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RESEARCH PAPER |
Isolation and characterization of four ethylene signal transduction elements in plums (Prunus salicina L.)
1University of Guelph, Department of Plant Agriculture, 4890 Victoria Ave. N., PO Box 7000, Vineland Station, ON L0R 2E0, Canada
2Agriculture and Agri-Food Canada, Southern Crop Protection and Food Research Centre, 4902 Victoria Ave. N., PO Box 6000, Vineland Station, ON L0R 2E0, Canada
3Agriculture and Agri-Food Canada, Southern Crop Protection and Food Research Centre, 1391 Sandford St, London, ON N5V 4T3, Canada
* To whom correspondence should be addressed. E-mail: jsubrama{at}uoguelph.ca
Received 19 June 2007; Revised 13 August 2007 Accepted 14 August 2007
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Abstract |
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Plums are climacteric fruits: their ripening is associated with a burst of ethylene production and respiration rate. Stone fruits, including plum, have a distinct pattern of growth and development, described as a double sigmoid pattern. In order to understand the developmental control of ethylene perception in plum, four ethylene perception and signal transduction components (EPSTCs) were characterized, including two ETR1-like proteins (Ps-ETR1 and Ps-ERS1), a CTR1-like protein, and an ethylene-responsive element-binding factor (ERF). Their regulation was studied throughout fruit development and ripening in early and late cultivars. Analysis of transcript levels revealed that only Ps-ERF1 and Ps-ERS1 accumulated immediately after fertilization. Increases in Ps-ETR1 and Ps-CTR1 transcript levels could not be detected before S3 of fruit development. Marked differences associated with the ripening behaviour of early (‘Early Golden’) and late (‘Shiro’) Japanese plum cultivars were observed. The early cultivar showed ripening patterns typical of climacteric fruits accompanied by sharp increases of the four transcript levels in an ethylene-dependent manner. However, the late cultivar exhibited a suppressed-climacteric pattern, with a slight increase in ethylene production related to ripening. The accumulation of the Ps-ETR1 (and not Ps-CTR1) mRNA in the late cultivar was ethylene independent. Ps-ERS1 mRNA was expressed at low, constant levels, while, Ps-ERF1 remained undetectable. The differences between the two plum cultivars in the date and rate of ripening in relation to the differences in the accumulation patterns of the four mRNAs are discussed.
Key words: Double sigmoid curve, ethylene perception, fruit development and ripening, gene expression, 1-MCP
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Japanese plum (Prunus salicina L.) has been categorized as a climacteric fruit, with increases in both ethylene production and respiration rate during ripening. However, this is not a general behaviour, since some cultivars such as ‘Shiro’ (‘SH’) and ‘Golden Japan’ show a suppressed-climacteric pattern, while ‘Early Golden’ (‘EG’) and ‘Santa Rosa’ behave as typical climacteric fruit (Abdi et al., 1997; Zuzunaga et al., 2001; Pérez-Vicente et al., 2002).
A double sigmoid curve is characteristic of the growth of many drupes and some berries. This phasic pattern of mesocarp growth is customarily divided into four stages (Chalmers and Van Den, 1975). Stone fruits (Prunus spp.), including plums, exhibit a typical double sigmoid growth pattern during fruit development and ripening (Tonutti et al., 1997). Within this developmental period, four distinct stages (S1–S4) are clearly recognized. The first stage (S1) is characterized by a rapid increase in cell division and elongation, and is referred to as the first exponential growth phase. In the second stage (S2), there is hardly any increase in fruit size but the endocarp hardens to form a solid stone (pit hardening). The third stage (S3) is accompanied by rapid cell division resulting in a significant increase in fruit size. This stage is also known as the second exponential growth phase. The last stage (S4) comprises the fruit ripening or climacteric stage. Some others divide the last stage into two substages: substage 4-1 in which the fruit arrives at its full size with changes in colour without having any ethylene production; and substage 4-2, where the fruit continues to ripen in an ethylene-dependent manner (Trainotti et al., 2003).
During fruit development, both the biosynthesis and the perception of the phytohormone ethylene are modulated by various factors. In climacteric fruits, such as plum, tomato, and pear, in which ripening is accompanied by a peak in respiration and a concomitant burst of ethylene, the majority of ripening processes are driven by ethylene-regulated changes in gene expression (Abeles et al., 1992; Giovannoni, 2001; El-Sharkawy et al., 2003; Martínez-Romero et al., 2003).
