JXB Advance Access originally published online on April 28, 2003
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Journal of Experimental Botany, Vol. 54, No. 387, pp. 1615-1625,
June 1, 2003
© 2003 Oxford University Press
Isolation and characterization of four ethylene perception elements and their expression during ripening in pears (Pyrus communis L.) with/without cold requirement
Received 22 October 2002; Accepted 26 February 2003
1 UMR No. 990 INRA/INPT-ENSAT ‘Génétique et Biotechnologie des fruits’, Av. de l’Agrobiopole, 31326 Castanet-Tolosan Cédex, France
2 PCBRC School of Botany University of Melbourne, Parkville 3010, Australia
3 To whom correspondence should be addressed. Fax: +33 5 62 19 35 73. E-mail: sharkawy{at}ensat.fr
Abbreviations: PC, Passe-Crassane; OH, Old Home; ACC, 1-aminocyclopropane-1-carboxylic acid; ACO, ACC oxidase; ORF, open reading frame.
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Pear (Pyrus communis L.) are climacteric fruit: their ripening is associated with a burst of autocatalytic ethylene production. Some late pear cultivars, such as Passe-Crassane (PC) require a long (80 d) chilling treatment before the fruit will produce autocatalytic ethylene and ripen. As the cold requirement is linked to the capacity to respond to ethylene (or its analogue, propylene), three pear cDNAs homologous to the Arabidopsis ethylene receptor genes At-ETR1, At-ERS1, and At-ETR2, designated Pc-ETR1a (AF386509 [GenBank] ), Pc-ERS1a (AF386515 [GenBank] ), and Pc-ETR5 (AF386511 [GenBank] ), respectively, have been isolated. A pear homologue of the Arabidopsis ethylene signal transduction pathway gene At-CTR1, called Pc-CTR1 (AF386508 [GenBank] ) has also been isolated. The search of the genomic sequences for Pc-ETR1a and Pc-ERS1a resulted in the isolation of four related genomic clones Pc-DETR1a (AF386525 [GenBank] ), Pc-DETR1b (AF386520 [GenBank] ), Pc-DERS1a (AF386517 [GenBank] ), and Pc-DERS1b (AF386522 [GenBank] ). Analysis of transcript levels for the four cDNAs in PC and pear fruit genotypes with little or no cold requirement revealed that Pc-ETR1a expression increased during chilling treatment, and Pc-ETR1a, Pc-ERS1a, Pc-ETR5, and Pc-CTR1 expression increased during fruit ripening and after ethylene treatment. Whether the differences in the ethylene response elements studied here are the cause or an effect of the cold requirement in PC fruit is discussed.
Key words: Cold requirement, ethylene perception elements, exogenous ethylene, fruit ripening, pear.
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Both the biosynthesis and the perception of the phytohormone ethylene are modulated during development of plant tissues. In climacteric fruit, such as the pear, avocado, and melon a majority of ripening processes are driven by ethylene-regulated changes in gene expression (Abeles et al., 1992; Lelièvre et al., 1997a; Giovannoni, 2001). However, in some fruit species such as pears, cold treatment can hasten the rate of ripening on or off the tree (Knee, 1993; Itai et al., 1999). Late ripening pears require several weeks at low temperatures and will then ripen rapidly at higher temperatures (Gerasopoulos and Richardson, 1997b). In a few genotypes such as Passe-Crassane (PC), unchilled fruit will generally not ripen normally (Ulrich, 1961; Leblond, 1975). In all cases, cold treatment stimulates ethylene biosynthesis that leads to ripening (Knee, 1993; Gerasopoulos and Richardson, 1997a). Treatment of cold-treated PC fruit with ethylene or its analogue, propylene, is able to advance ripening. However, the duration of propylene treatment, necessary to induce ripening-related changes in PC fruit is, unusually, quite long, from 20 d to 35 d. Non-chilled PC fruit often remain unable to produce autocatalytic ethylene during propylene treatment even though fruit softening and the induction of ACO1, the final enzyme in the ethylene biosynthetic pathway, can be observed (Lelièvre et al., 1997b). These results suggest that the capacity for autocatalytic ethylene production and ripening is developmentally and/or environmentally controlled in mature PC and related pear fruit. The mechanisms underlying the induction by low temperature of autocatalytic ethylene production are not well understood. In this work, the object was to determine whether the cold requirement is related to changes in ethylene perception and signal transduction.
