JXB Advance Access originally published online on June 27, 2005
Journal of Experimental Botany 2005 56(418):2183-2193; doi:10.1093/jxb/eri218
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RESEARCH PAPER |
Involvement of ethylene biosynthesis and perception in the susceptibility of citrus fruits to Penicillium digitatum infection and the accumulation of defence-related mRNAs
Departamento de Ciencia de los Alimentos, Instituto de Agroquímica y Tecnología de Alimentos (IATA), CSIC, Apartado de Correos 73, Burjassot, E-46100 Valencia, Spain
* To whom correspondence should be addressed. Fax: +34 96 3636301. E-mail: jmarcos{at}iata.csic.es
Received 21 December 2004; Accepted 4 May 2005
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Abstract |
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Citrus fruits infected with the fungus Penicillium digitatum substantially increase the production of the plant hormone ethylene. In this study, the regulation of ethylene biosynthesis in Citrus sinensis-infected fruits and its putative involvement in an active defence response against P. digitatum infection is examined. Ethylene production is demonstrated as being the result of the co-ordinated and differential up-regulation of at least three ethylene biosynthetic genes: ACS1, ACS2, and ACO. Blocking ethylene perception by 1-MCP resulted in an increased ethylene production and ACS2 expression during infection and mechanical wounding, suggesting that this gene is negatively regulated by ethylene. ACO expression was induced by ethylene in the absence of wounding or infection, although further results indicate that its induction during the course of infection may not be primarily mediated by ethylene. Treatment with 1-MCP also increased susceptibility to Penicillium decay, showing an involvement of ethylene perception in promoting defence responses in citrus fruits. The changes in the expression of two defence-related genes up-regulated during infection were also studied: the ones coding for phenylalanine ammonia-lyase (PAL) and an acidic class II chitinase (ACR311). The onset of PAL expression after mechanical wounding or inoculation was not changed in 1-MCP-pretreated fruits, while its later increase during the course of infection was abolished. Chitinase gene induction was more related to mechanical damage and was partially repressed by ethylene. These studies indicate distinct possible regulatory mechanisms of plant fruit defence genes in the context of fungal infection and ethylene perception.
Key words: Citrus fruits, defence response, ethylene biosynthesis, ethylene perception, Penicillium digitatum, regulation
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Introduction |
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The infection of plants by pathogens activates complex defence responses that have been mostly studied in vegetative organs, while mature fruits have received less attention. However, post-harvest diseases of fruits due to pathogen infection are responsible for major crop losses (Eckert and Brown, 1986
). Citrus green and blue moulds (caused by the fungi Penicillium digitatum Sacc. and P. italicum Wehmer, respectively) are the main post-harvest diseases of citrus worldwide. Little is known about the molecular mechanism involved in the defence response of citrus fruits against fungal infection. Several reports describe the induction of expression of defence-related genes, pathogenesis-related (PR) proteins, and/or phytoalexins and other antifungal secondary metabolites, either after fungal infection or by treatments that induce protection against fungal infection (Afek et al., 1999; McCollum, 2000; Porat et al., 2001, 2002; Del Río et al., 2004). Nevertheless, the signals eliciting these responses are largely unknown.
Citrus fruits infected with Penicillium release the gaseous molecule ethylene, from both fungal and fruit origin (Achilea et al., 1985a, b). Enhancement of ethylene production commonly occurs in most diseased or injured plants (Williamson, 1950). Ethylene is a plant hormone that is pivotal in many developmental and stress processes, and exerts its signalling role through regulation of its biosynthesis, perception, and signal transduction, to modulate a wide array of responses that include gene expression changes (Wang et al., 2002). In higher plants, ethylene is produced from methionine through its precursor 1-aminocyclopropane-1-carboxylic acid (ACC), and the last two biosynthetic steps are catalysed by ACC synthase (ACS) and ACC oxidase (ACO), both coded by complex multigene families (Wang et al., 2002). Environmental and endogenous signals regulate ethylene biosynthesis primarily, but not solely, through differential expression of distinct ACS and ACO genes (Bleecker and Kende, 2000). In the case of defence against pathogen infection, the role of ethylene is complex and somewhat controversial (Thomma et al., 2001; Diaz et al., 2002; Kunkel and Brooks, 2002), since ethylene may either promote susceptibility, resistance or tolerance, depending on the plant–pathogen interaction under study (Bent et al., 1992; Hoffman et al., 1999; Thomma et al., 1999; Berrocal-Lobo et al., 2002, 2004; Geraats et al., 2003). Other signalling molecules and pathways can also interact with ethylene in a complex network to regulate defence responses (Kunkel and Brooks, 2002).
