JXB Advance Access originally published online on April 25, 2008
Journal of Experimental Botany 2008 59(8):2191-2203; doi:10.1093/jxb/ern088
RESEARCH PAPER |
The organ-dependent abundance of a Solanum lipid transfer protein is up-regulated upon osmotic constraints and associated with cold acclimation ability
bowicz-Matuk1
1Institute of Plant Genetics, Polish Academy of Sciences, Strzeszy
ska 34, 60-479 Pozna, Poland
2 CEA, IBEB, SBVME, LEMP, Laboratoire d'Ecophysiologie Moléculaire des Plantes, UMR 6191 CNRS-CEA-Université de la Méditerranée, 13108 Saint-Paul-lez-Durance, Cedex, France
* To whom correspondence should be addressed. E-mail: tror{at}igr.poznan.pl
Received 12 December 2007; Revised 19 February 2008 Accepted 3 March 2008
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Abstract |
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The expression of a gene isolated from cDNA differential screening and encoding a lipid transfer protein, designated as SsLTP1, was analysed at the protein level in two groups of Solanum species and lines differing in cold acclimation capacity. Under control conditions, the SsLTP1 was localized in all aerial organs of S. sogarandinum and S. tuberosum plants. Western analysis of subcellular extracts indicated that the protein possesses an intracellular localization. The protein abundance was found to vary as a function of organ type, the highest levels being observed in flowers, stems, and young leaves. During low temperature treatment, no change in protein level was noticed in either the S. tuberosum cv. Irga, which displays a low capacity for cold acclimation, or in a S. sogarandinum line which has lost its cold acclimation capacity. By contrast, low temperature induced a noticeable increase in SsLTP1 level in stems and leaves of S. sogarandinum and S. tuberosum cv. Ursus plants, which are able to acclimate to cold, indicating that SsLTP1 could participate in the processes leading to freezing tolerance. In other respects, SsLTP1 accumulation was observed both in cold-acclimating and in non-acclimating Solanum species when subjected to water deficit or to salt treatment. These data indicate that SsLTP1 gene expression is regulated in an organ-dependent manner and through distinct pathways under non-freezing low temperature and during osmotic treatments.
Key words: Cold acclimation, gene expression, lipid transfer protein, Solanum tuberosum, Solanum sogarandinum
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Introduction |
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Low temperature is one of the major abiotic factors that impairs plant distribution and affects agricultural productivity (Bray et al., 2000; Mittler, 2006). Many plants from temperate regions have the ability to sense low non-freezing temperatures and to activate mechanisms leading to an increase in freezing tolerance (Thomashow, 2001; Heino and Palva, 2003). This adaptive process known as cold acclimation is associated with numerous biochemical alterations including changes in membrane lipid composition (Örvar et al., 2000), calcium movements (Knight, 2000), accumulation of compatible osmolytes (Murelli et al., 1995), and changes in cell wall properties (Rajashaker and Lafta, 1996). These changes are accomplished through the expression of a number of cold-regulated genes classified into two groups (Shinozaki and Yamaguchi-Shinozaki, 2000). The first group includes genes encoding proteins involved in signal transduction pathways or in regulation of gene expression (Seki et al., 2003). The second group codes for proteins functioning in stress tolerance such as water channels, detoxification enzymes, antifreeze proteins, chaperones, and enzymes required for biosynthesis of osmolytes (Cushman and Bohnert, 2000).