Ethylene perception and signal transduction have been extensively studied at the biochemical and molecular genetic levels in Arabidopsis and a few other species (Chang et al., 1993; Knee, 1993; Lanahan et al., 1994; Bleecker and Schaller, 1996; Giovannoni, 2001). To date, five ETR-like genes, At-ETR1 (Chang et al., 1993), At-ERS1 (Hua et al., 1995), At-ETR2 (Sakai et al., 1998), At-EIN4, and At-ERS2 (Hua and Meyerowitz, 1998), have been identified in Arabidopsis. All these receptors are characterized by having a sensor domain which contains at least three hydrophobic putative transmembrane stretches (Rodrìguez et al., 1999; O'Malley et al., 2005). Three of the receptors, At-ETR2, At-ERS2, and At-EIN4, are predicted to have a fourth membrane-spanning domain. Ethylene binding occurs within this N-terminal hydrophobic region (Hua and Meyerowitz, 1998; Sakai et al., 1998). The C-terminal half of the receptors consists of (i) a His kinase domain and (ii) a receiver domain. The His kinase domain consists of five subdomains that define the catalytic core of His kinases. While At-ETR1 and At-ERS1 contain all of these subdomains, the other three receptors lack one or more of them. The receiver domain has sequence identity to the response regulators of the two-component system in prokaryotes (Chang et al., 1993). Importantly, At-ERS1 and At-ERS2 are structurally similar to the At-ETRs, except that the response regulator region is lacking (Hua et al., 1998). Based on these distinguishing features and the overall sequence similarity, the five receptors can be classified into two subfamilies: subfamily I (At-ETR1 and At-ERS1) and subfamily II (At-ETR2, At-EIN4, and At-ERS2). Despite the extensive structural differences between them, all are receptors, as defined by their ability to bind ethylene (O'Malley et al., 2005).
Downstream of the receptors is the Raf-like protein kinase (MAPKKK), At-CTR1 (Kieber et al., 1993). According to the model, the ETR family of ethylene receptors and CTR1 act to regulate ethylene response pathways negatively in the absence of ethylene. Ethylene binding inhibits the activity of the receptor–CTR1 complex, leading to activation of response pathways (Hua and Meyerowitz, 1998). Further downstream of the receptor–CTR1 complex is an Nramp-related integral membrane protein, At-EIN2, which is absolutely required for ethylene signalling (Alonso et al., 1999). At the end of the signalling pathway are the transcription factors At-EIN3 and At-ERF1. At-EIN3 protein belongs to a family of transcription factors that work downstream from At-EIN2 (Chao et al., 1997; Alonso et al., 1999). At-EIN3 binds in a sequence-specific manner to the promoter of At-ERF1, an ethylene-inducible gene that belongs to the ethylene-responsive element-binding protein (EREBP) family. The AP2/EREBP-type transcription factors act at the last step of the ethylene signalling pathway (Ohme-Takagi and Shinshi, 1995; Riechmann et al., 2000). At-ERF1 protein specifically binds the so-called GCC box with a strictly conserved GCCGCC core domain to modulate transcription of a wide variety of ethylene-responsive pathogenesis-related genes, indicating that a transcriptional cascade is involved in ethylene signalling (Ohme-Takagi and Shinshi, 1995; Gu et al., 2002). In this study, the isolation and characterization of four putative EPSTC elements, two ethylene receptors [Ps-ETR1 (EF585294) and Ps-ERS1 (EF585295)], a CTR1-like protein [Ps-CTR1 (EF585298)], and one ethylene-responsive element-binding factor, Ps-ERF1 [referred to Pd-ERF1 (EF607278)] from plum are described. Their expression was studied during fruit development and ripening. The molecular and physiological results have been used to build a preliminary genetic model.