Exogenous ethylene promotes protein and mRNA changes in the peel of orange fruits (Alonso et al., 1992, 1995). It also raises the accumulation of carotenoids (Stewart and Whitaker, 1972) and induces the degradation of chlorophyll (Purvis and Barmore, 1981), and ethylene treatment is a common industrial procedure to induce the degreening of citrus fruits harvested before the full colour of the peel is accomplished. However, commercial practice indicates that prolonged or excessive ethylene fumigations might increase post-harvest deterioration. Elucidating the role of ethylene in citrus post-harvest decay is therefore relevant from both a practical and a basic point of view. Ethylene pretreatment before inoculation with P. italicum resulted in a minor reduction of lesion size (El-Kazzaz et al., 1983). On the other hand, compounds preventing ethylene perception have been developed and are useful not only for practical reasons but also for understanding its biological role (Sisler and Serek, 2003). The inhibitor 1-methylcyclopropene (1-MCP) blocks ethylene perception and action by irreversibly binding to cell receptors. The effect of 1-MCP on citrus decay has been addressed with contradictory conclusions. It has been reported that a pretreatment with 1-MCP increased decay due to natural infections over a 4 week period (Porat et al., 1999). However, it was also reported that progression of P. digitatum on artificially infected grapefruits was not affected by the presence of 1-MCP following inoculation (Mullins et al., 2000).
The present study was undertaken in order to understand the implication of ethylene in the response of Citrus sinensis fruits to P. digitatum infection. As far as is known, only three distinct genes involved in ethylene biosynthesis have been isolated from citrus fruits: two ACS and one ACO (Mullins et al., 1999; Wong et al., 1999; Zacarías et al., 2003; Katz et al., 2004). Their expression changes upon fruit infection have been analysed, including the effect on these changes of blocking ethylene perception by 1-MCP. Two defence-related genes were also selected to evaluate their regulation by ethylene during infection. One of them codes for phenylalanine ammonia-lyase (PAL), a key enzyme of the phenylpropanoid biosynthesis pathway that is responsive to stress conditions in plants, leading to lignin production and the synthesis of phytoalexins and other secondary metabolites (Dixon and Paira, 1995). The other codes for an acidic class II chitinase and was identified in mandarins subjected to temperature stress (Sánchez-Ballesta et al., 2003). Expression changes after ethylene or 1-MCP pretreatments are shown, and the data are discussed in the context of the effect of the treatments on ethylene production during infection, disease incidence, and gene expression.
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Materials and methods |
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Biological material
Mature oranges (Citrus sinensis L. Osbeck) from the cultivar ‘Navelate’ harvested from a commercial orchard were used throughout this study. Fruits were washed and dried immediately after harvest as previously described by López-García et al. (2000)
Penicillium digitatum Sacc., isolate PHI-26 (López-García et al., 2000) was cultured on potato dextrose agar (Difco, Detroit, USA) plates at 24 °C. Conidia were collected from 1-week-old plates by scraping them with a sterile spatula, and transferring them to sterile water. Conidia were then filtered, and titrated with a haemocytometer.
Fruit inoculation
Fruits were first wounded by making punctures with a nail (approximately 3 mm in depth). Ten µl of the inoculum were applied to each wound. Inocula consisted of suspensions of conidia in sterile water, either at 106 or 104 conidia ml–1 depending on the experiment (see below). After inoculation, fruits were maintained at 20 °C and 90% relative humidity.