Lipid transfer proteins, designated as non-specific lipid transfer proteins (ns-LTPs), are a group of ubiquitous small proteins able to bind several classes of lipids in vitro (Kader, 1996). Ns-LTPs contain an N-terminal signal peptide, indicating that they follow the secretory pathway (Schultz et al., 2000; Boutrot et al., 2005). They are thought to facilitate the transfer of lipids (Kader, 1996; Cheng et al., 2004). Nevertheless their function in vivo remains very elusive. Two main groups of ns-LTPs, ns-LTP1 and ns-LTP2, have been identified, with molecular masses of about 9 kDa and 7 kDa, respectively (Kader, 1996). Genes encoding plant ns-LTPs constitute redundant families (Arondel et al., 2000; Horvath et al., 2002; Pasquato et al., 2006). For example, 15 ns-LTP genes are present in Arabidopsis (Arondel et al., 2000). Lipid transfer proteins have been purified and characterized from several monocotyledon and dicotyledon species (Charvolin et al., 1999; Da Silva et al., 2005). Ns-LTPs share a basic pI and possess a hydrophobic cavity surrounded by four helices connected through disulphide bonds, which are partly covered by a long C-terminal arm with several turns (Charvolin et al., 1999). The hydrophobic cavity is likely to constitute an important feature for their biological function since it is able to bind diverse types of lipid molecules such as fatty acids (Han et al., 2001; Cheng et al., 2004) and phosphatidylglycerol (Sodano et al., 1997). Further, isothermal titration calorimetry experiments indicated that barley ns-LTP1 binds two 
-hydroxypalmitic acids, a compound found in cutin monomers (Douliez et al., 2001), suggesting the involvement of the protein in the formation of cutin layers. In other respects, the antimicrobial properties of ns-LTP1 observed in vitro indicate a possible role in the defence of plants against bacterial pathogens (Garcia-Olmedo et al., 1995). Accordingly, wheat ns-LTP1 can compete with elicitin for a receptor in tobacco plasma membranes (Buhot et al., 2001; Blein et al., 2002). Ns-LTPs could also be involved in numerous other processes such as somatic embryogenesis (Thoma et al., 1994), development (Edqvist and Farbos, 2002; Eklund and Edqvist, 2003), signalling pathways (Liu et al., 2001; Wang et al., 2005), and plant responses to environmental stress (Jung et al., 2003, 2005).
LTP family members show distinct expression patterns depending on developmental stage and on tissue type. Ns-LTP transcripts are detected in the epidermal tissue of aerial organs in the early stages of plant development (Kader, 1997). Ns-LTP expression has also been observed in young tissues of vascular zones in broccoli (Pyee et al., 1994), in phloem regions of stems, stolons, and tubers in potato (Horwath et al., 2002), and in shoot apex and floral meristems in tobacco (Canevascini et al., 1996). Ns-LTP1 transcripts are induced in response to low temperature (Pearce et al., 1998; Gaudet et al., 2003; Yubero-Serrano et al., 2003), drought and salt treatments (Torres-Schumann et al., 1992; Treviño and O'Connell, 1998; Wu et al., 2004; Jung et al., 2005), heat shock (Wu et al., 2004), or heavy metals (Gorjanovi
et al., 2004). Some ns-LTPs genes are induced by abscisic acid and salicylic acid, hormones mediating plant responses to abiotic stress (Garcia-Garrido et al., 1998; Yubero-Serrano et al., 2003; Wu et al., 2004). Recently, Jung et al. (2005) reported that Arabidopsis plants overexpressing a pepper ns-LTP1 displayed an increased tolerance to biotic stress in vivo and to osmotic treatments in vitro. These data suggest that ns-LTP1s participate in plant adaptation to variations in environmental conditions.
In the present study, the abundance of an ns-LTP belonging to group 1 (SsLTP1) was thoroughly analysed in four Solanum lines and species differing in their ability to acclimatize to cold. It is reported that under control conditions SsLTP1 abundance varies greatly as a function of organ type. Upon cold treatment, a noticeable SsLTP1 accumulation was observed only in plants able to acclimate to low temperature. Under water deficit or exposure to high salinity, an increased SsLTP1 abundance was observed both in cold-acclimating and in non-acclimating Solanum species, providing evidence that distinct signals mediate the SsLTP1 gene expression under low temperature and during osmotic treatments.
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Materials and methods |
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Plant material, growth conditions, and environmental constraints
Solanum sogarandinum (ochoa), PI 230510 plants, lines 1 and 2 and Solanum tuberosum L. cv. Irga and Ursus plants were propagated in vitro on solid MS medium at 20 °C/15 °C (day/night) under a PPFD of 150 µmol m–2 s–2 and a 14 h photoperiod as previously described (Rorat and Irzykowski, 1996), and in vivo in a phytotron under a PPFD of 200 µmol m–2 s–2 at 21 °C/18 °C (day/night) and a 12 h photoperiod for 2–3 weeks (Rorat et al., 2004). For cold treatment, in vitro and phytotron-grown plants were exposed for 0 min, 5 min, 20 min, 2 h, 4 h, 8 h, 1 d, and 3 d at 4 °C/3 °C (day/night) under the same PPFDs. A gradually increasing water deficit was applied on phytotron-grown plants by withholding watering for
9 d. Leaf relative water content (RWC) was determined on leaves as described by Pruvot et al. (1996). For salt treatment, 2-week-old phytotron-grown plants were watered with a nutritive solution containing 0.15 M NaCl for 8 h, 1 d, and 3 d. For ABA treatment in vitro plantlets were grown on MS medium supplemented with ABA (75 µM). Two independent experiments were performed for each environmental constraint and all cultivars and lines.
