Journal of Experimental Botany, Vol. 54, No. 383, pp. 633-645,
February 1, 2003
© 2003 Oxford University Press
Cloning and characterization of two ripening-related strawberry (Fragaria x ananassa cv. Chandler) pectate lyase genes
Received 10 June 2002; Accepted 26 September 2002
Departamento de Bioquímica y Biología Molecular, Edificio Severo Ochoa (Edificio C-6), Campus Universitario de Rabanales, Universidad de Córdoba, 14071 Córdoba, Spain
1 These authors contributed equally to this paper.
2 To whom correspondence should be addressed. Fax: +34 957 211079. E-mail: bb1mublj{at}uco.es
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Abstract |
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Two genomic clones corresponding to putative pectate lyase genes (plA and plB) were isolated and characterized in strawberry (Fragariaxananassa cv. Chandler). The corresponding ORFs for the plA and plB genes revealed deduced proteins of 451 and 439 amino acids, respectively, that differ from that of the previously isolated strawberry plC gene. Southern blot analysis has shown that while the plB gene is a single copy gene, the plA gene is probably encoded by a small multigene family. By using specific probes corresponding to the untranslated 3' terminal region of the pl genes, and QRT-PCR methodology, the spatio-temporal expression pattern of both strawberry pl genes have been compared with that of the plC gene. The three transcripts were specifically expressed only in fruit and mainly during the ripening stages. Moreover, the expression of the plA and plB genes was induced in green de-achened fruit, but this increase was reduced by the external application of auxins as was the expression of plC. The expression of both pl genes was also strongly reduced in harvested fruit kept in controlled atmosphere (CA) containing high CO2 levels. Immunolocalization studies using antibodies raised against the strawberry PL proteins placed the proteins in the cell wall of parenchymatic cells of the fruit receptacle. The role of pl genes in cell-wall disassembly and fruit ripening softening is discussed.
Key words: Fruit-ripening, pectate lyase, softening, strawberry.
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Introduction |
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Soft fruit ripening is characterized by both a pronounced swelling of the cell-wall and a progressive decrease of fruit firmness during the ripening process. There is a correlation between cell-wall swelling and structural changes of the cell-wall structure, including pectin solubilization and modifications of the cellulose–xyloglucan frame (Redgwell et al., 1997). In many fruits the depolymerization of cell-wall hemicellulose is a common characteristic of fruit ripening (Rose and Bennett, 1999). For instance, a recent study in Charentais melon showed a relationship between the selective degradation of xyloglucans and fruit softening (Rose et al., 1998).
In strawberry, fruit firmness decreases rapidly during ripening. Such fruit softening has been related to both an increase in the hydration and disorganization of the cell-wall, and the enhanced solubility of the middle lamella and wall matrix of cortical parenchyma cells (Manning, 1993; Perkins-Veazie, 1995). In this sense, softening of the strawberry fruit coincides with a marked increase in the solubilization of polyuronides from the cell-wall (Knee et al., 1977). In strawberry, it has also been proposed that there is a relationship between the softening of the fruit and the degradation of the middle lamella and cell-wall, that mostly occurs during the last stages of the ripening process (Knee et al., 1977). The observed increases in both total soluble polyuronides and their associated neutral sugars are indicative of possible alterations in the cross-linking of carbohydrates in the cell-walls (Huber, 1984; Manning, 1993). The loss of pectin stabilization by Ca2+, which is influenced by the degree of pectin methyl esterification, may also contribute to fruit softening. Moreover, the induction of several cell-wall hydrolytic enzymes has been demonstrated during strawberry fruit ripening. A clear increase in pectin methylesterase (PME) (Archer, 1979; Barnes and Patchett, 1976) and cellulase activities in ripe and over-ripe strawberry fruit has been described, which suggests that these enzymes could play a role in the fruit softening process (Barnes and Patchett, 1976; Abeles and Takeda, 1990).