To assess the incidence of disease, each fruit was wounded at four sites around the equator. For each treatment, three replicas (five fruits per replica, four wounds per fruit) were prepared in each experiment. Symptoms were scored at different days post-inoculation (dpi) as the number of infected wounds in each replica. The percentage of infected wounds and mean values ±SD for each treatment were calculated. Statistical analyses were carried out with the software package StatGraphics Plus 4.0 (Manugistics Inc., Rockville, USA). The F-test was applied to test if difference between the treatment means was significant, and the Tukey's honestly significant difference (HSD) procedure was used for mean separation.
To collect tissue samples for time-course RNA analysis, each fruit was wounded at eight sites around the equator. In these experiments, control mock-inoculations were carried out with sterile water. Additional controls consisted of fruits that had not been wounded. At either 24, 48, or 72 h post-inoculation, peel tissue discs of 5 mm in diameter around the inoculation site (containing flavedo and albedo, but not pulp) were sampled by using a cork borer.
In another set of experiments, samples from different zones (as defined from their distance/location from the maceration front) were collected from heavily diseased fruits (6 dpi). At this time, the maceration front was clearly visible as a distinct line between peel tissue with no visible symptoms and the soft water-soaked peel characteristic of macerated tissue (Achilea et al., 1985b). Zone 1 was healthy peel opposite to the inoculation site (and distant from the maceration front), zone 2 consisted of the 5 mm of healthy peel just outside the maceration front, zone 3 consisted of the first 5 mm of diseased peel inside the maceration front, and zone 4 was heavily decayed tissue that showed visible white mycelial growth and sporulation. Zone 0 was a control sampled from non-inoculated fruits. In this experiment, the flavedo (F, the outer orange-coloured part of the peel) and albedo (A, the inner white) were separated for each zone. In all the cases, tissue was immediately frozen in liquid nitrogen, ground to a fine powder, and stored at –80 °C until RNA isolation.
Treatment with ethylene or 1-MCP
In some experiments fruits were fumigated with either ethylene or 1-MCP (Rohm and Haas, Mozzanica, Italy) prior to inoculation. Treatments were carried out at 20 °C for 16 h in sealed 25 l glass jars (30 fruits/jar). Gaseous ethylene was injected to reach the desired 2 or 10 ppm (µl l–1) concentrations. Active 1-MCP was released by dissolving the appropriate amount of the compound in 2 ml of warm distilled water, which was immediately placed inside the containers. Controls consisted of fruits treated with atmospheric air. In all cases, calcium hydroxide was included inside the containers to absorb CO2. After the treatment, fruits were aireated for 30–60 min before proceeding to inoculation.
Ethylene production
Ethylene production from whole fruits was determined at 24, 48, and 72 h after inoculation by incubating two replicates of four fruits in sealed 1.7 l glass jars at 20 °C. After 3 h of incubation, two replicates of 1 ml of headspace gas were withdrawn from each jar and analysed with a gas chromatograph equipped with a flame ionization detector and an alumina column (Lafuente et al., 2001).
Northern blot analysis of mRNA accumulation
Total RNA was extracted from tissue samples essentially as described previously (Rodrigo et al., 2004), and 8 µg per sample were electrophoresed through formaldehyde gels and transferred to Hybond N+ membranes (Amersham Biosciences) following standard protocols. cDNA clones of the ethylene biosynthetic genes ACS1, ACS2, and ACO (Zacarías et al., 2003) (Table 1), PAL (Sánchez-Ballesta et al., 2000a), the basic chitinase clone ACR311 (Sánchez-Ballesta et al., 2003), and the D1–D2 region of the P. digitatum 26S rDNA (L González-Candelas et al., unpublished data), were labelled with [
-32P]dATP by using the Strip-EZ PCR Kit (Ambion, Cambridge, UK) following the instructions of the manufacturer. An equivalent number of counts (106 cpm ml–1) were used for hybridization with ULTRAhyb buffer (Ambion). Hybridization signals were quantified by exposure to a phosphor imaging plate, scanning with a FLA-3000 laser scanner (Fujifilm, Tokyo, Japan) and quantification with the software ImageGauge 4.0 (Fujifilm). After stripping off the probe following the instructions in the Strip-EZ PCR kit, membranes were rehybridized at least twice with different probes.
