Journal of Experimental Botany, Vol. 53, No. 368, pp. 429–437, March 2002
Ethylene-responsive genes are differentially regulated during abscission, organ senescence and wounding in peach (Prunus persica) Benedetto Ruperti1, Luigi Cattivelli2, Silvana Pagni1 and Angelo Ramina1,3 1
Department of Environmental Agronomy and Crop Science, University of Padova, Via Romea, 16, Agripolis, 35020 Legnaro (Padova), Italy 2 Istituto Sperimentale per la Cerealicoltura, via S. Protaso 302, 29017 Fiorenzuola d’Arda (PC), Italy Received 8 June 2001; Accepted 2 November 2001
Abstract
Introduction
Ethylene-responsive genes from peach (Prunus persica, L. Batsch) were isolated by differential screening of a cDNA library constructed from abscission zones in which cell separation had been evoked by treatment with the ethylene analogue propylene. DNA and deduced protein sequences of four selected clones, termed Prunus persica Abscission zone (PpAz), revealed homology to thaumatin-like proteins (PpAz8 and PpAz44), to proteins belonging to the PR4 class of pathogenesis-related (PR) proteins (PpAz89), and to fungal and plant b-D-xylosidases (PpAz152). Expression analyses conducted on embrioctomized and CEPA-treated fruitlets as well as on fruit explants have shown that PpAz8, PpAz44 and PpAz89 are preferentially transcribed in the cells of the fruit abscission zone rather than in the non-zone tissues. The PpAz152 transcript showed a different accumulation pattern being consistently and promptly induced by wounding and only slightly stimulated by propylene. By contrast, a complex pattern of transcript accumulation was found for the four genes in response to the wounding of leaves and during organ development and senescence. Based on this evidence, the existence of multiple regulatory pathways underlying the differential expression of the four PpAz genes in the different tissues and physiological processes is hypothesized.
The plant hormone ethylene plays a major role in the regulation of many developmental and stress response events in plants. Among these are important diverse physiological processes such as ripening, senescence, abscission, seed germination, cell elongation, wounding, and pathogen response (Abeles et al., 1992; Kitajima and Sato, 1999). Ethylene exerts its action through a complex regulation of its own biosynthesis, perception and signal transduction (Nakatsuka et al., 1998; Barry et al., 2000), finally leading to dramatic changes in gene expression (Chang and Shockey, 1999). Evidence supporting the major role played by the hormone in regulating important developmental processes comes from the fact that the inhibition of its biosynthesis or perception results in delayed ripening, senescence and abscission (Hamilton et al., 1990; Oeller et al., 1991; John et al., 1995). As a consequence, ethylene-responsive genes are expected to play a role in the physiological processes stimulated by the hormone and their isolation will help elucidate the mode of ethylene action in these processes. To this end, a number of developmentally and ethylene-responsive genes have been isolated on the basis of their differential transcription through screening of cDNA libraries (Slater et al., 1985; Lincoln et al., 1987; Clendennen and May, 1997; Davies and Robinson, 2000; Hadfield et al., 2000), differential display (Zegzouti et al., 1999; Ruperti et al., 1999; Hajouj et al., 2000) and subtractive techniques (Quirino et al., 1999). The analysis of the ethylene-inducible gene expression has suggested that the action of the hormone on gene regulation is complex and varies in relation to the tissueuorgan type, stage of devel-
Key words: b-D-xylosidase, cDNA, pathogenesis-related, thaumatin-like. 3
To whom correspondence should be addressed. Fax: q39 049 827 2850. E-mail:
[email protected] Abbreviations: PR, pathogenesis-related; AZ, abscission zone; NZ, distal non-zone; 1-MCP, 1-methylcyclopropene; C3H6, propylene; CEPA, 2-chloroethyl-phosphonic acid. ß Society for Experimental Biology 2002
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opment and interaction with other regulatory factors (Johnson and Ecker, 1998; Chang and Shockey, 1999). For example, as far as the pathogen response is concerned, it has been demonstrated that ethylene promotes the overexpression of several plant defence genes through a synergistic interaction with methyl jasmonate (Xu et al., 1994; O’Donnel et al., 1996; Pieterse and van Loon, 1999), pointing out the existence of a cross-talk between ethylene and jasmonic acid-dependent signal transduction pathways. In addition, some ethylene-responsive genes and their promoters have been reported to respond to developmental or environmental stimuli, through ethyleneindependent pathways, thus suggesting that complex regulatory mechanisms control the expression of these genes (reviewed in Ohme-Takagi et al., 2000). In order to evaluate how the expression of ethyleneresponsive genes varies in different ethylene-regulated processes (abscission, ripening and senescence) in peach (Prunus persica, Batsch), several cDNAs have been cloned from fruitlet abscission zones treated with the ethylene analogue propylene. In this paper the isolation, characterization and analysis of expression of four genes that were selected as ethylene-responsive are reported. Expression analyses have been conducted on different organs at different stages of development and on abscising fruits and leaves to provide evidence on the differential regulation of the four genes in response to developmental, environmental and hormonal cues.
