ACC Synthase Activity Research Articles

Effect of Tropolone and Hinokitiol on in Vitro Activities of 1-Aminocyclopropane-1-carboxylate Synthase and Oxidase in Wounded Winter Squash Mesocarps.

Effects of tropolone and hinokitiol on ethylene production, ACC content and in vitro activities of ACC synthase and oxidase were investigated by using wounded mesocarp tissues of winter squash fruit (Cucurbita maxima Duch. cv. Ebisu). Tropolone over the range from 1 to 500 ppm inhibited ethylene production by 33 to 98% and reduced ACC content by 48 to 83%, whereas hinokitiol treatment reduced them by 17 to 97% and by 34 to 83%, respectively. Both tropolone and hinokitiol were found to inhibit ethylene production through suppression of activities of the two key enzymes.

Ethylene-induced rise of abscisic acid levels and ACC oxidase activity in immature melons

SummaryImmature cantaloupe charentais melons (Cucumis melo L. cv. Alpha) were harvested ten days before their optimum picking date, treated for 2 d with 100 ppm ethylene and kept at 21°C 85% r.h. for 10 d for further ripening. The treated immature fruits were compared with untreated immature or mature melons. C02, ethylene, ACC oxidase activity and abscisic acid levels were measured. Ethylene treatment induced ACC oxidase activity in immature fruits, whereas ACC and MACC levels remained low, which may indicate a lack of ACC synthase activity. Ethylene treatment also induced an increase in ABA levels in both skin and pulp. Relationships between ethylene and ABA content is discussed.

Promotion of ripening of `Gebhard' red `d'Anjou' pears by treatment with ethylene

Pears (Pyrus communis L., `d'Anjou'), `Gebhard' red strain, harvested at commercial maturity with flesh firmness (FF) of 64.5 N, did not ripen normally at 20°C even though the chilling requirement had been met by storage at −1°C. Treating fruit with 100 μl l−1 ethylene at 20°C for 3 days, followed by 14 days of a simulated transit period at −1°C, induced normal ripening at 20°C, with a climacteric-like rise in ethylene production, fruit softening, and reduction of extractable juice. Fruit did not ripen under these conditions without the ethylene treatment. A 3 day treatment with 100 μl l−1 ethylene readily induced pulp tissue to convert 1-aminocyclopropane-1-carboxylic acid (ACC) to ethylene, regardless of the length of chilling. Tissue conversion of ACC to ethylene was also induced by holding fruit at −1°C for 2 months or longer. ACC synthase activity was induced only by ethylene treatment, and did not increase until the fruit had been transferred to 20°C for 3 days. Promotion of normal ripening in `Gebhard' red `d'Anjou' pears by ethylene treatment could be primarily attributed to the coordinate induction of ACC to ethylene conversion and ACC synthase, followed by increasing ACC synthase activity at 20°C.

Ozone induction of ethylene emission in tomato plants: regulation by differential accumulation of transcripts for the biosynthetic enzymes

SummaryStress ethylene emission is positively correlated with ozone sensitivity in various plant species, indicating that ethylene may be involved in the control of ozone damage. This study shows that ozone exposure of tomato plants for 5 h at 85 nl l−1 and above leads to leaf injury within 24 h. 1‐aminocyclopropane‐1‐carboxylic acid (ACC) content and ACC synthase activity were accordingly elevated within 1–2 h. Pre‐treatment of leaves with inhibitors of ACC synthase and ACC oxidase significantly inhibited the evolution of ethylene and reduced ozone‐induced visible damage. Transcript levels for only one out of three S‐adenosyl‐l‐methionine (SAM) synthetase genes (SAM3), and one out of four ACC synthase genes (LE‐ACS2) were induced by ozone (maximum at 2 h). Treatment with protein kinase (K‐252a) and phosphatase inhibitors (calyculin A) revealed that ACC synthase activity was additionally regulated by protein phosphorylation/dephosphorylation. Transcripts of ACC oxidase (pTOM13 cDNA probe) displayed the fastest response of the parameters tested (maximum at 30 min), suggesting a regulatory role for ACC oxidase in ethylene formation of ozone‐exposed plants. The results demonstrate a highly selective ozone response by ethylene biosynthetic genes which resembles that of plant—pathogen interactions.