Pectate lyases (EC 4.2.2.2) were formerly described as pathogen-secreted extracellular enzymes that help pathogenesis by cleaving polygalacturonate blocks in the plant host cell-wall (Davis et al., 1984; Collmer and Keen, 1986). The enzyme randomly cleaves ß-1,4 linked galacturonosyl residues of pectins from the middle lamella and primary cell-walls of higher plants, resulting in the maceration of plant tissues (Collmer and Keen, 1986). Enzymatic cleavage of glycosidic bonds occurs through a ß elimination reaction, at a pH optimum of 8–11. This results in unsaturated C4–C5 bonds in the galacturonosyl moieties at the non-reducing ends of the polysaccharides, generating 4,5 unsaturated oligogalacturonates (Yoder et al., 1993). However, pectate lyases are distinguished both by their specificity for a glycosidic linkage next to a free carboxyl group rather than to an esterified carboxyl group, and by their different pH optima (Pilnik, 1990). In pathogens, there are multiple and independently regulated pectate lyase isoenzymes which are encoded by different genes (Kelemu and Collmer, 1993; Alfano et al., 1995). Such isoenzymes are 27–80% identical in amino acid sequence and have subtle differences in substrate specificity. So far, two endo- and exopectate lyase activities have been described; the endoenzymes being much more active in producing maceration (Kobayashi et al., 1988).
A Japanese cedar pollen protein with pectate lyase activity has been reported (Taniguchi et al., 1995). While no biochemical functions have been shown for any plant genes with sequence similarity to pectate lyase genes, the sequence conservation suggests that they encode this enzyme.
Recently, a Zinnia elegans cDNA that shows homology to pectate lyase genes from higher plants was isolated and characterized. Pectate lyase activity for the recombinant protein was also shown (Domingo et al., 1998). In situ hybridization studies have determined the expression pattern of a pectate lyase gene (ZePel gene) in different Zinnia organs (Domingo et al., 1998). In this study, the pectate lyase gene expression was located in the recent products of cambial divisions of both phloem and xylem and conspicuously localized in the xylem parenchyma cells of young vascular bundles. In roots, the ZePel mRNA was located in the outer (most recently formed) part of the xylem and in phloem parenchyma cells (Domingo et al., 1998). Thus, in Zinnia, ZePel expression was correlated with sites of vascular differentiation and with cells that are recent products of meristematic divisions (Domingo et al., 1998).
In strawberry and banana fruits, a fruit-specific cDNA with sequence homology to that of pectate lyase genes from higher plants has been isolated and characterized (Dominguez-Puigjaner et al., 1997; Medina-Escobar et al., 1997; Medina-Suarez et al., 1997). The expression of these genes was restricted to the fruit-ripening stages (Domingez-Puigjaner et al., 1997; Medina-Escobar et al., 1997; Medina-Suarez et al., 1997) where fruit softening is evident, suggesting a close relationship between fruit softening and gene expression.
In this paper the isolation and molecular characterization of two strawberry fruit genes that show a high similarity with pectate lyase (pl) genes of higher plants is reported. The spatio-temporal and hormonal expression pattern of each of three strawberry pl genes is shown. The immunolocalization of the three strawberry pectate lyase proteins (PL) is also shown. The putative relationship between the expression of these genes and strawberry fruit ripening is discussed.
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Plant material
The strawberry fruit (Fragariaxananassa cv. Chandler, an octoploid cultivar) was harvested at different developmental stages: small-sized green fruits (G1), middle-sized green fruits (G2), full-sized green fruits (G3), white fruits with green achenes (W1), white fruits with red achenes (W2), turning stage fruits (T), and fully ripe red fruits (R).
For controlled atmosphere (CA) conditions, freshly red ripe strawberry fruits were harvested and stored at 25 °C in containers ventilated continuously with a high-CO2 atmosphere: air enriched with 60% (v/v) CO2. Control fruits were kept in air under the same conditions.
Cloning of the strawberry pectate lyase genes
The isolation of the strawberry pectate lyase genomic clones was performed by plaque hybridization screening of about 1.5x105 pfu of a strawberry genomic library (Fragariaxananassa cv. Chandler) in the phage 
-FixII (Stratagene), using the 32P-labelled pectate lyase cDNA, previously isolated from strawberry, as a probe (Medina-Escobar et al., 1997). Filters were prehybridized and hybridized at 65 °C in hybridization solution: 5x SSC, 5x Denhardt’s solution, 200 µg ml–1 salmon sperm, and 0.5% SDS. After hybridization, filters were washed twice, 15 min each, at room temperature in a 0.2x SSC, 0.5% SDS solution. Afterwards, the filters were washed for 15 min at 65 °C, in a 0.2x SSC, 0.1% SDS solution. Five positive clones ranging between 12 and 17 kb in size were isolated and their inserts were analysed by restriction mapping. Suitable clones for DNA sequencing were obtained by subcloning into pBluescript vector and their DNA inserts were completely sequenced in both strands.