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Materials and methods |
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Plant material and post-harvest treatments
Fruits were harvested from Japanese plum (Prunus salicina) cultivars ‘EG’ and ‘SH’ grown at the experimental farm of the University of Guelph (Vineland Station, ON, Canada). These two varieties were chosen according to their maturity times, early and late, respectively. Fruit were picked at stages S1 (first exponential growth phase), 22–37 days after bloom (DAB); S2 (pit hardening), 42–52 DAB; and S3 (second exponential growth phase), 57–77 DAB. Fruits from S4 (climacteric stage) were collected from ‘EG’ between 78 and 83 DAB and from ‘SH’ between 90 and 105 DAB. Ethylene action was inhibited by applying 1-methylcyclopropene (1-MCP; 1 µl l–1) overnight immediately after harvest for pre-climacteric ‘EG’ and ‘SH’ fruits at 76 DAB and 88 DAB, respectively, before the onset of endogenous ethylene production. Fruit tissues from five fruits exhibiting similar ethylene production, as determined by gas chromatography, were used for mRNA extraction and analysis. Since the stone (seed) is not separable in S1 and S2, the whole fruit tissue was used for RNA extraction, while in the S3 and S4 stages fruit pulp and seed were carefully separated for RNA analysis. Other tissues such as shoot apex, axillary bud, young leaves, and flowers were collected from the same trees. All plant material was frozen in liquid nitrogen and stored at –80 °C.
RNA isolation
Total RNA from fruit samples was extracted using the methods described by Boss et al. (1996). For vegetative tissues and flowers, total RNA was extracted using the PureLink Plant RNA reagent (Invitrogen, Burlington, ON, Canada), as per the manufacturer's instructions. All RNA extracts were treated with DNase I (Promega, Madison, WI, USA) then cleaned up with an RNeasy mini kit (Qiagen, Mississauga, ON, Canada).
Isolation and in silico analysis of plum cDNA sequences
For the isolation of plum homologues of EPSTC cDNAs, first-strand cDNA synthesis was carried out using 20 µg of total DNase-treated RNA in a 50 µl aliquot. A 1 µl aliquot of cDNA was used in a PCR with the appropriate degenerate primers. In order to isolate ethylene perception components from plum, eight PCR primers (Table 1) were designed from the conserved regions of At-ETR1 and At-ERS1 of Arabidopsis (Chang et al., 1993; Hua et al., 1995), Le-NR and Le-ETR1 of tomato (Wilkinson et al., 1995; Zhou et al., 1996), CTR1 of Arabidopsis and tomato (Kieber et al., 1993; Clark et al., 1998; Zegzouti et al., 1999), and ERF1 of Arabidopsis and tomato (Riechmann et al., 2000; Tournier et al., 2003). The isolated fragments were cloned by using the pGEM-T easy vector (Promega, Madison, WI, USA), sequenced, and compared with database sequences using the BLAST program (Altschul et al., 1997). Extension of the partial cDNA clones was carried out using a 3'- and 5'-RACE kit (Invitrogen, Burlington, ON, Canada). A high fidelity PCR system (BMB Indianapolis, IN, USA) was used as described previously (El-Sharkawy et al., 2003). Alignments of the predicted protein sequences were performed with ClustalX (Jeanmougin et al., 1998) and GeneDoc (Nicholas and Nicholas, 1997). Finally, a cDNA clone with homology to a β-actin sequence (EF585293) was isolated. Northern blot analysis was used to check that the β-actin mRNA level was similar in all treatments (data not shown). The gene was, thereafter, used as an internal control in subsequent gene expression studies.
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Real-time quantitative RT-PCR
DNase-treated RNA (5 µg) was reverse transcribed in a total volume of 50 µl using SuperScript III Reverse Transcriptase (Invitrogen, Burlington, ON, Canada). Real-time quantitative PCR was performed using 10 ng of total RNA in a 20 µl reaction volume with SYBR GREEN PCR MasterMix (Qiagen, Mississauga, ON, Canada) on a Mx4000® multiplex Quantitative PCR system (Stratagene, La Jolla, CA, USA). Mx4000® v 4.20 software (Stratagene, La Jolla, CA, USA) was used to design gene-specific primers (Table 2). For all the genes studied here, the optimal primer concentration was 200 nM. RT-PCR conditions were as follow: 95 °C (15 min), then 45 cycles of 95 °C (15 s) and 60 °C (30 s). The products were further analysed by a dissociation curve program at 95 °C to 60 °C (16 s). All RT-PCR experiments, for each gene, were run in triplicate with different cDNAs synthesized from three biological replicates. Each sample was run in three technical replicates on a 96-well plate. For each sample, a Ct (threshold constant) value was calculated from the amplification curves by selecting the optimal
Rn (emission of reporter dye over starting background fluorescence) in the exponential portion of the amplification plot. Relative fold differences were calculated based on the comparative Ct method using the β-actin as an internal standard. To demonstrate that the efficiencies of the different gene primers were approximately equal, the absolute value of the slope of log input amount versus Ct was calculated for Ps-ETR1, Ps-ERS1, Ps-CTR1, Ps-ERF1, and β-actin genes, and was determined to be <0.1. To determine relative fold differences for each sample in each experiment, the Ct value for the four studied genes was normalized to the Ct value for β-actin and was calculated relative to a calibrator (fruits 52 DAB for Ps-ETR1 and Ps-CTR1, and 1-MCP ‘EG’ treated fruits, 83 DAB for Ps-ERS1 and Ps-ERF1) using the formula 2Ct.
