Low-level atmospheric ozone exposure induces protection against Botrytis cinerea with down-regulation of ethylene-, jasmonate- and pathogenesis-related genes in tomato fruit

The aim of this study was to determine if ozone exposure could prevent spoilage in tomato fruit by fungal infection and to explore concomitant changes in expression of genes involved in signal transduction (ethylene, jasmonic acid and C 6 -aldehydes) and defence-related (chitinases, glucanases and defensin) pathways. Tomato fruit ( Lycopersicon esculentum Mill. cv. Mareta) were exposed to low-level ozone enrichment (0.05 μmol mol −1 ) for up to 6 days and then wounded and/or inoculated with Botrytis cinerea (grey mould) and transferred for one or two weeks’ post-fumigation exposure to ‘clean’ (i.e. Charcoal/Purafil ® -filtered) air in chilled storage (13 °C). Control fruit were maintained throughout in ‘clean’ air. Pre-exposure to ozone resulted in a marked reduction in lesion development when fruit were subsequently wounded and inoculated with a mycelial plug. Tomato fruit subjected to ozone-enrichment not only showed enhanced protection against fungal infection, but also retained firmness in comparison with fruit maintained in ‘clean’ air. Ozone treatment resulted in strong inhibition of expression of both signal transduction (1-aminocyclopropane-1-carboxylic acid oxidase, allene oxide synthase and hydroperoxide lyase), and defence-related (acidic chitinase, basic chitinase, acidic glucanase, basic glucanase, plant defensin) genes, and the pattern of change was consistent with suppression of fungal growth. Overall, ozone exposure would appear to enhance tomato resistance to B. cinerea infection and has potential commercial applications. Keywords Botrytis cinerea Gene expression Induced resistance Microbial spoilage Ozone Tomato 1 Introduction Postharvest research has focused on improvement in handling and preservation of fresh produce. Growing health and environmental concerns have arisen over current treatment practices using agrochemicals that can result in potentially carcinogenic residues on or in the treated produce ( Spotts and Peters, 1980 ). Considerable interest therefore is currently being expressed in safer and ‘environmental-friendly’ sanitizing agents, including ozone ( Rice, 2002 ). Ozone is a powerful oxidant, and leaves no detectable residues on or in treated produce ( Graham et al., 1997 ). Ozone has been granted full approval as a direct contact food sanitizing agent by the US-FDA ( Palou et al., 2003 ). Moreover, the gas is effective against a much wider spectrum of microorganisms than other disinfectants ( Neff, 1998 ) and is safer to use [lower Threshold Limit Value – Long Term Exposure Limit (TLV-LTEL)] than many other chemical treatments ( Pryor, 1999 ). Much research interest has been expressed in the deployment of gaseous or aqueous ozone for disinfection of fresh produce ( Graham et al., 1997; Xu, 1999; Rice, 2002 ), with significant reductions in spoilage documented in a variety of fruit stored in an ozone-enriched atmosphere e.g. peaches ( Palou et al., 2002 ), table grapes ( Sarig et al., 1996; Xu, 1999; Palou et al., 2002 ), carrots ( Liew and Prange, 1994 ), citrus ( Palou et al., 2003; Tzortzakis et al., 2007a ), strawberries, raspberries, apples ( Xu, 1999 ), tomatoes ( Roberts, 2005; Tzortzakis et al., 2007a, 2008 ) and kiwifruit ( Minas et al., 2010 ). Moreover, work on the impacts of ozone on foliage indicates that ozone may influence plant responses to a variety of biotic and abiotic stresses, including diseases ( Tonneijck and Leone, 1993 ). Ozone appears to act by inducing a hypersensitive response (HR) in plant tissue mimicking biochemical and molecular events induced by pathogens and other stresses ( Rao et al., 2000 ). Similar to plant pathogen attack, ozone has been shown to activate an oxidative burst in leaves which results in the accumulation of reactive oxygen species (ROS) such as superoxide radicals (O 2 − ) and hydrogen peroxide (H 2 O 2 ) ( Rao et al., 2000 ). The mechanisms underlying the suppression of pathogens on ozone-treated fruit are poorly understood. The major focus to date has been on the impacts of ozone on fungal pathogens per se , with less attention paid to the impacts of ozone on fruit in terms of ozone-induced resistance to pathogens. Plant hormones, including salicylic acid (SA), jasmonate (JA), ethylene, and ascorbic acid (AA), have been implicated in mediating defence responses under biotic and abiotic stresses via