Materials and methods Plant material and treatments Peach (Prunus persica L. Batsch, cv. Springcrest) fruitlets were harvested 45 d after full bloom from an experimental orchard in Legnaro (Padova, Italy). This stage coincides with the end of the early growth exponential phase (S1) (Tonutti et al., 1991) and with the highest sensitivity of fruitlet abscission zones to treatment with exogenously applied propylene (Ramina et al., 1986; Golding et al., 1998; Nakatsuka et al., 1998). Explants of fruitlets were prepared as described previously (Ruperti et al., 1998) by fixing segments of shoots, each bearing an abscission zone (AZ) and a small part of pericarp (distal non-zone, NZ), upright on 0.8% (wuv) agar plates. Fruitlet abscission was induced by flushing explants with humidified streams of the ethylene analogue propylene (500 ppm) or with air (as a control) and tissues from AZ and distal NZ were excised after 12 h and 24 h as described previously (Ruperti et al., 1998). Abscission zone and non-zone tissues were collected from untreated fruits to give a time zero control. The abscission process was also induced in vivo by embrioctomy or 2-chloroethyl phosphonic acid (CEPA) treatment in fruitlets and by deblading in leaves. Embrioctomy was performed on healthy fruits on the plant, at the S1 stage, by cutting the pericarp transversally and removing the seed (Ramina et al., 1986). Embrioctomized fruits were collected and sections of AZ and NZ were excised and frozen 0, 4 and 24 h after embrioctomy. Treatment with the ethylene releaser compound CEPA (2-chloroethyl-phosphonic acid) was performed by spraying intact healthy fruitlets at the S1 stage with a 100 ppm solution (Ramina et al., 1986). Tissues from AZ
and NZ were collected from treated fruits after 0, 4, 24, and 96 h. Longer experimental times were required in the CEPAtreated samples due to the slower overall induction of the abscission process (Ramina et al., 1986). Leaf abscission was induced by deblading healthy fully expanded leaves located in the central portions of shoots. Tissues from the AZ and distal NZ were collected 0, 2 and 5 d after treatment. All excised tissues were frozen in liquid nitrogen immediately after excision and kept at 80 8C for later analysis. Mesocarp from fruits at different stages of development and ripening coincident with the S1, S2, S3, early, intermediate, and late S4 stages as described by Tonutti et al., was collected and frozen in liquid nitrogen (Tonutti et al., 1991). Over-ripe mesocarp was collected from fruits at the late S4 stage which were subsequently kept at room temperature for 3 d. Whole flowers were sampled at the stages of closed bud, anthesis and senescence. Healthy leaves at different stages ranging from young unexpanded to fully expanded and from early to late senescence were washed in sterile water and immediately frozen in liquid nitrogen. Leaf senescence was evaluated according to chlorophyll content quantified as described earlier (Welburn and Lichtenthaler, 1984). For experiments with wounded leaves, shoots bearing intact mature fully expanded leaves were incubated for 1 h in sealed jars, before wounding, in the presence or not of the inhibitor of ethylene action 1-MCP (1-methylcyclopropene) (Bouquin et al., 1997; Nakatsuka et al., 1998; Golding et al., 1998), at a saturating concentration (10 ppm). Leaves from untreated and 1-MCP-treated shoots were wounded by cutting into 0.5 cm sections and flushed in humidified streams of air or propylene as described for fruitlet explants. 1-MCP was a gift of Rohm and Haas, MI, Italy. RNA isolation and blotting