Ethylene perception and signal transduction (Bleecker and Schaller, 1996) have been extensively studied at the biochemical and molecular genetic levels in Arabidopsis thaliana and other species (Chang et al., 1993; Knee, 1993; Lanahan et al., 1994; Giovannoni, 2001). To date, five ETR1-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. Functional ETR1 homologues have been isolated from several plant species (Lanahan et al., 1994; Müller et al., 2000; Sato-Nara et al., 1999). Ethylene receptor genes are differentially regulated throughout plant development in Arabidopsis and tomato (Hua and Meyerowitz, 1998; Lashbrook et al., 1998). Importantly, At-ERS1 (Hua and Meyerowitz, 1998) and NR (Wilkinson et al., 1995; Payton et al., 1996) are involved in the autoregulation of ethylene perception as their expression is up-regulated by the phytohormone. Ethylene/receptor binding leads to a plant response through the ethylene signal transduction pathway. The Arabidopsis protein, At-CTR1, is one of the early elements of this pathway (Kieber et al., 1993). It has been shown to be a Raf-like Ser/Thr protein kinase (MAPKK kinase) and, as with the ethylene receptor proteins, is a negative regulator of the ethylene response. Mutant forms of ctr1 confer a constitutive ethylene response in air.
In this paper the isolation and characterization of four putative pear ethylene perception response elements, three ethylene receptors (Hua and Meyerowitz, 1998; Bleecker and Kende, 2000) and a CTR1-like protein (Kieber et al., 1993; Clark et al., 1998) are described. Their expression was studied during ripening in PC fruit and in pears that have a reduced or no cold requirement in order to determine whether there are dissimilarities that could account for the diversity in ripening behaviour. The molecular and physiological results have been used to build a preliminary genetic model.
|
Materials and methods |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Plant material and post-harvest treatments
Pear (Pyrus communis L. cv. Passe-Crassane) fruits were harvested and treated as described previously (Lelièvre et al., 1997b) except that, for the 1-methylcyclopropene (1-MCP) treatment, the 1-MCP (1 µl l–1) was applied overnight immediately before cold treatment. Fruit of Old Home (OH) and Old HomexPC hybrid trees P7R7A16 (A16) and P7R6A50 (A50) were obtained from INRA Angers (Garonne valley), France. PC, OH, A16, and A50 were grafted onto quince rootstock BA27. Preclimacteric fruit were harvested as late as possible before fruit ethylene production had risen and the fruit had abscised. After measurements of ethylene production, fruit tissue was frozen as previously described (Lelièvre et al., 1997b). Mixed tissues of two fruit displaying a similar ethylene production at the same age were used for mRNA extraction and analysis.
Isolation and in silico analysis of pear cDNA sequences
RNA extraction and the PC cDNA library were as previously described (Lelièvre et al., 1997b). For the isolation of pear homologues of ethylene perception and signal transduction components, the RNA was treated with DNase (Promega) and then first strand cDNA synthesis was carried out using 10 µg of total RNA in a 50 µl. One µl of cDNA was used in a PCR with the appropriate degenerate primers (see below). In order to isolate ethylene perception components from pear, six PCR primers ETR1-F and ETR1-R, ETR2-F and ETR2-R, and CTR1-F and CTR1-R were designed from the conserved regions of At-ETR1, At-ERS1 of Arabidopsis (Chang et al., 1993; Hua et al., 1995, 1998), NR, Le-ETR1 of tomato (Wilkinson et al., 1995; Zhou et al., 1996), At-ETR2, At-ERS2, At-Ein4 of Arabidopsis (Sakai et al., 1998; Hua et al., 1998), and CTR1 of Arabidopsis and tomato (Kieber et al., 1993; Clark et al., 1998; Zegzouti et al., 1999). The oligonucleotides used were ETR1-F (5'-GAGACGGG[ATC]AG[AG]CATGT[AGCT]AG[AG]ATG-3'); ETR1-R (5'-CATGGG[AC]GTTCTCATTTCATG[AG]TTCAT-3'); ETR2-F (5'-CAGAATTGTGCGGTTTG GATGCCG-3'); ETR2-R (5'-CACAACTTTAACAATCTCAATCTCCTG-3'); CTR1-F (5'-ATGGAGCAAGA[CT]TT[CT]CATGCTGAGCG-3'); CTR1-R (5'-ATCTCG[AC]T[GT]AACTTC[AGCT] GGTGCCATCC-3'). A high fidelity PCR system (BMB Indianapolis, IN) was used with the following PCR parameters: 1 min 30 s template denaturation at 95 °C, 2 min primer annealing at 58 °C, and 1 min primer extension at 72 °C for five cycles, followed by 15 cycles at 95 °C (30 s), 58 °C (1 min), and 72 °C (1 min 30 s), then 20 cycles at 95 °C (30 s), 58 °C (1 min), and 72 °C (2 min) with a final 7 min extension step at 72 °C. The isolated sequences were compared with database sequence using the BLAST program (Altschul et al., 1997). Alignments and comparison of the predicted protein sequences were performed with ClustalX (Jeanmougin et al., 1998), and GeneDoc (Nicholas and Nicholas, 1997), and the tree was visualized with the Treeview program (http://taxonomy.zoology. gla.ac.uk/rod/rod.html).
Two full-length and one partial cDNA clone putatively coding for ethylene receptors, and a partial cDNA homologue of Arabidopsis At-CTR1 were isolated. Genomic sequences for four putative ethylene receptors were also isolated by PCR. Extension of the partial cDNA clones was carried out by screening a pear fruit cDNA library and by using the 3' and 5' RACE kit from Gibco BRL.
