/mg protein, showed a single band of M _r 48,000 in SDS-polyacrylamide gel electrophoresis. The molecular mass of the native enzyme by gel filtration was 96,000 Da. The ACC synthase had the K _m for S-adenosyl-L-methionine of 1.74 mM and k _cat of 0.56 s^-1 per monomer. The purified ACC deaminase, with a specific activity of 4.7 unit/mg protein, showed one band in SDS-polyacrylamide gel electrophoresis of M _r 41,000. The molecular mass of the native ACC deaminase was 68,000 Da by gel filtration. The enzyme had a K _m for ACC of 4.8 mM and k _cat of 3.52 s^-1. The presence of 7 mM Cu²⁺ in alkaline buffer solution was effective for increasing the stability of the ACC deaminase in the process of purification.     

Immunocytolocalization of 1-aminocyclopropane-1-carboxylic acid oxidase in tomato and apple fruit

The subcellular localization of 1-aminocyclopropane-1-carboxylic acid oxidase (ACC oxidase), an enzyme involved in the biosynthesis of ethylene, has been studied in ripening fruits of tomato (Lycopersicum esculentum Mill.). Two types of antibody have been raised against (i) a synthetic peptide derived from the reconstructed pTOM13 clone (pRC13), a tomato cDNA encoding ACC oxidase, and considered as a suitable epitope by secondary-structure predictions; and (ii) a fusion protein overproduced in Escherichia coli expressing the pRC13 cDNA. Immunoblot analysis showed that, when purified by antigen affinity chromatography, both types of antibody recognized a single band corresponding to ACC oxidase. Superimposition of Calcofluor white with immunofluorescence labeling, analysed by optical microscopy, indicated that ACC oxidase is located at the cell wall in the pericarp of breaker tomato and climacteric apple (Malus × domestica Borkh.) fruit. The apoplasmic location of the enzyme was also demonstrated by the observation of immunogold-labeled antibodies in this region by both optical and electron microscopy. Transgenic tomato fruits in which ACC-oxidase gene expression was inhibited by an antisense gene exhibited a considerable reduction of labeling. Immunocytological controls made with pre-immune serum or with antibodies pre-absorbed on their corresponding antigens gave no staining. The discrepancy between these findings and the targeting of the protein predicted from sequences of ACC-oxidase cDNA clones isolated so far is discussed.     

Light- and temperature-entrained circadian regulation of activity and mRNA accumulation of 1-aminocyclopropane-1-carboxylic acid oxidase in Stellaria longipes

Stem and leaf tissues of Stellaria longipes Goldie (prairie ecotype) exhibit circadian rhythmicity in the activity and mRNA abundance for 1-aminocyclopropane-1-carboxylic acid oxidase (EC 1.4.3). The steady-state mRNA levels and enzymatic activity levels fluctuated with a period of approximately 24 h and reached their maxima by the middle of the light phase and minima by the middle of the dark phase. The oscillations showed damping under constant light, constant dark and constant temperature conditions, indicating that the rhythm is entrained by an external signal. The results indicate that light/dark cycles have greater entraining effects than temperature cycles. A 15-min red light pulse, but not a blue light pulse, could reset rhythm in continuous darkness, suggesting the possible role of a red-light signal transduction pathway in the circadian regulation of 1-aminocyclopropane-1-carboxylic acid oxidase.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - DD continuous dark - LD light-dark - LL continuous light - ZT Zeitgeber time (start of light period for circadian entrainment) This study was supported by operating grants to C.C.C., and D.M.R. from the Natural Sciences and Engineering Research Council of Canada.The authors gratefully acknowledge the award of a Bettina Bahlsen memorial Graduate Scholarship by University of Calgary to A.K. We are grateful to Dr. M.M. Moloney for allowing the use of his laboratory facilities.