RNA isolation and gene expression analysis
Total RNA from a pool of six to seven strawberry fruits at different ripening stages and from roots, leaves, flowers, and runners was isolated according to Manning (1991), and the remaining carbohydrates were removed by passing RNA through a cellulose column (Speirs and Longhurst, 1993). Poly A+ RNA was obtained using an Oligo-dT cellulose column (Speirs and Longhurst, 1993).
To investigate the differential expression of the strawberry pl genes during the strawberry ripening process, northern analysis was carried out. In order to differentiate between the expression patterns of the three pl genes, non-cross-hybridizing radioactive specific probes corresponding to the 3' non-translated end of each pl gene were generated by PCR. The PCR mixture reaction contained: 1x Taq buffer, 1.25 mM MgCl2, 0.4 mM each dNTP (with the exception of dCTP), 5 µl of dCTP32, 5 mM of sequence-specific primers and 2.5 U of Taq polymerase. The thermal cycling conditions were: 94 °C for 2 min, 72 °C for 30 s followed by 35 cycles of 55 °C for 30 s, 72 °C for 30 s, and 94 °C for 1 min. A final step of 55 °C for 30 s and 72 °C for 5 min was also added. The sequence-specific primers used for specific amplification of each one of the PL amplicons were: 5'-ACTTGGATGCCGCAGAGGA-3' and 5'-GAGGTGGGAGGGAAATGG-3' for PLA; 5'-ATTACTGCTGGTGCTGGTG-3' and 5'-ACTGCAAACTCACCAATAA-3' for PLB, and 5'-GCGAAAGAGGTGACACATAGA-3' and 5'-TTCTGGAACTTGTATATTATG-3' for PLC.
Twenty micrograms of total RNA per sample were routinely used for the northern analysis. Filters (Hybond N+, Amersham Pharmacia Biotech) were prehybridized at 65 °C for 1 h, in 15 ml of hydridization solution (0.25 M NaH2PO4, 7% SDS, 0.1 mM Na2EDTA, and 1% BSA). Denatured probes were added to the same hybridization solution and hybridization was carried out at 65 °C for 14–16 h. Filters were washed (twice) at 65 °C, for 15 min in 100 ml of 0.2x SSC, 0.1% SDS, and then exposed to X-ray film, at –70 °C for 24–48 h.
A cDNA corresponding to 18S ribosomal RNA was used to control equal loading of RNA samples. The probes were labelled to a specific activity of approximately 108 cpm µg–1 using a commercial random priming kit (Amersham Pharmacia-Biotech).
In addition, RT-PCR experiments were performed, using these specific primers. The RT reactions contained: 1x RT buffer, 10 mM DTT, 1 mM each dNTP, specific PL primers (0.2 µM), 1 µg of DNaseI-treated total RNA and 40 U of MMLV-RT. The reaction mixtures were heated at 65 °C for 5 min and cooled to room temperature. Afterwards, the reaction mixtures were incubated for 60 min at 42 °C and then at 95 °C for 5 min. An aliquot (1 µl) of each cDNA product was used for the corresponding PCR reaction. The PCR mix contained: 1x PCR core mix (1x Taq buffer, 1.25 mM MgCl2, 50 µM each dNTP, 0.2 µM sequence-specífic primers, and 2.5 U Taq polymerase). The thermal cycling conditions were: 94 °C for 2 min, followed by 40 cycles of 94 °C for 15 s, 55 °C for 30 s, and 72 °C for 1 min. A final step of 70 °C for 5 min was also included.
The amplicons corresponding to each pl gene were gel purified and subcloned into pGEMT-easy vectors (Promega), according to the manufacturer’s protocol. The DNA inserts were sequenced and the sequences were compared with those of the 3' untranslated region of the corresponding pl genes.
For the quantitative real time PCR (QRT-PCR) analysis of gene expression the iCycler (BioRad) system was used. The PCR reaction consisted of 25 µl of a mixture containing: 1x PCR buffer, 1.5 mM MgCl2, 0.2 mM dNTPs, 0.2 µM of each sequence-specific primers 3 µl SYBR Green I (1:15 000 diluted), 3 µl of transcribed cDNA, and 0.5 U of Taq polymerase. The PCR program was as previously described.