distinct, yet overlapping signal transduction pathways ( Foyer and Noctor, 2005 ). Changes in the levels of these hormones, alongside their perception and signalling, are known to produce shifts in local and systemic resistance ( Thomma et al., 1998; Audenaert et al., 2002 ), against some necrotrophic pathogens ( Fidantsef et al., 1999 ). Synthesis of ethylene is one of the earliest responses in plant tissues exposed to ozone ( Overmyer et al., 2000; Nakajima et al., 2001 ). Application of 1-methylcyclopropene (1-MCP) shows both ethylene-dependent and -independent pathways mediate ozone-induced gene expression involved in pathogenesis (β-1,3 glucanases) and phytoalexin biosynthesis ( Vst1 ) ( Grimmig et al., 2003 ). Jasmonate is associated with up-regulation of plant defences against pest attack, mechanical injury and different developmental processes ( Froehlich et al., 2001 ) while hydroperoxide lyase ( Hpl ) C 6 -aldehyde products act as signals for the regulation of defence-related genes ( Bate and Rothstein, 1998 ). Several glucanohydrolases found in plants, such as chitinases and β-1,3 glucanases, are also considered important in constitutive and inducible resistance against pathogens ( Ghaouth, 1994 ). These enzymes, frequently referred to as pathogenesis-related (PR) proteins, hydrolyse principal components of fungal cell walls, resulting in inhibition of fungal growth ( Schlumbaum et al., 1986 ). Another class of anti-pathogen components are collectively known as plant defensins (e.g. PDF 1.2), which are small (5 kDa) cysteine-rich peptides, apparently ubiquitous in plants, that are potent inhibitors of microbial growth ( Broekaert et al., 1995 ). These peptides are weakly expressed in organs of healthy plants, but are induced strongly in leaves challenged by fungal pathogens ( Penninckx et al., 1996 ). Ozone-induced ROS are believed to activate distinct signalling pathways dependent on SA, JA and ethylene that induce a wide array of defence reactions including HR-associated cell death plus the induction of PR-proteins and anti-microbial defences ( Overmyer et al., 2000; Rao et al., 2000 ). To date, little or no attention has been paid to the downstream benefits (i.e. ‘memory effects’) of storing fresh produce in an ozone-enriched atmosphere. One notable exception is the study of Sarig et al. (1996) who showed that moderate ozone-enrichment (0.2 μmol mol −1 ) resulted in enhanced levels of antifungal compounds (e.g. resveratrol and pterostilbene) in table grapes, and this effect was associated with pronounced benefits in terms of shelf-life when fruit were subsequently removed from the ozone-enriched environment in which they had been stored. The storage life of normally ripened tomato ( Lycopersicon esculentum Mill) fruit is limited to a few days due to both fruit softening and the development of postharvest diseases. Different strategies have been tested to extend shelf-life, including use of transgenic tomato expressing anti-ripening genes ( Xiong et al., 2004 ). Tomato is easy to grow, and together with the wealth of knowledge of the physiology and genetics of this species, this has led to its use as a model fruit for studying preservation of fresh produce and postharvest physiology. In this work we examined potential downstream benefits of treating tomato fruit with gaseous ozone. The specific aims of the study were to (i) assess whether ‘memory effects’ resulting from ozone treatment lead to the suppression of fungal spoilage following the removal of produce from an ozone-enriched atmosphere, and (ii) examine if ozone exposure affected the expression of key genes involved in signalling and defence against pathogens. 2 Materials and methods 2.1 Plant material Freshly harvested fully ripe (firmness 37.4 N, total carbohydrate content 199.7 μmol g −1 f.wt, ascorbate content 1312 nmol g −1 f.wt,) tomato fruit ( L. esculentum Mill. cv Mareta) were purchased from Paradise Foods (Orchard House Foods Ltd., UK), selected for uniformity in size (47–57 mm diameter) and appearance, absence of injury and physical defects. Fruit were used immediately in the different experiments. 