Selective Induction of 1-Aminocyclopropane-1-carboxylic Acid (ACC) Synthase Activity Is Involved in the Selectivity of the Auxin Herbicide Quinclorac between Barnyard Grass and Rice

Selective accumulation of cyanide, derived from the stimulation of 1-aminocyclopropane-1-carboxylic acid (ACC) production in ethylene biosynthesis, was demonstrated to be the primary mode of action of the auxin herbicide quinclorac in the control of barnyard grass in rice (Pestic. Biochem. Physiol.51,150 (1995)). The influence of quinclorac on the induction of ACC synthase activity was studied in both plant species. After hydroponic treatment of barnyard grass plants (Echinochloa crus-galliL.) with 10 and 100 μM of the herbicide, a transient rise in ACC synthase activity and concomitantly ACC levels was detectable in the root tissue as early as 1 h. Peak levels of an approximately threefold increase, relative to control tissue, were observed at 3 h. Since quinclorac did not influence thein vitroactivity of ACC synthase, the compound appears to interfere with the induction process of the enzyme. In the shoot tissue, ACC synthase activity began to increase after 2 to 3 h and proceeded during the investigation period of 7 h, dependent on the herbicide concentration. The rise in ACC levels and ethylene formation preceded that of ACC synthase activity. Interorgan transport of ACC from the root to the shoot is assumed. When detached shoots and roots of barnyard grass were treated with quincloracin vitro,ACC synthesis was stimulated only in the root tissue. Since no qualitative or quantitative differences between the metabolism of quinclorac in barnyard grass shoot and root tissue were found, the principal site of interaction of quinclorac with ACC synthase is localized in the root tissue. In the shoot tissue of intact plants, ACC synthase activity appears to be stimulated indirectly by quinclorac-induced ACC from the root and its oxidation product, cyanide. This is concluded from experiments with detached shoots of barnyard grass. Treatment with ACC or KCN via the vascular system led to an increase in ACC synthase activity in the shoot tissue. Intact plants or detached shoots and roots of rice (Oryza sativaL. cv. Thaibonnet) did not respond to quinclorac with changes in ethylene formation, ACC synthase activity, and ACC levels. It is suggested that species-selective induction of ACC synthase activity is implicated in the mechanism of quinclorac selectivity between barnyard grass and rice.

Bioregulatory Effects of the Fungicidal Strobilurin Kresoxim-methyl in Wheat (Triticum aestivum)

Apart from its fungicidal effect, the strobilurin kresoxim-methyl (BAS 490 F) was found to induce physiological and developmental alterations in wheat (Triticum aestivum L.) which are seen in connection with improved yield. In a series of biotests including heterotrophic maize and photoautotrophic algal cell suspensions, duckweed, isolated mustard shoots and germinating cress seeds, kresoxim-methyl showed a similar response pattern to standard auxins (e.g. indol-3-ylacetic acid, IAA; 2-(1-naphthyl)acetic acid, α-NAA). Auxin-like activity of kresoxim-methyl was also found when stem explants of tobacco were cultured on a hormone-free medium. Kresoxim-methyl stimulated shoot formation, particularly at 10-7 M. The same effect was induced by 10-8 M IAA. The determination of phytohormone-like substances in shoots of wheat plants foliar-treated with 7×10-4 M kresoxim-methyl revealed only slightly changed levels of endogenous IAA, gibberellins and abscisic acid. In contrast, the contents of dihydrozeatin riboside-type cytokinins increased to 160% of the control, while trans-zeatin riboside- and isopentenyladenosine-type cytokinins remained nearly unchanged. The most remarkable alterations were the reductions in 1-aminocyclopropane-1-carboxylic acid (ACC) levels and ethylene formation which were demonstrated in intact plants, leaf discs and the shoots of wheat subjected to drought stress. Kresoxim-methyl affected the induction of ACC synthase activity which converts S-adenosyl-methionine to ACC in ethylene biosynthesis. In shoots from foliar-treated wheat plants, 10-4 M kresoxim-methyl inhibited stress-induced increases in endogenous ACC synthase activity, ACC levels and ethylene formation by approximately 50%. Reductions in ACC synthase activity and ACC levels of 30% were also obtained at low concentrations of α-NAA (10-6 M). In contrast, ACC synthase activity in vitro was not influenced by adding the compounds. In wheat leaf discs, the inhibiting effect of kresoxim-methyl, α-NAA and IAA on ethylene formation was accompanied by delayed leaf senescence, characterized by reduced chlorophyll loss. However, in contrast to kresoxim-methyl which showed only inhibitory activity on ethylene synthesis over a wide range of concentrations applied, the auxins stimulated ethylene production at high concentrations of about 10-4 M. The inhibition of ethylene biosynthesis by kresoxim-methyl, together with an increase in endogenous cytokinins could explain the retardation of senescence and the intensified green leaf pigmentation in wheat exposed to this strobilurin. © 1997 SCI.

