Inhibition of ethylene production in fruit slices by a rhizobitoxine analog and free radical scavengers

The rhizobitoxine analog, L-2-amino-4-(2-aminoethoxy)-trans-3-butenoic acid (Ro), which effectively inhibits ethylene production in apple (Malus domestica Borkh.) and other tissues at concentrations at about 68 micromolar, inhibited ethylene production by about 50 to 70% in green tomato (Lycopersicon esculentum Mill.) fruit slices but only by about 15% in pink and ripe tomato tissue slices. Ethylene production in climacteric-rise and postclimacteric avocado slices was likewise relatively insensitive to 68 micromolar Ro. At 340 micromolar Ro, inhibition of ethylene production increased up to 50% in pink tomato slices, whereas 680 micromolar Ro was required to inhibit ethylene production by 30% in avocado slices. Incorporation of ¹⁴C from [¹⁴C]methionine into ethylene in green and pink tomato tissues was inhibited by Ro to about the same extent as inhibition of total ethylene production. Results thus far are inconclusive as to the mechanism of Ro resistance in tomato and avocado tissues. At 1 millimolar, free radical scavengers such as benzoate, propyl gallate, nordihydroguaiaretic acid, and to a lesser extent, eugenol, inhibited ethylene production in both Ro-sensitive (green tomato and apple) tissues and Ro-resistant (pink tomato and avocado) tissues. Therefore, free radical steps are suggested in the ethylene-forming systems.     

Does light inhibit ethylene production in leaves?

The effect of light on the rate of ethylene production was monitored using two different techniques—leaf segments incubated in closed flasks versus intact plants in a flow-through open system. Three different plants were used, viz sunflower (Helianthus annuus), tomato (Lycopersicon esculentum), and soybean (Glycine max). Experiments were conducted both in the presence and absence of 1-aminocyclopropane-1-carboxylic acid (ACC).

The results obtained indicate that, in all three species studied, light strongly inhibits ethylene production when cut leaf segments are incubated in the presence of ACC in closed flasks. When ethylene measurements are made with ACC-sprayed intact plants using a continuous flow system, the effect of light on ethylene production is only marginal. In leaf segments of sunflower and soybean incubated only in distilled H₂O in closed flasks, light promotes ethylene production. In tomato, there is no difference between the rate of ethylene production between light and darkness under such conditions. When measurements are made with intact plants in a continuous flow system, the rate of ethylene production is almost identical in light and darkness, in the three plants studied.

It is concluded that the effect of light on cut leaf segments incubated in the presence of ACC in closed flasks can be attributed to the techniques used for these measurements. Light has little effect on ethylene production by intact plants in an open system.

     

Effect of Waterlogged Soil Conditions on the Production of Ethylene and on Water Relationships in Tomato Plants

The roots of tomato plants (Lycopersicon esculentum Mill., cv.Moneymaker) were exposed to low concentrations of oxygen bywaterlogging the soil or by growing the plants in nutrient solutionflushed with nitrogen gas. After 24 h, the rate of ethyleneproduction by the petioles, main stem, and shoot apex was increasedby 4–6-fold and the petioles developed epinastic curvatures.Removing the roots did not reproduce these responses. The amountsof ethylene produced by shoot tissues in response to physicalwounding was greatly increased by waterlogging the soil. The production of ethylene by roots was suppressed by the absenceof oxygen. When the roots were transferred back to an aerobicenvironment ethylene production quickly exceeded that observedin roots maintained continuously in aerobic conditions. The enhanced rate of ethylene production in the shoots occurredin the absence of increased water stress as measured with aleaf pressure chamber; leaf water potentials were increasedrather than decreased by waterlogging for 30 h or more. Thiswas associated with stomatal closure and reduced transpiration.Resistance to water flow through the plant increased as transpirationdecreased in response to waterlogging. However, at similar ratesof transpiration, resistance was normally lower in waterloggedplants than in controls.     