2.2 Botrytis cinerea Botrytis cinerea isolated from tomato (strain number: 169558) was supplied by CABI (CABI Bioscience UK Centre, Bakeham Lane, Egham, England). The fungus was aseptically sub-cultured and purified by serial transfer on to standard triple-vented Petri dishes containing 20 mL of Potato Dextrose Agar (PDA, Oxoid Ltd., Hampshire, England) and incubated in the dark at 20 °C for 1 week. Cultures were stored at 4 °C for long-term use. 2.3 Ozone fumigation system Ozone treatments were administered in a system comprising eight Perspex ® chambers (each with an internal volume of 0.28 m 3 ), housed in a walk-in controlled environment chamber (see Tzortzakis et al., 2007a for details). Each chamber was ventilated with charcoal/particulate-filtered air (‘clean air’ [CFA <0.005 μmol mol −1 ozone]) at a flow rate sufficient to achieve 2 complete air changes per min in each chamber, and turbulence within each chamber was created by an integral 12V fan. Ozone, generated by electric discharge from pure oxygen (model SGA01 Pacific Ozone Technology Inc., Brentwood, CA, USA) to avoid the generation of impurities, was introduced into the CFA airstream entering four of the chambers. The introduction of ozone to each chamber was controlled via stainless steel needle-valved gap flow meters. The ozone concentration in each chamber was recorded every 6 min by a photometric analyser (model 450, manufactured by Advanced Pollution Instrumentation Inc., San Diego, CA) via a multi-channel PTFE ® -sample/logging system (ICAM Ltd., Worthing, Sussex, UK). Chambers were maintained at 13 °C, 95% RH, with both temperature and RH monitored and logged during experiments with the aid of cross-calibrated temperature/humidity sensors (Vaisala HMI 32, Vaisala OY, Helsinki, Finland). 2.4 Impacts of ozone-enrichment on disease development Equivalent tomato fruit were divided into four replicate batches (each batch containing 3–4 fruit) per treatment and were fumigated with CFA or ozone at 0.05 μmol mol −1 ( Fig. 1 ). Following 6 days of exposure, replicate batches of fruit were wounded (by sterilized scalpel) and then inoculated with a mycelial plug (2.5 mm diameter) of B. cinerea taken from the advancing margins of 2-day-old fungal cultures grown on PDA at 25 °C. Other fruit were transferred to CFA and stored at 13 °C, 95% RH in the dark prior to inoculation after one or two weeks of storage. A batch of these fruit was wounded, but no fungi were introduced, as the control for the potential effects of wounding on disease development. Lesion development was measured on duplicate batches of fruit every day for 7 days after inoculation and/or wounding. Moreover, samples were taken 24 h after inoculation (or wounding) to probe shifts in targeted gene expression. The genes were selected based on previous studies on the impact of ozone in the leaf and because of their relevance to the physiological processes suspected to be involved in the observed fruit response to ozone. To probe rapid shifts in gene expression, fruit were incubated in CFA or ozone (0.05 μmol mol −1 ) for up to 24 h, with samples collected after 0, 0.5, 1, 3, 6, 9, 12 and 24 h. Tissue half pericarp (the healthy tissue in the case of the wound-inoculated tomatoes) of six individual fruit per treatment was sampled, snap-frozen in liquid nitrogen, then stored at −80 °C prior to extraction of RNA (see Section 2.6 ). 2.5 Impact of ozone-enrichment on tomato fruit firmness Fruit were exposed to CFA or ozone (0.05 μmol mol −1 ) for up to 6 days and/or transferred and stored in CFA up to 18 days. Fruit firmness was measured at 2 points on the shoulder of each 5–6 tomato fruit for each treatment using a penetrometer FT 011 (TR Scientific Instruments, Forli, Italy) with a 8 mm diameter plunger. The amount of force (N) required to compress the radial pericarp (i.e. surface) of each tomato was recorded at ambient temperature. 2.6 RNA extraction and RT-PCR Total RNA was isolated by mixing 200 mg of ground, frozen fruit tissue in 1 mL of TRIzol Reagent (Tri-Reagent, Helena Bioscience, Sunderland, UK) as described by Taybi and Cushman (1999) . Spectrophotometric quantification of RNA at 260 nm was conducted after DNase treatment of RNA extracts and dilution to100 ng μL −1 made after denaturation at 65 °C for 10 min prior to RT-PCR. After RNA isolation and quality determination, RNA extracts were treated with amplification grade DNase I (Invitrogen, Paisley, UK) according to manufacturer's instructions to eliminate DNA contamination. RNA were diluted in diethyl pyrocarbonate–water to 100 ng μL −1 and used for RT-PCR amplification. RT (reverse transcription) and PCR were performed as a single tube reaction using 100 ng of total RNA. Reactions containing primer sets (see Table 1 ) for genes encoding 1-aminocyclopropane-1-carboxylic acid oxidase ( Aco1 , involved in ethylene signalling), allene oxide synthase ( Aos1 , jasmonate