Acid Oxidase Research Articles

Fungal Elicitor Induces Singlet Oxygen Generation, Ethylene Release and Saponin Synthesis in Cultured Cells of Panax ginseng C. A. Meyer

Singlet oxygen is a high-energy molecular oxygen species. As one of the most active intermediates involved in chemical and biochemical reactions, singlet oxygen plays essential roles in plant responses to UV and strong light. Here, we report that Cle, an elicitor derived from fungal cell walls, induces the generation of singlet oxygen in cell cultures of ginseng, Panax ginseng. Cle treatment also triggers the activation of plasma membrane NADPH oxidase and 1-aminocyclopropane-1-carboxylic acid oxidase (ACO), subsequently leading to ethylene release and increased saponin synthesis, as shown by increased mRNA expression of squalene synthase (SQS) and squalene epoxidase (SQE), and accumulation of beta-amyrin synthase (beta-AS). Suppression of Cle-induced singlet oxygen generation or inhibition of ethylene production blocks saponin synthesis, whereas treatment of ginseng cells with ethylene or singlet oxygen induces the synthesis of saponin. Together, these results indicate that Cle-induced production of both singlet oxygen and ethylene is required for saponin synthesis, and that singlet oxygen may function upstream of ethylene during Cle-induced saponin synthesis.

Involvement of a Flavosemiquinone in the Enzymatic Oxidation of Nitroalkanes Catalyzed by 2-Nitropropane Dioxygenase

2-Nitropropane dioxygenase (EC 1.13.11.32) catalyzes the oxidation of nitroalkanes into their corresponding carbonyl compounds and nitrite. In this study, the ncd-2 gene encoding for the enzyme in Neurospora crassa was cloned, expressed in Escherichia coli, and the resulting enzyme was purified. Size exclusion chromatography, heat denaturation, and mass spectroscopic analyses showed that 2-nitropropane dioxygenase is a homodimer of 80 kDa, containing a mole of non-covalently bound FMN per mole of subunit, and is devoid of iron. With neutral nitroalkanes and anionic nitronates other than propyl-1- and propyl-2-nitronate, for which a non-enzymatic free radical reaction involving superoxide was established using superoxide dismutase, substrate oxidation occurs within the enzyme active site. The enzyme was more specific for nitronates than nitroalkanes, as suggested by the second order rate constant k(cat)/K(m) determined with 2-nitropropane and primary nitroalkanes with alkyl chain lengths between 2 and 6 carbons. The steady state kinetic mechanism with 2-nitropropane, nitroethane, nitrobutane, and nitrohexane, in either the neutral or anionic form, was determined to be sequential, consistent with oxygen reacting with a reduced form of enzyme before release of the carbonyl product. Enzyme-monitored turnover with ethyl nitronate as substrate indicated that the catalytically relevant reduced form of enzyme is an anionic flavin semiquinone, whose formation requires the substrate, but not molecular oxygen, as suggested by anaerobic substrate reduction with nitroethane or ethyl nitronate. Substrate deuterium kinetic isotope effects with 1,2-[(2)H(4)]nitroethane and 1,1,2-[(2)H(3) ethyl nitronate at pH 8 yielded normal and inverse effects on the k(cat)/K(m) value, respectively, and were negligible on the k(cat) value. The k(cat)/K(m) and k(cat) pH profiles with anionic nitronates showed the requirement of an acid, whereas those for neutral nitroalkanes were consistent with the involvement of both an acid and a base in catalysis. The kinetic data reported herein are consistent with an oxidasestyle catalytic mechanism for 2-nitropropane dioxygenase, in which the flavin-mediated oxidation of the anionic nitronates or neutral nitroalkanes and the subsequent oxidation of the enzyme-bound flavin occur in two independent steps.