Ethylene production by Botrytis cinerea in vitro and in tomatoes

A laser-based ethylene detector was used for on-line monitoring of ethylene released by the phytopathogenic fungus Botrytis cinerea in vitro and in tomato fruit. Ethylene data were combined with the results of a cytological analysis of germination of B. cinerea conidia and hyphal growth. We found that aminoethoxyvinylglycine and aminooxyacetic acid, which are competitive inhibitors of the 1-aminocyclopropane-1-carboxylic acid pathway, did not inhibit the ethylene emission by B. cinerea and that the fungus most likely produces ethylene via the 2-keto-4-methylthiobutyric acid pathway. B. cinerea is able to produce ethylene in vitro, and the emission of ethylene follows the pattern that is associated with hyphal growth rather than the germination of conidia. Ethylene production in vitro depended on the L-methionine concentration added to the plating medium. Higher values and higher emission rates were observed when the concentration of conidia was increased. Compared with the ethylene released by the fungus, the infection-related ethylene produced by two tomato cultivars (cultivars Money Maker and Daniela) followed a similar pattern, but the levels of emission were 100-fold higher. The time evolution of enhanced ethylene production by the infected tomatoes and the cytological observations indicate that ethylene emission by the tomato-fungus system is not triggered by the ethylene produced by B. cinerea, although it is strongly synchronized with the growth rate of the fungus inside the tomato.     

The effect of competition from neighbours on stomatal conductance in lettuce and tomato plants

Competition decreased transpiration from young lettuce plants after 2 days, before any reductions in leaf area became apparent, and stomatal conductance (g(s) ) of lettuce and tomato plants was also reduced. Stomatal closure was not due to hydraulic signals or competition for nutrients, as soil water content, leaf water status and leaf nitrate concentrations were unaffected by neighbours. Competition-induced stomatal closure was absent in an abscisic acid (ABA)-deficient tomato mutant, flacca, indicating a fundamental involvement of ABA. Although tomato xylem sap ABA concentrations were unaffected by the presence of neighbours, ABA/pH-based stomatal modulation is still likely to underlie the response to competition, as soil and xylem sap alkalization was observed in competing plants. Competition also modulated leaf ethylene production, and treatment of lettuce plants with an ethylene perception inhibitor (1-methylcyclopropene) diminished the difference in g(s) between single and competing plants grown in a controlled environment room, but increased it in plants grown in the greenhouse: ethylene altered the extent of the stomatal response to competition. Effects of competition on g(s) are discussed in terms of the detection of the absence of neighbours: increases in g(s) and carbon fixation may allow faster initial space occupancy within an emerging community/crop.     

Dynamics of Acetaldehyde Production during Anoxia and Post-Anoxia in Red Bell Pepper Studied by Photoacoustic Techniques

Acetaldehyde (AA), ethanol, and CO2 production in red bell pepper (Capsicum annum L.) fruit has been measured in a continuous flow system as the fruit was switched between 20% O2 and anaerobic conditions. Minimum gas phase concentrations of 0.5 nL L-1, 10 nL L-1, and 1 mL L-1, respectively, can be detected employing a laser-based photoacoustic technique. This technique allows monitoring of low production rates and transient features in real time. At the start of anaerobic treatment respiration decreases by 60% within 0.5 h, whereas AA and ethanol production is delayed by 1 to 3 h. This suggests a direct slow-down of the tricarboxylic acid cycle and a delayed onset of alcoholic fermentation. Reexposure of the fruit to oxygen results in a 2- to 10-fold upsurge in AA production. A short anoxic period leads to a sharp transient peak lasting about 40 min, whereas after numerous and longer anoxic periods, post-anoxic AA production stays high for several hours. High sensitivity of the fruit tissue to oxygen is further evidenced by a sharp decrease in post-anoxic AA production upon an early return to anaerobic conditions. Ethanol oxidation by the "peroxidatic" action of catalase is proposed to account for the immediate post-anoxic AA upsurge.     