Finally, a cDNA was isolated with homology to actin genes from a wide range of species.
Relative quantification of mRNA based on RT-PCR
A semi-quantitative RT-PCR method was used for the relative quantification of mRNA levels in fruit tissues (Wang et al., 1989; Zegzouti et al., 1999). Complementary DNA was synthesized with a first strand cDNA synthesis kit (Promega) using 1 µl (50 U) M-MuLV Reverse Transcriptase and 10 µg of total RNA. The number (n) of cycles necessary for exponential, but non-saturated, PCR amplification (in the presence of 200 µM dNTPs, 2.5 mM Mg2+, and 2.5 U of Taq DNA polymerase) was determined for each clone using the cDNA from the highest expressing sample. For actin, the required number of PCR cycles was 25. Ethylene signal component gene-specific primers (Table 1) were added to the PCR either before or with the actin primers, depending on the relative abundance of the two mRNA species. The following PCR parameters were used: 1 min 30 s template denaturation at 95 °C, 1 min primer annealing at 58 °C, and 1 min primer extension at 72 °C for five cycles, followed by 15 cycles at 95 °C (30 s), 58 °C (1 min), and 72 °C (1 min 30 s), then the required number of cycles at 95 °C (30 s), 58 °C (1 min), and 72 °C (2 min) with a final 5 min extension step at 72 °C.
|
The PCR products were separated on a 2% agarose gel, transferred to a nylon membrane, and hybridized with an
32P dCTP-labelled gene-specific probe in 50% (w/v) deionized formamide, 4x SSPE, 1% SDS, 5x Denhardt’s solution, 10% Dextran sulphate-Na salt (MW 500 000), and 100 µg ml–1 denaturated DNA (salmon sperm) at 55 °C. The PCR fragments were labelled by incorporation of [32P] dCTP using Ready-To-Go DNA Labelling Beads (Amersham Pharmacia Biotech). The membranes were finally washed at 68 °C in 4x SSPE for 5 min and radioactivity corresponding to each band was directly counted with the Ambis 100 ß-counter. The expression level for each cDNA was given as the percentage relative to the maximum expression level (MAX) and the expression ratio for actin: % Relative abundance of transcript X in situation S=100x(number of total counts for gene X in situation S) (number of total counts for actin in situation S)–1 (number of total counts for actin in situation MAX) (number of counts for gene X in situation MAX)–1. |
Results |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Ethylene perception genes structure and organization
PCR amplifications using degenerate oligonucleotide primers resulted in the isolation of partial cDNAs of the expected sizes for fragments of Pc-ETR1 (625 bp), Pc-ETR2 (350 bp), and Pc-CTR1 (320 bp). The amplified fragments were cloned into the pGEM-T vector, sequenced and found to have strong nucleotide sequence similarity to the Arabidopsis At-ETR1, At-ERS1, At-ETR2, and At-CTR1 mRNAs.
A cDNA library from fruit stored for 100 d at 0 °C (Lelièvre et al., 1997b) was screened with the Pc-ETR1 probe in order to isolate the full length cDNA sequences. Two full-length cDNA clones were isolated. The first cDNA was 2852 bp in length with a predicted open reading frame (ORF) encoded a protein of 741 amino acids. The 5'-non-coding, 3'-non-coding, and poly (A+) sequences were 324, 282, and 22 bp, respectively. The predicted ORF was more than 82% identical in amino acid sequence to other ethylene receptor genes (Table 2). The predicted amino acid sequence contained putative hydrophobic and histidine (His) kinase regions and a receiver domain, indicating that it is an At-ETR1–like ethylene receptor. This clone was therefore designated Pc-ETR1a.
|
The second clone isolated with the Pc-ETR1 probe had strong sequence similarity to the Arabidopsis At-ERS1 mRNA. The isolated cDNA was 2510 bp in length with a predicted ORF encoding a protein of 638 amino acids. The 5'-non-coding, 3'-non-coding, and poly (A+) sequences were 364, 241, and 18 bp, respectively. The full length predicted protein shared more than 67% amino acid identity with other ethylene receptor genes (Table 2). The predicted amino acid sequence contained the three hydrophobic domains and the His kinase domain, but lacked a receiver domain, indicating that it is a homologue of the Arabidopsis At-ERS1 protein. It was designated Pc-ERS1a. Ethylene receptors can be divided into two subgroups based upon sequence divergence in the conserved domains (Bleecker, 1999) (Fig. 2). Pc-ETR1a and Pc-ERS1a belong to the ETR1 subgroup. The ETR1 subfamily includes pear Pc-ETR1a/b, Pc-ERS1a/b, the Arabidopsis At-ETR1, At-ERS1 genes, the apple Md-ETR1 (AF032448 [GenBank] ) gene, and the tomato Le-ETR1, Le-ETR2, and NR genes.