Effects of methyl jasmonate (MeJA) on the dark-induced senescence in oat (Avena sativa L.) leaf segments

To investigate the relationship between methyl jasmonate (MeJA) and ethylene in leaf senescence, we studied the effects of MeJA on ethylene production and ethylene biosynthetic enzyme activities in oat(Avena sativa L.) leaf segments incubated in darkness. MeJA promoted dark-induced senescence judged from the contents of chlorophyll and protein, and increased ethylene production 6 times of the control. MeJA also increased the activities of ethylene biosynthetic enzymes, 1-aminocyclopropane carboxylic acid (ACC) synthase and ACC oxidase as compared to control. In MeJA-treated leaf segments, ACC synthase activity reached its maximum level at 24 h of incubation and ACC oxidase activity peaked at 6 h of incubation. Aminoethoxyvinylglycine (AVG) and Co2+, inhibitors of ACC synthase and ACC oxidase respectively, reduced MeJA-induced ethylene production. They also delayed leaf senescence that was promoted by the treatment of MeJA. From these results, we can suggest that MeJA increased the activities of ACC synthase and ACC oxidase, these increased activities lead to increase in ethylene production and this increased ethylene production might promote dark-induced leaf senescence.

Expression and internal feedback regulation of ACC synthase and ACC oxidase genes in ripening tomato fruit.

We have examined whether or not a positive feedback regulation of gene expression for 1-aminocyclopropane-1-carboxylate (ACC) synthase and ACC oxidase also operates in ripening tomato (Lycopersicon esculentum) fruit during the burst of ethylene production. Two cDNA fragments for ACC synthase and one for ACC oxidase were cloned with high homology to already known genes involved in ethylene biosynthesis in ripening tomato fruit. Accumulation of mRNAs which hybridize to these cDNA probes were induced in mature green fruit within two days by treatment with propylene. In the fruit ripened from the turning stage, red color development, ethylene production, ACC content, and activities of ACC synthase and ACC oxidase increased as maturity progressed. The abundance of two ACC synthase and one ACC oxidase mRNAs in the fruit increased from the turning to pink stage and were followed by a slight decline towards the red stage. These increases in mRNAs abundance with ripening were prevented to a large extent by treatment with the ethylene action inhibitor, 1-methylcyclopropene (MCP). This was most pronounced in the fruit treated with MCP at the turning stage, in which the accumulation of ACC synthase and ACC oxidase transcripts was almost completely eliminated in the first two d, precisely the same stage at which the control fruit had the greatest level of each mRNA accumulation. The inhibition of transcript accumulation recovered to the control level within two to four d. MCP also decreased ethylene biosynthetic activity, although this decrease did not reflect the reduction in the mRNAs accumulation. These results suggest that a strong positive feedback regulation is involved in ethylene biosynthesis at the gene transcriptional level in tomato fruit, even at the stage with a burst of ethylene production.

Effects of chilling on the expression of ethylene biosynthetic genes in Passe-Crassane pear (Pyrus communis L.) fruits.