Chelator used in pectin extraction triggers ethylene production by tomato fruit

In our search for an endogenous ethylene trigger from tomato ( Lycopersicon esculentum Mill. ev. Rutgers) fruit cell wall alkaline soluble pectin (ASP), we purified an active component using DEAE-Sepharose chromatography followed by elution on Bio-Gel P-100 or Superose 12. The purified active fraction produced a single band on silver-stained SDS-PAGE of approximately M_r 20000. Using two-dimensional proton-proton and proton-carbon correlation spectroscopy, we identified the repeating sub-unit as trans-1,2-diamino-cyclohexane- N,N,N',N'-tetraacetic acid (CDTA), a chelator used to extract ASP. Although the ASP undergoes extensive dialysis during its extraction which should remove CDTA, the CDTA apparently forms a large molecular weight polymer which does not diffuse out of the dialysis tubing. Infiltration of commercially prepared CDTA into mature green tomato fruit stimulated ethylene production. The ethylene stimulatory effect of CDTA was not affected by the presence of equimolar amounts of CaCl₂, or nmol g^-1 amounts of the calcium channel blockers, nifedipine or verapamil. EDTA, EGTA, and diethylenetriaminepentaacetic acid, other divalent cation chelators, also stimulated ethylene production when they were infiltrated into tomato fruit. Neither the purified material nor commercial CDTA stimulated ethylene production when they were infiltrated into leaf tissue.     

Ethylene plays multiple nonprimary roles in modulating the gravitropic response in tomato.

Ethylene is known to interact with auxin in regulating stem growth, and yet evidence for the role of ethylene in tropic responses is contradictory. Our analysis of four mutants of tomato (Lycopersicon esculentum) altered in their response to gravity, auxin, and/or ethylene revealed concentration-dependent modulation of shoot gravitropism by ethylene. Ethylene inhibitors reduce wild-type gravicurvature, and extremely low (0.0005-0.001 microliter L-1) ethylene concentrations can restore the reduced gravitropic response of the auxin-resistant dgt (diageotropica) mutant to wild-type levels. Slightly higher concentrations of ethylene inhibit the gravitropic response of all but the ethylene-insensitive nr (never-ripe) mutant. The gravitropic responses of nr and the constitutive-response mutant epi (epinastic) are slightly and significantly delayed, respectively, but otherwise normal. The reversal of shoot gravicurvature by red light in the lz-2 (lazy-2) mutant is not affected by ethylene. Taken together, these data indicate that, although ethylene does not play a primary role in the gravitropic response of tomato, low levels of ethylene are necessary for a full gravitropic response, and moderate levels of the hormone specifically inhibit gravicurvature in a manner different from ethylene inhibition of overall growth.     

1-甲基环丙烯采后处理对樱桃番茄果实成熟过程的影响

研究了不同浓度(0、0.035、0.07和0.11μL/L)的乙烯受体竞争性抑制剂1-甲基环丙烯(1-MCP)采后处理对绿熟期樱桃番茄的乙烯合成、果实软化、果实色素(叶绿素、茄红素、β-胡萝卜素)含量消长的影响.0.07 μL/L及其以上浓度的1-MCP降低了前期乙烯合成,同时推迟了乙烯释放高峰,但0.035 μL/L浓度的1-MCP处理并不能抑制内源乙烯合成.1-MCP显著延迟了果实软化和叶绿素降解,但并不影响这两个过程的启动.茄红素合成的启动和积累均受到了1-MCP抑制,而1-MCP并不推迟β-胡萝卜素合成的启动,只抑制其积累.这些结果提示了乙烯调节成熟生理过程的不同机制.对于绿熟期的樱桃番茄,0.07～0.11μL/L的1-MCP是实用的有效处理浓度.1-MCP有效浓度可能用于了解果实的乙烯受体水平和乙烯敏感性.     

Suppression by Citrus Exocortis Viroid Infection of the Naturally Occurring Inhibitor of the Conversion of 1-aminocyclopropane-1-carboxylic Acid to Ethylene by Tomato Microsomes

Citrus exocortis viroid (CEVd) infection of tomato cell cultures suppresses the constitutive inhibitor which blocks the conversion of 1-aminocyclopropane-1-carboxylic acid (ACC) to ethylene by tomato microsomes. The inhibitor is associated to microsomal membranes and is also found in the water soluble fraction co-isolated from the cells. The inhibitory effect is concentration dependent, heat stable and could be removed from solution by dialysis. Its possible relationship with regulation of the viroid-induced ethylene production is discussed.     