|
Oligonucleotide primers were designed from the 350 bp (Pc-ETR2) sequence and were used in 3' and 5' RACE. This resulted in the isolation of a cDNA with strong nucleotide sequence similarity to Arabidopsis At-ETR2, At-EIN4 and At-ERS2 (Sakai et al., 1998; Hua et al., 1998) and tomato Le-ETR4 and Le-ETR5 (Tieman and Klee, 1999) mRNAs. The sequence was labelled Pc-ETR5 and appears to be incomplete. The predicted Pc-ETR5 protein (582 amino acid residues) lacks the C-terminal region but has the four hydrophobic domains and the degenerate histidine kinase-encoding domain typical of the second subgroup of ethylene receptors, the ETR2 subgroup. The ETR2 subfamily includes Pc-ETR5, Arabidopsis At-ETR2, At-EIN4, At-ERS2, and tomato Le-ETR4, and Le-ETR5 genes.
Cloning of the genomic sequences for the putative ethylene receptors, Pc-ETR1a and Pc-ERS1a resulted in the isolation of four related sequences: Pc-DETR1a (4067 bp), Pc-DETR1b (4033 bp), Pc-DERS1a (2512 bp), and Pc-DERS1b (2361 bp). The Pc-DETR1a gene corresponds to the Pc-ETR1a cDNA and is closely related to a second gene, Pc-DETR1b (Fig. 1; Table 3a). There is also a closely related gene to Pc-DERS1a (Pc-ERS1a cDNA) (Fig. 1; Table 3b). This gene has been labelled Pc-DERS1b. It is unknown whether of Pc-DETR1b and Pc-DERS1b are expressed as all attempts to isolate their cDNAs were unsuccessful.
|
|
A partial sequence encoding a predicted polypeptide of 520 amino acid residues was also isolated. The predicted protein displayed strong homology (63% identity, 77% similarity) (Fig. 3) to the Arabidopsis At-CTR1 protein (Kieber et al., 1993). The C-terminal region of Pc-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 (Fig. 3).
|
Finally, one actin sequence (AF386514 [GenBank] ) was isolated. Northern analyses (data not shown) verified that the actin mRNA level remained similar in all treatments. The actin sequence was used as an internal control in RT-PCR gene expression studies.
Separating the competence for climacteric ripening from the cold-induced stimulation of ethylene biosynthesis in Passe-Crassane
Post-harvest 1-MCP treatment immediately before refrigeration reduced the cold-induced increase in ethylene production (Fig. 4A) and abolished the ethylene burst in rewarmed fruit (Fig. 4B). PC fruit previously chilled for 30 d after harvest produced the ethylene burst, i.e. the climacteric, and fruit ripened after approximately 17 d of propylene treatment (Fig. 4C). In non-chilled fruit, propylene treatment did not result in an ethylene burst after 20 d (Fig. 4C).
|
Effects of chilling and ethylene on the expression of the four ethylene perception cDNAs in Passe-Crassane
Semi-quantitative RT-PCR analysis of steady-state mRNA levels were carried out to determine mRNA accumulation patterns during chilling and ripening in pear fruit. Pc-ETR1a mRNA accumulate to higher levels and at a greater rate during the cold treatment than Pc-ERS1a, Pc-ETR5, and Pc-CTR1 (Fig. 5A). When normal ripening was inhibited (fruit in air at 20 °C) the expression of the four mRNAs remained at basal levels (data not shown). Ethylene is essential for the increase in mRNA levels for Pc-ETR1a, Pc-ERS1a, Pc-ETR5, and Pc-CTR1 during the cold treatment, as inhibition of ethylene action by 1-MCP resulted in the elimination of any increase (Fig. 5C). Pc-ETR5 and Pc-CTR1 mRNA peaked at
15 d after fruit were placed at 20 °C for fruit previously chilled for 80 d, whereas Pc-ETR1a and Pc-ERS1a mRNA continued to increase past this peak. The peak of Pc-ETR5 and Pc-CTR1 mRNA coincided with the climacteric peak in ethylene production (Figs 4B, 5B). Post-cold-treatment increases were dependent on ethylene action as they were not present when the fruit were pre-treated with 1-MCP (Fig. 5C, D).
|
Propylene induced changes in mRNA levels for the four clones after a lag phase of about 17 d following rewarming. This coincided with the onset of the ethylene climacteric (Fig. 5F). The propylene treatment had no effect in the absence of cold pretreatment (Fig. 5E).