In QRT-PCR analysis, quantification is based on Ct values. The Ct (threshold cycle) is a measurement taken during the exponential phase of amplification when limiting reagents and small differences in starting amount have not yet influenced the PCR efficiency. Ct is defined as the cycle at which fluorescence is first detectable above background and is inversely proportional to the log of the initial copy number. In this system, each 10-fold difference in initial copy number produced a 3.2 cycle difference in Ct. Each reaction was performed in triplicate and the corresponding Ct values were determined. The Ct values of each QRT-PCR reaction were normalized in relation to the Ct value corresponding to an interspacer 26S-18S strawberry RNA gene (housekeeping gene). Moreover, the efficiency of each particular QRT-PCR was also calculated. All these values were then used to determine the fold changes of gene expression between the different fruit ripening stages and, according to the following expression:
Fold change=2
(Ct)
Ct=Ct (target)–Ct (constitutive gene)
and (Ct)=Ct (problem)–Ct (control)
Auxin treatment
Achenes of two sets of G2 strawberry fruits on the plant were carefully removed, using the tip of a scalpel blade. One set of deachened fruits were treated with the synthetic auxin, 1 naphthaleneacetic acid (NAA) as a lanolin paste with 1 mM NAA in 1% (w/v) DMSO. The other set of deachened fruits (control group) were treated with the same paste, but without NAA. Both treatments were applied over the whole fruit surface. Fruits were harvested at 0 h, 48 h, 96 h, and 120 h after treatment, immediately frozen in liquid nitrogen and stored at –80 °C.
DNA extraction and Southern blot analysis
Strawberry genomic DNA was extracted as previously described (Medina-Escobar et al., 1997). Genomic DNA (2 µg) was digested with the restriction enzymes BamHI, BglII, EcoRI, and HindIII, fractionated on 0.7% agarose gels and then transferred to Hybond-N+ membranes. DNA was fixed by UV light using the Stratalinker (Stratagene) and the blot was hybridized using the specific radioactive probe for each gene. Hybridization and washing conditions were as in the northern blot experiments.
DNA sequencing and computer analysis of sequences
DNA was sequenced by the dideoxy-chain termination method using T3, T7 and specific primers within the DNA inserts. An automated DNA sequencer (AbiPrism, Applied Biosystems) was used to generate the sequence data. The DNA sequence was analysed for ORFs using the Codon Preference program from the GCG-Wisconsin package (version 9.0; December 1996). BLAST at http://www.ncbi.nlm.nih.gov/BLAST/ was used to compare sequences with the available data bases. Amino acid sequences were analysed and compared using Tfasta, Bestfit, Compare, Dotplot programs, and Prettybox from the GCG package. For alignment the Clustal Method from MegaAlign 4.05/00 (LaserGene 99) for Mac OS from DNAStar (Madison, WI, USA) was used.
Antibody production
To obtain antibodies against PL proteins, four conserved antigenic peptides deduced from the putative PL protein sequences were selected and synthesized (CSSMADRSNDHWNEHAVDNPE, CDCKPTGNAMVRSSP, CDDCWRCDPQWQRHRKRPANCG, and CGKYYWSDPGHDDPVNPRPG). The peptides were individually conjugated to a keyhole limpet haemocyanin (KLH) carrier protein by the l-ethyl-3-(3 dimethylaminopropyl) carbodiimide hydrochloride. All the conjugated peptides were injected together in white rabbits as antigens for antibody production.
Removal of cross-reactive anti-carrier KLH antibodies
The presence of anti-KLH antibodies in the serum may cause non-specific background in immunohistochemical staining of tissues of some plant species (Prorankiewicz et al., 2000). The problem was avoided by removing anti-KLH antibodies from the immune serum, by passing the serum through an affinity column coupled with KLH, according to Prorankiewicz et al. (2000). Proteins were extracted from the G1 and R stages (Harpster et al., 1998), and were used in order to test the specificity of the anti-PL antibodies. A band of 46 kDa, corresponding to the deduced size of the PL proteins was found (Fig. 7).