New Amperometric Biosensors Based on Diamond Paste for the Assay of L‐ and D‐Pipecolic Acids in Serum Samples

Monocrystalline natural diamond, L‐amino acid oxidase (L‐AAOD), D‐amino acid oxidase (D‐AAOD), and paraffin oil were used for the design of the modified diamond paste. The technique used for the direct voltammetric assay was differential pulse voltammetry (DPV) with applied potential pulse amplitude of 25 mV vs. Ag/AgCl. Using the new amperometric biosensors L‐pipecolic acid (L‐PA) and D‐pipecolic acid (D‐PA) were determined reliably from serum samples at 700 and 200 mV vs. Ag/AgCl, respectively, with low limits of detection.

Crystal Structure and Mechanistic Implications of 1-Aminocyclopropane-1-Carboxylic Acid Oxidase—The Ethylene-Forming Enzyme

The final step in the biosynthesis of the plant signaling molecule ethylene is catalyzed by 1-aminocyclopropane-1-carboxylic acid oxidase (ACCO). ACCO requires bicarbonate as an activator and catalyzes the oxidation of ACC to give ethylene, CO2, and HCN. We report crystal structures of ACCO in apo-form (2.1 A resolution) and complexed with Fe(II) (2.55 A) or Co(II) (2.4 A). The active site contains a single Fe(II) ligated by three residues (His177, Asp179, and His234), and it is relatively open compared to those of the 2-oxoglutarate oxygenases. The side chains of Arg175 and Arg244, proposed to be involved in binding bicarbonate, project away from the active site, but conformational changes may allow either or both to enter the active site. The structures will form a basis for future mechanistic and inhibition studies.

Developmental regulation of peach ACC oxidase promoter--GUS fusions in transgenic tomato fruits.

A genomic DNA sequence (PpACO1) encoding 1-aminocyclopropane-1-carboxylic acid oxidase (ACO) from peach (Prunus persica L. Batsch cv. Loring) was isolated. It has four exons interrupted by three introns and 2.9 kb of flanking region 5' of the translational start codon. Previous work with the cDNA demonstrated that accumulation of the peach ACO message correlated with increasing amounts of ethylene synthesized by the fruit as they ripened. To identify regulatory elements in the peach ACC oxidase gene, chimeric fusions between 403, 610, 901, 1319, 2141, and 2919 bp of the 5' flanking region of the PpACO1 sequence and the beta-glucuronidase (GUS) coding sequence were constructed and used to transform tomato (Lycopersicon esculentum [Mill] cv. Pixie). Fruits from the various promoter lines were analysed for GUS expression by histochemical GUS staining, GUS quantitative enzyme activity determination, and measuring the relative amounts of GUS mRNA. Constructs with the smallest promoter of 403 bp had significant GUS expression in fruit, but not in other tissues, indicating the presence of a region that affects tissue-specific expression. An increase in GUS expression was observed with promoters longer than 901 bp, indicating an enhancer region between -1319 and -901. The full-length promoter of 2919 bp directed GUS expression in the green stage of fruit development, and increased GUS expression as fruit matured, indicating a regulatory region between -2919 and -2141 that controls the temporal expression of the gene in fruit. Only the full-length promoter sequence demonstrated responsiveness to ethylene.

Ascorbic acid biosynthesis in hens producing strong and weak eggshells

1. An experiment was conducted with two strains of layers to ascertain whether the reduction in eggshell strength occurring at the end of the production cycle is the result of reduced ascorbic acid biosynthesis. 2. Hens producing strong and weak eggshells were identified within each strain and egg production, egg weight, per cent shell, shell surface density, plasma, adrenal and hepatic ascorbic acid and renal L-gulonolactone oxidase activity were measured. 3. The strains differed in ascorbic acid synthesis, as measured by L-gulonolactone oxidase activity, and tissue ascorbic acid concentration. 4. Comparison of results from birds producing eggs of similar weight but markedly different in shell strength detected neither a shell strength group × strain interaction nor an effect of shell strength group on plasma and hepatic ascorbic acid and activity of L-gulonolactone oxidase. 5. The results did not support the hypothesis that tissue ascorbate and ascorbic acid biosynthesis are reduced in old hens producing weak eggshells.