Passe-Crassane pears require a 3-month chilling treatment at 0 degrees C to be able to produce ethylene and ripen autonomously after subsequent rewarming. The chilling treatment strongly stimulated ACC oxidase activity, and to a lesser extent ACC synthase activity. At the same time, the levels of mRNAs hybridizing to ACC synthase and ACC oxidase probes increased dramatically. Fruit stored at 18 degrees C immediately after harvest did not exhibit any of these changes, while fruit that had been previously chilled exhibited a burst of ethylene production associated with high activity of ACC oxidase and ACC synthase upon rewarming. ACC oxidase mRNA strongly accumulated in rewarmed fruits, while ACC synthase mRNA level decreased. The chilling-induced accumulation of ACC synthase and ACC oxidase transcripts was strongly reduced when ethylene action was blocked during chilling with 1-methylcyclopropene (1-MCP). Upon rewarming ACC synthase and ACC oxidase transcripts rapidly disappeared in 1-MCP-treated fruits. A five-week treatment of non-chilled fruits with the ethylene analog propylene led to increased expression of ACC oxidase and to ripening. However, ethylene synthesis, ACC synthase activity and ACC synthase mRNAs remained at very low level. Our data indicate that ACC synthase gene expression is regulated by ethylene only during, or after chilling treatment, while ACC oxidase gene expression can be induced separately by either chilling or ethylene.

Regulation of ethylene biosynthesis in climacteric apple fruit by elevated CO2 and reduced O2 atmospheres

Abstract Autocatalytic (System II) C 2 H 4 biosynthesis in climacteric ‘Golden Delicious’ apples ( Malus domestica Borkh) was effectively inhibited at 20°C by atmospheres of 20% CO 2 -enriched air (17% O 2 + 63% N 2 ) or 0.25% O 2 (balance N 2 ). In vitro 1-aminocyclopropane-1-carboxylic acid (ACC) synthase (ACC-S) activity of apples held in atmospheres of air + 20% CO 2 or 0.25% O 2 was significantly inhibited when compared to apples kept in air, and correlated well with fruit C 2 H 4 production rates. In vivo and in vitro ACC oxidase (ACC-O) activity of fruit held in atmospheres of air, air + 20% CO 2 or 0.25% O 2 were similar when the assays were performed under standard assay conditions (i.e., in vivo assay performed in air, in vitro assay performed in air + 6% CO 2 ). However, if the in vivo or in vitro ACC-O enzyme activity assays were performed in an atmosphere of 0.25% O 2 , ACC-O catalytic competency and activity were significantly reduced. When the in vivo or in vitro ACC-O enzyme activity assays were performed in an atmosphere of air + 20% CO 2 , ACC-O enzyme activity was actually stimulated. These data indicate that elevated levels of CO 2 do not inhibit ACC-O catalytic competency. Western blot analysis revealed that ACC-O protein abundance was not significantly affected by any of the treatments tested, and only the 0.25% O 2 atmosphere significantly inhibited ACC-O activity. ACC-S activity was significantly reduced by atmospheres of air + 20% CO 2 or 0.25% O 2 but not via direct inhibition of ACC-S catalytic competency.

Li(+)-regulated 1-aminocyclopropane-1-carboxylate synthase gene expression in Arabidopsis thaliana.

In Arabidopsis thaliana, 1-aminocyclopropane-1-carboxylate synthase (ACS) is encoded by a multigene family consisting of at least five members whose expression is induced by hormones, developmental signals, and protein synthesis inhibition. Li+, known to interfere with the phosphoinositide (PI) second messenger system by inhibiting the activity of inositol-phosphate phosphatases, is one of the strongest inducers of ACC synthase activity in plants. Treatment of etiolated Arabidopsis seedlings with LiCl results in a rapid induction of the ACS5 gene. Also, LiCl represses the cycloheximide (CHX)-induced accumulation of the ACS2 mRNA. The effects of Li+ on the expression of ACS5 and ACS2 are specific, dose-dependent, and can be reversed by Ca2+ and mimicked by the protein kinase inhibitor K-252a. The results suggest that the regulation of some ACS genes by various inducers may involve protein kinase activity, which in turn may be controlled through an inositol 1,4,5-triphosphate (IP3)-mediated Ca2+ mobilization. Since plants contain no Li+, the cation appears to unmask pre-existing biochemical capacity that may be utilized by various unknown transducers during plant growth and development.

