Journal of Experimental Botany, Vol. 54, No. 389, pp. 1865-1877,
August 1, 2003
© 2003 Oxford University Press
Identification of a strawberry gene encoding a non-specific lipid transfer protein that responds to ABA, wounding and cold stress*
Received 12 December 2002; Accepted 7 May 2003
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Departamento de Bioquímica y Biología Molecular e Instituto Andaluz de Biotecnología. Campus Universitario de Rabanales, edificio Severo Ochoa (C-6). Universidad de Córdoba, 14071-Córdoba. Spain
* The gFxaltp nucleotide sequence (njjs46) will appear in the EMBL/GenBank nucleotide sequence databank under the accession numbers AJ315844 and FRX315844. To whom correspondence should be addressed. E-mail: bb1carej{at}uco.es
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cDNA and genomic clones encoding a strawberry (Fragariaxananassa cv. Chandler) non-specific lipid transfer protein (Fxaltp gene) were isolated and characterized. The spatio-temporal expression pattern and structural features of this gene were studied for the first time in strawberry, a non-climacteric fruit of agricultural importance. The architecture and the encoded amino acid sequence of this non-climacteric fruit ltp gene were similar to those of other plant LTPs previously reported, and presents the eight cysteine residues and other features characteristic of plant LTPs. In addition, the deduced protein posseses an N-terminal signal peptide and lacks the K/HDEL retention signal, indicating that the strawberry LTP protein would enter the secretory pathway. In situ studies have shown that the Fxaltp gene is expressed in the epidermal cell layer of the strawberry fruit receptacle and achenes, flowers, and within the cell layer surrounding the endosperm. These results suggest that this Fxaltp gene promoter could be used as an endogenous promoter for biotechnological purposes in strawberry. Computer analysis using the PLACE database predicted the presence of several putative cis-regulatory sequences in response to abscisic acid and cold or wounding stresses within the Fxaltp 5'-flanking region. Accordingly, the strawberry gene responds to ABA and SA, but not to salt and heat stresses. It is also reported that ltp gene expression in strawberry is stimulated by wounding and repressed by cold stresses.
Key words: ABA, gene regulation, LTPs, strawberry fruit, stress.
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Introduction |
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Lipid transfer proteins (LTPs) are small, basic proteins that have been purified from both monocotyledons and dicotyledons. These proteins were so named because of their ability to transfer phospholipids between membranes and to bind fatty acids in vitro and, based on that, a cytoplasmic role of the LTPs was first proposed (Kader et al., 1984). However, taking into account that they are synthesized as precursors with typical signal peptides as well as the existence of a small family of related genes for most of the plant species thus far analysed (Kader, 1997; Trevino and Ma, 1998), a wide range of other extracellular roles have been suggested for these proteins. Indeed, two families of plant non-specific LTP (nsLTP) have been described so far: the ns-LTP1 (9 kDa) and the ns-LTP2 (7 kDa). Both families present the characteristic eight cysteine residues whose position is highly conserved in all plant LTPs although their amino acid sequences share weak similarity (García-Garrido et al., 1998). They have been proposed to be involved in cutin biosynthesis or surface wax formation (Kader, 1997), pathogen-defence reactions (Garcia-Olmedo et al., 1995; Kader, 1997), embryogenesis and developmental stages (Kader, 1997; Soufleri et al., 1996), such as pollen adherence to the stigma during pollen elongation (Park et al., 2000) or the adaptations of plants to environmental changes (Dunn et al., 1998; Kader, 1997; Trevino and Ma, 1998). Despite the many studies on LTPs, no direct evidence has been demonstrated for most of their suggested functions and the in vivo role of these proteins remains unclear. Nevertheless, a clear and specific defensive role in in vivo enhancing tolerance to bacterial and fungal pathogens has been reported for non-specific lipid transfer proteins (Garcia-Olmedo et al., 1995; Molina et al., 1996) and, very recently, a putative biological function in the early recognition of intruders in plants and in systemic resistance signalling has been proposed (Blein et al., 2002; Buhot et al., 2001; Maldonado et al., 2002). As different patterns of expression have been described for the ltp genes it is possible that different gene family members account for the observed diversity in patterns of expression, each one perhaps performing a different function. Indeed, in some cases, these patterns of expression are specific for a cell layer (Aguirre and Smith, 1993), over exposed surfaces or in the vascular tissue (Fleming et al., 1992). A systematic study of all ltp genes within a plant species is of great importance and has already been suggested as it can provide a clear picture of their regulated pattern of expression in the plant (Kader, 1997; Sabala et al., 2000; Sohal et al., 1999).