Total RNA was isolated as described previously (Bonghi et al., 1992), with the inclusion of an ethylene glycol monobutyl ether precipitation to purify samples from contaminating carbohydrates prior to the lithium chloride precipitation of RNA (Manning, 1991). RNA was quantified by spectrophotometric reading and quality was assayed by agarose gel electrophoresis. Poly(A)q selection was performed according to Sambrook et al. (Sambrook et al., 1989). For Northern blotting, 10 mg of total RNA were separated by electrophoresis on 1.2% (wuv) agarose gels containing formaldehyde and transferred on GeneScreen nylon membranes (NEN Life Science, Boston, MA, USA) as described previously (Sambrook et al., 1989). Membranes were fixed for 2 h at 80 8C and prehybridized for 2 h. Prehybridization and hybridization (overnight) were carried out at 42 8C in 5 3 SSC, 5 3 Denhardt’s, 25 mM potassium phosphate, pH 7.4, 50% formamide (vuv), and 10 mg l 1 (wuv) salmon sperm DNA. Membranes were washed with 2 3 SSC and 0.1% SDS (wuv) for 15 min at room temperature and once in 1 3 SSC and 0.1% SDS (wuv) at 65 8C, followed by a 0.1 3 SSC and 0.1% (wuv) SDS wash at 65 8C (15 min each) before exposure to X-ray film. Filters with RNA from fruitlet explants were exposed for 48 h and those from embryoctomized and CEPA-treated fruits for 3 d, filters with RNA from leaves, flowers and fruits at different developmental stages and from debladed leaves were exposed for 7 d and filters with RNA from wounded leaves were exposed for 2 weeks. cDNA library construction and differential screening
The peach fruit abscission zone cDNA library was constructed from 5 mg of poly(A)q RNA purified from abscission zones
Regulation of ethylene-responsive genes in peach of fruitlet explants which had been treated for 24 h with propylene. cDNA synthesis and cloning was performed by using the l-ZAP cDNA kit from Stratagene (La Jolla, California, USA) according to the manufacturer’s instructions. Differential screening was performed on a total of 50 000 clones (Sambrook et al., 1989). Filters (GeneScreen, NEN Life Science, MA, USA) were hybridized in duplicate to cDNA probes obtained from 1 mg of poly(A)q RNA from untreated and propylene-treated (24 h) fruitlet abscission zones. Fixing of membranes, prehybridization, hybridization and washing conditions were those described for Northern blotting. Positive differential clones from the primary screen were confirmed by two additional rounds of screening and plasmids from single isolated plaques were in vivo excised and sequenced. DNA sequence analysis of clones and homology searches
Clones selected from the differential screening were sequenced on both strands with T3 and T7 universal primers or with internal synthetic primers using a ThermoSequenase sequencing kit (Amersham-Pharmacia Biotech, UK). Sequences were compared to the National Center for Biotechnology Information (NCBI) non-redundant sequence database using Blastx and Blastn programs. Nucleic acid labelling and synthesis of gene-specific probes
Labelled (32P) DNA probes were prepared from the selected clones via the random primer labelling method (Feinberg and Vogelstein, 1984). Templates for labelling reactions from the PpAz89 and PpAz152 clones were excised by EcoRIuXhoI digestion and gel purification. Gene specific probes from the two thaumatin-like clones PpAz8 and PpAz44 were selected from the 39 untranslated region by PCR on purified plasmids using an oligodT primer (59-GCGCAAGCTTTTTTTTTTTTV-39) in combination with the following primers respectively: 59-TACTTTCTGCCCATGATGAGCATAC-39 (PpAz8); 59-TACCTCATCACATTCTGCCCATAAG-39 (PpAz44). PpAz8 and PpAz44 gene-specific probes were cross-hybridized in order to exclude reciprocal recognition (data not shown).