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Immunolocalization of the strawberry PL proteins
For the cytolocalization of the PL polypeptides, tissue sections were prepared as follows: small portions of strawberry fruits were fixed in ethanol–acetic acid (3:1, v/v), dehydrated through an ethanol–tertiary butanol series, and embedded in Paraplast Plus (Sherwood Medical/St Louis, MO). Sections of about 5 µm were cut with a microtome, mounted on slides covered with gelatin, deparaffinized in xylene, and rehydrated through an ethanol series. For immunolocalization purposes, sections were blocked with 2% non-fat dried milk in TBS. Immunological detection was then performed using the primary polyclonal anti-strawberry PL antiserum diluted 1/25, and a secondary anti-rabbit alkaline phosphatase-conjugated antibody (Sigma) diluted 1/500. The reaction of alkaline phosphatase was developed with nitroblue tetrazolium and 5-bromo-4-chloro-3-indol-phosphate for 15–30 min. The sections were dehydrated through a graded ethanol series, cleared in xylene and mounted in Entellan New (Merck). An Olympus AH-2 photomicroscope was used for sample visualization and photography. Immunolocalization studies were performed with tissues corresponding to the G1/G2 and R stages of fruit growth and ripening.
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Structural features of the strawberry pectate lyase genes
By screening a strawberry genomic library, five positive genomic clones were isolated. Restriction analysis showed three different patterns, suggesting that three different genes were isolated. Afterwards, a 4.2 kb EcoRI-XbaI fragment (for the plA gene), and a 5.365 kb EcoRV-XbaI fragment (for the plB gene), containing the promoter and complete coding region of both genes, were isolated. A clone containing a partial coding region of plC gene was also isolated and the three clones were sequenced in both strands.
Comparison of the strawberry full-length plC cDNA sequence (Medina-Escobar et al., 1997) with the genomic sequence of plA and plB genes, revealed the presence of six introns and seven exons in both strawberry pl genes. The exon–intron junctions obey the rule GT-AG (Gallie, 1993) and, as in higher plant genes, the introns are relatively AT-rich compared with the coding regions. These genomic structures are clearly different to those described for other higher plant pectate lyase genes that are organized in two introns and three exons (Wing et al., 1989; Rogers et al., 1992; Wu et al., 1996; Kulikauskas and McCormick, 1997).
Computer analysis has revealed the existence of putative cis regulatory elements within the plA and plB gene upstream promoter regions. Thus, in the plA gene promoter, a Ph3 myb cis-sequence repeats (GTTA) 5' side flanked by an A-rich sequence (Solano et al., 1995) at nt. 511, 578, 586, 593, 614, 621, and 630 was found, as well as a myc consensus sequence (CANNTG) (Busk and Pagès, 1998; Abe et al., 1997) at nt. 5, 20, 69, and 357 were found. Similarly, for the plB gene promoter, a myb consensus sequence (YAAC(G/T)G) (Busk and Pagès, 1998) at nt. 818 and c-myc binding site (CANNTG) (Busk and Pagès, 1998; Abe et al., 1997) at nt. 849, 1032, and 1123 were also observed. Pollen-specific box described within promoters of plant pl genes were not found in both strawberry pl genes.
Coding regions of pectate lyase genes
Computer analysis of the ORFs corresponding to plA and plB genes revealed a deduced protein of 451 and 439 amino acids, respectively. Both proteins contain a signal peptide (28 and 20 amino acid residues for the PLA and PLB, respectively). Two potential cleavage sites for signal peptides were located between Gly28 and Arg29, and Gly41 and Gly42 residues for the PLA protein, and between Ala20 and Ser21, and Ser30 and Arg31 residues for PLB protein (Fig. 1). Considering the former one, the cleavage of the signal peptides results in mature proteins of 46.4 kDa with a pI of 7.37 for PLA, and of 46.63 kDa with a pI of 8.04 for PLB.
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Both proteins contain two putative glycosylation sites NXS/T. Thus, PLA protein showed two motifs: NSS (amino acid residues 51–53) and NST (amino acid residues 87–89). Similarly, the PLB protein also presents two motifs NSS (amino acid residues 44–56 and 76–78), potentially involved in glycosylation processes.
The amino acid residues conserved in all pectate lyase proteins involved in Ca2+ co-ordination (Asp251, Asp253, Asp273, and Asp277 for PLA; Asp240, Asp242, Asp262, and Asp266 for PLB) and the conserved residues related to the active site conformation of pectate lyases (Arg329, Pro331, Arg334 for PLA and Arg318, Pro320, Arg323, for PLB) are also present in both strawberry pectate lyase proteins (Fig. 1). Besides, the conserved motifs described in all pectate lyases were also present in both strawberry proteins (motif I: WVDH, motif II: DGLVDAVMGCSTAITISNNHL, motif III: LYQRMPRCRHGYFHVVWNDY for PLA protein, and motif I: WVDH, motif II: DGLIDAIMGSTAITISNNYF, motif III: LIQRMPRCRHGYFHVVNNDY for PLB protein) (Fig. 1).