Ethylene synthesis regulated by biphasic induction of 1-aminocyclopropane-1-carboxylic acid synthase and 1-aminocyclopropane-1-carboxylic acid oxidase genes is required for hydrogen peroxide accumulation and cell death in ozone-exposed tomato

We show that above a certain threshold concentration, ozone leads to leaf injury in tomato (Lycopersicon esculentum). Ozone-induced leaf damage was preceded by a rapid increase in 1-aminocyclopropane-1-carboxylic acid (ACC) synthase activity, ACC content, and ethylene emission. Changes in mRNA levels of specific ACC synthase, ACC oxidase, and ethylene receptor genes occurred within 1 to 5 h. Expression of the genes encoding components of ethylene biosynthesis and perception, and biochemistry of ethylene synthesis suggested that ozone-induced ethylene synthesis in tomato is under biphasic control. In transgenic plants containing an LE-ACO1 promoter-beta-glucuronidase fusion construct, beta-glucuronidase activity increased rapidly at the beginning of the O(3) exposure and had a spatial distribution resembling the pattern of extracellular H(2)O(2) production at 7 h, which coincided with the cell death pattern after 24 h. Ethylene synthesis and perception were required for active H(2)O(2) production and cell death resulting in visible tissue damage. The results demonstrate a selective ozone response of ethylene biosynthetic genes and suggest a role for ethylene, in combination with the burst of H(2)O(2) production, in regulating the spread of cell death.     

Ethylene production and toxigenicity of methionine and its derivatives with riboflavin in cultures of Verticillium, Fusarium and Colletotrichum species exposed to light

Ethylene was produced by Verticillium dahliae Kleb. grown in liquid Czapek's medium. The rate of ethylene production was enhanced by light but was not affected by shaking or the growth rate of the cultures. L-, D- and DL-methionine, DL-ethionine and a -keto- y -methylthiobutyric acid (KMBA) were good substrates for ethylene production. KMBA may be an intermediate in ethylene production and it appears to be degraded to ethylene either enzymatically by peroxidase or photochemically in the presence of riboflavin. Addition of riboflavin or briefly heating the cultures to 100°C enhanced ethylene production greatly, while the addition of sodium azide, potassium cyanide and catalase were very inhibitory. The SS4 (non-defoliating) pathotype of V. dahliae produced significantly more ethylene (up to 108.4 nl ethylene h₁ from 20 ml-10-day-old cultures) than did the T9 (defoliating) pathotype with all substrates tested. The results suggest that the in vitro rate of ethylene production is not related to the relative virulence of pathotypes of V. dahliae on cotton. A number of Verticillium species, Fusarium oxysporum f. sp. vasinfectum and Colletotrichum dematium var. truncatum were able to produce ethylene in liquid Czapek's medium containing 1 m M L-methionine under continuous light. Riboflavin, although highly stimulatory to ethylene production, caused a fungicidal reaction to all the fungi tested in Czapek's medium containing L-methionine under continuous light. The fungicidal effect of the riboflavin-methionine-light combination occurred at concentrations of riboflavin and methionine less than 1.33 μ M and 0.5 m M , respectively. No fungicidal activity was detected when the cultures were grown in total darkness or when either methionine or riboflavin was omitted from the culture medium.     

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.     

Biosynthesis of ethylene: the effect of phosphate*

Abstract Biosynthesis of ethylene in tomato and avocado fruit slices, carrot root, pea seedling and tomato shoot segments, Penicillium expansum and Escherichia coli was found to be inhibited by inorganic phosphate. Compared with microbial systems, relatively high concentrations of phosphate in the incubating medium were necessary to bring about a significant inhibition of ethylene production in higher plants. The degree of inhibition in higher plants correlated with the increased internal cellular concentration of phosphate and not with that of the incubating medium. Phosphate concentrations inhibitory for ethylene biosynthesis did not affect the respiration of tomato fruit slices. The phosphate effect was reversible, confined to only the biological systems and was not due to a change in the ionic strength. The differential inhibitory effects of aminoethoxyvinylglycine on ethylene biosynthesis in tomato fruit slices of various stages of ripening, were markedly influenced by high phosphate concentrations. The data indicate a biological significance to the phosphate control of ethylene biosynthesis.     