The fact that LTPs seem to be externally associated with the cell wall (Kader, 1997) as well as the putative plant defensive role against pathogens described for some of them, makes this study and the characterization of different LTPs of major interest because of their biotechnological implications. Thus, plant tolerance to specific pathogens overexpressing a particular ltp gene could be increased or its promoter region might be used to express other related or unrelated genes of agronomical interest in a particular tissue or type of cells, in a controlled and programmed manner. An important goal of plant improvement through genetic engineering is to use novel genes to develop disease-resistant agricultural plants. So far, a common strategy used to enhance disease resistance in plants has been constitutively to express genes thought to be involved in the plant defence response (Alexander et al., 1993; Hain et al., 1993; Terras et al., 1995). Although this approach is well documented, the use of constitutive expression promoters like the CaMV-35S promoter can cause non-desired effects in the plant. Furthermore, the use of this kind of heterologous promoter has also been seriously discussed due to its putative environmental implications. Therefore, the alternative use of endogenous species-specific promoters might overcome all these disadvantages, having the additional benefit of being able to express genes of interest in specific tissues or developmental stages of the plant.
Strawberry is an important crop of agronomical interest worldwide. Apart from its commercial importance, this fruit presents quality components of great significance for the consumers: the content of L-ascorbic acid (vitamin C) can be the highest (depending on the cultivar) among fruits currently available in the market (Haffner and Vestrheim, 1997). Consequently, strawberry fruit may be an important source of vitamin C for health (Agius et al., 2003). However, pests and diseases can be responsible for important losses in strawberry production and research in strawberry has been increased in the past 10 years with the emphasis on developing alternatives to chemical treatments (Maas and Galletta, 1997). In this paper, the isolation and characterization of a gene encoding a non-specific lipid transfer protein from strawberry (Fxaltp gene) with the aim of using its promoter region for biotechnological purposes is reported. The 5'-flanking region of this gene is described, and the fruit developmental and spatio-temporal expression pattern of this gene is investigated. To date, no known studies have been reported for these sorts of genes in non-climacteric fruit of agronomical interest. The Fxaltp gene expression pattern under ABA and SA treatments, and salt and temperature stress conditions, is also reported.
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Materials and methods |
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Plant material
Strawberry fruits (Fragariaxananassa cv. Chandler, an octaploid cultivar) were 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 full ripe red fruits (R), immediately frozen in liquid nitrogen, and stored at –80 °C.
Fruit stress treatments
For in vitro treatments, the strawberry in vitro system previously described was essentially followed (Perkins-Veazie and Huber, 1992), except that asparagine (10 mM) and glutamine (10 mM) were used as nitrogen source, and KCl (1 mM) was incorporated in the nutrient solution. Briefly, after harvesting, fruit peduncles were trimmed to uniform length with a scalpel, and each fruit was immediately placed in an autoclaved container with a nutrient solution. The base of each peduncle was recut to remove 1–2 mm every day during all the experiments. After maintaining the fruits for one night in this nutrient solution at room temperature to acclimate them, the fruits were subjected to different treatments. For the salt-stress treatment, 170 mM NaCl was added to the same new nutrient solution and fruits were maintained in these conditions in the dark at 25 °C for 24 h. For the heat-shock treatment, fruits were maintained for 6 h to 37 °C in the dark in new nutrient solution, as previously described for other ltp genes in tomato (Torres-Schumann et al., 1992). For the cold-shock treatment, fruits were maintained for 4 d and 14 d at 4 °C in the dark, and in the same new nutrient solution or without nutrient solution. Control fruits were maintained at 25 °C in similar conditions and for the same period of time in all treatments. After treatments, all fruits were collected and immediately frozen at –80 °C in liquid nitrogen.
ABA, SA and wounding treatments
Strawberry fruits (green G2-stage) and leaves from field-growing plants were sprayed either with abscisic acid (0.1 mM ABA) or sodium salicylate (1 mM SA), at 6 h intervals for 5 d as described by Molina and Garcia-Olmedo (1993), and fruit and leaf samples were collected at 1 d and 5 d, and immediately frozen at –80 °C in liquid nitrogen.
The wounding treatment was performed as previously described (Sohal et al., 1999), and essentially is as follow: fruits and leaves from field-grown plants were pricked several times by introducing the tip of a scalpel blade inside these tissues and samples were collected at 1 d and 3 d, and immediately frozen at –80 °C in liquid nitrogen.