Expression of the four ethylene perception mRNAs in pear fruit without cold requirement
Accumulation of transcripts during fruit ripening were measured on three other pear genotypes. Fruit from OH, A16 and A50 do not have the cold requirement of PC fruit. OH fruit are characterized by an early, rapid ripening, and an early and brief (maximal at 6 d) ethylene production maximum. A16 fruit display an early but slow ripening pattern compared to OH. A16 fruit ethylene production peaks at 15 d after it begins to rise. Finally, A50 fruit ripen at a slow rate, except if first chilled. A50 fruit chilled for 45 d display a peak of ethylene production 10 d after removal from the cold (Fig. 6A).
|
In common with ethylene levels, Pc-ETR1a and Pc-ERS1a mRNA abundance increased during the ripening process (Fig. 6B, C). By contrast, Pc-ETR5 and Pc-CTR1 mRNA decreased during ripening of the early pears, OH and A16, but not for the late A50 pear (Fig. 6D, E). Pc-ETR5 and Pc-CTR1 basal expression levels were relatively high while ethylene production was low in early pears (OH and A16).
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Pear fruit are climacteric, producing a burst of autocatalytic ethylene that triggers and maintains many aspects of the ripening process. The fruit from late ripening pear varieties such as PC require a long cold treatment before they can ripen (Gerasopoulos and Richardson, 1997b). Cold treatment enables the fruit to produce autocatalytic ethylene (Lelièvre et al., 1997b), and hence to ripen. The aim of this work was to isolate components of the ethylene response in order to come to an understanding of the developmental control of ethylene perception and production in pears.
Four pear clones were isolated that closely matched known Arabidopsis ethylene response element genes. Pc-ETR1a, Pc-ERS1a, Pc-ETR5, and Pc-CTR1 correspond to the Arabidopsis At-ETR1, At-ERS1, At-ETR2, and At-CTR1 genes, respectively (Chang et al., 1993; Hua et al., 1995, 1998; Kieber et al., 1993). Pc-ETR1a, Pc-ERS1a, and Pc-ETR5 are predicted to code for ethylene receptors, and Pc-CTR1 for a pear homologue of the Arabidopsis Raf-like histidine kinase, At-CTR1. Both At-CTR1 and the ethylene receptors act as negative regulators of the ethylene signal response pathway (Kieber et al., 1993).
The complete Pc-ETR1a and Pc-ERS1a predicted proteins are 741 and 638 amino acids long, respectively. Pc-ERS1a, like its Arabidopsis homologue, At-ERS1, lacks the C-terminal receiver domain that forms part of Pc-ETR1a and the Arabidopsis, At-ETR1 protein. However, Pc-ETR1a and Pc-ERS1a both contain the conserved motifs (H, N, G1, F, and G2) that characterize two component bacterial His protein kinases (Bleecker, 1999). In addition to Pc-ETR1a and Pc-ERS1a, a partial cDNA sequence, Pc-ETR5, was isolated that has a protein sequence closer to the Arabidopsis At-ETR2, At-EIN4, At-ERS2 (Sakai et al., 1998; Hua et al., 1998), and tomato LeETR4 and LeETR5 (Tieman and Klee, 1999) proteins. Although several of the histidine kinase residues present in At-ETR1 are degenerate in At-ETR2 it has been shown to be a fully active ethylene receptor (Sakai et al., 1998). The predicted Pc-ETR5 protein (582 amino acid residues) lacks its C-terminal region, and has a histidine kinase domain which lacks four of the five histidine kinase residues conserved in the ETR1-like subfamily. In theory, these residues are necessary for transmitting the signal to downstream components of the ethylene pathway. However, as At-ETR2 is a fully functional ethylene receptor, all members of this subgroup, including Pc-ETR5, are potentially functional (Bleecker, 1999; Klee, 2002). The three predicted proteins contain all the residues that have been shown to be important for ethylene receptor function (Schaller et al., 1995; Bleecker and Kende, 2000). The predicted Pc-ETR1a protein also contains the aspartate residue in the receiver domain that has been predicted to be the target of autophosphorylation in Arabidopsis At-ETR1, At-ETR2, and other receiver sequences.
The N-terminal sequence of Pc-ETR5 has four hydrophobic domains, as do the Arabidopsis At-ETR2, At-ERS2, At-EIN4 (Sakai et al., 1998; Hua et al., 1998), and tomato Le-ETR4 and Le-ETR5 proteins (Tieman and Klee, 1999). Three of these domains share high sequence similarity with the three hydrophobic domains present in the Arabidopsis At-ETR1/At-ERS1 proteins that are associated with ethylene binding activity (Chang et al., 1993; Hua et al., 1995, 1998).Together, these results strongly indicate that the three isolated sequences code for functional ethylene receptors in pear.
In the process of isolating the corresponding genomic sequences, two sequences Pc-DETR1b and Pc-DERS1b, were isolated that putatively code for close homologues of Pc-ETR1a and Pc-ERS1a, respectively. Pc-DETR1b is closely related in sequence to the genomic clone of Pc-ETR1a, Pc-DETR1a. Similarly, Pc-DERS1b is related to Pc-DERS1a. Nucleotide sequence similarity between both genes is significant and in the case of Pc-DETR1a and Pc-DETR1b sequence similarity between the genes is higher for the introns than for the exons, suggesting the possibility that they are duplicated genes and that the introns are important for correct expression of the genes. The sequences of Pc-DETR1b and Pc-DERS1b also contain all the residues that have been shown to be important for ethylene receptor function (Schaller et al., 1995; Bleecker and Kende, 2000). Attempts to reverse transcribe and amplify sequences corresponding to Pc-DETR1b and Pc-DERS1b from PC mRNA have been unsuccessful, suggesting that in PC these sequences may be pseudogenes.