Regulation of Tomato Fruit Polygalacturonase mRNA Accumulation by Ethylene: A Re-Examination

Polygalacturonase (PG) is the major enzyme responsible for pectin disassembly in ripening fruit. Despite extensive research on the factors regulating PG gene expression in fruit, there is conflicting evidence regarding the role of ethylene in mediating its expression. Transgenic tomato (Lycopersicon esculentum) fruits in which endogenous ethylene production was suppressed by the expression of an antisense 1-aminocyclopropane-1-carboxylic acid (ACC) synthase gene were used to re-examine the role of ethylene in regulating the accumulation of PG mRNA, enzyme activity, and protein during fruit ripening. Treatment of transgenic antisense ACC synthase mature green fruit with ethylene at concentrations as low as 0.1 to 1 μL/L for 24 h induced PG mRNA accumulation, and this accumulation was higher at concentrations of ethylene up to 100 μL/L. Neither PG enzyme activity nor PG protein accumulated during this 24-h period of ethylene treatment, indicating that translation lags at least 24 h behind the accumulation of PG mRNA, even at high ethylene concentrations. When examined at concentrations of 10 μL/L, PG mRNA accumulated within 6 h of ethylene treatment, indicating that the PG gene responds rapidly to ethylene. Treatment of transgenic tomato fruit with a low level of ethylene (0.1 μL/L) for up to 6 d induced levels of PG mRNA, enzyme activity, and protein after 6 d, which were comparable to levels observed in ripening wild-type fruit. A similar level of internal ethylene (0.15 μL/L) was measured in transgenic antisense ACC synthase fruit that were held for 28 d after harvest. In these fruit PG mRNA, enzyme activity, and protein were detected. Collectively, these results suggest that PG mRNA accumulation is ethylene regulated, and that the low threshold levels of ethylene required to promote PG mRNA accumulation may be exceeded, even in transgenic antisense ACC synthase tomato fruit.     

Ethylene and methane in the upper water column of the subtropical Atlantic

The vertical distributions of ethylene and methane in the upper water column of the subtropical Atlantic were measured along a transect from Madeira to the Caribbean and compared with temperature, salinity, oxygen, nutrients, chlorophyll-a, and dissolved organic carbon (DOC).Methane concentrations between 41.6 and 60.7 nL L-1 were found in the upper 20 m of the water column giving a calculated average flux of methane into the atmosphere of 0.82 g m-2 h-1. Methane profiles reveal several distinct maxima in the upper 500 m of the water column and short-time variations which are presumably partly related to the vertical migration of zooplankton.Ethylene concentrations in near surface waters varied in the range of 1.8 to 8.2 nL L-1. Calculated flux rates for ethylene into the atmosphere were in the range of 0.41 to 1.35 g m-2 h-1 with a mean of 0.83 g m-2 h-1. Maximum concentrations of up to 39.2 nL L-1 were detected directly below the pycnocline in the western Atlantic. The vertical distributions of ethylene generally showed one maximum at the pycnocline (about 100 m depth) where elevated concentrations of chlorophyll-a, dissolved oxygen, and nutrients were also found; no ethylene was detected below 270 m depth. This suggests that ethylene release is mainly related to one, probably phytoplankton associated, source, while for methane, enhanced net production occurs at various depth horizons. For surface waters, a simple correlation between ethylene and chlorophyll-a or DOC concentrations could not be observed. No considerable diurnal variation was observed for the distribution and concentration of ethylene in the upper water column.     

Effect of suppression of ethylene biosynthesis on flavor products in tomato fruits

To elucidate the role of ethylene in the production of flavor compounds by tomato fruits, wild-type tomato (Lycopersicon esculentum L., cv. Lichun) and its transgenic antisense LeACS2 line with suppressed ethylene biosynthesis were used. The metabolism of individual sugars was ethylene-independent. However, citric acid and malic acid were under ethylene regulation. The content of these acids was higher in transgenic tomato fruits and returned to normal level after transgenic fruits were treated with ethylene. Because most of amino acids, which are important precursors of volatiles, were shown to be correlated with ethylene, we surmise that amino acid-related aroma volatiles were also affected by ethylene. Headspace analysis of volatiles showed a significant accumulation of aldehydes in wild-type tomato fruits during fruit ripening and showed a dramatic decrease in most aroma volatiles in transgenic tomato fruits as compared with wild-type fruits. The production of hexanal, hexanol, trans-2-heptenal, cis-3-hexanol, and carotenoid-related volatiles, except β-damascenone and β-ionone, was inhibited by suppression of ethylene biosynthesis. No remarkable differences were observed in the concentrations of cis-3-hexenal and trans-2-hexenal between transgenic and wild-type tomato fruits, indicating these two volatiles to be independent of ethylene. Thus, there are various regulation patterns of flavor profiles in tomato fruits by ethylene. Published in Russian in Fiziologiya Rastenii, 2007, Vol. 54, No. 1, pp. 92–101. The text was submitted by the authors in English. Both authors equally contributed to this work.     