Cloning of the strawberry cDNA (Fxaltp clone) and the Fxaltp gene
A strawberry cDNA subtractive library (red stage versus green stage) was screened as previously described (Medina-Escobar et al., 1997b). Briefly, to generate the cDNA subtractive library a modified magnet-assisted subtraction technique was used. Tracer ds-cDNA (ds-cDNA synthesized from RNA isolated from the full-red stage of fruit ripening) was subject to two rounds of subtraction versus Driver ss-cDNA (ss-cDNA synthesized from RNA isolated from the G2-green stage of fruit ripening). The product of subtraction was ligated to commercial EcoRI/NotI adapters and PCR-amplified so that only a yield of ds-cDNA (red-stage) differentially expressed was obtained. The amplified product was EcoRI digested and ligated to 
ZapII/EcoRI arms to produce the cDNA subtractive library. The screening of the library combines the differential screening technique with a Southern blot screening by means of the polymerase chain reaction and using labelled cDNA from G2-stage and R-stage as probes (Medina-Escobar et al., 1997b). A 643 bp cDNA clone was isolated (njjs46 clone, renamed Fxaltp clone) and the cDNA insert was sequenced in both strands. The isolation of the strawberry Fxaltp genomic clone was performed by plaque hybridization screening of 1.5x105 pfu of a strawberry genomic library (Fragariaxananassa cv. Chandler), in the phage -FixII (Stratagene), and using the 32P-labelled Fxaltp cDNA as a probe. Filters were prehybridized and hybridized at 65 °C in hybridization solution: 5x SSC, 5x Denhardt’s, 200 µg ml–1 salmon sperm, and 0.5% SDS. After hybridization, filters were washed (twice) for 15 min at room temperature in 0.2x SSC, 0.5% SDS. Afterwards, the filters were washed for 15 min, at 65°C, in 0.2x SSC, 0.1% SDS. A positive clone of about 17 kb in size was isolated and the DNA insert was analysed by restriction mapping and Southern hybridization using the Fxaltp cDNA probe. Then, appropriate subclones for DNA sequencing were obtained by subcloning into pBluescript vectors and their DNA inserts were fully sequenced in both strands.
RNA isolation and northern analysis
Total RNA extraction and northern analysis at high stringency was performed as previously described for other strawberry genes (Medina-Escobar et al., 1997a), but using the Fxaltp cDNA as a probe. A cDNA corresponding to 18S ribosomal RNA was always used to control equal loading of RNA samples. The probe (
500 bp in length) was labelled to a specific activity of approximately 108 cpm µg–1 using a commercial random priming kit (Amersham Pharmacia-Biotech).
In situ hybridization
Probes were made from Fxaltp cDNA cloned in pBluescript SK (–) using T7 or T3 promoters to generate sense or antisense RNA. The methods used for digoxigenin labelling of RNA probes, tissue preparation, and in situ hybridization are as described by Jackson (1991) and Coen et al. (1990) with the following modifications: in the treatment of tissues prior to hybridization, samples were incubated with 3 µg ml–1 of proteinase K for 30 min, and 5 min with 0.2% glycine to block the protease.
Hybridization of the samples were performed at 50 °C for 12–14 h in hybridization solution (50% formamide, 6x SSC, 3% SDS, 100 µg ml–1 tRNA, 100 µg ml–1 Poly-A). Afterwards, the samples were washed with 2x SSC, 50% formamide, at 50 °C for 30 min, and twice with the same wash solution for 1 h 30 min. Then, the samples were rinsed twice for 5 min with NTE solution (10 mM TRIS-HCl, pH 7.5, 500 mM NaCl, 1 mM EDTA), at 37 °C, and incubated at 37 °C for 30 min, in a prewarmed NTE solution containing RNase A (20 µg ml–1). Samples were then rinsed twice for 5 min with NTE solution, and then washed twice for 20 min with 1x SSC and once for 1 h with 0.1x SSC, at room temperature. The hybridized probes were detected using an alkaline phosphatase antibody conjugate. After final colour development, slides were then dehydrated through an ethanol series, dried, mounted with Entellan (Merck) and viewed using brightfield microscopy.
DNA extraction and Southern blot analysis
Strawberry genomic DNA was extracted as previously described (Medina-Escobar et al., 1997a). Genomic DNA (5 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 (Amersham). DNA was fixed by UV light using the Stratalinker (Stratagene) and the blot was hybridized using the Fxaltp cDNA probe. Hybridization and washing conditions at high stringency were performed 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. DNA and amino acid sequences were analysed and compared with the GenBank (release 100 4/97) and EMBL Nucleic Acid database (release 50.0, 3/97) and the Pir (release 52.0, 3/97) and Swisprot database (release 34.0, 3/97), using programs from the GCG-Wisconsin package (version 9.0, December 1996, (Devereux et al., 1984). For alignment the Clustal Method from MegaAlign 4.05/00 (LaserGene Navigator 99) for MacOS from DNAStar (Madison, WI, USA) was used.
PLACE database (http://www.dna.affrc.go.jp/htdocs/PLACE/) (Higo et al., 1999) was used as a tool for homology searches of nucleotide sequence motifs from plant cis-acting regulatory DNA elements (cis-elements).