Postharvest ripening and ethylene biosynthesis in purple passion fruit

Abstract A study was undertaken to investigate the pattern of changes in some chemical constituents, ethylene biosynthesis, and the effect of ethylene treatment during postharvest ripening in purple passion fruit (Passiflora edulis Sims.). During ripening, sucrose content decreased while fructose and glucose contents increased. Citric and malic acid contents slightly increased during the early stage of ripening and decreased thereafter. Amino acids did not change significantly, except for proline, which increased rapidly towards the late stage of ripening. While 1-aminocyclopropane-1-carboxylic acid (ACC) content and ACC synthase activity increased in parallel with ethylene production, ACC oxidase activity was already high when harvested at the turning stage and further increased during ripening. Application of 1000 ppm ethylene for 24 h did not induce earlier onset of ethylene production when applied on harvest day, but was effective when applied one day or five days after harvest. The results indicate that purple passion fruit produces ethylene with the same biosynthetic pathway as other tissues in higher plants; its ethylene biosynthesis is regulated mainly by ACC synthase activity, and the sensitivity to ethylene might have changed after harvest as the fruit ripened.

Controlled-atmosphere Suppression of ACC Synthase and ACC Oxidase in `Golden Delicious' Apples during Long-term Cold Storage

Preclimacteric `Golden Delicious' apples (Malus domestica Borkh.) were stored at 0 °C in: air; air + 5% CO2; 2% O2 + 98% N2; or 2% O2 + 5% CO2 + 93% N2, and sampled monthly for 4 months to investigate the mechanism(s) by which reduced O2 and/or elevated CO2 atmospheres inhibit C2H4 biosynthesis. Ethylene biosynthesis rates and in vitro ACS activity were closely correlated in all treatments, while in vitro ACO activity significantly increased over time regardless of the treatment. Only a small amount of C2H4 biosynthesis inhibition by lowered O2 and/or elevated CO2 atmospheres could be accounted for by suppressed induction of ACO activity. Western blot analysis demonstrated that apples held for 2 months in lowered O2 and/or elevated CO2 atmospheres had significantly reduced abundance of ACO protein, compared to fruit held in air. Northern blot analysis of ACS and ACO transcript abundance revealed that reduced O2 and/or elevated CO2 atmospheres delay induction and reduce the abundance of both transcripts. Reduced O2 and/or elevated CO2 atmospheres reduce C2H4 biosynthesis by delaying and suppressing expression of ACS at the transcriptional level and by reducing the abundance of active ACO protein. Chemical names used: 1-aminocyclopropane-1-carboxylic acid (ACC), ACC synthase (ACS), ACC oxidase (ACO), ethylene (C2H4), S-adenosylmethionine (AdoMet).

The Role in Ozone Phytotoxicity of the Evolution of Ethylene upon Induction of 1-Aminocydopropane-1-carboxylic Acid Synthase by Ozone Fumigation in Tomato Plants

The rate of evolution of ethylene by tomato plants was rapidly increased by O3 fumigation. The time course of the increase in 1-aminocyclop ropane-l-carboxylic acid (ACC) synthase activity was the same as that in the rate of evolution of ethylene, suggesting that ACC synthase activity might be a rate-limiting step in the evolution of ethylene that is caused by O3 fumigation. The rate of the O3-induced evolution of ethylene was increased by the application of ACC to tomato plants, suggesting the involvement of ACC oxidase in the O3-induced evolution of ethylene. Treatment of plants with tiron inhibited the evolution of ethane, but not of ethylene. These results indicated that evolution of ethylene in O 3-treated tomato plants might result from enzymatic reactions catalyzed by both ACC synthase and ACC oxidase, but not from stimulation by O3 of the peroxidation of lipids mediated by free radicals. Pretreatment of leaves with aminoethoxyvinylglycine (AVG), an inhibitor of ACC synthase, significantly inhibited the evolution of ethylene that was induced by O3 and concomitantly reduced the extent of O3-induced visible damage to leaves. Treatment with 2,5-norbonadiene, an inhibitor of the action of ethylene, strongly reduced the extent of visible damage caused by O3, even though it did not suppress the evloution of ethylene. These results indicate that ethylene acts on certain metabolic processes to cause visible damage.