Results Isolation and sequence analysis of ethylene-responsive genes
Ethylene-responsive genes were isolated through differential screening of a peach cDNA library made from poly(A)q RNA isolated from abscission zones of fruitlet explants treated with the ethylene analogue propylene. Four ethylene-responsive PpAz (Prunus persica Abscission zone) clones: PpAz8, PpAz44, PpAz89, and PpAz152, exhibiting a stronger expression after propylene treatment, were isolated and sequenced. The corresponding nucleotide and the deduced amino acid sequences were then compared with the GeneBanku EMBL databases. Three clones (PpAz8, PpAz44 and PpAz89) were related to genes coding for pathogenesisrelated proteins (PR-like). The clones PpAz8 and PpAz44 were 981 and 946 bp in length with an ORF of 242 and 248 amino acids, respectively. Both clones shared a high
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level of identity with a number of thaumatin-like proteins from tree species. PpAz8 showed the highest identity (79% at amino acid level) with a cherry thaumatin like protein (Fils-Lycaon et al., 1996), whereas identity for PpAz44 was highest (80% at amino acid level) with a thaumatin-like protein from apple (accession number CAC10270.1). A third clone, PpAz89 (509 bp length, ORF of 107 amino acids), showed significant homology to wound-inducible genes from potato (Stanford et al., 1989) and tomato (Harris et al., 1997) which encode pathogenesis-related proteins belonging to the PR4 class. Finally, the PpAz152 cDNA clone (1413 bp in length) contains a putative ORF of 461 amino acids showing extended identity (up to 64% at amino acid level) with putative b-D-xylosidase proteins from Arabidopsis (accession number AC022521), Aspergillus niger (accession number Z84377) and Japanese pear (Pyrus pyrifolia) (Itai et al., 1999). Southern blot analysis showed that PpAz89 and PpAz152 are encoded by single genes, while PpAz8 and PpAz44 belong to the same multigene family (data not shown). 39 untranslated regions were therefore used to assess PpAz8 and PpAz44 gene expression in all further analyses. Gene expression during fruitlet and leaf abscission
The involvement of the ethylene-responsive genes in ethylene-regulated processes was investigated by Northern analysis. Since ethylene is known to promote abscission (Gonzales-Carranza et al., 1998), the expression of the four PpAz genes was analysed during both leaf and fruitlet abscission. In a first experiment, Northern analyses were performed on RNA samples from abscission zones (AZ) and surrounding tissues (NZ) of fruitlet explants treated with propylene or kept in air (Fig. 1). The results showed that all clones are stimulated by propylene, nevertheless the expression behaviour of the four clones is different. PpAz8, PpAz44 and PpAz89 corresponding mRNAs are induced to some extent in the AZ and NZ tissues kept in air for 12 h and 24 h after setting the explants, although the patterns of expression were slightly different (Fig. 1, AIR). The PpAz8 transcripts were higher in AZ tissues kept in air, with respect to the corresponding NZ tissues. The PpAz44 clone was equally expressed in AZ and NZ while the PpAz89 clone was preferentially expressed in the latter. Propylene treatment up-regulated the expression of the three genes in both AZ and NZ. The transcription rate reached the maximum expression level 12 h after treatment with the exception of PpAz8 transcripts in NZ tissues (Fig. 1, C3H6). In fact, PpAz8 mRNAs were fully expressed after 12 h of propylene treatment in AZ tissue, while in the NZ the expression of the same gene was still weak after 24 h, suggesting a different sensitivity of AZ and NZ to propylene.
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The pattern of PpAz152 transcription is remarkably different from that of the genes previously considered, being already significantly induced after 12 h in both AZ and NZ tissues in explants kept in air and only slightly stimulated by propylene. These results point out that, although the same set of genes is induced in AZ and NZ cells, the mechanisms of regulation of these genes in the two regions differ. The involvement of PpAz genes in the abscission process was also investigated in vivo on fruitlets embryoctomized on the tree in which abscission was induced by
Fig. 1. Northern analysis carried out on RNA extracted from abscission zone (AZ) and non-zone (NZ) tissues of fruitlet explants. Samples were collected immediately after setting the explants (0) and after 12 h and 24 h of air (AIR) or airq500 ppm of propylene (C3H6) flushing. Filters were exposed for 2 d at 80 8C. Probes used are reported on the right. Bottom panel shows a representative hybridization with 18S rDNA probe to confirm equal loading and transfer of samples.