production), acidic chitinase ( Chi3a , PR 3a), basic chitinase ( Chi9b , PR 9b), acidic glucanase ( Gluac , PR 2a), basic glucanase ( Glubs , PR 2b), hydroperoxide lyase ( Hpl1 ), and plant defensin ( Pdf1.2 ), were set on ice in a single reagent master-mix for each primer set containing 1× PCR buffer, 0.1 M DTT, 2.5 mM MgCl 2 , 1 mM dNTPs, 500 μM forward primer, 500 μM reverse primer, 15 U RNase out (Invitrogen, Paisley, UK), 100 U Superscript II RT (Invitrogen, Paisley, UK) and 1 U Taq DNA polymerase (Bioline, UK) and 100 ng of RNA. Glyceraldehyde-3-phosphate dehydrogenase ( Gapdh 1 ) was used as control gene to report equivalent RNA input and RT-PCR conditions. The RT reaction was conducted at the appropriate annealing temperature for each primer set (see Table 1 ) for 30 min, followed by 2 min heat denaturation at 94 °C. RT was followed by PCR using the following thermal cycle: 94 °C (denaturation) for 30 s (strand separation), 50 °C for 30 s (primer annealing), 72 °C 45 s (strand extension) for different numbers of cycles depending on the primer set (see Table 1 ). The programme ended with a 5 min extension step at 72 °C. After amplification, the reaction products were resolved by electrophoresis on a 1.5% (w/v) agarose gel (at 140 V for 60–80 min) stained with ethidium bromide. Separated fragments were visualised using a Gel-Doc 1000 DNA Gel Analysis and Documentation System (Bio-Rad Laboratories, Hercules, CA) which enabled band quantification and image optimisation using the Multi-Analyst software package (Bio-Rad Laboratories, Hercules, CA). To confirm the specificity of the RT-PCRs, the product obtained with each primer set was cloned in PCR2.1 vector (Invitrogen, Paisley, UK) and its sequence verified. Primer sets for genes encoding Actin , G6pdh and Polyubiquitin were tested for suitability as controls, but were found unsuitable as they appeared to be regulated by the treatments deployed. All RT-PCRs were within the linear dynamic range of amplification and all were repeated at least three times. Representative data are shown. 2.7 Statistical analysis Data were analysed using SPSS (SPSS Inc., Chicago, USA) and graphs produced using Prism v.2.0 (GraphPad Inc., San Diego, USA). Data were first tested for normality, then subject to Repeated Measures Analysis of Variance (RM-ANOVA) while significant differences between mean values were determined using Duncan's Multiple Range Test. In the case of gene expression, at least 3 repeats were conducted and a representative gel-image was shown. 3 Results 3.1 Effect of pre-exposure to ozone on disease development in wound-inoculated tomato fruit To determine the influence of pre-exposure to an ozone-enriched environment on subsequent disease development (i.e. ‘memory effect’ induced by ozone treatment), tomato fruit were exposed to ‘clean air’ (CFA) or ozone (0.05 μmol mol −1 ) for 6 days in the dark at 13 °C, 95% RH, then inoculated with B. cinerea either immediately following ozone-treatment or after one or two weeks of storage in CFA. ANOVA revealed lesion development to be significantly ( P < 0.01) reduced (up to 50%) in fruit previously maintained in an ozone-enriched atmosphere and this reduction was consistent throughout the 7 days ( Fig. 2 ). The ozone effect persisted in ozone-treated fruit exposed to gas for 6 days and then transferred and stored in CFA for one or two weeks before wound-inoculation with the fungus ( Fig. 2 ). 3.2 Impacts of ozone-enrichment on tomato fruit firmness Ozone-enrichment resulted in no change in fruit firmness during 6 days’ storage in ozone ( Fig. 3 ). However, when treated fruit were subsequently transferred to CFA, fruit previously exposed to ozone remained significantly ( P < 0.01) more firm (i.e. in better condition) than fruit subjected to traditional storage conditions and the effect persisted up to 18 days’ storage in CFA ( Fig. 3 ). 