Mechanistic studies of 1-aminocyclopropane-1-carboxylic acid oxidase: single turnover reaction.

The final step in the biosynthesis of the plant hormone ethylene is catalyzed by the non-heme iron-containing enzyme 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase (ACCO). ACC is oxidized at the expense of O(2) to yield ethylene, HCN, CO(2), and two waters. Continuous turnover of ACCO requires the presence of ascorbate and HCO(3)(-) (or an alternative form), but the roles played by these reagents, the order of substrate addition, and the mechanism of oxygen activation are controversial. Here these issues are addressed by development of the first functional single turnover system for ACCO. It is shown that 0.35 mol ethylene/mol Fe(II)ACCO is produced when the enzyme is combined with ACC and O(2) in the presence of HCO(3)(-) but in the absence of ascorbate. Thus, ascorbate is not required for O(2) activation or product formation. Little product is observed in the absence of HCO(3)(-), demonstrating the essential role of this reagent. By monitoring the EPR spectrum of the sample during single turnover, it is shown that the active site Fe(II) oxidizes to Fe(III) during the single turnover. This suggests that the electrons needed for catalysis can be derived from a fraction of the initial Fe(II)ACCO instead of ascorbate. Addition of ascorbate at 10% of its K(m) value significantly accelerates both iron oxidation and ethylene formation, suggesting a novel high-affinity effector role for this reagent. This role can be partially mimicked by a non-redox-active ascorbate analog. A mechanism is proposed that begins with ACC and O(2) binding, iron oxidation, and one-electron reduction to form a peroxy intermediate. Breakdown of this intermediate, perhaps by HCO(3)(-)-mediated proton transfer, is proposed to yield a high-valent iron species, which is the true oxidizing reagent for the bound ACC.

CHILLING STORAGE TEMPERATURE AFFECTS ETHYLENE BIOSYNTHESIS IN MANGO FRUIT DURING RIPENING

Effects of non-chilling and chilling storage temperatures on ethylene biosynthesis during fruit ripening in 'Kensington Pride' mango (Mangifera indica) were studied. Mature mango fruit were stored at chilling (5 deg C) and non-chilling (15 deg C) temperatures for two weeks, were ripened at 22 plus/minus 1 deg C and then assessed for chilling injury (CI), ethylene production, 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase and ACC synthase activities and ACC content during the fruit ripening period. CI symptoms were observed during fruit ripening in the fruit stored at 5 deg C and significantly increased as the ripening period was extended. Ethylene production, ACC oxidase, ACC synthase and ACC content in the skin and pulp were reduced in the fruit which were stored at 5 deg C compared with those stored at 15 deg C prior to ripening. These results showed that the suppression of ethylene biosynthesis during ripening in fruit stored at chilling temperature was due to reduced activities of ACC oxidase, and ACC synthase, and may be associated with the development of CI symptoms in 'Kensington Pride' mangos.

The simultaneous determination of 1-aminocyclopropane-1-carboxylic acid and cyclopropane-1,1-dicarboxylic acid in Lycopersicum esculentum by high-performance liquid chromatography--electrospray tandem mass spectrometry.

Varying concentrations of cyclopropane-1,1-dicarboxylic acid (CDA), an inhibitor of 1-aminocyclopropane-1-carboxylic acid oxidase, added to the solid culture medium of tomato nodal shoot segments resulted in a reduction in the level of endogenous ethylene according to the concentration of inhibitor applied. Following treatment with inhibitor, plants were homogenised and the concentrations of CDA and of 1-aminocyclopropane-1-carboxylic acid (ACC) were measured simultaneously in the resulting juice using an HPLC-ESI/MS-MS method. The levels of CDA and ACC measured in the plant tissues were associated with the concentration of inhibitor added to the solid medium. The HPLC-ESI/MS-MS method described produced limits of detection of 0.8 pmol for ACC and of 4 pmol for CDA.