wounding the fruits and removing the seed as well as on intact fruits treated with the ethylene releaser CEPA. Results obtained from embryoctomized fruitlets still confirmed the accumulation of PpAz mRNAs in the AZ and distal NZ 24 h after wounding (Fig. 2A). PpAz8 mRNAs also showed a marked preferential accumulation in the AZ being almost undetectable in the NZ cells. PpAz44, PpAz89 and PpAz152 mRNAs accumulated to a similar amount in AZ and distal NZ cells. Treatment of intact fruits with CEPA resulted in a slower induction of the abscission process in comparison with embryoctomized fruitlets (Ramina et al., 1986). The delay in the CEPA-induced abscission was reflected by a reduced and delayed accumulation of PpAz homologous transcripts (Fig. 2A, B). PpAz8 (the gene displaying highest expression under CEPA treatment) PpAz44 and PpAz89 were induced preferentially in the AZ when compared with the distal NZ. A nearly equal induction in both the AZ and NZ tissues was found for PpAz152 transcripts (Fig. 2B). The expression of PpAz8, PpAz44 and PpAz89 was also found associated with the abscission of leaves, induced by deblading (Fig. 3). The expression kinetics of the four PpAz genes during leaf abscission were similar to those found during fruit abscission, although the level of expression was generally lower. PpAz8 is nearly equally expressed both in AZ and NZ tissue, while PpAz44 and PpAz89 transcripts are accumulated to a higher extent in AZ. PpAz152 is almost undetectable in AZ, while its transcript level increases in the NZ tissues 5 d after deblading. Differently from fruit abscission PpAz44 transcripts remain almost undetectable throughout the experiments in NZ tissue.
Fig. 2. Transcript accumulation in fruitlet abscission zone (AZ) and non-zone (NZ) tissues following induction of abscission by embrioctomy (A) or treatment of intact fruits with the ethylene releaser compound CEPA (B). (A) Fruitlets were cut transversely in order to remove the seed and left on the tree. Tissues from AZ and NZ were collected before embryoctomy (0) and 4 h and 24 h later. (B) Intact healthy fruitlets were sprayed on the tree with a 100 ppm solution of CEPA and samples were collected after 4, 24 and 96 h. All filters were exposed for 3 s at 80 8C. Probes are indicated in the right. Each blot was hybridized with 18S rDNA to check equal loading and transfer, with the bottom panel showing a representative result.
Regulation of ethylene-responsive genes in peach
Gene expression during fruit, leaf and flower development and senescence
The expression of the four PpAz genes was also investigated in fruits, leaves and flowers at different stages of development and senescence. Results shown in Fig. 4
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highlight that a number of tissue-specific factors are also involved in the regulation of PpAz genes. Fruit ripening and leaf senescence are both promoted by ethylene (Abeles et al., 1992), nevertheless the four PpAz genes are differentially regulated in the two processes (Fig. 4A, B). The transcription of PpAz8 is associated with leaf senescence (Fig. 4B, lanes 3–6), but not with fruit ripening. Conversely, PpAz44 corresponding mRNAs are induced during fruit ripening (Fig. 4A, lane 7), but not in senescent leaves. The transcripts of PpAz89 and PpAz152 accumulated to significant levels during the late stages of fruit ripening (Fig. 4A, lane 7), as well as during leaf senescence (Fig. 4B, lanes 5–6). Notably, transcripts of PpAz44 and PpAz152 were also found in the early stages of fruit development (Fig. 4A, lanes 1, 2). Messenger RNAs corresponding to PpAz8, PpAz89 and PpAz152 genes were also found in RNA samples from flowers at all developmental stages (Fig. 4C, lanes 1–3), while PpAz44 transcripts were detected only in senescing flowers (Fig. 4C, lane 3). Gene expression in response to wounding, propylene treatment and inhibition of ethylene perception in leaf blades
Fig. 3. Gene expression in abscission zone (AZ) and distal non-zone (NZ) tissues sampled from debladed leaves. Fully expanded leaves were debladed on the tree to induce abscission and samples were collected immediately before deblading (0) and 2 d and 5 d later. Filters were exposed for 7 d at 80 8C. The probes used are indicated in the right. Bottom panel shows a representative hybridization with 18S rDNA probe to confirm equal loading and transfer of samples.
Since the natural abscission process can be interpreted as a self-inflicted wound, the expression of the PpAz genes was investigated in response to wounding of mature leaves in the presence or absence of saturating concentrations of an inhibitor of ethylene action (1-MCP). Transcription of
Fig. 4. PpAz gene expression in fruits, leaves and flowers at different stages of development, evaluated by Northern blot analysis. (A) Fruit developmental stages were those described previously (Tonutti et al., 1991): 1 (S1), 2 (S2), 3 (S3), 4 (early S4), 5 (intermediate S4), 6 (late S4), 7 (overripe fruit). (B) Leaf: 1 (young unexpanded), 2 (partially expanded), 3 (fully expanded), 4 (early senescence), 5 (intermediate senescence), 6 (late senescence). (C) Flower: 1 (closed bud), 2 (anthesis), 3 (senescent). All hybridizations were exposed for 7 d at 80 8C. Probes used are on the right. Bottom panel shows a representative hybridization with 18S rDNA probe to confirm equal loading and transfer of samples.