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Results |
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Ethylene biosynthetic gene expression during infection of citrus fruits by Penicillium digitatum
Two partial ACS and one ACO cDNAs have been isolated in our laboratory by RT-PCR from the flavedo of Fortune mandarin (C. clementinaxC. reticulata) (Zacarías et al., 2003
). The three clones are highly homologous or identical (in the overlapping regions) to corresponding sequences previously isolated from other Citrus species, including C. sinensis (Table 1). The two ACS clones correspond to the same region of the protein and, although were 74% identical to each other, control experiments demonstrated discrimination of the corresponding mRNAs under the hybridization conditions used here (data not shown). The two ACC synthase transcripts showed a differential timing of accumulation upon P. digitatum infection of oranges (Fig. 1) and mandarins (data not shown). Figure 1 shows data from a representative experiment with a high inoculation dose (106 conidia ml–1), which resulted in 100% infection at 5 dpi. Expression of ACS1 preceded that of ACS2, and was clear within the first 24 h after inoculation. Fungal ribosomal RNAs just started to be noticeable at that time as judged by hybridization to a specific probe (Fig. 1). The specificity of gene expression in response to infection was more evident in the case of the two ACS than in ACO, as accumulation of ACO mRNA was very significant in mock-inoculated controls. Maximal accumulation of all the three ethylene biosynthetic genes occurred at 48 h post-inoculation, when infection symptoms started to appear (i.e. maceration visible around the inoculation site in 3–10% of the inoculated wounds). At this time (I48), fungal rRNAs were noticed even in methylene blue-stained northern membranes (see additional ribosomal bands in samples I48 and I72, Fig. 1). ACO mRNA accumulation was much higher than that of the ACS genes, since quantification of hybridization signals at 48 h showed that ACO expression was 25-fold higher than ACS1, while ACS1 was 2-fold higher than ACS2. It is also noticeable that the ACS2 peak of expression was sharper than the peak of the other two genes. After 72 h, maceration was evident in most of the inoculated wounds (90% of infection) and expression of ACS1, ACS2, and ACO substantially decreased. This decline in the amount of citrus mRNAs is most likely a consequence of cellular lysis and degradation of plant constituents.
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Analyses of the accumulation of these mRNAs in peel tissue harvested at 6 dpi at different distances from the inoculation point (Fig. 2) were also performed. At this time, the diameter of the macerated area was approximately 6 cm, and mycelial growth and esporulation were evident. Flavedo and albedo tissues were analysed separately in this experiment. Maximal accumulation of ACS1, ACS2, and ACO mRNAs occurred in symptomless areas adjacent to the maceration front (F2 and A2), similarly to that observed with cpACS in grapefruit (Mullins et al., 2000
). In macerated areas (F3 and A3), degradation of all the three mRNAs was evident. Expression levels of ethylene biosynthetic genes were higher in flavedo than in albedo, with the noticeable exception of zone 1, for which comparable amounts of mRNA were detected for ACO in both flavedo and albedo. In fact, ACO was induced in distal (and healthy) areas of the fruit (F1 and A1), whereas ACS genes were not. None of the mRNAs were detected in the flavedo or albedo of non-inoculated fruits held under the same conditions (F0 and A0). Taken together, all these data demonstrate a temporal, spatial, and tissue-specific regulation of these genes upon infection.
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Effect of ethylene or 1-MCP pretreatment on the susceptibility to infection and ethylene production
To test the hypothesis that ethylene mediates specific fruit responses to Penicillium infection, fruits were pretreated with either ethylene or the ethylene perception inhibitor 1-MCP, and subsequently infected to follow disease progression. Distinct concentrations of fungus, ethylene, and 1-MCP were tested in these experiments (data not shown). It was observed, and reasoned, that aggressive inoculum doses (i.e. 106 conidia ml–1 as in Fig. 1) gave a very high disease incidence that did not allow the effect of the treatments to be discriminated. Table 2 shows the results of two representative experiments with a low inoculum (104 conidia ml–1). Pretreatment with 1-MCP (from 50 ppb up to 1000 ppb) consistently increased disease incidence, which could be observed from 4 dpi.