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Promoter isolation
Genomic DNA was extracted from plum immature leaves according to the DNeasy Plant Maxi Kit (Qiagen, Mississauga, ON, Canada). Promoters of Ps-ETR1 (EF585296), Ps-ERS1 (EF585297), and Ps-ERF1 (data not shown) were isolated using the Universal Genome Walker Kit (Clontech, Palo Alto, CA. USA).
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Gene structure and organization
PCR amplifications using degenerate oligonucleotide primers resulted in the isolation of partial cDNAs with the expected sizes for Ps-ETR1 (1737 bp), Ps-ERS1 (1530 bp), Ps-CTR1 (513 bp), and Ps-ERF1 (462 bp). The amplified fragments were cloned into the pGEM-T easy vector and sequenced. The deduced nucleotide sequences exhibited strong similarity to the Arabidopsis ETR1, ERS1, CTR1, and ERF mRNAs, respectively. Extension of the partial cDNA clones revealed that Ps-ETR1 full-length cDNA was 2758 bp in length with a predicted open reading frame (ORF) encoding a protein of 741 amino acids (Fig. 1). The 5'-non-coding, 3'-non-coding, and poly(A)+ sequences were 318, 192, and 25 bp, respectively. The predicted ORF was 83% identical in amino acid sequence to At-ETR1 (Table 3). The full-length Ps-ERS1 sequence was 2469 bp in length with a predicted ORF encoding a protein of 642 amino acids (Fig. 1). The 5'-non-coding, 3'-non-coding, and poly(A)+ sequences were 301, 221, and 21 bp, respectively. The full-length predicted protein shared 69% amino acid identity with At-ERS1 (Table 3). Isolation of the promoter sequence of Ps-ETR1 (–831 bp) and Ps-ERS1 (–1070 bp) revealed the presence of a sequence motif, in Ps-ERS1 and not in the Ps-ETR1 promoter sequence, that shows 88.9% identity with a regulatory element found in the promoter of At-ERF1 (Solano et al., 1998).
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Ethylene receptors can be divided into two subfamilies based upon sequence divergence in the conserved domains (Bleecker, 1999). Ps-ETR1 and Ps-ERS1 belong to subfamily I. Multiple alignments of the amino acid sequences with other reported homologues and the structural similarities between the receptors are shown in Fig. 1. Both plum ethylene receptor sequences contain the ethylene-binding domain which requires copper as a cofactor for high affinity ethylene-binding activity (Rodrìguez et al., 1999), the three predicted ethylene sensor domains, a conserved His kinase domain, and a region that shows homology to the GAF domain (Schaller and Bleecker, 1995; Hall et al., 1999; Rodrìguez et al., 1999; Wang et al., 2006). Only the Ps-ETR1 sequence contains the ethylene response regulator domain (Fig. 1).
A partial sequence encoding a predicted polypeptide of 701 amino acid residues was also isolated. The predicted protein displayed strong homology (71% identity, 79% similarity) to the Arabidopsis CTR1 protein (Kieber et al., 1993). The C-terminal region of Ps-CTR1 has all the features of a Ser/Thr-specific protein kinase, including an ATP-binding site (VGAGSFGTV) and a ‘IVHWDLKSPN’ Ser/Thr kinase domain.