Southern blot analysis
the specific PLs probes used in the gene expression analysis were used to carry out Southern blot experiments of genomic strawberry DNA digested with BamHI, BglII, EcoRI, and HindIII. As shown in Fig. 2, hybridization fragments of a large size were detected. In the case of plB and plC, single hybridization fragments were observed with some enzymes, suggesting that both genes are single copy genes (Fig. 2; plB and plC). However, several hybridization fragments were observed for the plA gene suggesting that this gene is probably encoded by a small multigene family (Fig. 2 plA).
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Gene expression studies
The strawberry pl specific DNA probe corresponding to the 3' terminal non-coding region of each strawberry pl gene (plA, plB, and plC) was used in order to study the spatio-temporal and hormonal expression patterns. The specificity of each probe was determined by Southern-blot cross-hybridization studies among the three probes. No cross-hybridization at high stringency conditions was observed (data not shown).
As shown in Fig. 3A, northern analysis detected three pectate lyase transcripts of an estimated size of 1.8 kb for plA, 1.65 kb for plB and 1.6 kb for plC. Moreover, a fruit-specific expression pattern of these three strawberry pl genes was observed and no expression in vegetative tissues such as roots, leaves, flowers, and runners was found for such three pl genes (Fig. 3A). Thus, the three pl genes were strongly and predominantly expressed in the fruit-ripening stages W2, T and R, showing the highest transcript level in the full ripe red stage (R stage) (Fig. 3A). However, gene expression in fruit-growth stages as G1, G2, G3, and W1 was not detected. The plB gene presented a higher transcript level than the plA or plC genes in all fruit-ripening stages studied. Such a gene expression pattern presents a close correlation between the physiological role of the three pl genes and the fruit ripening process, probably related to the degradation of cell-wall pectins during strawberry fruit ripening and softening. The specific expression of the three strawberry pl genes in the fruit receptacle was also corroborated by RT-PCR analysis. Thus, by using sequence-specific primers corresponding to the non-coding 3' regions within the mRNA of each pl gene, three different amplicons were obtained (data not shown), which were identical to sequences found within the 3' end of each pl gene, clearly indicating that the three strawberry pl genes are expressed during fruit-ripening stages. The expression of these genes was assayed by QRT-PCR methodology and a similar expression pattern of the three strawberry pl genes was obtained to the one observed by northern blot analysis (Fig. 3B), corroborating that the three pl genes are expressed in the strawberry fruit receptacle. However, gene expression in G1, G2, G3, and W1 fruit growth stages was also detected by this methodology, although at low levels (Fig. 3B).
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It has previously been shown that the expression of the plC gene was directly or indirectly regulated by the auxins produced by the achenes (Medina-Escobar et al., 1997). In order to ascertain if the two newly isolated pl strawberry genes (plA and plB) were also under this hormonal control, northern blot experiments were performed and QRT-PCR analysis with mRNA isolated from deachened fruits and from deachened fruits treated with the auxin NAA. As is shown in Fig. 4, a clear increase in plA, plB and plC transcript level was found in the strawberry G2 stage with deachened fruits after 5 d. These increments in PL transcript level were partially reverted in G2 deachened fruit treated with NAA (Fig. 4), suggesting that the expression of the three strawberry pl genes was auxin-regulated.
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The amount of transcript corresponding to each strawberry pl gene in red stage fruit from transgenic strawberry plants (line R39) containing the antisense cDNA of pelC (Jiménez-Bermúdez et al., 2002) was also determined. It has been demonstrated previously that these plants produced fruits with an increased firmness. In agreement with this observation, a clear decrease in the amount of transcript for each pl gene was detected (Fig. 5).
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It has also been demonstrated previously that carbon dioxide-enriched atmospheres (CA) promote strawberry fruit firmness (Siriphanich, 1998). In order to determine if this effect could potentially be related to a decrease in the expression of pl genes, an experiment was performed with strawberry fruits kept in CA. As shown in Fig. 6, a clear decrease in the expression of the three pl genes was observed in red fruit kept in a 60% CO2-enriched CA, compared to control fruit kept in air.
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Immunolocalization of the strawberry pl genes
The antiserum raised against common antigenic epitopes from the three putative PL proteins was used for western blot analysis of proteins extracted from different stages of fruit growth and ripening (G1, G2, W2, T, and R stages). The western blot showed a major cross-reacting polypeptide of 46 kDa that is coincident with the expected size range of the deduced PL proteins (Fig. 7).