The phylogenetic analysis indicates that, similar to Arabidopsis sequences (Bleecker, 1999), the pear sequences can be divided into two subfamilies. The pear Pc-ETR1a/b and Pc-ERS1a/b sequences belong to the ETR1 subfamily, as do the Arabidopsis At-ETR1 and At-ERS1 (Chang et al., 1993; Hua et al., 1995), the apple Md-ETR1 (AF032448 [GenBank] ), and the tomato Le-ETR1, Le-ETR2, and NR proteins (Wilkinson et al., 1995; Lashbrook et al., 1998; Zhou et al., 1996). Sequence divergence is greater in the second group that includes the predicted Pc-ETR5, Arabidopsis At-ETR2, At-EIN4, At-ERS2 (Sakai et al., 1998; Hua et al., 1998) and the tomato Le-ETR4 and Le-ETR5 (Tieman and Klee, 1999) proteins. In tomato, there is a significant increase in Le-ETR4 and Le-ETR5 mRNA as fruits mature and ripen. Antisense inhibition of Le-ETR4 results in a constitutive ethylene response and substantially earlier fruit ripening (Tieman et al., 2000). This indicates that Le-ETR4 is critical for maintaining pre-climacteric ethylene insensitivity, but also that other developmentally-regulated factors are able to override its influence during ripening.
The predicted Pc-CTR1 protein has all the features of a Ser/Thr-specific protein kinase, including the ATP binding site (VGAGSFGTV) and the Ser/Thr kinase domain (IVHWDLKSPN) (Kieber et al., 1993). CTR1, similar to the ethylene receptors, acts as a negative regulator of the ethylene response. Assuming that the sequences isolated in this study encode functional ethylene response regulators, the aim was to determine their expression patterns during pear fruit development in order to determine their involve ment in ethylene sensitivity and the capacity to ripen.
Depending on growing season and location, PC fruit can require cold treatment from 0 d to 16 weeks in order to ripen (Ulrich, 1961; Leblond, 1975). This suggests that cold can replace endogenous signal(s) in late pear fruit. Changes in ethylene sensitivity, measured as the ability of PC fruit to generate autocatalytic ethylene in response to exogenous propylene, occurs early (30 d) in the cold treatment of PC fruit. Ethylene-dependent ethylene production increases during long-term cold storage of PC fruit. It has been proposed for Anjou pears (Gerasopoulos and Richardson, 1997a) that a threshold level of endogenous ethylene may be required to sustain autocatalytic ethylene production. Early removal of the fruit from the cold leads to a fall of ethylene production because the endogenous ethylene level is too low. In PC fruit, cold treatment leads to a gradual increase in ethylene production, but there is a commensurate increase in receptor expression. Ethylene/receptor binding changes the conformation of the receptor and effectively ‘switches off’ its inhibition of the signal response pathway (Moller and Chua, 1999). It is probable that a minor increase in ethylene production would be counteracted by the inhibitory effect of an increasing synthesis of ethylene receptors. There is the potential that these changes are involved in the attainment of a capacity to ripen, but given the magnitude of the changes it is unlikely that they are the primary cause of the changes in ethylene sensitivity.
The four putative ethylene perception elements are differentially regulated throughout pear fruit ripening. Pc-ETR1a mRNA accumulation is up-regulated by cold and during ripening. Pc-ERS1a, Pc-ETR5, and Pc-CTR1 are less affected by cold treatment, but all increase during post-cold-treatment, ethylene-dependent ripening. This is similar to what has been observed for ethylene response elements in tomato and suggests that their primary role during this process is to temper the ethylene response (Klee, 2002).
The ethylene climacteric and ripening occur without cold treatment in early season pears (A16 and OH) and after a brief cold treatment in winter pears (A50). There is a sharp peak of Pc-ETR1a and Pc-ERS1a mRNA accumulation during ripening in the early season pears, in contrast to the gradual increase seen in PC fruit. A more pronounced difference between early cultivars and PC fruit can be seen in the behaviour of Pc-ETR5 and Pc-CTR1 transcript accumulation. Transcript levels for Pc-ETR5 and Pc-CTR1 diminish sharply before and during the ethylene climacteric and ripening of OH and A16 fruit, whereas in PC fruit they increase sharply. A decrease in the expression of a negative regulator could result in an increase in ethylene sensitivity early in the ripening phase of early fruit development. However, given the potential for redundancy in the ethylene receptor family it remains to be determined whether reduced levels of Pc-ETR5 affect the overall ethylene sensitivity of early pear fruit.