The role of ethylene during the infection of Nicotiana tabacum by Colletotrichum destructivum.

Two periods of increased ethylene production were observed after inoculation of Nicotiana tabacum by Colletotrichum destructivum. This pathogen exhibits an intracellular hemibiotrophic infection process, with a biotrophic phase followed by a necrotrophic phase. Ethylene production first increased during the biotrophic phase with a peak at 24 h before the necrotrophic phase. A second increase in ethylene occurred late in the necrotrophic phase when the lesions were expanding. Two different 1-aminocyclopropane-1-carboxylic acid synthase genes showed increased expression after the first ethylene peak with a maximum at 24 h before the second ethylene increase. Expression of an 1-aminocyclopropane-1-carboxylic acid oxidase (ACO) gene increased during the first ethylene peak and then declined at the beginning of the second ethylene increase. A second ACO gene showed relatively little change in expression during infection with slightly higher expression at 24 h before the second ethylene increase, and a third ACO gene showed a progressive decline in expression with a major decrease occurring before the second ethylene increase. Inoculation of ethylene-insensitive tobacco with C. destructivum revealed that it was more susceptible than the wild type. The changes in ethylene production and associated gene expression as well as the increased disease susceptibility of ethylene-insensitive tobacco indicate that ethylene plays a role in this interaction, perhaps as a signalling molecule to trigger defense mechanisms.

Synergistic effect of auxins and polyamines in hairy roots of Cichorium intybus L. during growth, coumarin production and morphogenesis

Hairy roots of Cichorium intybus obtained by infecting with different Agrobacterium rhizogenes strains (LMG-150 and A20/83) were studied for total endogenous indole-3-acetic acid (IAA) levels and indole-3-acetic acid oxidase (IAAO) activity. The roots initiated by LMG- 150 showed higher endogenous IAA levels as well as IAAO activity as compared to the roots from A20/83. Coumarin production in roots obtained by both of these strains strictly correlated with growth, with higher content in the roots obtained by LMG- 150. Moreover roots from LMG-150 showed increased growth index, length of primary roots and number of secondary and tertiary roots. The roots derived from LMG-150 were studied for total endogenous IAA and IAAO activity under the exogenous administration of polyamines and fungal elicitors. The treatment with putrescine (Put) at 1.5 mM level showed maximum endogenous IAA levels and IAAO activity as compared to the control and other polyamine administration, it also supported faster growth in terms of biomass accumulation, and total coumarin production. Of the various treatments, mycelial extract (ME) and culture media filtrate (CMF) of Pythium aphanidermatum and Phytopthora parasitica var. nicotiana, the treatment with 1 % CMF of P. parasitica var. nicotiana, resulted in maximum IAA levels and IAAO activity, which was supported by maximum biomass, coumarin production as compared to the control and other elicitor treatments. Two different regenerants of chicory obtained through A. rhizogenes LMG-150 designated as T-I and T-II, were studied for total endogenous IAA levels and IAAO activity. T-II showed higher titers of IAA with higher activity of IAAO as compared to T-I. Endogenous titer of IAA and IAAO activity was found to be maximum in transformed roots as compared to T-I, T-II, normal roots and normal plants. Our work showed a variation in endogenous auxin levels in these transformed plants. There exists a synergistic effect of endogenous IAA titers and polyamines in regulating root morphogenesis. Fungal elicitors influenced growth and coumarin production and an elicitor preparation of 1 % CMF of P. parasitica var. nicotiana gave spontaneous regeneration of shoots. The implications of these results are discussed.