Assessment of freezing tolerance
The freezing tolerance of 2-week-old S. tuberosum cv. Ursus plants, grown in a phytotron at 21 °C/18 °C (day/night) and exposed to cold treatment at 4 °C (day/night) for 7 d, was determined by a conductivity method as described earlier (Rorat et al., 2006). Briefly, detached well-expanded leaves were placed into borosilicate glass tubes containing ultrapure water and cooled for 30 min in a circulating-cooling thermostat at –2 °C. Afterwards, crushed ice was added to the tubes to start tissue ice formation. Subsequently the temperature was lowered by 2 °C h–1 to –11 °C. Every hour, leaves were removed and slowly thawed overnight on ice. After the treatment and subsequent freezing to –70 °C, the leakage conductivity was measured. Freezing tolerance was expressed as the temperature resulting in 50% ion leakage (killing temperature, LT50). Three samples were used for each treatment at different temperature points. The value of LT50 was statistically analysed using the ‘Statgraphic plus’ program.
Isolation of total cellular RNA
Total cellular RNA was prepared from control (20 °C) and cold-hardened plantlets of both Solanum species at 4 °C for 0, 5, or 20 min and 1, 2, 4, or 8 h by the guanidine isothiocyanate method as previously described (Rorat et al., 1997). For RNA isolation, the samples collected at the time points indicated above were combined.
Construction of a cDNA library and isolation of clones corresponding to cold-induced transcripts
A cDNA library was constructed with poly(A)+ mRNA prepared using the Uni-ZAP-cDNA synthesis kit (Stratagene, La Jolla, CA, USA) and cold-induced cDNAs were screened through two steps of differential screening as described previously (Kiebowicz-Matuk et al., 2007).
DNA sequencing, computer analysis, and alignment
DNA sequencing was performed using the dideoxynucleotide chain-termination method, the BigDyeTM and dRhodamine terminator cycle DNA sequencing kits (AbiPrism), and the ABIPRISM 310 genetic analyser. Partial nucleotide sequences of the 5' and 3' termini of the cDNA clones were identified by the NCBI BLAST program (http:/www.ncbi.nlm.nih.gov/BLAST/; Altschul et al., 1997). A cDNA clone homologous to genes encoding lipid transfer protein, designated as SsLTP1, was sequenced completely. The theoretical isoelectric point (pI) and the predicted molecular mass of the SsLTP1 were calculated using the DNAStart program. The signal peptide and putative cleavage site were predicted using the SignalP program (http://www.cbs.dtu.dk/services/SignalP; Nielsen et al., 1997). Amino acid sequence alignment was generated using the ClustalW program, version 1.81 (Thompson et al., 1994).
Northern blot hybridization
Total cellular RNA extracted from in vitro plantlets of both Solanum species was fractionated on a 1.2% (w/v) denaturing formaldehyde agarose gel and transferred onto a hybridization membrane (GeneScreen, DuPont, NEN) in 20x SSC. RNA samples (15 µg) were loaded onto the gel. Equal loading of RNA in each lane was verified using ethidium bromide staining. After transfer, RNA was fixed to the membrane by baking at 80 °C for 2 h. [32P]dCTP-labelled SsLTP1 cDNA of 621 bp was used as a probe for northern blot hybridization. Pre-hybridization, hybridization, and washing were performed as described by Rorat et al. (1998).
Preparation of protein extracts
Soluble and membrane proteins were prepared from in vitro grown plantlets or from organs of plants grown in a phytotron (in vivo). Soluble proteins were extracted in a buffer containing 10 mM Na2HPO4, 15 mM NaH2PO4, 100 mM KCl, and 4 mM EDTA, pH 5.9, as described by Carvalho et al. (2001). Membrane proteins were extracted in a buffer containing 50 mM TRIS–HCl, pH 8.0, 2% SDS, 0.1 M β-mercaptoethanol, and 1 mM PMSF (Gillet et al., 1998). Apoplastic proteins were prepared from young and well-expanded leaves of phytotron-grown plants in 20 mM ascorbic acid and 20 mM calcium chloride, pH 3.0, for 30 min by vacuum infiltration followed by centrifugation at 800 g for 30 min as described by Hon et al. (1994). Protein content was determined using a modified Lowry method (Pierce).