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Sequence comparison and structural features of the Fxaltp strawberry gene and protein
By differential screening of a strawberry subtractive cDNA library using labelled cDNA from the G2-stage and the R-stage as probes, a cDNA (Fxaltp clone) has been isolated that shows significant similarity with genes encoding low molecular weight lipid transfer proteins from higher plants. This cDNA clone was used as a radioactive probe to isolate the corresponding genomic gene. The genomic and cDNA nucleotide sequences together with the predicted amino acid sequence of the protein are shown in Fig. 1. The strawberry cDNA insert (Fxaltp cDNA) is 643 bp long and agrees with the estimated size of mRNA detected in northern hybridization experiments (see below), thus indicating that this cDNA is probably a full length cDNA. The start codon boundaries (ATGGCT) of the first of two putative start codons (nt positions 1699 and 1717), match the consensus sequence of initiation codons of higher plants perfectly (Gallie, 1993) and, taking into consideration the amino acid comparison studies with other LTP proteins (Fig. 2), it is proposed that this first ATG is the initiation codon for the strawberry predicted protein (FxaLTP). However, the second ATG must not be discarded. Therefore, a major open reading frame of 356 bp extends from position 1699 to 2170. The Fxaltp cDNA contains a 3'-untranslated region of 229 bp including the stop codon and 9 residues of the poly(A+) tail. The stop codon (TGAA) also matches the preferred one in plants (Gallie, 1993). No clear polyadenylation and cleavage sequence that matches the consensus motif (AATAAA) can be found in the 3'-UTR sequence, except the sequence on position 2227-2232 (Fig. 1). However, this putative polyadenylation signal might not be the correct one as the cleavage site for this gene can be deduced from the corresponding isolated cDNA sequence that is located between nucleotides 2389 and 2390 in the genomic sequence, far away (157 bp downstream, nucleotide position 2389) from the consensus position (10–15 bp downstream AATAAA) reported for this kind of signal. It is also followed by a GU-rich region motif (Gallie, 1993; Weaver, 1999). Therefore, another non-canonical plant polyadenylation signal must be located in closer proximity to this deduced cleavage site. Plant polyadenylation signals allow more variation in this region than mammalian ones (Weaver, 1999). The deduced translation product is 118 amino acids long with a predicted molecular mass of 12 800 Da and a pI of 8.9.
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Comparison of the gFxaltp nucleotide sequence with that of the corresponding Fxaltp cDNA revealed the presence of a 120 nt length intron within the Fxaltp gene (nt 2043 to 2162) (Fig. 1) that was located within the 3'-end of the coding region just two codons before the stop codon. In this sense, the strawberry ltp gene structure agrees with that of almost all of the Ltp1 genomic sequences so far characterized from other higher plants as they also have an intron placed in the region corresponding to the C-terminus of the protein, generally two codons before the stop codon (Kader, 1996). No intron has been found in Ltp2 genes (García-Garrido et al., 1998; Pelese-Siebenbourg et al., 1994; White et al., 1994). These results allow the proposal that the strawberry Fxaltp gene belong to type 1 of plant LTPs (nsLTP1).
Sequence analysis and comparison with data banks revealed high similarity and identity of this strawberry FxaLTP deduced protein with LTP1 proteins from other higher plants (Fig. 2). Thus, sequence similarities and identities ranged, respectively, from 56.88% and 49.54% (N. tabacum) to 72.65% and 66.66% (P. amigdalus), at the amino acid level. The strawberry FxaLTP lacks tryptophan and contains the eight cysteine residues strictly conserved in all LTPs described so far which are engaged in four disulphide bridges (Kader, 1997). The presence of a characteristic signal peptide at the amino terminal end of all LTPs is also found in the FxaLTP. The length of this FxaLTP signal peptide (26 residues) agrees both with the range (Kader, 1996; Soufleri et al., 1996) and with the hydrophobic predictions for this kind of region within the deduced protein (data not shown).
Possible regulatory elements in the 5'-flanking region of the strawberry Fxaltp gene
The 5'-flanking region of the strawberry Fxaltp gene has been computer analysed by searching for known cis regulatory sequences. Inspection of 1636 bp upstream of the putative transcription start site (+1) revealed the presence of several sequences that match cis regulatory elements described in other plant genes. No clear TATA box can be deduced from the Fxaltp promoter sequence, but a TA-rich sequence (TATTTAAT) close to the consensus TATAAT box is located at the –35 region (nt 1593). However, some putative abscisic acid (ABRE) cis elements (Busk and Pages, 1998) were located at positions 1452 (–184, sequence TACGTA), and 437, 464, and 707 (–1199, –1173, and –929; sequence GACGTC). An ACGT motif-like sequence (GTACGTG, complementary strand), shown to be a cis element that modulates the level of expression of some genes in rice seed (Washida et al., 1999) was found at position 1139 (–497). In addition, sequences that match the consensus E-box sequence (ACACNNG) found in the DC3 carrot lea class gene, and shown to be the consensus binding site for the plant bZIP-like factors DPBF-1 and 2 (Dc3 promoter binding factors 1 and 2) (Kim et al., 1997), were located at positions 495 (–1141, sequence ACACCAG), 755 (–880; sequence ACACACG, complementary lower strand), and 956 (–680; sequence TACACATG, complementary lower strand). Several low temperature responsive element (LTRE)-like sequences (Dunn et al., 1998; Jiang et al., 1996) were also found at nucleotides 623 (–1013; sequence GCCGAC, complementary lower strand), 1303 and 944 (–333 and –692; sequence CCGAC, complementary lower strand), and 1435 (–201, sequence CCGAAA). The LTRE sequence CCGAAA was required for the blt4.9 ltp gene from barley and other higher plant genes to respond to low temperatures (Dunn et al., 1998; Jiang et al., 1996).