Calcium requirement for ethylene‐dependent responses involving 1‐aminocyclopropane‐1‐carboxylic acid oxidase in radicle tissues of germinated pea seeds*

ABSTRACTThe Ca2+ requirements of ethylene‐dependent responses were investigated in germinating seeds of Pisum sativum L. using 1‐aminocyclopropane‐1‐carboxylic acid (ACC) oxidase (Ps‐ACO1), ACC synthase (Ps‐ACS2) and class I β‐1,3‐glucanase as molecular markers. Ethylene biosynthesis and responsiveness are localized to the elongation and differentiation zones of the pea radicle. Ethylene treatment induced ectopic root hair formation in the cell elongation zone and promoted root hair elongation growth in the radicles of germinated seeds. Characterized Ca2+ antagonists, including EGTA, lanthanum, verapamil, ruthenium red, W‐7, lithium and neomycin, were used to test for the involvement of the apoplastic and the intracellular Ca2+‐pool, the Ca2+/calmodulin complex and the phoshoinositide (PI) cycle in the ethylene responses. Ca2+ release from internal pools, but no appreciabe apoplastic Ca2+, is involved in the transcriptional induction by ethylene of Ps‐ACO1 and in ectopic root hair formation in the radicle elongation zone of germinated pea seeds. Furthermore, the Ca2+/calmodulin complex and the PI cycle seem to be involved in these ethylene responses. In contrast, both the intracellular and the apoplastic Ca2+‐pools are required for the negative and positive ethylene responses to the gene expression of PS‐ACS2 and class I β‐1,3‐glucanase, respectively; and, apoplastic Ca2+ also promotes root hair elongation growth. Tissues from adult plants and germinating seeds exhibit temporal and spatial differences in the signal/response coupling by Ca2+ of ethylene‐regulated processes.

Coronatine and Abscission in Citrus

Coronatine is a polyketide phytotoxin produced by several plant pathogenic Pseudomonas spp. The effect of coronatine on abscission in Citrus sinensis L. Osbeck `Hamlin' and `Valencia' orange fruit, leaves, fruitlets, and flowers was determined. Coronatine at 200 mg·L-1 significantly reduced fruit detachment force of mature fruit, and did not cause fruitlet or flower loss in `Valencia'. Cumulative leaf loss was 18% with coronatine treatment. Coronafacic acid or coronamic acid, precursors to coronatine in Pseudomonas syringae, did not cause mature fruit abscission. Ethylene production in mature fruit and leaves was stimulated by coronatine treatment, and 1-aminocyclopropane-1-carboxylic acid oxidase (ACO) and 12-oxo-phytodienoate reductase (12-oxo-PDAR) gene expression was upregulated. A slight chlorosis developed in the canopy of whole trees sprayed with coronatine, and chlorophyll content was reduced relative to adjuvant-treated controls. Leaves formed after coronatine application were not chlorotic and had chlorophyll contents similar to controls. Comparison of coronatine to the abscission compounds methyl jasmonate, 5-chloro-3-methyl-4-nitro-pyrazole and ethephon indicated differences in ethylene production and ACO and 12-oxo-PDAR gene expression between treatments. Leaf loss, chlorophyll reduction and low coronatine yield during fermentation must be overcome for coronatine to be seriously considered as an abscission material for citrus.

Contribution of the dimeric state to the thermal stability of the flavoprotein D-amino acid oxidase.

The flavoenzyme DAAO from Rhodotorula gracilis, a structural paradigm of the glutathione-reductase family of flavoproteins, is a stable homodimer with a flavin adenine dinucleotide (FAD) molecule tightly bound to each 40-kD subunit. In this work, the thermal unfolding of dimeric DAAO was compared with that of two monomeric forms of the same protein: a Deltaloop mutant, in which 14 residues belonging to a loop connecting strands betaF5-betaF6 have been deleted, and a monomer obtained by treating the native holoenzyme with 0.5 M NH(4)SCN. Thiocyanate specifically and reversibly affects monomer association in wild-type DAAO by acting on hydrophobic residues and on ionic pairs between the betaF5-betaF6 loop of one monomer and the alphaI3' and alphaI3" helices of the symmetry-related monomer. By using circular dichroism spectroscopy, protein and flavin fluorescence, activity assays, and DSC, we demonstrated that thermal unfolding involves (in order of increasing temperatures) loss of tertiary structure, followed by loss of some elements of secondary structure, and by general unfolding of the protein structure that was concomitant to FAD release. Temperature stability of wild-type DAAO is related to the presence of a dimeric structure that affects the stability of independent structural domains. The monomeric Deltaloop mutant is thermodynamically less stable than dimeric wild-type DAAO (with melting temperatures (T(m)s) of 48 degrees C and 54 degrees C, respectively). The absence of complications ensuing from association equilibria in the mutant Deltaloop DAAO allowed identification of two energetic domains: a low-temperature energetic domain related to unfolding of tertiary structure, and a high-temperature energetic domain related to loss of secondary structure elements and to flavin release.