In order to detect the cellular location of the PL proteins in strawberry tissues, immunolocalization studies were also performed. A clear presence of PL proteins in the cell-wall of receptacle parenchymatic cells was always detected in ripe strawberry fruit R-stage (Fig. 8A). However, no immunolocalization in the equivalent cells of G1/G2 fruit developmental stages was found (Fig. 8B), indicating a close relationship between PL gene expression pattern and deposition of the corresponding proteins in parenchymatic cells walls of the strawberry fruit ripening stages. These results support a role for these PL enzymes in cell-wall degradation.
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Discussion |
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The promoters of plant pl genes expressed in pollen, contain a pollen-specific box (Kulikauskas and McCormick, 1997; Twell et al., 1990, 1991). This box is absent in both promoters of strawberry pl genes suggesting that these genes are not expessed in pollen. Also, no expression in pollen was found for both pl genes (data not shown). In addition, putative myb and myc cis sequences have been identified within the strawberry pectate lyase A and B gene promoters. Recently, a strawberry myb transcriptional factor whose expression is fruit ripening-related has been isolated and characterized (Aharoni et al., 2001), indicating that this family of plant transcription factors can play regulatory roles in strawberry fruit ripening. Also, a similar myb cis sequence directing specific gene fruit ripening expression has already been described within the promoter region of an apple cell-wall hydrolase gene (Atkinson et al., 1998).
In Figure 3, the three strawberry pectate lyase genes present a similar pattern of gene expression. The three corresponding mRNAs are only detected in fruit, while no mRNA was detected in vegetative tissues. This gene expression is mainly confined to ripening fruit stages when fruit softening is occurring (W2 to R). The northern blot results were confirmed by specific QRT-PCR methodology, clearly indicating that the three strawberry pl genes are being expressed during fruit development at low levels and in ripening stages at higher levels. These results indicate that the expression of pectate lyase genes is significantly correlated with fruit stages where evident cell-wall pectin degradation and softening occurs. In tomato, other cell-wall modifyng enzymes have been implicated effecting cell-wall changes during ripening. Thus, it has been suggested that the disassembly of the cell-wall structural network probably involves the concerted and synergistic action of several different enzymatic activities, where one family of cell-wall modifying proteins might mediate the activity of another, thus resulting in ordered cell-wall degradation (Rose and Bennett, 1999). Tomato (PG) and pectinmethylesterase (PME) seems to act synergistically, with PME generating sites for PG action (Steele et al., 1997). Thus, the PME activity produces de-esterification of cell-wall pectin and renders de-esterified polygalacturonosyl backbones more susceptible to the activity of PG (Steele et al., 1997). In this fruit, polygalacturonase (PG) activity is high, but cellulase activity has been found to be low (Hadfield and Bennett, 1998). However, down-regulation of polygalacturonase does not abolish softening in tomato, indicating that more than one enzyme plays a dominant role in the changes of texture associated with fruit ripening. Accordingly, in strawberry, an increase in both gene expression and in the activity of cellulase (Abeles and Takeda, 1990; Dominguez-Puigjaner et al., 1997; Manning, 1998; Trainotti et al., 1999) and PME (Perkins-Veazie, 1995, and references herein) during fruit ripening has been reported. However, little or no endo- or exo-PG activity has been found in the strawberry receptacle (Perkins-Veazie, 1995). Thus, Nogata et al. (1993) isolated three forms of exo-PG from strawberry fruit, but the PG activity decreased with fruit ripening. However, recently, a fruit-specific endopolygalacturonase gene, mainly expressed in the first ripening stages from strawberry, has been isolated (Redondo-Nevado et al., 2001).