Effects of N-terminal deletions on 1-aminocyclopropane-1-carboxylate synthase activity

A series of nested N-terminal deletions were made on the full-length ( wt) and C-terminal deleted (Cdel) 1-aminocyclopropane-1-carboxylate synthase cDNAs. These wt and mutant ACC synthases were over-expressed in a heterologous E. coli expression system. It was found that removal of an amino acid region (residues 2–12) from the non-conserved N-termini of wt and Cdel ACC synthases led to a slight increase in both in vivo ACC production and in vitro ACC synthase activity. Further deletion of 11 amino acids through Glu-23 from the N-termini of both wt and Cdel ACC synthases resulted in a substantial reduction in both in vivo ACC production and in vitro enzyme activity. Deletion of an amino acid region, residues 3 through 27, from the N-terminus of ACC synthase abolished enzyme activity completely. Kinetic analysis of a highly purified double-deletion mutant (NCdel-1) of ACC synthase demonstrated that the K m of this mutant is 42 μM, which is much smaller than that of the corresponding Cdel (280 μM) and closer to that of wt (22 μM) reported previously, suggesting a clear effect of the non-conserved N-terminal region on its ACC synthase function.

Ethylene Biosynthesis and Its Involvement in Senescence of Broccoli Florets.

Broccoli (Brassica oleracea, var. italica) florets (flower buds) senesce rapidly after har-vest at ambient temperatures. During senescence of harvested broccoli kept at 20°C, the rate of ethylene production significantly increased concurrent with a rapid yellowing ascribed to chlorophyll degradation with the rate reaching a maximum and declining thereafter. ACC (1-aminocyclopropane-1-carboxylic acid) synthase activity gradually de-creased as senescence progressed and, in contrast, ACC content in florets increased from the low level at the early stage of senescence and reached the high level at a later stage. ACC oxidase activity in florets rapidly increased to reach a peak, which was followed by a sharp decline to a low level as senescence progressed, in parallelling the increased pat-tern of ethylene production. The increase in ACC oxidase activity was promoted by the continous supply of exogenous ethylene as well as the enhanced rate of senescence. On the contrary, the rise in ACC oxidase activity was strongly suppressed by the treatment with 2, 5-norbornadiene (NBD), an inhibitor of ethylene action; concurrently NBD strongly retarded the progress in senescence. These results may indicate that endogenous ethylene that is involved in senescence of broccoli florets simultaneously regulates the induction of ACC oxidase activity.

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Bamboo shoots are highly nutritious but possess poor shelf life after harvest due to rapid decline of tenderness mostly result from lignification processes. Present investigation was performed to evaluate the potential of gamma radiation as a postharvest technology to preserve bamboo shoots. The effects of 0.5 kGy gamma radiation on quality and lignification of bamboo shoots during storage at 2 °C were investigated. The results showed that gamma radiation reduced the decay rate of bamboo shoots by 71% at the end of storage. It also significantly retarded ethylene production through controlling the activities of ACC synthase (ACS) and ACC oxidase (ACO), which may contribute to inhibition of lignin synthesis. Meanwhile, gamma radiation decreased the activities of phenylalanine ammonia-lyase (PAL), cinnamyl alcohol dehydrogenase (CAD) and peroxidase (POD), which are associated with lignin accumulation, thus reduced lignin content by 12.5% than that of the control after twenty-eight days of storage. These results demonstrated the potential of the gamma radiation as a promising postharvest technique to maintain the quality and inhibit the lignification of bamboo shoots.

ASC4, a Primary Indoleacetic Acid-responsive Gene Encoding 1-Aminocyclopropane-1-carboxylate Synthase in Arabidopsis thaliana: STRUCTURAL CHARACTERIZATION, EXPRESSION IN ESCHERICHIA COLI, AND EXPRESSION CHARACTERISTICS IN RESPONSE TO AUXIN