Putative plant Myb- (MACCWAMC) and Myc-consensus (CACATG)-like sequences have also been identified within the 5'-flanking region of the strawberry Fxaltp gene at nucleotide positions 652 (–984; sequence CACCAAAC, complementary lower strand) and 871 (–765; sequence AACCAAAC, complementary lower strand), and 641, 955, and 1490 (–995, –682, and –146; sequence CACATG, complementary lower strand), respectively. MYB and MYC recognition sites have been identified in stress-responsive plant genes such as those in the abscisic acid (ABA)-mediated dehydration-responsive expression of the rd22 promoter gene from Arabidopsis (Abe et al., 1997).
Other sequences identical to the consensus motif of the wounding-responsive element (AACGTGT) described within the promoter region of the extA extensin gene from Brassica napus (Elliott and Shirsat, 1998) were also identified within the strawberry Fxaltp gene promoter region at nucleotide positions 414 (–1223) and 748 (–889).
Tissues and developmental gene expression by northern analysis
The spatial and temporal expression pattern of the Fxaltp gene has been studied. Thus, total RNA isolated from fruits (receptacle plus achenes) at different development stages and from roots, leaves, flowers, and stolons were analysed by northern hybridization (Fig. 3). A mRNA transcript of about 0.65 kb in size was observed during all the fruit development and ripening stages, and in all the tissues analysed except in the roots. Hybridization signals were observed after washing using high stringency conditions and 24 h of exposure to X-film, and a slight decrease in the expression of the gene was observed during the intermediate and red stages of the strawberry fruit development.
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Effects of physical and chemical treatments on Fxaltp gene expression
As putative ABA-responsive (ABRE)-like elements, low-temperature responsive (LTR)-like elements, and wounding-like elements were located within the promoter region of the strawberry Fxaltp gene (Fig. 1), the expression pattern of this gene has been studied in strawberry plants under different in vivo and in vitro treatments. Ltp gene expression has been described to be induced in plants in response to various environmental conditions including high and low temperatures (Kader, 1996; White et al., 1994), drought and salt stresses (Kader, 1996; Torres-Schumann et al., 1992; Trevino and Ma, 1998; White et al., 1994), and abscisic acid (ABA) treatments (Kader, 1996), etc.
As shown in Fig. 4A, Fxaltp transcripts in strawberry leaves were found to increase after 24 h of ABA treatment (0.1 mM) and this increase was preserved during the 5 d of this treatment. Similar increases in the expression of this gene were observed in leaves after 24 h of either SA (1 mM) or wounding treatments. However, this increased expression was reduced after 5 d of SA and wounding treatments to nearly comparable levels to those observed in the control leaves untreated (Fig. 4A). This suggests that ABA treatment must activate a different and more permanent mechanism of gene induction than SA and wounding treatments, producing a long-lasting effect on the Fxaltp gene expression.
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Strawberry fruits were also subjected to the same treatments as above. These results are shown in Fig. 4B. No comparable increases in gene expression for ABA were observed in fruits. Thus, contrary to the effect observed in leaves, the expression of the Fxaltp gene was reduced in fruits 24 h after ABA treatment and this reduction was maintained over the 5 d of this treatment. As in leaves, SA and wounding treatments also produced a different pattern of Fxaltp gene expression than ABA treatment, but these expression patterns were also different from the ones observed in leaves for the same two treatments. Therefore, a progressive and slight increase and accumulation of the transcript mRNA during the 5 d of SA and wounding treatments was observed in both cases.
These results clearly demonstrate that ABA treatment mediates different regulatory mechanisms for the Fxaltp gene expression than SA or wounding treatments in fruit.