3.3 Effect of pre-exposure of tomato fruit to ozone on defence-related gene expression in wound-inoculated tomato fruit Steady-state transcript abundance for Aco1 , Aos1 , Hpl1 (signalling genes) and Chi3a , Chi9b and Glubs (defence genes) as well as a plant defensin gene, Pdf1.2 was monitored in fruit stored in ‘clean air’ (13 °C, 95% RH) for 24 h or 1 week. Transcript levels of Aos1 , Hpl1 , Chi3a , Chi9b , Glubs and Pdf1.2 increased significantly ( P < 0.05) after 1-week storage in the absence of any stress ( Fig. 4 ). Fungal infection and/or wounding resulted in a strong increase in the transcript levels of these target genes. The steady-state level of transcripts for Aco1 , Aos1 , Chi3a , Chi9b , Gluac , Glubs and Pdf1.2 genes increased ( P < 0.05 or P < 0.001) dramatically in response to wounding and/or fungal attack ( Figs. 5 and 6 ). Moreover the increase ( P < 0.05) in transcript abundance remained apparent after 2 weeks of storage of fruit in CFA ( Fig. 5 ). Hpl1 transcript levels also increased ( P < 0.05) in response to wounding and/or fungal attack and remained higher after 2 weeks of storage in CFA ( Fig. 7 B ). Ozone-enrichment resulted in a strong reduction ( P < 0.05 or P < 0.001) in transcript levels of the signalling genes in fruit transferred to CFA and/or those infected with fungus and/or wounded ( Aco1 , Aos1 and Hpl1 , Figs. 5 and 7B ). A similar decline in transcript abundance ( P < 0.05 or P < 0.001) was apparent in defence-related genes ( Chi , Glu and Pdf1.2 ) in ozone-treated fruit ( Figs. 5 and 6 ). To probe rapid changes in gene expression induced in tomato tissue upon exposure to ozone, fruit were exposed to ozone or maintained in CFA for up to 24 h. No significant changes in the steady-state levels of signalling genes Aco1 , Aos1 or PR genes ( Chi3a , Chi9b , Gluac and Glubs ) were observed. Expression of Hpl1 , however, exhibited ( P < 0.05) a transient induction (between 0.5 and 9 h, Fig. 7 A). Transcript levels for other monitored genes appeared unaffected by ozone treatment. This was confirmed (results not shown) by Real-Time RT-PCR using a RNA amplification kit employing SYBR Green I for single tube RT-PCR (LightCycler instrument, Roche Molecular Biochemicals, Germany). 4 Discussion The present study demonstrated that prior exposure to low level ozone-enrichment markedly reduces subsequent spoilage by B. cinerea of tomato (see Fig. 2 ). Ozone-exposure resulted in reduced fungal development on fruit, and the effects persisted for up to 2 weeks of storage, yet no effects of ozone were observed on colony dimensions in vitro ( Tzortzakis et al., 2007a ). This suggests that the reduced fungal growth induced by ozone is controlled via changes taking place in the fruit. Ozone enrichment seems to exert a ‘memory’ effect by ‘priming’ fruit tissue against subsequent challenge ( Conrath et al., 2002 ). The mechanism underpinning this response remains unclear. It has been observed previously that ozone prevented lesion development in fruit that were wounded using sterile scalpels but not infected ( Roberts, 2005 ). Our main focus was to assess the effect of ozone on fruit responses to fungal infection and wounding (as a physical signal). It is known ( Rice, 2002 ) that ozone reacts rapidly with ethylene so this ripening agent is effectively destroyed (i.e. removed) in ozone-enriched atmospheres ( Owino et al., 2005 ). This effect would be expected to delay ripening and cell wall disassembly in climacteric fruit, such as tomato, as demonstrated through the retention of fruit firmness in ozone-treated fruit in accordance with previous studies where levels of soluble sugars (glucose, fructose) and fruit firmness were maintained in ozone-treated tomato cv. Carousel following transfer to CFA ( Tzortzakis et al., 2007b ). This effect would likely reduce susceptibility to disease proliferation ( Giovannoni and Rakshy, 2005 ). The effective removal of ethylene by ozone-enrichment is consistent with the dramatic decrease in transcript abundance observed in the targeted gene set including genes linked with ethylene biosynthesis per se . Consistent with this hypothesis, reduced ethylene production in antisense transgenic tomato fruit, in which ACC oxidase expression had been silenced, exhibited prolonged shelf-life (>120 days; Xiong et al., 2004 ). To determine responses of fruit to low-level ozone-enrichment, we monitored the expression of genes involved in the synthesis of ethylene ( Aco1 ), JA ( Aos1 ), C 6 -aldehydes and traumatin ( Hpl1 ) as well as genes directly involved in defence i.e. chitinases ( Chi3a , Chi9b ), glucanases ( Gluac , Glubs ) and a defensin ( Pdf1.2 ). Transcript abundance of the targeted genes increased significantly following one week's exposure to ‘clean air’ (i.e. naturally) consistent with shifts in the expression patterns of the same gene set observed by others ( Wang et al., 2002 ) in ripening fruit and/or induced by exposure to ethylene/JA or wounding and fungal attack/bacterial elicitors ( Kim and Hwang, 1997 ). Ripening-associated shifts in the targeted gene set were enhanced by the introduction of pathogen and/or wounding ( Figs. 5–7 ) consistent