Generation of an antiserum raised against SsLTP1 and western blot analysis
Proteins (40–80 µg) were separated using SDS–PAGE containing 10% or 15% acrylamide and electroblotted onto a 0.22 µm polyvinyl membrane (Hybond-PVDF, Amersham) using a semidry blotting apparatus (Pal Gelman Laboratory, Saint Germain en Laye, France). Membranes were stained with Ponceau red to ensure that equal protein amounts were transferred in each lane. A 273 bp fragment of the coding region of the SsLTP1 cDNA from nucleotides 112 to 384, encoding a 90 residue polypeptide, was cloned in the pQE-30Xa expression vector (Qiagen) to produce a recombinant protein fused to a 6x His-tag in the M15[pRep4] Escherichia coli strain. The recombinant protein, containing an N-terminal 6x His-tag, was purified on an Ni-NTA resin (Qiagen) and used to raise antibodies in a rabbit. Western blot analysis was performed using the serum raised against SsLTP1 diluted 1:1000. Sera raised against cytochrome b559 (Cyt b559), UDP-glucose pyrophosphorylase, and ascorbate oxidase were purchased from Agrisera (Vännäs, Sweden) and used diluted 1:10 000, 1:1000, and 1:2000, respectively. Bound antibodies were detected using an anti-rabbit immunoglobulin G (horseradish peroxidase conjugate) diluted 1:10 000 (Amersham) and a chemiluminescent substrate (Isacsson and Wettermark, 1974). Autoradiography was performed by exposing PVDF membranes to X-ray film. Band intensities (relative) were analysed by computerized densitometry using the Image Master program (Amersham-Pharmacia). Western blot experiments were carried out using plant protein extracts originating from two independent stress experiments and were replicated at least once for each experiment and each cultivar or species.
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Sequence analysis of the SsLTP1
cDNA clones corresponding to cold-induced mRNAs were screened from the S. sogarandinum, line 2 cDNA library by two steps of differential screening. The isolated clones were designated Sslti (Solanum sogarandinum low temperature induced) (Kie
bowicz-Matuk et al., 2007). One clone of 621 nt designated as SsLTP1 displayed high homology with genes encoding plant lipid transfer proteins (ns-LTPs). The SsLTP1 clone displays an open reading frame of 345 nt encoding a basic protein of 114 amino acids with a calculated molecular weight of 11.65 kDa and a pI of 8.0 (Fig. 1). Analysis of the SsLTP1 sequence using the SignalP program revealed a putative cleavage site for a signal peptide located between residues 24 (Ala) and 25 (Leu). The first N-terminal 24 residues comprise a hydrophobic region closely related to typical signal peptides for the secretory pathway (Thoma et al., 1993; Schultz et al., 2000). The calculated molecular mass of mature SsLTP1 is 9.07 kDa. Mature SsLTP1 contains eight cysteine residues (Fig. 1, lower denotation) highly conserved in ns-LTP1s from other plant species (Charvolin et al., 1999; Douliez et al., 2000; Samuel et al., 2002). The conserved cysteines form four disulphide bonds, which stabilize ns-LTP1 folding, and are essential for their function (Samuel et al., 2002). As in other ns-LTP1s, mature SsLTP1 contains four putative 
-helices, a threonine (Thr48) separating two cysteines in the -CXC- motif, and well-conserved hydrophobic or positively charged residues (Fig. 1). Altogether, these sequence features unambiguously classify SsLTP1 into the ns-LTP1 family.
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The database search revealed that the SsLTP1 sequence shares the highest homology with ns-LTP1s from other Solanaceae species such as Lycopersicon pennellii LpLTP2 and LpLTP1 (93% and 83% identity, respectively) (Fig. 1). A much lower homology was scored with ns-LTP1s from more distant species, for example Hordeum vulgare (58% identity) or Arabidopsis thaliana (52% identity). It is noteworthy that genes encoding ns-LTP1s constitute multigenic families, as reported in Arabidopsis (Arondel et al., 2000), and display some divergence in their sequence as shown for the two ns-LTP1 genes described in L. pennellii (Fig. 1).
As shown in Fig. 1, SsLTP1 shares a low homology (<25% identity) with proteins belonging to the ns-LTP2 family, mainly limited to the eight conserved Cys residues and to some important hydrophobic or positively charged amino acids. Instead of a hydrophilic residue, ns-LTP2s possess a hydrophobic residue in the -CXC- motif (Fig. 1).