In order to gain more information on ethylene signal transduction, a plum homologue of the Arabidopsis ethylene-responsive factor (At-ERF), called Ps-ERF1, has also been isolated. The partial Ps-ERF1 sequence encoded a putative protein of 154 amino acid residues. Analysis of the deduced amino acid sequence revealed that this protein has a typical AP2/EREBP DNA-binding domain involved in the activity to bind the GCC box in the promoter of ethylene-responsive genes. The isolation of the Ps-ERF1 promoter sequence (data not shown) revealed the presence of two sequence motifs (ERE boxes) that show 100% identity with a regulatory element found in the promoter of At-ERF1 (Solano et al., 1998). Taken together, the results suggest that Ps-ERF1 is a member of the ERF subfamily among the AP2/EREBP proteins (Sakuma et al., 2002; Feng et al., 2005).
Ethylene production during fruit development and ripening
Low levels of ethylene production, ranging from 0.8 nl g–1 h–1 to 2 nl g–1 h–1 ±0.2, have been detected during the S1 stage of fruit development (22–37 DAB). In both studied cultivars, ‘EG’ and ‘SH’, the whole fruit produced ethylene throughout S4, i.e. fruit ripening (Fig. 5A, B). No ethylene production was detected from the separated S3 and S4 seeds (data not shown). ‘EG’ fruit displayed an early, rapid ripening, and a short and rapid (maximal 5 d) ethylene production profile (Fig. 5A). ‘SH’ fruit exhibited a suppressed climacteric phenotype. The fruit ripened more slowly and later than ‘EG’; ethylene production in ‘SH’ fruit reached a maximum at 
11 d after the onset of ethylene emission (Fig. 5B). 1-MCP treatment immediately after harvest, before the onset of endogenous ethylene, abolished the ethylene burst and ripening in the treated fruits (data not shown).
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The expression of the four EPSTC elements during fruit development and ripening
To understand the possible role of the various EPSTC elements in fruit physiology, real-time PCR analysis of steady-state mRNA levels was carried out to determine mRNA accumulation patterns throughout fruit development and ripening.
Figures 2 and 3 show the expression patterns of the four cDNAs in vegetative tissues, and during flower and early fruit development, respectively. All of the studied mRNAs accumulated in a constitutive manner in vegetative tissues (Fig. 2). In flowers and during early fruit development, the expression of Ps-ERS1 mRNA slightly increased after fertilization (7–15 DAB) as did the Ps-ERF1 transcript level, which peaked at 7 DAB (Fig. 3), while those for Ps-ETR1 and Ps-CTR1 mRNAs were constitutively accumulated.
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During S1 and S2 of fruit development, Ps-ERF1 mRNA was expressed at a basal constant level, whereas mRNAs for Ps-ETR1, Ps-ERS1, and Ps-CTR1 decreased slightly to reach the basal level (Ps-ERS1) or to be undetectable (Ps-ETR1 and Ps-CTR1) at the end of S2 (Fig. 4). Throughout the S3 stage, transcript levels of Ps-ERS1 and Ps-ERF1 were detected in the whole fruit (pulp and seed) but remained at a basal level (Fig. 4). On the other hand, Ps-ETR1 transcripts were between 11% and 30% of their maximum levels (Fig. 4), and those of Ps-CTR1 increased slightly to reach
18% of their maximum levels at the end of S3 (Fig. 4).
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‘EG’ fruit exhibited a dramatic increase in the transcript levels of the four mRNAs during S4, i.e. fruit ripening (78–83 DAB). Ps-ERS1 and Ps-ERF1 cDNAs peaked
82 DAB (Fig. 5E, I), and declined thereafter, whereas Ps-ETR1 and Ps-CTR1 transcript levels continued to increase past this peak (Fig. 5C, G). The peak of Ps-ERS1 and Ps-ERF1 mRNAs coincided with the climacteric peak of ethylene production (Fig. 5A, E, I). The four mRNAs accumulated in ‘EG’ seeds in the same manner as in pulp, but at lower levels (Fig. 5C, E, G, I). Throughout ripening of the late cultivar ‘SH’ (90–105 DAB), the expression patterns of Ps-ETR1 and Ps-CTR1 had the same trend as in ‘EG’ fruit but their transcript levels were much lower (Fig. 5D, H). The only difference between the two cultivars was that Ps-ETR1 mRNA accumulation in ‘SH’ seeds peaked at the climacteric peak (101 DAB) of ethylene production (Fig. 5B, D). Ps-ERS1 mRNA was expressed in low, constant levels in comparison with the ‘EG’ cultivar and did not respond to the presence of autocatalytic ethylene during ‘SH’ fruit ripening (Fig. 5F). Ps-ERF1 mRNA remained at a basal level or was almost undetectable in the whole ‘SH’ fruit during ripening (Fig. 5J).