There are clear differences between early pears and PC fruit in the mRNA accumulation patterns of the ethylene response components isolated in this study. 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. Differences between ethylene responsiveness between early season and winter pears may determine the ability to ripen, but it is more likely that these differences reflect a combination of developmentally or cold-induced changes that occur in the fruit. Further work is needed to determine the precise role of the isolated genes and their products in the capacity to ripen in pear fruit.
|
References |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Abeles FB, Morgan PW, Saltveit ME. 1992. Ethylene in plant biology, 2nd edn. San Diego: Academic Press.
Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ. 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Research 25, 3389–3402.
Bleecker AB. 1999. Ethylene perception and signaling: an evolutionary perspective. Trends in Plant Science 4, 269–274.[CrossRef][Web of Science][Medline]
Bleecker AB, Kende H. 2000. Ethylene: a gaseous signal molecule in plants. Annual Review of Cell and Developmental Biology 16, 1–18.[CrossRef][Web of Science][Medline]
Bleecker AB, Schaller GE. 1996. The mechanism of ethylene perception. Plant Physiology 111, 653–660.[Web of Science][Medline]
Chang C, Kwok SF, Bleecker AB, Meyerowitz EM. 1993. Arabidopsis ethylene-response gene ETR1: similarity of product to two-component regulators. Science 262, 539–544.
Clark KL, Larsen PB, Wang X, Chang C. 1998. Association of the Arabidopsis CTR1 Raf-like kinase with the ETR1 and ERS ethylene receptors. Proceedings of the National Academy of Sciences, USA 95, 5401–5406.
Gerasopoulos D, Richardson DG. 1997a. Ethylene production by ‘d’Anjou’ pears during storage at chilling and non-chilling temperature. HortScience 32, 1092–1094.
Gerasopoulos D, Richardson DG. 1997b. Storage-temperature-dependent time separation of softening and chlorophyll loss from the autocatalytic ethylene pathway and other ripening events of ‘Anjou’ pears. Journal of the American Society for Horticultural Science 122, 680–685.
Giovannoni J. 2001. Molecular biology of fruit maturation and ripening. Annual Review of Plant Physiology and Plant Molecular Biology 52, 725–749.[CrossRef][Web of Science][Medline]
Hua J, Chang C, Sun Q, Meyerowitz EM. 1995. Ethylene insensitivity conferred by Arabidopsis ERS gene. Science 269, 1712–1714.
Hua J, Meyerowitz EM. 1998. Ethylene responses are negatively regulated by a receptor gene family in Arabidopsis thaliana. Cell 94, 261–271.[CrossRef][Web of Science][Medline]
Hua J, Sakai H, Nourizadeh S, Chen QG, Bleecker AB, Ecker JR, Meyerowitz EM. 1998. EIN4 and ERS2 are members of the putative ethylene receptor gene family in Arabidopsis. The Plant Cell 10, 1321–1332.
Itai A, Kawata T, Tanabe K, Tamura F, Uchiyama M, Tomomitsu, Shiraiwa N. 1999. Identification of 1-aminocyclo propane-1-carboxylic acid synthase genes controlling the ethylene level of ripening fruit in Japanese pear (Pyrus pyrifolia Nakai). Molecular and General Genetics 261, 42–49.
Jeanmougin F, Thompson JD, Gouy M, Higgins DG, Gibson TJ. 1998. Multiple sequence alignment with Clustal X. Trends in Biochemical Science 23, 403–405.
Kieber JJ, Rothenberg M, Roman G, Feldmann KA, Ecker JR. 1993. CTR1, a negative regulator of the ethylene response pathway in Arabidopsis, encodes a member of the raf family of protein kinases. Cell 72, 427–441.[CrossRef][Web of Science][Medline]
Klee HJ. 2002. Control of ethylene-mediated processes in tomato at the level of receptors. Journal of Experimental Botany 53, 2057–2063.
Knee M. 1993. Pome fruits. In: Seymour G, Taylor J, Tucker G, eds. Biochemistry of fruit ripening. London: Chapman and Hall, 325–346.
Lanahan MB, Yen HC, Giovannoni JJ, Klee HJ. 1994. The Never ripe mutation blocks ethylene perception in tomato. The Plant Cell 6, 521–530.[Abstract]
Lashbrook CC, Tieman DM, Klee HJ. 1998. Differential regulation of the tomato ETR gene family throughout plant development. The Plant Journal 15, 243–252.[CrossRef][Web of Science][Medline]
Leblond C. 1975. Effect of different temperatures on respiratory intensity, ethylene synthesis and some organoleptic characters in Passe-Crassane pears. Physiologie Végétale 13, 651–665.