Strawberry fruits were also subjected either to salt stress or high and low temperature treatments as described in the Materials and methods. Then, total RNA was extracted from treated fruits from W1, W2, T, and R-stages of fruit development, and subjected to northern analysis with the Fxaltp probe. The results are shown in Fig. 5. The Fxaltp transcript levels detected with this in vitro system were similar to the ones detected in vivo (data not shown). However, no significant changes in the mRNA transcript levels were detected after salt stress treatments in all the fruit stages analysed (Fig. 5A). The same results were obtained after high-temperature treatments (data not shown). By contrast, the level of Fxalpt transcripts was significatively lower than the controls in the red fruit stage after cold treatments, either in the presence or the absence of nutrient solution, indicating gene repression under these 4 °C stress conditions (Fig. 5B).
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Fruit in situ studies
The authors were interested in endogenous species-specific promoters as an alternative to the use of the CaMV 35S promoter to express particular genes in a precise location and specific tissues in transgenic strawberry plants (i.e. defence genes against pathogens, ripening-related genes etc.). Therefore, the in situ expression patterns of the Fxaltp gene have been studied in strawberry fruits and flowers as they are the primary tissue targets of infection by Botrytis, Colletotrichum spp. and other strawberry pathogenic organisms (Bristow et al., 1986; Smith and Black, 1987). Figure 6 shows that a strong hybridization signal can be detected within the epidermal cells of the fruit receptacle and achenes when green fruits and the antisense Fxaltp probe was used (Fig. 6B, C, D). Hybridization was also observed within the cell layer surrounding the endosperm (Fig. 6E). The same results were found when white fruit stages instead of green fruit stages were used (data not shown). When flower tissue was analysed, strong hybridization was also observed within the epidermal cell layer of flower envelopes and young achenes (Fig. 6G, H, I, J), corroborating the cell type and tissue specificity of the ltp gene in strawberry.
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Southern studies
Strawberry genomic DNA was digested with the restriction enzymes BamHI, BglII, EcoRI, and HindIII and analysed by Southern blot hybridization using the same Fxaltp cDNA probe previously used in northern blot studies. Hybridization and washing conditions were performed under high stringency according to the Materials and methods. Only one hybridization fragment above 8 kb was detected in the BamHI digestion, and only a fragment of size 1,6 kb was also detected in the EcoRI digestion (data not shown).
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Despite the large amount of information related to the LTPs, the attribution of an in vivo role applicable to all of them is extremely difficult. In fact, they are expressed in a complex pattern within an organism, and a thorough characterization of individual ltp genes and a complete overview of the gene family within a species is important and has already been suggested in order to provide the means to understand the in vivo role(s) of the LTPs (Kader, 1997; Soufleri et al., 1996). The isolation and characterization of a strawberry Fxaltp gene that showed significant similarity with genes encoding LTPs from other higher plants has been undertaken. Sequence analysis and comparisons with sequences from known LTPs, have revealed that the strawberry FxaLTP protein presents all the characteristics of a secreted and cell wall externally associated non-specific LTP protein. Accordingly, it lacks the ER retention signal (H/KDEL) (Munro and Pelham, 1987) and contains a hydrophobic amino terminal signal peptide (26 amino acid long) (Kader, 1997). The genomic structure of the strawberry Fxaltp gene is similar to that found in most plant type 1 ltp genes for which a genomic sequence is available (Kader, 1996, 1997; Trevino and Ma, 1998). Thus, although the length of the intron varies from one gene to another the size of the strawberry Fxaltp intron (120 nt) is in agreement and within the range of most of them (Kader, 1996; Trevino and Ma, 1998). These results allow the proposal that the strawberry Fxaltp gene belongs to the type 1 nsLTP family.
Computer analysis of 1635 bp of the 5'-flanking region of this Fxaltp gene using the PLACE database (Higo et al., 1999) identified several nucleotide sequence motifs that significantly matched known plant cis-regulatory DNA elements (cis-elements). The presence of an ACGT-like sequence within the 5'-flanking region of the strawberry Fxaltp gene, identical to that present in the rice GluB-1 gene (GTACGTG), suggests that this nucleotide sequence could be involved in the expression of the gene in the strawberry seeds (the achenes), since this sequence has recently been shown to be important for GluB-1 promoter specific activity in the endosperm of rice seed functioning in co-operation with other seed-specific motifs (i.e. ACAA and GCN4) (Washida et al., 1999). In fact, the in situ results have shown that a strong hybridization is detected in achenes of green fruits that is preferentially located both within the cell layer surrounding the endosperm and within the external epidermal cell layer of the achene, thus, supporting this prediction.