Effect of 1-MCP on the expression of the four EPSTC elements during fruit ripening
Increases in the four transcripts in ‘EG’ fruit were dependent on the action of ethylene as their mRNA levels were strongly down-regulated when the fruit were pre-treated with 1-MCP (Fig. 6A, C, E, G); however, the accumulation of Ps-CTR1 mRNA in the seed did not respond significantly to 1-MCP treatment (Fig. 6E). In contrast to the ‘EG’ cultivar, 1-MCP treatment did not affect the accumulation patterns and/or levels of Ps-ETR1, Ps-ERS1, and Ps-ERF1 mRNA in MCP-treated ‘SH’ fruit during ripening (Fig. 6B, D, H). Ps-CTR1 accumulation was inhibited in the pulp and did not respond significantly to 1-MCP treatment in the seed (Fig. 6F).
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Discussion |
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In climacteric fruits such as plum, pear, and tomato, most aspects of the ripening process are triggered and maintained by ethylene (Lelièvre et al., 1997). However, some fruit species such as plum show marked differences that distinguish them from the other climacteric fruits. Stone fruits, including plum, exhibit a typical double sigmoid growth pattern during fruit development and ripening (Tonutti et al., 1997). Within this developmental period, four distinct stages (S1–S4) are clearly recognized. The aim of this work was to isolate and characterize components of the ethylene response in order to understand the developmental control of ethylene perception and production in terms of the double sigmoid growth pattern.
In this study, the molecular cloning, expression, and regulation of four cDNAs encoding two ethylene receptors, a Raf-like histidine kinase and an ethylene-responsive element binding-factor, from plum are reported. All the isolated clones exhibited close homology with known Arabidopsis ethylene response and signal transduction element genes. Ps-ETR1, Ps-ERS1, Ps-CTR1, and Ps-ERF1 correspond to At-ETR1, At-ERS1, At-CTR1, and At-ERF1, respectively (Chang et al., 1993; Hua et al., 1995; Kieber et al., 1993; Ohme-Takagi and Shinshi, 1995). Both At-CTR1 and the ethylene receptors act as negative regulators of the ethylene signal response pathway (Kieber et al., 1993). At-ERF1 transcription factor acts at the last step of the ethylene signalling pathway by modulating transcription of a wide variety of ethylene-responsive genes (Ohme-Takagi and Shinshi, 1995; Riechmann et al., 2000).
Previous work with Arabidopsis has indicated at least five different genes encoding ethylene receptors which can be divided into two subfamilies based on structural and sequence similarities (Hua et al., 1998). Ps-ETR1 and Ps-ERS1 are predicted by sequence identity and by structural analysis to code for the ETR1 subfamily. Ps-ETR1 and Ps-ERS1 predicted proteins contain all the residues that have been shown to be important for ethylene receptor function (Schaller and Bleecker, 1995; Bleecker, 1999; Hall et al., 1999; Rodrìguez et al., 1999; Wang et al., 2006). Ps-ERS1, like its Arabidopsis homologue, At-ERS1, lacks the C-terminal receiver domain that forms part of Ps-ETR1 and At-ETR1 proteins. The aspartate residue found in the receiver domain has been predicted to be the target of autophosphorylation in At-ETR1 and other receiver sequences.
The predicted Ps-CTR1 protein has all the features of a Ser/Thr-specific protein kinase, including the ATP-binding site and the Ser/Thr kinase domain (Kieber et al., 1993). CTR1, similarly to the ethylene receptors, acts as a negative regulator of the ethylene response. The isolated Ps-ERF1 sequence showed high sequence identity with the other known ERF proteins. The predicted Ps-ERF1 protein has a typical AP2/EREBP DNA-binding domain involved in the activity of binding the GCC box in the promoter of ethylene-responsive genes (Sakuma et al., 2002; Feng et al., 2005). Assuming that the sequences isolated in this study encode functional ethylene response regulators, their expression patterns were studied during fruit development and ripening in order to determine their involvement in ethylene sensitivity and the capacity of the fruit to ripen.