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Conversely, ethylene pretreatment resulted in a minor reduction of susceptibility, an effect that was only observed at earlier times after inoculation (up to 3–4 dpi), and was lost later on (Table 2). Besides the absence of infection for the 2 ppm ethylene pretreatment at 3 dpi, no statistically significant differences were observed between the two ethylene concentrations used during the course of the experiments.
In wounded mock-inoculated controls, ethylene production from the whole fruit reached a maximum at 48 h and decreased later (Fig. 3). In infected fruits, ethylene production increased continuously, being after 72 h six times higher than in wounded fruits and confirming previous reports (Achilea et al., 1985a; Mullins et al., 2000). Pretreatment with 1-MCP substantially increased the evolution of ethylene, even in mock-inoculated fruits monitored after 72 h of fumigation. This behaviour is characteristic of the autoinhibitory production of ethylene (Riov and Yang, 1982). Pretreatment with ethylene had a moderate effect in inhibiting ethylene production in both wounded and inoculated fruits (Fig. 3), which was observed as a slight reduction only at 48 h post-treatment and lost later on at 72 h.
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Effect of ethylene or 1-MCP pretreatment on ethylene biosynthetic gene expression
Ethylene biosynthetic gene expression was also monitored in mock-inoculated or inoculated fruits that had been held in air or pretreated with either 1-MCP or ethylene before inoculation. Prior to inoculation, the only noticeable effect observed was an increase in ACO mRNA after treatment with 10 ppm of ethylene for 16 h (Fig. 4A). Gene expression analyses were carried out in low dose inoculation experiments, as changes in susceptibility (Table 2) and ethylene production had been shown for them (Fig. 3). Gene expression in control air-pretreated fruits (Fig. 4) followed the same pattern as described above (Fig. 1), with a delayed progression of changes due to the lower progression of disease at lower inoculum dose (Table 2). Thus, ACS1 mRNA accumulation preceded that of ACS2 (Fig. 4B, C). In these analyses, the hybridization signals were quantified and referred to the signal in the air-pretreated control for each gene (Fig. 4).
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An increase of ACS2 mRNA accumulation was observed in 1-MCP-treated and mock-inoculated fruits (Fig. 4C). Noticeably, ACS2 mRNA was boosted in inoculated fruits that had been pretreated with 1-MCP. On the other hand, these data did not indicate a substantial increase of ACS1 in either mock-inoculated or infected tissue upon 1-MCP treatment, as compared with the air-pretreated control (Fig. 4B). This difference between the two ACS genes was better visualized in a plot of their induction after 72 h, following each pretreatment (Fig. 5). Regarding ACO, 1-MCP promoted a modest increase of its mRNA accumulation after subsequent wounding, and a negligible effect after infection (Figs 4D, 5). Thus, data from 1-MCP pretreated samples are consistent with a negative regulation of ACS2 mRNA by ethylene.
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Ethylene pretreatment had a low and comparable effect in the expression of these three genes after wounding or infection, as concluded from the quantification of hybridization signals (Fig. 4B–D). The much lower mRNA induction that is apparent in ethylene versus air pretreatment after 72 h (Fig. 5) is due to the higher expression of the three genes after the 16 h ethylene treatment (Fig. 4A), the sample that was used as a reference for the presentation of data in Fig. 5.