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Fig. 5. Expression analysis in fully expanded wounded leaves kept in air (A, AIR), in airq10 ppm of 1-MCP (B, AIRqMCP) and in airq500 ppm of propylene (C, C3H6). Samples were collected after 0, 4, 12, 24, and 48 h and the accumulation of transcripts was assessed by hybridization of Northern blots. Filters were exposed for 2 weeks at 80 8C. Probes used are indicated in the right. Hybridization with 18S probe was performed to assess the correct loading and transfer of samples (bottom panel).
the four genes is stimulated by wounding to a different extent and with different kinetics. The transcripts of PpAz8 and PpAz89, which were previously shown to be present in the mature leaf, displayed a transient downregulation 4 h after wounding, then underwent a progressive accumulation from 12–48 h after wounding (Fig. 5A). Indeed, the inhibition of ethylene perception accelerated and enhanced the accumulation of mRNAs corresponding to these two genes (Fig. 5B), which was not significantly affected by propylene treatment (Fig. 5C). PpAz44 and PpAz152 transcripts showed a later induction, not significantly modified by the inhibition of ethylene perception or propylene treatment (Fig. 5A, B, C). These results suggest that the signal transduction pathways controlling the expression of PpAz genes during wounding are ethylene independent.
Discussion PR-like genes are expressed during abscission
In this paper the isolation of four genes from peach (Prunus persica, Batsch), that were up-regulated during propylene-induced abscission of young fruit, is reported. Based on sequence homology two clones were found to code for thaumatin-like proteins (PpAz8 and PpAz44), one for a PR4-like protein (PpAz89), while a fourth clone,
PpAz152, showed significant homology to b-D-xylosidases from fungi and plants. Abscission is a highly co-ordinated process in which ethylene plays a crucial role by stimulating cell separation at specific sites, finally leading to organ detachment (Gonzales-Carranza et al., 1998). Beside shedding, the localized accumulation of PR-like and other stress-related proteins at the sites of separation is an important event to protect the exposed fracture surface arising after organ detachment from pathogen infection (GonzalesCarranza et al., 1998). A range of PR proteins such as b-1,3-glucanases, multiple isoforms of chitinases and a thaumatin-like protein were first shown to accumulate in response to ethylene treatment in bean abscission zones (del Campillo and Lewis, 1992). More recently, the accumulation of transcripts corresponding to genes encoding a PR4-like protein, a metallothionein-like protein and an allergen-like protein has been reported to occur during tomato and Sambucus nigra leaf abscission (Coupe et al., 1995; Harris et al., 1997; Ruperti et al., 1999). In this work the abscission process has been studied through four independent experimental systems, either in vivo (embryoctomized fruitlets, CEPA-treated fruitlets and debladed leaves) or in vitro (fruitlet explants flushed with air or airq500 ppm propylene), since each experimental condition reproduces only partially natural abscission. These data show that a spectrum of PR-like genes,
Regulation of ethylene-responsive genes in peach
induced by ethylene, is transcribed during both peach fruit and leaf abscission, although the expression levels are lower in leaves. Among them, a clone (PpAz8) coding for a thaumatin-like protein was preferentially expressed in fruit AZ cells in all experimental conditions in which abscission was induced. PpAz8 can therefore be considered to be a gene regulated by ethylene preferentially, although not exclusively, in the AZ tissue. The different expression level of this gene in AZ and NZ cells cannot be attributed to different ethylene concentrations since previous research showed that the AZ and the NZ tissues produce similar amounts of endogenous ethylene (Ruperti et al., 1998). While PpAz8 represents a PR-like gene inducible by ethylene in an AZ-specific manner, PpAz44 and PpAz89 are PR-like sequences inducible by the hormone in a tissue-independent fashion. In vivo experiments conducted on intact fruits treated with CEPA confirmed that the PpAz genes are ethyleneinducible and that the stimulatory effect exerted by the hormone on PpAz8 transcription takes place almost exclusively in the abscission zone. Embryoctomy, which by itself is a severe wounding, dramatically enhanced the expression of PpAz8, PpAz44 and PpAz89 genes in both AZ and NZ tissues, demonstrating that these genes, beside being ethylene-responsive, are also strongly induced by wounding. The different sensitivity to ethylene accumulation of fruit AZ and NZ cells does not affect the expression of PpAz152, which is already fully stimulated after 12 h in explants kept in air, before the synthesis of endogenous ethylene reaches the highest levels (Ruperti et al., 1998). This shows that PpAz152 expression is largely ethyleneindependent and associated with a different regulatory pathway possibly related to wounding, being only slightly stimulated by propylene. A similar clone was reported to be expressed during fruit ripening and leaf senescence in Japanese pear (Itai et al., 1999). Different regulatory mechanisms affect PpAz gene expression