The isolated cDNAs are differentially regulated throughout fruit development and ripening. They are expressed at constant low levels in the different vegetative tissues. The expression levels of Ps-ERS1 and Ps-ERF1 mRNAs are up-regulated after fertilization and then decrease. Such behaviour has been observed for Le-NR mRNA in tomato ovaries at anthesis (Klee, 2002, 2004). This pattern of expression defines the role of ethylene in petal wilt and flower abscission. Slight increases of Le-ERF2 and Le-ERF3 transcripts have been shown in tomato open flowers (Tournier et al., 2003).
During S1 and S2 stages, all the four cDNAs were slightly down-regulated to reach a basal level at the end of S2, indicating only a minor role for ethylene during these two developmental stages. The accumulation of Ps-ETR1 with a concomitant increase of Ps-CTR1 mRNAs during S3 occurs in an ethylene-independent manner, as ethylene emission was not detected during this stage of fruit development.
As mentioned earlier, the differences in ripening behaviour between various plum cultivars is due to the differences in their capacity to produce and respond to ethylene. Early plum cultivars showed ripening patterns typical of climacteric fruits, while late cultivars exhibited a suppressed-climacteric phenotype, with only a slight increase of autocatalytic ethylene production associated with ripening (Abdi et al., 1997). Mature ‘EG’ fruit displayed an early and rapid ripening, accompanied by a sharp increase in autocatalytic ethylene production after 82 DAB. In contrast, ‘SH’ fruit exhibited a very low ethylene production, which perhaps resulted in slower ripening. Ethylene production in ‘SH’ fruit reached a maximum 101 DAB. Late cultivars, including ‘SH’, seem to result from an inability of the fruit to produce sufficient quantities of ethylene to co-ordinate ripening. However, treatment with propylene restored the typical climacteric pattern in such late cultivars (Abdi et al., 1997). Taken together, the data suggest that there is a minimum requirement for ethylene (endogenous and/or exogenous) in late cultivars, but, once this minimum requirement is met, the developmental changes required for ripening will be accelerated.
In both cultivars, Ps-ETR1 and Ps-CTR1 expression levels dramatically increased during fruit ripening (S4). Both transcripts continued to increase past the climacteric peak of ethylene production, as observed during pear fruit ripening (El-Sharkawy et al., 2003). The transcript levels of Ps-ETR1 and Ps-CTR1 in ‘SH’ were at least two and three times, respectively, lower than in ‘EG’. Application of the ethylene antagonist 1-MCP abolished the fruit ripening and inhibited the ethylene burst in both cultivars. 1-MCP treatment strongly down-regulated Ps-ETR1 and Ps-CTR1 mRNA levels in ‘EG’ fruit; whereas, it inhibited only Ps-CTR1 mRNA accumulation and did not significantly affect the level of Ps-ETR1 transcripts in ‘SH’. An earlier study in peach also suggests that the Pp-ETR1 mRNA level was not affected by 1-MCP treatment (Rasori et al., 2002). Expression of ETR1 under different conditions and/or in different tissues appears to be primarily constitutive in Arabidopsis and tomato (Zhou et al., 1996; Lashbrook et al., 1998) and in mume (Prunus mume; Mita et al., 1999). The two exceptions are the muskmelon gene, Cm-ETR1 (Sato-Nara et al., 1999), whose expression is markedly higher in ripening fruit than in fully enlarged fruit, and the pear gene, Pc-ETR1, which strongly accumulated due to cold treatment and throughout ripening (El-Sharkawy et al., 2003). The present results indicate that Ps-ETR1 is essentially constitutive during fruit development, but increases in abundance during fruit ripening. Since CTR1 is a negative regulator of ethylene responses, its expression would decrease during ripening if it is a key regulatory element in ethylene signalling. However, it was shown that in tomato and pear, CTR1 expression is higher in ripe fruits (Leclercq et al., 2002; El-Sharkawy et al., 2003). Thus, tomato, pear, and plum CTR1 behave much like the ethylene receptors in that their expression increases in response to ethylene.