Effect of ethylene or 1-MCP pretreatment on mRNA accumulation of defence-related genes
The hybridization analyses were also carried out with two probes derived from citrus genes putatively involved in defence responses. Citrus PAL has been involved in temperature abiotic stress (Sánchez-Ballesta et al., 2000a, b) and its enzymatic activity and gene expression are induced by ethylene (Lafuente et al., 2001, 2003). P. digitatum also enhanced PAL mRNA accumulation in grapefruits (McCollum, 2000). These experiments confirmed that a 16 h ethylene pretreatment (in the absence of infection) induced PAL mRNA accumulation (Fig. 6A), and showed a subtle time-course induction during infection, above the level observed in mock-inoculated controls (Fig. 6B). Remarkably, 1-MCP pretreatment reduced the induction of PAL mRNA in subsequently infected oranges (Figs 6B, 7), thus demonstrating that PAL expression changes during infection are, to some extent, mediated by ethylene perception. Despite this reduced induction of PAL expression in 1-MCP-treated fruits, ethylene treatment did not result in increased expression, but rather mRNA levels remained almost constant during infection and at a level similar to that reached after the treatment (Figs 6B, 7). This behaviour might be due to the high expression already achieved after ethylene application (Fig. 6A), that could fulfil the requirements of mRNA for protein translation.
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Chitinases are among the PR proteins induced by pathogen attack and are suspected to promote protection against fungi through degradation of their cell wall (Neuhaus, 1999
). Clone ACR311 was identified in a survey of genes from the flavedo of mandarins subjected to temperature stress, and codes for an acidic class II chitinase (Sánchez-Ballesta et al., 2003). Data showed a clear induction of ACR311 mRNA, in both wounded and infected fruits, and a higher expression in the former (Fig. 6C). Changes in ACR311 following either 1-MCP or ethylene treatments prior to infection were not observed (Fig. 6A). However, subsequent time-course evolution of ACR311 mRNA in these pretreated fruits pointed to a negative inhibition by ethylene in mock-inoculated and inoculated fruits (Fig. 7). |
Discussion |
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Plant pathogen infection activates a complex network of signals that, through perception and transduction, result in the stimulation of defence-related responses. It should be mentioned that these processes have mostly been studied in vegetative organs of plants, while mature fruits have received less attention. In order to gain a better understanding of the defence mechanism against post-harvest decay in citrus fruits, the focus was on their response to P. digitatum infection.
Regulation of ethylene biosynthesis during infection of citrus fruits by Penicillium
Penicillium-infected citrus fruits release significant amounts of ethylene from fruit and fungal origin. It was shown previously that citrus produce a minor amount of ethylene in healthy areas adjacent to macerated diseased tissues (similar to F2 and A2 samples in Fig. 2) (Achilea et al., 1985b). The data reported here extend this observation showing that the burst in ethylene production upon Penicillium infection is the result of the co-ordinated temporal and spatial distinct up-regulation of ACS1, ACS2, and ACO (Figs 1, 2, 4). The possibility that other members of these two gene families from citrus might also be induced in fruits by fungal infection can not be ruled out, although it is noteworthy that these are the only ethylene biosynthesis genes so far isolated in citrus fruits by different research groups (Mullins et al., 1999; Wong et al., 1999; Zacarías et al., 2003; Katz et al., 2004).
It is interesting to note that induction of ACS1 during infection preceded that of ACS2. In other examples, distinct members of ethylene biosynthetic gene families are differentially regulated upon pathogen attack or elicitor action (Oetiker et al., 1997; Cooper et al., 1998). It has also been described that citrus ACS1 mRNA did not accumulate upon the storage of fruits at a low chilling temperature, while ACS2 and ACO increased their expression very significantly (Zacarías et al., 2003). In conjunction with the data reported here, this observation would indicate that ACS1 shows a differential response in citrus fruits depending on the type of stress.
Inhibition of ethylene perception by 1-MCP increased ethylene production in infected grapefruits (Mullins et al., 2000), and this is in agreement with the autoinhibition of wound-induced ethylene production in citrus peel (Riov and Yang, 1982). This study's results show that 1-MCP fumigation was effective in promoting not only susceptibility to infection (Table 2), but also ethylene production (Fig. 3), and gene expression changes (Figs 4–7
). A 10-fold difference in ethylene production was observed between infected fruits pretreated with air or 1-MCP (Fig. 3, data at 72 h), while the evolution of ACS1 or ACO mRNA accumulation in the same samples did not change substantially (Fig. 5). The burst in ethylene after 1-MCP treatment was correlated with an increase in the accumulation of ACS2 mRNA in both wounded and infected fruits (Figs 4, 5). Therefore, this study's data are consistent with an autoinhibitory regulation of ethylene production during the response to wounding and Penicillium infection, acting primarily on ACS2 gene expression in a negative feedback.