The core ACGT is also present in other identified regulatory cis-elements like ABRE, but no clear consensus for the sequence flanking this core has been described (Busk and Pages, 1998). ABRE elements have been described to be present in promoter regions of ltp genes that respond to ABA in tomato (Torres-Schumann et al., 1992; Trevino and Ma, 1998), barley (Molina and Garcia-Olmedo, 1993), and other plants (Kader, 1996, 1997). However, it is not known whether transcript induction by ABA occurs with all or only specific members of the LTP family and other factors have been suggested as being involved in regulating these genes (Busk and Pages, 1998; Kader, 1996; Trevino and Ma, 1998). Five ABRE-like sequences have been predicted by computer analyses in the strawberry Fxaltp promoter region and, therefore, ABA-responsiveness could be expected for this strawberry gene. Accordingly, the strawberry Fxaltp gene responds to ABA as shown in Fig. 4. Furthermore, the expression pattern of this gene during fruit development and ripening correlates well with the corresponding pattern of abscisic acid already described for strawberry fruit development (Perkins-Veazie et al., 1995). Thus, the expression of this gene starts to decline after the white-2 stages (Fig. 3), and maximum concentrations of ABA were found in achenes from strawberry red-ripe fruits (Perkins-Veazie et al., 1995). Indeed, this inverse correlation suggests that the Fxaltp gene might be negatively regulated by the increasing amount of ABA in fruit development. In fact, ABA negative regulation of this Fxaltp gene during fruit development is also supported by ABA treatment experiments in Fig. 4B. Thus, transcript levels in green fruit declining after ABA treatment to lower levels which are similar to those found in control red-ripe fruits (see lane 10, control red-ripe fruit in Figs 3, 4B). Accordingly, the addition of exogenous ABA to green fruits has the effect of increasing the steady-state levels of ABA already present in the green fruits probably to the levels found in red fruit stages and, consequently, the Fxaltp gene is repressed.
The strawberry Fxaltp ABA-responsiveness is tissue dependent and this gene is repressed in green fruits but it is induced in leaves, strongly supporting either independent regulatory mechanisms for the same gene in these two tissues or the presence of two similar genes with different regulation. Examples where two similar ABA-responsive cis elements within the same 5'-flanking region function differently have been described (Abe et al., 1997). This is the case for two MYC recognition sites, one functioning as a positive element and the other as a negatively repressing element. Co-operation of ABA-induced MYC and MYB transacting factors seem to be involved in this response (Abe et al., 1997). In this sense it is noteworthy that MYC and MYB-like sequences have also been predicted within the promoter region of the strawberry Fxaltp gene.
Although several studies report regulation by biotic and abiotic external stimuli of ltp gene expression, little or no information on the effects of several important environmental factors like wounding, cold stress, etc. on ltp gene expression has so far been reported (Sohal et al., 1999). Two wounding responsive-like sequences (Elliott and Shirsat, 1998) have been predicted and are located within the Fxaltp promoter region. Therefore, the Fxaltp gene expression has been studied under wounding treatment. Consequently, the strawberry Fxaltp gene has been shown by northern analysis to respond to damaging produced by wounding (Fig. 4). Damaging by wounding has been shown to have no effect in other LTPs (Sohal et al., 1999) although very recently a rice ltp1 gene was shown to be induced by wounding and pathogen attack (see note at the end of Discussion). To date, no previous data in the literature regarding the wounding-responsiveness of ltp gene expression in non-climacteric fruit have been found. In addition, the observed Fxaltp gene expression pattern found after wounding treatment in strawberry was different from that found under ABA treatment where the gene is expressed in leaves but repressed in fruits. This was also the case for the SA treatment. However, as in ABA treatment, the expression pattern of both wounding and SA treatments was also different in leaves and fruits. These results suggest the independent regulation of the strawberry Fxaltp gene by ABA, SA and wounding treatments both in leaves and fruits. However, there is the possibility that SA does not penetrate fruit tissues as easily as leaf tissues, and therefore, the increase observed in fruit gene expression during the 5 d of SA treatment may be due to the low levels of SA reached inside the fruit cells earlier during treatment. Nonetheless, this last possibility does not apply to wounding treatment and a similar increase in transcripts was observed in fruits under this treatment with a different expression pattern being detected in leaves, thus reinforcing the idea that different regulatory mechanisms must act in both tissues. Alternatively, different ltp genes can be expressed in leaves and fruits in response to these treatments.
Since four putative low-temperature-responsive-ele ment (LTRE)-like sequences have also been detected within the Fxaltp 5'-flanking region, a cold-responsiveness of this gene was also predicted. The experiments in Fig. 5B have shown a clear decrease of the Fxaltp transcripts when strawberry red fruits were maintained for 4 d and 14 d at 4 °C, both with and without nutrient solution. However, no decline was seen in the level of transcripts in control red fruits maintained at 25 °C for 14 d, thus indicating gene repression provoked by cold stress. Although these results demonstrate that the strawberry Fxaltp gene can respond to cold stress, they also show that this response is opposite to the expected one as the expression of a number of low-temperature-responsive genes, including ltp genes from barley, containing the pentanucleotide CCGAC and closely related sequences have been reported to increase in response to low-temperature treatments (Hughes and Dunn, 1996; Molina et al., 1996; White et al., 1994). Nevertheless, no increase in expression has also been reported for a ltp gene promoter from Brassica in response to cold (Sohal et al., 1999). These results illustrate the differences between species or even between ltp genes in the low-temperature regulation of ltp expression. As far as is known, no repression in response to cold stress has so far been reported for an ltp gene.