A more pronounced difference between early and late plum cultivars could be seen in the behaviour of Ps-ERS1 and Ps-ERF1 transcript accumulation throughout fruit ripening. Transcript levels of Ps-ERS1 and Ps-ERF1 increased sharply with the climacteric peak of ‘EG’ fruit in an ethylene-dependent manner, since 1-MCP treatment inhibited the accumulation of both transcripts. In contrast, Ps-ERS1 appears to be constitutively expressed and Ps-ERF1 mRNA accumulation remained at a basal level in the ‘SH’ cultivar. Their expression did not respond to the slight increase in autocatalytic ethylene production and was not significantly affected by 1-MCP treatment. It seems that the quantity of ethylene produced throughout ripening of the late cultivar ‘SH’ was not enough to enhance the expression of both transcripts. ‘SH’ fruits pre-treated with propylene showed ripening patterns typical of climacteric fruits and displayed rapid ripening and a sharp increase in autocatalytic ethylene production (Abdi et al., 1997). Dramatic increases in endogenous ethylene production accompanied by higher ERS-type transcripts have been detected in peach fruit (Pp-ERS1; Rasori et al., 2002) and in late pear cultivars (Pc-ERS1; El-Sharkawy et al., 2003) in response to propylene treatment. Increases of ERS-type transcripts have been shown during ripening in tomato (Le-NR), muskmelon (Cm-ERS1), and passion fruit (Pe-ERS2) (Payton et al., 1996; Sato-Nara et al., 1999; Mita et al., 2002); these genes are up-regulated by ethylene, and the accumulation of specific mRNA parallels the increase in tissue sensitivity to ethylene. ERFs mediate ethylene-regulated responses to both biotic and abiotic stresses (Riechmann and Meyerowitz, 1999; Singh et al., 2002). In tomato, Le-ERF1–4 transcript levels are up-regulated by ethylene and wounding (Tournier et al., 2003). Among the four tomato Le-ERFs, only Le-ERF2 shows ripening-associated expression (Tournier et al., 2003). Taken together, the results suggest that there is a positive correlation between the Ps-ERS1 and Ps-ERF1 mRNA accumulation pattern. An increase in the transcript levels of one leads to an increase in the expression of the other. Interestingly, isolation of the promoter sequence of Ps-ETR1, Ps-ERS1, and Ps-ERF1 revealed the presence of two motifs (ERE boxes), only in Ps-ERS1 and Ps-ERF1 promoter sequences, that show 88.9% and 100% identity, respectively, with a regulatory element found in the promoter of At-ERF1 (Solano et al., 1998). This sequence has also been found in the promoter regions of tomato E4 and TLC1, peach Pp-ERS1, and carnation GST1 genes, which are up-regulated by ethylene (Montgomery et al., 1993; Itzhaki and Woodson, 1993; Rasori et al., 2002; Tapia et al., 2005). The presence of the two boxes in the promoter is necessary for full promoter activity (Tapia et al., 2005).
This investigation clearly shows that the EPSTC elements characterized are consistent, both in sequence identity and in function, with already known genes of the ethylene perception pathway. Furthermore, we have also demonstrated the modulation of the transcripts of these members in an ethylene-responsive manner, thus confirming their role in ethylene transduction. Clear differences between early and late plum cultivars in the mRNA accumulation patterns of the ethylene response components isolated in this study are shown. The absence of Ps-ERS1 and Ps-ERF1 transcripts, which play a major role in fruit ripening in the late cultivar, may have resulted in a reduced response to ethylene and ripening. It has been shown that the ethylene receptors and CTR1 act as negative regulators of the ethylene response. It has also been shown that, despite a degree of redundancy in the ethylene receptor family, the expression of ethylene receptors is related to function and therefore to the capacity of plant tissues to respond to ethylene. In this investigation, differences between ethylene responsiveness of the two plum cultivars may determine the ability of the fruit to ripen, and clearly provide an explanation for such differences; however, it is more likely that these differences reflect a combination of developmental changes that occur in the fruit. The results of this study can potentially be used to screen lines for maturity and ripening in plum breeding programmes. With additional work on these lines, it may also be practically possible to control fruit ripening artificially to meet the market needs.
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Acknowledgements |
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We thank Dr Dennis Murr (University of Guelph) for providing generous use of the GC equipment, Peter Alm for technical assistance, and Dr Gopi Paliyath for helpful discussions. Financial assistance from the Canadian Foundation for Innovation, Ontario Innovation Trust, and Ontario Tender Fruit Marketing Board (SJ), and an NSERC-Visiting Fellowship (IES) is also gratefully acknowledged.
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Abbreviations |
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DAB, days after bloom; ‘EG’, ‘Early Golden’; EPSTC, ethylene perception and signal transduction component element; 1-MCP, 1-methylcyclopropene; ‘SH’, ‘Shiro’.
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