Comparison of ethylene production and mRNA accumulation (Figs 3, 4) also suggest a post-transcriptional regulation of ACS, as occurs with other plant systems described in the literature (Oetiker et al., 1997). It is widely recognized that ACS is the key regulatory step in ethylene biosynthesis, by receiving signals that act positively or negatively at the gene transcription and post-translational level (Wang et al., 2002). Post-translational regulation of ACS activity involves phosphorylation or modulation of protein turnover, among others. In this case, ACS mRNA levels are higher in I48 and I24 than in the corresponding W48 and W24 (Fig. 4), while ethylene production was not significantly different between wounded and inoculated samples within the first 48 h in all three pretreatments assayed (Fig. 3). These observations are particularly relevant for ACS1, for which an early and very substantial increased mRNA in infected versus wounded tissues was observed: I48 has a 223-fold increased expression over the reference value and 13-fold (223/17) over the corresponding wounded control. All together, this study's analyses indicate that the expression that best correlates with ethylene production in different samples and pretreatments is that of ACS2. In order to counteract the inhibition of ACS2 expression by ethylene (see above), this gene must therefore be up-regulated by other unidentified mechanisms at the onset of the appearance of symptoms (72 h in the experiments shown in Figs 3–7; Table 2).
Neither ACS1 nor ACS2 showed very significant changes upon pretreatment with ethylene or 1-MCP, while ACO mRNA was induced by ethylene (Fig. 4A). ACO mRNA accumulation in distal healthy areas of fruits (F1 and A1 in Fig. 2) might therefore be explained as the induction by the ethylene produced in those heavily diseased fruits. However, it is significant that blocking ethylene perception did not down-regulate ACO during infection or wounding (Figs 4D, 5). This observation indicates that this gene is primarily regulated by other signals throughout the course of infection, despite its responsiveness to ethylene in the absence of wounding/infection. Indeed, F2 zones are exposed to the same gaseous ethylene as F1, but show much higher ACO mRNA (Fig. 2). Also, the early response of both ACS1 and ACO after 24 h of inoculation (50–70-fold induction over the reference control and 2–3-fold over the wounded control) (Fig. 4), even 24 h before the rise in ethylene production and 48 h before the appearance of symptoms, also suggests the involvement of additional signals regulating their expression.
Role of ethylene perception in susceptibility of citrus fruits to Penicillium
It has been described that ethylene pretreatment reduced citrus decay severity, although a continuous treatment during the course of infection increased fungal biomass (El-Kazzaz et al., 1983). A similar protective effect has also been observed with ethylene, which lasted for 3–4 d after inoculation and was lost afterwards (Table 2). Although no significant differences were observed between 2 ppm and 10 ppm ethylene doses, the lower dose seemed better able to induce defence against infection. It must be taken into account that ethylene is also known to promote senescence that could in fact facilitate fungal degradation of plant tissues. Therefore it was reasoned that a suitable way to investigate the involvement of ethylene in citrus susceptibility to infection was by blocking its perception (and action), and this was achieved by 1-MCP.
It has been shown that blocking ethylene perception increases susceptibility of oranges to P. digitatum infection (Table 2). Concentrations as low as 50 ppb of 1-MCP result in higher incidence of infection. This extends previous observations obtained by following the post-harvest quality of oranges after 1-MCP fumigation, in which the incidence of natural infections was also enhanced (Porat et al., 1999). This reasoning is in accordance with those obtained from experiments with plants treated with ethylene or 1-MCP (Diaz et al., 2002) or using mutants and genetically modified plants insensitive to ethylene (Thomma et al., 1999; Geraats et al., 2003), and led to the conclusion that ethylene activates in citrus fruits defence-related processes against fungal attack and therefore inhibition of its action results in more severe disease.