SsLTP1 subcellular localization and organ distribution
To produce a serum against SsLTP1, a 272 bp fragment of the SsLTP1 cDNA from nucleotide 112 to nucleotide 384, encoding mature SsLTP1, was used to synthesize a His-tag recombinant protein in E. coli. The serum raised against the recombinant protein revealed in bacterial cells a band 12 kDa in size corresponding to that of the His-tag-LTP1 protein, 11.85 kDa (Fig. 2A, lane 1), and a single protein band of 9.0 kDa in protein extracts from S. sogarandinum plantlets (Fig. 2A, lane 2). The higher mass of the E. coli protein originates from the presence of the His-tag and of 21 additional amino acids encoded by the pQE-30Xa expression vector. The upper protein band of 24 kDa detected by the immune serum in E. coli extracts (Fig. 2A, lane 1) might correspond to an LTP dimer formed in bacteria through a disulphide bond as observed for other recombinant proteins (Vieira Dos Santos et al., 2005). Note also that ns-LTPs can form dimers in plant tissues (Gorjanowi et al., 2005). The size of the protein, 9 kDa, specifically recognized in extracts from S. sogarandinum plants is in agreement with that calculated for predicted mature SsLTP1. The pre-immune serum detected neither the His-tag recombinant SsLTP1 protein, nor any protein in plant extracts (Fig. 2A, lanes 3, 4). Based on these data, it was concluded that the band recognized by the serum in plant extracts corresponds to SsLTP1.
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All known plant LTPs are synthesized with N-terminal extensions, indicating that they follow the secretory pathway in agreement with their location in extracellular space, i.e. the apoplast (Federico et al., 2005) or in vacuolar structures (Kader, 1996). As SsLTP1 possesses a putative 24 residue signal peptide, the presence of SsLTP1 in apoplast, soluble and membrane protein fractions prepared from leaves of control and cold-hardened phytotron-grown S. sogarandinum plants was investigated using western analysis. To ensure the purity of the prepared fractions, three sera were used raised against: (i) an apoplastic enzyme, ascorbate oxidase, as a positive marker of the apoplastic fraction (Pignocchi and Foyer, 2003; Pignocchi et al., 2006); (ii) a cytosolic enzyme, UDP-glucose pyrophosphorylase, as a marker of the soluble fraction (Martz et al., 2002); and (iii) Cyt b559, as a marker of the membrane fraction (Haley and Bogorad, 1989). When performing western analysis of S. sogarandinum subcellular fractions, it was observed that the serum raised against ascorbate oxidase revealed only one band, at the expected size, 45 kDa, in the apoplastic fraction and no protein in the two other fractions. Similarly, single protein bands at 51 kDa and 9 kDa, corresponding to UDP-glucose pyrophosphorylase and Cyt b559, respectively, were specifically revealed in the soluble and membrane fractions (Fig. 2B). With regard to SsLTP1, western blot analysis showed that the protein was detected only in the soluble fraction (Fig. 2B). It was also noticed that the SsLTP1 abundance was higher in cold-hardened plants than in control plants. Together these data show that SsLTP1 is not localized in the apoplast and displays an intracellular localization, which is likely to be in the vacuole as reported for other plant LTPs (Kader, 1996).
The SsLTP1 abundance was then investigated in soluble proteins from different organs: young leaves (including very young developing leaves and terminal shoots), well-expanded leaves, old leaves, stems, open flowers, and roots of 2-month-old S. sogarandinum plants at the flowering stage grown in the phytotron. As shown in Fig. 2C, a substantial SsLTP1 abundance was observed in extracts from open flowers, stems, and young leaves, the highest level being noticed in flowers. The protein amount was much lower in well-expanded and old leaves, and the protein was not detected in roots (Fig. 2C). These data show that SsLTP1 abundance is differentially regulated as a function of organ type and developmental stage.
Expression of the SsLTP1 gene in response to low temperature
The expression of the SsLTP1 gene was analysed in two S. sogarandinum lines, 1 and 2, differing in cold tolerance. As previously shown (Rorat et al., 2006), S. sogarandinum line 2 is able to acclimatize to cold whereas line 1 lost its cold acclimation capacity after a few years of in vitro culturing. First, SsLTP1 gene expression was analysed in in vitro grown plantlets using northern blot. A noticeable increase in SsLTP1 mRNA level had already been observed after 4 h of cold treatment in the cold-acclimating S. sogarandinum line 2 plants (Fig. 3A). No visible change in transcript level was observed in S. sogarandinum line 1 (Fig. 3A). In the cold-acclimating S. sogarandinum line 2, a noticeable increase in SsLTP1 abundance was observed after 8 h of low-temperature treatment (Fig. 3B). The SsLTP1 level was 3-fold higher after 8 h of low-temperature treatment compared with that observed in control plants. By sharp contrast, no change in SsLTP1 amount was observed during cold-condition treatment in the non-acclimating S. sogarandinum line 1 (Fig. 3B). These data show that the SsLTP1 gene is induced at both transcript and protein levels upon cold treatment only in the cold-acclimating S. sogarandinum line 2.