Data on the expression of the four genes during peach organ development and senescence confirmed previous findings showing that pathogenesis and stress-related genes are also developmentally regulated in a pathogenindependent fashion. Thaumatin-like proteins have been found to accumulate in a fruit and ripening-specific manner in grape (Davies and Robinson, 2000; Tattersall et al., 1997), cherry (Fils-Lycaon et al., 1996) and banana (Clendennen and May, 1997). In addition, the transcription of a number of defence-related genes has been shown to be induced during melon fruit ripening (Hadfield et al., 2000), grape leaf senescence (Davies and Robinson, 2000) and Arabidopsis leaf senescence (Quirino et al., 1999). These data show that the four PpAz genes from peach
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described in this paper, besides being ethylene-inducible during abscission are also differentially expressed during ethylene-dependent processes, pointing out the existence of tissue-specific and developmentally dependent pathways. Despite the high levels of ethylene production associated with peach fruit ripening and leaf and flower senescence (Miller et al., 1988; Callahan et al., 1992; Tonutti et al., 1991; Ruperti et al., 2001), PpAz8 transcripts remained undetectable throughout ripening while those of PpAz44 were absent in senescent leaves. Ethylene-independent regulatory mechanisms controlling expression of the PpAz genes are also active during the wounding response. In fact, transcript accumulation following wounding, as highlighted by experiments with the inhibitor of ethylene action 1-MCP, appeared ethylene independent. A number of ethylene-independent pathways controlling wounding-inducible gene expression and relying on jasmonate and salicylate signalling have been described (Reymond and Farmer, 1998). The same signals might be involved in the regulation of the expression of the four PpAz genes described in this paper. Remarkably, the induction of PpAz8 and PpAz89 transcript accumulation is up-regulated by exogenous propylene during fruit abscission, while in wounded leaves it occurs through ethylene-independent pathways. Even though the physiological role of the PpAz genes described in this paper is unknown, their involvement in pathogen response may be speculated. This is suggested by the fact that three of these genes (PpAz8, PpAz44 and PpAz89) share significant identity in terms of deduced amino acid sequences and pattern of expression with known pathogenesis-related proteins. The fact that expression of these genes is induced following wounding and associated to abscission, which can be interpreted as a self-inflicted wound, strongly suggests that their products may be involved in protecting plant tissues from pathogen infection. In fact, wounding can be fully regarded as a pathogenesis-related state per se (van Loon et al., 1994). As far as the physiological role of PpAz152 is concerned it can be hypothesized that this gene may be involved in cell wall metabolism, related to senescence. For a similar clone, reported to be associated to fruit ripening and senescence in Japanese pear, a role in cell wall disassembly and consequent fruit softening was hypothesized (Itai et al., 1999). This study’s expression data show that the wound-inducible nature of PpAz152 gene expression in young fruits may also indicate some involvement of its product in the response to pathogens (van Loon et al., 1994). As a concluding remark, the four clones described in this paper represent an example of genes involved in a range of different physiological processes, in which tissuespecific, developmental and hormonal factors interact in modulating their action. This confirms results reported
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by others pointing out that expression of Arabidopsis genes, encoding the transcription factors responsible for the regulation of some defence genes, are under the control of complex pathways both dependent on and independent from ethylene, leading to tissue and stage-specific expression (Fujimoto et al., 2000). Future work will be needed to shed light on the cis-acting elements and on factors controlling the signal transduction pathways leading to differential gene activation in the different tissues, during development and in response to exogenous stimuli.
Acknowledgements The authors would like to thank Dr Jeremy Roberts and Dr Cathy Whitelaw for kind technical assistance in cDNA library construction and Professor Giorgio Casadoro for his helpful discussion of results and critical reading of the manuscript.
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