In this paper, putative cis elements within the Fxaltp promoter region that match consensus sequences of ABREs, wounding-, and cold-responsive elements already described in other higher plants have been located by computer analysis. Accordingly, it has been shown that the Fxaltp gene responds to these treatments in a complex and different way, thus strongly suggesting that some of these or other cis elements could act regulating the expression of this strawberry gene. A further analysis, using expression in both transient assays and transgenic strawberry plants of 5'-promoter truncations linked to a GUS reporter gene to identified controlling cis acting regions, should provide insights into the regulation of this Fxaltp gene and is currently in progress.
Lipid transfer protein genes are expressed in a range of tissues within a plant (Kader, 1997; Molina and Garcia-Olmedo, 1993; Molina et al., 1997; Park et al., 2000; Pyee et al., 1994; Sohal et al., 1999), for example, in rape, two different ltp genes have been shown to be expressed differentially, one in the entire cotyledon and another in the seed tapetal cell layer (Kader, 1997). The in situ hybridization analyses described here have shown that the strawberry Fxaltp gene is expressed in the epidermal cell layers of flowers, fruit receptacles and achenes, and also in the cell layer surrounding the endosperm in achenes. Although no direct demonstration of the role of this strawberry Fxaltp gene can be deduced from these studies and clear evidence of extracellular lipid transfer activity is lacking for most of the LTPs so far described (Kader, 1997; Sohal et al., 1999), these results are consistent with the postulated roles of some LTPs both in defence and external wax deposition (Kader, 1996, 1997) (Molina and Garcia-Olmedo, 1997). Furthermore, these results open the possibility of exploiting the promoter of this Fxaltp gene for biotechnological purposes in strawberry. These strawberry tissues are one of the primary infection sites of strawberry pathogens such as Colletotrichum and Botrytis (Denoyes and Baudry, 1995; Grove et al., 1985) and the expression of particular defensive genes in the strawberry fruit epidermal cell layer using specific endogenous promoters such as the Fxaltp promoter, is of potential interest and is in progress.
The possibility that the observed differences in the pattern of expression of this gene in fruit and leaves are due to the presence of two or more members of the same LTP family in strawberry is plausible and has to be contemplated. The entire Fxaltp cDNA insert has been used as a radioactive probe to detect the spatial and temporal expression of this gene in strawberry, but very rarely have gene-specific probes been used to monitor differential patterns of expression of ltps (Molina et al., 1996; Trevino and Ma, 1998). However, with the high stringency conditions used during the hybridization and washing steps in these experiments (65 °C and 0.2% SSC, 0.1% SDS washing solutions) only sequences with homologies higher than 90.64% are expected to appear on the filters (Casey and Davidson, 1977) and, therefore, to be due mostly to the expression of the Fxaltp gene. Only one ltp gene was detected in carrot and spinach using high stringency hybridization conditions (Kader, 1996). Never theless, positive signals due to cross-hybridization with mRNAs corresponding to gene members from the same LTP family with a high degree of identity must not be discarded. However, if this is the case, both genes must code for similar transcripts in size, as only one transcript has been detected in all northern experiments so far carried out. Furthermore, either no expression of genes from this gene family except the Fxaltp gene is taking place in strawberry fruit during the G2-green stages of elongation or these genes are equally repressed by ABA. This can be deduced from the experiment in Fig. 4, where ltp transcripts are reduced and do not accumulate in fruit at the G2-green stage after 1 d and 5 d of ABA treatment, showing that transcript levels were lower than the characteristic basal ones normally detected at the G2-stage in untreated fruits (Fig. 4, lanes 1, 2, 3, 6).
Note: During the process of reviewing this manuscript another article has been published elsewhere showing that a rice ltp1 gene was induced by wounding and pathogen attack (Guiderdoni et al., 2002).
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Acknowledgements |
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This work was supported by grant FEDER IFD97-0843-CO5-03 (DGESIC, Spain), grant BIO1998-0496-C02-02 (DGICYT, Spain) and Junta de Andalucía (Grupo 3249). The equipment of Instituto Andaluz de Biotecnología, Andalucía, Spain, is also acknowledged. E-M Yubero-Serrano thanks the Junta de Andalucía, Spain, for a predoctoral fellowship. The authors also thank the collaboration of JM López Aranda and JJ Medina (CIDA ‘Churriana’, Málaga, Spain, and Finca Experimental ‘El Cebollar’, Huelva, Spain) for their help in strawberry field cultivation.
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