Synthesis of [14C]‐l‐tryptophan and [14C]‐5′‐hydroxy‐l‐tryptophan labeled in the carboxyl group

AbstractThe synthesis of selectively 14C‐labeled l‐tryptophan and its derivative 5‐hydroxy‐l‐tryptophan using chemical and multienzymatic methods is reported. The mixture containing [1‐14C[‐dl‐alanine, indole or 5‐hydroxyindole has been converted to [1‐14C]‐l‐tryptophan or 5′‐hydroxy‐[1‐14C]‐l‐tryptophan, respectively, in a one‐pot multienzymatic reaction using four enzymes: d‐amino acid oxidase, catalase, glutamic‐pyruvic transaminase and tryptophanase. Copyright © 2003 John Wiley & Sons, Ltd.

Induction of chill-induced ripening in Fuji apples is a function of both temperature and time

Ethylene production is stimulated by chilling in some cultivars of apples, citrus, cucumbers and pears. The apple varieties Granny Smith, Lady Williams and Fuji can be induced to ripen by a period of chilling at 0°C in air, which hastens the onset of the rise in ethylene production.Fuji apples were susceptible to rapid ripening if stored in air at low temperatures (<10°C). The induction of rapid ripening occurred in both early and late harvested fruit and was correlated with increased activity of 1-aminocyclopropane-1-carboxylic acid oxidase during low temperature storage. The induction of rapid ripening caused the fruit to soften more quickly after cold storage than after continuous exposure to 20°C. Rapid ripening could be prevented by storing fruit in controlled atmosphere (CA) storage (2%�O2 and <1% CO2). Fruit had lower internal ethylene levels following CA storage than following low temperature air storage. The practical solution for growers would therefore be to store Fuji apples in CA, even for short periods, to optimise fruit quality for the consumer.

Subcellular Localization of 1-Aminocyclopropane-1-Carboxylic Acid Oxidase in Apple Fruit

1-Aminocyclopropane-1-carboxylic acid (ACC) oxidase catalyzes the oxidation of ACC to the gaseous plant hormone, ethylene. Although the enzyme does not contain a typical N-terminal consensus sequence for the transportation across the endoplasmic reticulum (ER), it has recently been shown to locate extracellularly by immunolocalization study. It was of interest to examine whether the enzyme contains a signal peptide that is overlooked by structure prediction. We observed that the in vitro translated apple ACC oxidase was not co-processed or imported by the canine pancreatic rough microsomes, a system widely used to identify signal peptide for protein translocation across ER, suggesting that apple ACC oxidase does not contain a signal peptide for ER transport. A highly specific polyclonal antibody raised against the recombinant apple ACC oxidase was used to examine the subcellular localization of the enzyme in apple fruit (Malus domestica, var. Golden Delicious). The location of ACC oxidase appeared to be mainly in the cytosol of the apple fruit pericarp tissue as was demonstrated by electron microscopy using immunogold-labeled antibodies. The pre-immune serum or pre-climacteric fruit control gave essentially no positive signal. Based on these observations, we conclude that ACC oxidase is a cytosolic protein.