The presence of predicted signal peptides and potential glycosylation sites within the three deduced strawberry PL proteins suggests that the strawberry pectate lyase proteins may enter the secretory pathway and become part of the extracellular matrix. Curiously, a banana pectate lyase gene also encodes a protein with a similar predicted signal peptide and glycosylation site (Dominguez-Puigjaner et al., 1997; Medina-Suarez et al., 1997). The banana pectate lyase mRNA levels increased in early climacteric fruit and reached a maximum at the climacteric peak, declining thereafter in overripe fruit. Also, as in strawberry, the gene is under hormonal control and its induction is regulated by a rapid increase in ethylene production at the onset of ripening. A role in the loss of mesocarp firmness during fruit ripening by degrading cell-wall pectins has been proposed for that protein (Domínguez-Puigjaner et al., 1997). The immunolocalization studies performed here show a clear subcellular localization of the strawberry PL proteins which are restricted to the cell-walls of the fruit receptacle cells from ripe fruit. No immunolabelling in the cell-walls of G2 stage unripe fruit receptacle was found (Fig. 8). These results also support the role of a cell-wall degrading enzyme for the strawberry pectate lyase enzymes and thus their functional involvement in strawberry fruit softening. In agreement with that, both in banana and strawberry, pectate lyase clones were among of the most abundant ripening-related sequences obtained from ripe fruit cDNA libraries (Medina-Escobar et al., 1997; Medina-Suarez et al., 1997).
The expression of three different strawberry pectate lyase genes during fruit ripening could indicate a common function, but a different cellular location for each gene. Alternatively, the product of each gene could act on different cell-wall domains. It has previously been shown that de-esterification of pectins and deposition of a polygalacturonase protein (PGII) occurs in block-like domains within the cell-wall (Steele et al., 1997). The boundaries of these domains are distinct and persistent, implying strict and spatial regulation of enzyme activities (Steele et al., 1997). A strong association between a PGII enzyme and the cell-wall involves binding to particular pectic polysaccharides (Steele et al., 1997). In this sense, it has been demonstrated that pectic polysaccharides fall into three classes (HGA, RGI and RGII) depending on polysaccharide composition (Carpita and Gibeaut 1993; Steele et al., 1997) and that pectins are degraded in block-like domains in tomato (McLauchlan and Brandy, 1994).
Nearly all previously identified ripening-related genes in strawberry are negatively regulated by auxins (Manning, 1994). Thus, as in the case of other strawberry ripening-related genes, the removal of strawberry fruit achenes produced a clear increase in gene expression for the three pectate lyases. The expression was partially reverted by the application of exogenous auxins (Fig. 4). Similar results have already been obtained for other ripening-related genes, including some encoding cell-wall hydrolases (Medina-Escobar et al., 1998; Manning, 1998; Harpster et al., 1998). These results indicate a negative relationship between fruit auxin content and ripening-related gene expression and supports the idea that, in strawberry, a declining of the auxin content with the ripening process activates the expression of a number of ripening-associated genes, which in turn initiate the process of fruit ripening (Manning, 1994, 1998).
Transgenic plants that incorporate an antisense sequence of strawberry pectate lyase gene (pel C) under the control of the 35S promoter present a significant decrease in fruit softening (Jiménez-Bermúdez et al., 2002). Though these antisense plants only contain the antisense cDNA corresponding to the pel C gene, a general reduction in the level of the corresponding three pel gene transcripts was observed. Therefore, these results strongly suggest a close relationship between pectate lyase genes and fruit softening.
It is noteworthy that carbon dioxide-enriched atmospheres used to reduce the incidence and severity of decay extends the post-harvest life of strawberries and increases fruit firmness. Thus, post-harvest treatment of the strawberry fruit with CA enriched in CO2 (20% v/v) produced a 20–30% increase in fruit firmness that was accompanied by a decrease in soluble pectins (Siriphanich, 1998). The decrease observed in the expression of pectate lyase genes in ripe red strawberry fruits stored in high CO2 CA agrees with the reported increases in fruit firmness in this condition and also suggests a close relationship between strawberry fruit firmness and pl gene expression. Similarly, it was shown that the storage of tomato fruit in high CO2 concentration CA atmosphere for 3 d decreased the polygalacturonase cell-wall hydrolase gene expression in fruit, supporting the notion that high CO2 levels reduce the expression of genes encoding cell-wall degrading enzymes (Rotham et al., 1997) as found in this case.
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Acknowledgement |
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This work was supported by grant BIO1998-0496-C02-02 (DGICYT, Spain) and grant FAIR CT97 3005 (UE). The equipment of Instituto Andaluz de Biotecnología, Andalucía, Spain is also acknowledged. The authors thank the collaboration of JM López Aranda and JJ Medina (CIDA ‘Churriana’, Málaga, Spain and Finca Experimental ‘El Cebollar’ JA, Huelva, Spain) for his help with strawberry field experiments. The technical assistance of JL Gónzalez and JM Villalba in the immunolocalization experiments is also acknowledged. The gene accession numbers are plA gene: AF339025; plB gene: AF339024.
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