with previous reports on a variety of plant tissues ( Kim and Hwang, 1997; Gomi et al., 2003 ). Several reviews emphasize the role played by ethylene and/or JA in regulating the expression of the targeted gene set and in the induction of non-specific disease resistance through signalling pathways that are distinct from the classic systemic response regulated by SA ( Feys and Parker, 2000 ). The product of the monitored defensin, PDF1.2, does not correlate with susceptibility to Botrytis infection ( Ferrari et al., 2003 ), but the protein accumulates following exposure to challenge by pathogens and/or oxidative stress ( Tierens et al., 2002 ). The pattern of accumulation of Pdf1.2 transcripts was similar to that for signalling and PR genes, suggesting independent JA-, SA- and ethylene-regulated signalling pathways ( Rao et al., 2002; Foyer and Noctor, 2005 ). In contrast, ozone-treatment resulted in the suppression of ripening/pathogen/wound-induced shifts in expression of the targeted signalling- and defence-related gene set in tomato fruit and this effect persisted when fruit were transferred to ‘clean air’ with the extent of suppression of the targeted gene set declining over two weeks following transfer to CFA (see Fig. 5 ). To our knowledge, this is the first time that long-lasting effects (i.e. ‘memory effects’) on disease susceptibility have been reported in fruit treated with ozone. In foliage, it is accepted that ozone induces a rapid increase in transcript accumulation for ethylene-, SA- and JA-sensitive signalling genes as well as defence-related genes ( Overmyer et al., 2000; Nakajima et al., 2001 ) which act as second or third messengers regulating ozone-induced gene expression ( Örvar et al., 1997 ). However, ethylene and JA are considered to play opposing roles in mediating cell death ( Koch et al., 2000 ), implying that distinct pathways are activated by ozone. Our findings suggest profound differences in responses at the molecular level when fruit and leaves are exposed to ozone. For example, in contrast to the pattern of change in Aco1 gene expression monitored in ozone-treated fruit, exposure of foliage to ozone has been shown to display a dramatic increase in Aco1 expression and the activity of ACC oxidase, paralleled by a burst in ethylene emission and a corresponding increase in activity of key biosynthetic enzymes, including ACC synthase and ACC oxidase ( Tuomainen et al., 1997 ). In Arabidopsis , Nakajima et al. (2001) have shown that the induction and sustained expression of ACC oxidase also regulate the rate of ozone-induced ethylene biosynthesis ( Tamaoki et al., 2003 ). The pattern of shifts in gene expression and the lack of any rapid shifts in gene expression in fruit (within 24 h of exposure to ozone) suggest that low-level ozone-enrichment may result in a rapid burst in ROS production in leaves (as documented by Langebartels et al., 2002 ), but not in fruit, triggering different downstream consequences, but this needs to be verified more in detail in future. In a commercial context, the benefit accrued from ozone-enrichment would be reflected in increased shelf-life of tomato fruit. The findings of the present study suggest that exposure to low levels of ozone-enrichment may result in a significant extension in the shelf-life of tomato fruit. Observed shifts in the expression patterns of targeted signalling and defence-related genes seemed at odds with expectation from studies on plant foliage, suggesting differences in perception and response to ozone between leaves and fruit. The present study suggests that atmospheric ozone-enrichment suppress ethylene- and JA-induced shifts in gene expression associated with ripening, fungal-attack and wounding and this is reflected in the inhibition of fungal development and delayed ripening in ozone-treated fruit. Interestingly, ozone-treatment was shown to exert long-lasting (i.e. ‘memory’) effects following transfer of treated fruit to ‘clean air’, though the protection against pathogen attack induced by ozone dwindled over two weeks. This suggests that reversal of ozone-induced shifts in the metabolism of tomato fruit appear to be slow. Acknowledgements We are indebted to biofresh ( www.bio-fresh.co.uk ) for their support during commercial trials of the technology (covered by a trials licence from the UK-Pesticide Safety Directorate [COP 2005/00578 PP]). We also thank Dr. Eva Tallentire and Alan Craig for their respective technical inputs. 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