1-Aminocyclopropane-1-carboxylic acid (ACC) synthase is the key regulatory enzyme in the biosynthetic pathway of the plant hormone ethylene. The enzyme is encoded by a divergent multigene family in Arabidopsis thaliana, comprising at least five genes, ACS1-5 (Liang, X., Abel, S., Keller, J.A., Shen,N. N.F., and Theologis, A. (1992) Poc. Natl. Acad. Sci. U.S.A. 89, 11046-11050). In etiolated seedlings, ACS4 is specifically induced by indoleacetic acid (IAA). The response to IAA is rapid (within 25 min) and insensitive to protein synthesis inhibition, suggesting that the ACS4 gene expression is a primary response to IAA. The ACS4 mRNA accumulation displays a biphasic dose-response curve which is optimal at 10 microM of IAA. However, IAA concentrations as low as 100 microM are sufficient to enhance the basal level of ACS4 mRNA. The expression of ACS4 is defective in the Arabidopsis auxin-resistant mutant lines axr1-12, axr2-1, and aux1-7. ACS4 mRNA levels are severely reduced in axr1-12 and axr2-1 but are only 1.5-fold lower in aux1-7. IAA inducibility is abolished in axr2-1. The ACS4 gene was isolated and structurally characterized. The promoter contains four sequence motifs reminiscent of functionally defined auxin-responsive cis-elements in the early auxin-inducible genes PS-IAA4/5 from pea and GH3 from soybean. Conceptual translation of the coding region predicts a protein with a molecular mass of 53,795 Da and a theoretical isoelectric point of 8.2. The ACS4 polypeptide contains the 11 invariant amino acid residues conserved between aminotransferases and ACC synthases from various plant species. An ACS4 cDNA was generated by reverse transcriptase-polymerase chain reaction, and the authenticity was confirmed by expression of ACC synthase activity in Escherichia coli.

Elevated CO2 and/or Low O2 Atmospheres Influence ACC Synthase and ACC Oxidase during Long-term Storage of `Golden Delicious' Apple Fruit

The objective of this study was to compare and contrast the mode of action by which elevated carbon dioxide and/or reduced oxygen atmospheres inhibit ethylene biosynthesis. `Golden Delicious' apple fruit were placed at 0C in one of the following four atmospheres: 1) air; 2) air + 5% CO2; 3) 2% O2 + 98% N2; or 4) 2% O2 + 5% CO2 + 93% N2 and then sampled monthly for 4 months. Ethylene biosynthesis rates and in vitro ACC synthase activities were closely correlated in all treatments. In vitro ACC synthase activity and ethylene biosynthesis rates were lowest in fruit treated with 5% CO2 + 2% O2, while air-treated fruit had the highest ethylene biosynthesis rate and in vitro ACC synthase activity. Fruit treated with air + 5% CO2, or 2% O2 + 98% N2, had intermediate ethylene and in vitro ACC synthase activities. In vitro ACC oxidase was significantly different among treatments, but not as closely correlated with the ethylene biosynthesis rate as in vitro ACC synthase activity. Western blot analysis of the ACC oxidase protein was performed to determine if activity differences among treatments were correlated with the amount of enzyme present in vivo. ACC synthase and ACC oxidase mRNA transcript of abundance was determined via Northern blot analysis. Results will be discussed regarding how ethylene biosynthesis is inhibited at the molecular level by elevated CO2 and/or reduced O2.

Analysis of LE-ACS3, a 1-Aminocyclopropane-1-carboxylic Acid Synthase Gene Expressed during Flooding in the Roots of Tomato Plants

The plant hormone ethylene is produced in response to a variety of environmental stresses. Previous work has shown that flooding or anaerobic stress in the roots of tomato plants caused an increase in the production of the ethylene precursor 1-aminocyclopropane-1-carboxylate (ACC) in the roots, due to flooding-induced activity of ACC synthase (EC 4.4.1.14). RNA was extracted from roots and leaves of tomato plants flooded over a period of 48 h. Blot analysis of these RNAs hybridized with probes for four different ACC synthases revealed that the ACC synthase gene LE-ACS3 is rapidly induced in roots. LE-ACS2 is also induced, but at later times. The genomic clone for LE-ACS3 was isolated and sequenced. At all time points, the probe from the LE-ACS3 coding region hybridized to two bands in the RNA blots. Hybridization using the first and third introns of LE-ACS3 separately as probes indicate that flooding may inhibit processing of the LE-ACS3 transcript. Sequence homology analysis identified three putative cis-acting response elements in the promoter region, corresponding to the anaerobic response element from the maize adh1 promoter, the root-specific expression element from the cauliflower mosaic virus 35S promoter and a recognition element for chloroplast DNA binding factor I from the maize chloroplast ATP synthase promoter.