Lelièvre JM, Latché A, Jones B, Bouzayen M, Pech JC. 1997a. Ethylene and fruit ripening. Physiologia Plantarum 101, 727–739.[CrossRef]
Lelièvre JM, Tichit L, Dao P, Fillion L, Nam Y, Pech JC, Latché A. 1997b. Effects of chilling on the expression of ethylene biosynthetic genes in Passe-Crassane pear (Pyrus communis L.) fruits. Plant Molecular Biology 33, 847–855.[CrossRef][Web of Science][Medline]
Moller SG, Chua NH. 1999. Interactions and intersections of plant signaling pathways. Journal of Molecular Biology 293, 219–234.[CrossRef][Web of Science][Medline]
Müller R, Stummann BM, Serek M. 2000. Characterization of an ethylene receptor family with differential expression in rose (Rosa hybrida L.) flowers. Plant Cell Reports 19, 1232–1239.[CrossRef]
Nicholas KB, Nicholas HBJ. 1997. GeneDoc: a tool for editing and annotating multiple sequence alignments, distributed by the authors.
Payton S, Fray RG, Brown S, Grierson D. 1996. Ethylene receptor expression is regulated during fruit ripening, flower senescence and abscission. Plant Molecular Biology 31, 1227–1231.[CrossRef][Web of Science][Medline]
Sakai H, Hua J, Chen QG, Chang C, Medrano LJ, Bleecker AB, Meyerowitz EM. 1998. ETR2 is an ETR1 like gene involved in ethylene signaling in Arabidopsis. Proceedings of the National Academy of Sciences, USA 95, 5812–5817.
Sato-Nara K, Yuhashi KL, Higashi K, Hosoya K, Kubota M, Ezura H. 1999. Stage- and tissue-specific expression of ethylene receptor homolog genes during fruit development in muskmelon. Plant Physiology 119, 321–329.
Schaller GE, Ladd AN, Lanahan MB, Spanbauer JM, Bleecker AB. 1995. The ethylene response mediator ETR1 from Arabidopsis forms a disulphide-linked dimer. Journal of Biological Chemistry 270, 12526–12530.
Tieman DM, Klee HJ. 1999. Differential expression of two novel members of the tomato ethylene-receptor family. Plant Physiology 120, 165–72.
Tieman DM, Taylor MG, Ciardi JA, Klee HJ. 2000. The tomato ethylene receptors NR and LeETR4 are negative regulators of ethylene response and exhibit functional compensation within a multigene family. Proceedings of the National Academy of Sciences, USA 97, 5663–5668.
Ulrich R. 1961. Temperature and maturation: pears require preliminary cold treatment. Recent Advances in Botany 1961, 1172–1176.
Wang AM, Doyle MV, Mark DF. 1989. Quantitation of mRNA by the polymerase chain reaction. Proceedings of the National Academy of Sciences, USA 86, 9717–9721.
Wilkinson JQ, Lanahan MB, Yen HC, Giovannoni JJ, Klee HJ. 1995. An ethylene-inducible component of signal transduction encoded by Never-ripe. Science 270, 1807–1809.
Zegzouti H, Jones B, Frasse P, Marty C, Maitre B, Latché A, Pech JC, Bouzayen M. 1999. Ethylene-regulated gene expression in tomato fruit: characterization of novel ethylene-responsive and ripening-related genes isolated by differential display. The Plant Journal 18, 589–600.[CrossRef][Web of Science][Medline]
Zhou D, Kalaitzis P, Mattoo AK, Tucker ML. 1996. The mRNA for an ETR1 homologue in tomato is constitutively expressed in vegetative and reproductive tissues. Plant Molecular Biology 30, 1331–1338.[CrossRef][Web of Science][Medline]
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  X.-r. Yin, K.-s. Chen, A. C. Allan, R.-m. Wu, B. Zhang, N. Lallu, and I. B. Ferguson Ethylene-induced modulation of genes associated with the ethylene signalling pathway in ripening kiwifruit J. Exp. Bot., May 1, 2008; 59(8): 2097 - 2108. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
I. El-Sharkawy, W. S. Kim, A. El-Kereamy, S. Jayasankar, A. M. Svircev, and D. C. W. Brown Isolation and characterization of four ethylene signal transduction elements in plums (Prunus salicina L.) J. Exp. Bot., October 1, 2007; 58(13): 3631 - 3643. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Wang, D. Tan, A. Takahashi, T. Zhong Li, and T. Harada MdERFs, two ethylene-response factors involved in apple fruit ripening J. Exp. Bot., October 1, 2007; 58(13): 3743 - 3748. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Trainotti, A. Pavanello, and G. Casadoro Different ethylene receptors show an increased expression during the ripening of strawberries: does such an increment imply a role for ethylene in the ripening of these non-climacteric fruits? J. Exp. Bot., August 1, 2005; 56(418): 2037 - 2046. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y.-F. CHEN, N. ETHERIDGE, and G. E. SCHALLER Ethylene Signal Transduction Ann. Bot., May 1, 2005; 95(6): 901 - 915. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. J. Klee Ethylene Signal Transduction. Moving beyond Arabidopsis Plant Physiology, June 1, 2004; 135(2): 660 - 667. [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||