Ethylene responses to infection are manifested through the induction of metabolites, modulation of enzymatic activities, and gene expression changes, among others, including those linked with PR or defence genes. Citrus genes previously identified in this laboratory have been used as models to address the involvement of ethylene signalling in changes in gene expression upon Penicillium infection. Induction of the plant phenylpropanoid pathway by ethylene and fungal elicitors is well known (Chappell et al., 1984). In citrus, PAL is known to be up-regulated by ethylene (Lafuente et al., 2001, 2003) (Fig. 6A) and by P. digitatum infection (McCollum, 2000) (Fig. 6B). It is interesting to note that the blockage of ethylene perception by 1-MCP reduced, but not abolished, the infection-induced PAL transcript (Figs 6B, 7). In fact, the onset of PAL expression detected 24 h after wounding or inoculation was not substantially altered by 1-MCP, and the inhibitor effect was only noticeable later at 48 h and 72 h. It is concluded that the initial induction of PAL mRNA is largely ethylene-independent and would be regulated by other signal(s), while ethylene would promote transcript induction during infection once the response has been initiated. Given the correlation observed between 1-MCP pretreatment and increased susceptibility to fungal attack (Table 2), these data point towards an implication of lower PAL expression in this increased susceptibility and, therefore, of the phenylpropanoid metabolism as an active defence response of citrus fruits towards P. digitatum infection.
Chitinases have the potential to degrade the fungal cell wall. They are among the PR proteins that can be induced by a variety of chemical elicitors, abiotic stress or pathogens, and have been involved in plant defence (Neuhaus, 1999). They are coded by complex gene families and classified according to their primary structure and isoelectric point. ACR311 clone is an EST from an acidic class II chitinase associated to heat-induced chilling tolerance in mandarins (Sánchez-Ballesta et al., 2003). There are examples of acidic class II chitinases induced by fungal infection, wounding or elicitors (Buchter et al., 1997; Bravo et al., 2003). Regarding citrus, another gene from this class has been reported, which is 84% identical to ACR311 and was not expressed in flavedo (Nairn et al., 1997). Also, a citrus chitinase had increased message accumulation following P. digitatum infection (McCollum, 2000; L González-Candelas et al., unpublished data), while a chitinase protein band was also up-regulated with infection (Pavoncello et al., 2001). It has been shown that ACR311 has a substantial induction after infection, which is most likely a consequence of initial tissue damage, since its mRNA reached an even higher accumulation in mock-inoculated controls (Fig. 6C). This study's results also raise the possibility that ACR311 expression is partly repressed by ethylene, since it was lower in infected samples that produce higher amounts of ethylene, and also increased after 1-MCP treatment (Fig. 7).
In summary, these results support the hypothesis of the involvement of ethylene perception in promoting defence responses in diseased citrus fruits, and neatly summarize distinct possible regulations of plant defence genes in the context of fungal infection. ACS1, ACS2, ACO, PAL, and ACR311 are all induced upon infection. The former four have a higher induction over mock-inoculated controls while the latter (ACR311) would be more related to general cell or tissue damage, considering its expression in controls (Fig. 6) and stressed fruits (Sánchez-Ballesta et al., 2003). Two of them (ACO and PAL) are positively induced by ethylene, but also require additional signals to explain its induction during infection. Finally, ACS2 would be repressed by ethylene and, again, additional regulatory signals are likely acting to counteract and promote its induction despite the rise in ethylene production from diseased fruits. These are examples that summarize the complex array of responses to infection in citrus fruits. Hopefully, high throughput technologies and functional genomic tools will help to unveil this complex response.
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Acknowledgements |
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We thank Adolfo Garcia from REVA (Regadíos y Energía de Valencia, SA) for providing fruits from a commercial orchard. We acknowledge Natalia Alepuz, Ana Izquierdo, and Maria José Pascual for their excellent technical assistance. This work was supported by grant AGL2000-1443 from the Ministry of Science and Technology (MCyT), Spain.
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References |
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