Kinetic Measurements of Small Ethylene Changes in an Open System Designed for Plant Physiological Studies

Based upon the method we developed to measure ethylene in very low concentrations (as low as 0.01 ppb in the ambient atmosphere) an experimental chamber was constructed and integrated in the measuring system for plant physiological studies. Parameters influencing the accuracy of the technique are evaluated. The pressure in the plant chamber increased 0.05 atm at a flow rate of 10 1/h during ethylene trapping. At this flow rate the ethylene production per seedling is independent of the number of seedlings used. The ethylene measured per seedling is directly proportional to the length of trapping time. Under standard conditions of chamber configuration and volume very small changes in ethylene content can be detected accurately in a very short time range. Our experimental arrangement allows kinetic studies of ethylene evolution by biological objects in a qualitative and quantitative manner. All methods used heretofore are more complicated and less accurate compared to the measuring system presented here. Its versatility is demonstrated for both in vitro and in vivo studies of ethylene production by bean seedlings. The application fields of the apparatus are discussed.     

Inhibitor-resistant early ethylene production during tomato fruit development

In addition to the ethylene formed at the onset of tomato fruit ripening, three peaks of ethylene are produced during earlier periods of in vitro development of tomato flower to fruit. This is the first report characterizing ethylene production during early development of tomato fruit. Previous reports from this laboratory showed that VFNT Cherry tomato calyces are transformed into fruit tissue when cultured in vitro at lower temperatures (16–23 °C). Early ethylene production was also measured in these ripening calyces, as well as in fruit and calyces of other tomato cultivars cultured in vitro. Calyces from Ailsa Craig and rin tomato flowers, which are not transformed into fruit tissue at these lower temperatures, also form ethylene during early periods of in vitro culture, but to a much smaller extent. Unlike ethylene formed at the onset of fruit ripening, the earlier peaks are resistant to the inhibitors, aminovinylglycine (AVG) and CoCl₂. The data suggest that ethylene produced during earlier periods of tomato fruit development is formed by an alternative biosynthetic pathway.     

Suppression of Ripening-Associated Gene Expression in Tomato Fruits Subjected to a High CO2 Concentration

High concentrations of CO2 block or delay the ripening of fruits. In this study we investigated the effects of high CO2 on ripening and on the expression of stress- and ripening-inducible genes in cherry tomato (Lycopersicon esculentum Mill.) fruit. Mature-green tomato fruits were submitted to a high CO2 concentration (20%) for 3 d and then transferred to air. These conditions effectively inhibited ripening-associated color changes and ethylene production, and reduced the protein content. No clear-cut effect was observed on the expression of two proteolysis-related genes, encoding polyubiquitin and ubiquitin-conjugating enzyme E2, respectively. Exposure of fruit to high CO2 also resulted in the strong induction of two genes encoding stress-related proteins: a ripening-regulated heat-shock protein and glutamate decarboxylase. Induction of these two genes indicated that high CO2 had a stress effect, most likely through cytosolic acidification. In addition, high CO2 blocked the accumulation of mRNAs for genes involved in the main ripening-related changes: ethylene synthesis (1-aminocyclopropane-1-carboxylic acid synthase and 1-aminocyclopropane-1-carboxylic acid oxidase), color (phytoene synthase), firmness (polygalacturonase), and sugar accumulation (acid invertase). The expression of ripening-specific genes was affected by CO2 regardless of whether their induction was ethylene- or development-dependent. It is proposed that the inhibition of tomato fruit ripening by high CO2 is due, in part, to the suppression of the expression of ripening-associated genes, which is probably related to the stress effect exerted by high CO2.