Spectroscopic studies of 1-aminocyclopropane-1-carboxylic acid oxidase: molecular mechanism and CO(2) activation in the biosynthesis of ethylene.

1-Aminocyclopropane-1-carboxylic acid (ACC) oxidase (ACCO) catalyzes the last step in the biosynthesis of the gaseous plant hormone ethylene, which is involved in development, including germination, fruit ripening, and senescence. ACCO is a mononuclear non-heme ferrous enzyme that couples the oxidation of the cosubstrate ascorbate to the oxidation of substrate ACC by dioxygen. In addition to substrate and cosubstrate, ACCO requires the activator CO(2) for continuous turnover. NIR circular dichroism and magnetic circular dichroism spectroscopies have been used to probe the geometric and electronic structure of the ferrous active site in ACCO to obtain molecular-level insight into its catalytic mechanism. Resting ACCO/Fe(II) is coordinatively saturated (six-coordinate). In the presence of CO(2), one ferrous ligand is displaced to yield a five-coordinate site only when both the substrate ACC and cosubstrate ascorbate are bound to the enzyme. The open coordination position allows rapid O(2) activation for the oxidation of both substrates. In the absence of CO(2), ACC binding alone converts the site to five-coordinate, which would react with O(2) in the absence of ascorbate and quickly deactivate the enzyme. These studies show that ACCO employs a general strategy similar to other non-heme iron enzymes in terms of opening iron coordination sites at the appropriate time in the reaction cycle and define the role of CO(2) as stabilizing the six-coordinate ACCO/Fe(II)/ACC complex, thus preventing the uncoupled reaction that inactivates the enzyme.

Herbivore-induced volatiles induce the emission of ethylene in neighboring lima bean plants.

Herbivore attacks induce leaves to emit a specific blend of volatiles. Here we show that exposure to Tetranychus urticae-induced volatiles, as well as T. urticae infestation and artificial wounding, activates the transcription of the genes involved in the biosynthesis of ethylene [S-adenosylmethionine (SAM) synthetase and 1-aminocyclopropane-1-carboxylic acid oxidase] and a gene involved in the biosynthesis of polyamines from SAM (SAM decarboxylase) in lima bean leaves. Moreover, exposure of leaves to any one of the seven major chemical components of T. urticae-induced volatiles also induces expression of these genes. Furthermore, we found that, when lima bean plants were exposed to T. urticae-induced volatiles, they emitted ethylene. Lima bean plants infested by T. urticae and artificially wounded plants also emitted ethylene. Endogenous polyamine levels were not increased in the exposed leaves or the infested leaves, suggesting that polyamine production from SAM was only slightly promoted at the metabolic levels present in the leaves. We found that jasmonate (JA) accumulated in leaves exposed to T. urticae-induced volatiles, and that both JA and salicylate (SA) accumulated in leaves infested by T. urticae. These findings, as well as results of pharmacological analyses, suggest that, in leaves exposed to T. urticae-induced volatiles, ethylene biosynthesis might be regulated by pathways involving JA and the ethylene positive feedback loop. They also suggest that ethylene biosynthesis might be regulated by signaling pathways involving JA, SA and ethylene in T. urticae-infested leaves.

Physiological response and extension of vase life of cut carnation flowers treated with ethanol and acetaldehyde. II. Protein content and enzyme activity

Ethanol and acetaldehyde both prevent the formation of ethylene bysenescing cut carnation flowers. This is due to the almost complete inhibitionof the activity of 1-aminocyclopropane-carboxylic acid oxidase. Thesetreatmentsalso reduce the 1-aminocyclopropane-1-carboxylic acid content of the tissue andresult in a loss of protein. The protein content of treated flowers wassignificantly lower than that of control flowers, due to a general rather thanspecific loss of protein. This affects the metabolism of the flowers,preventingenzyme mediated reactions as well as cell growth and development. One enzymethat remained active was alcohol dehydrogenase, allowing for a constantshuttling between ethanol and acetaldehyde.