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SsLTP1 abundance in various organs during low-temperature treatment
Then, SsLTP1 abundance was investigated in various organs (young leaves, well-expanded leaves, i.e. the most expanded leaves, old leaves, and stems) of 2-week-old phytotron-grown plants subjected to low temperature. In addition to the two S. sogarandinum lines previously described, the present studies were extended using two S. tuberosum cultivars, Irga and Ursus, differing in their cold tolerance. The cv. Irga displays a very low capacity to acclimatize to low temperature (Rorat et al., 2006). The freezing tolerance of cv. Ursus plants grown for 2 weeks in a phytotron at 21 °C/18 °C (day/night) and then exposed to a low temperature treatment, 4 °C/3 °C (day/night) for 7 d (hardening period) was determined. Plants from this cultivar displayed a significantly higher tolerance during cold treatment [LT50 ranging from –5.35 (±0.81) to –7.75 °C (±1.05)] in comparison with that of Irga plants [LT50 ranging from –2.6 (±0.42) to –3.6 °C (±1.05)] (Rorat et al., 2006). These data indicate that cv. Ursus is capable of acclimatizing to cold.
Under control conditions, the highest protein abundances were observed in young leaves and in stems of all S. sogarandinum and S. tuberosum plants. As shown in Fig. 4A, B (upper parts for each organ), a noticeable increase in SsLTP1 abundance was observed, during low-temperature treatment, in all organs of cold-acclimating plants, i.e. S. sogarandinum line 2 and S. tuberosum cv. Ursus. No change in SsLTP1 abundance was noticed in plants from the same line and species grown under control conditions for the same duration (Fig. 4A, B, lower parts for each organ). In S. sogarandinum line 2 as well as in S. tuberosum cv. Ursus, the highest protein levels were reached after 3 d of treatment. In S. sogarandinum line 2, the most pronounced increase was noticed in well-expanded leaves (Fig. 4A), in which the SsLTP1 abundance was 4-fold higher after 3 d of cold exposure compared with that observed under control conditions. A similar increase in protein abundance was noticed in young, well-expanded, and old leaves of Ursus plants (Fig. 4B). Noticeable increases (2- to 4-fold) in SsLTP1 level were thus observed in all aerial organs of the cold-acclimating lines or species (Fig. 4A, B). Interestingly, in old leaves of cv. Ursus, a more pronounced accumulation of SsLTP1 (4-fold increase) was observed under cold conditions compared with that noticed in S. sogarandinum line 2 (2-fold). In sharp contrast, no noticeable change in SsLTP1 level was noticed during low temperature treatment in the non-acclimating S. sogarandinum line 1 (Fig. 4C) and S. tuberosum cv. Irga (Fig. 4D), whatever the organ analysed. Altogether, these data reveal that only cold-acclimating Solanum lines and species display a substantially increased SsLTP1 amount in aerial organs.
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SsLTP1 abundance in plants subjected to drought and salt treatments
When exposed to drought conditions, the four Solanum lines or species displayed similar responses. During the first 5 d of water deficit, plants remained turgid. After 9 d, the RWC of expanded leaves reached a value of
75% and the old leaves exhibited pronounced wilting and turned yellow (data not shown). In water-deprived S. sogarandinum line 2 and S. tuberosum cv. Ursus plants, a progressive increase in SsLTP1 amount was observed in all organs, the highest levels being reached after 9 d of water shortage when the RWC decreased to 75%. In S. sogarandinum line 2 plants, a strong increase in SsLTP1 amount was noticed in stems, the protein level being 3-fold higher after 9 d of water deficit than that in control conditions (Fig. 5). In young leaves and in well-expanded leaves, the elevation in SsLTP1 abundance was also noticeable, with 2.5- to 3-fold increases, respectively, compared with control plants (Fig. 5). Similarly, in water-deprived plants of S. tuberosum cv. Ursus the highest increases in protein amount were noticed in stems and in well-expanded leaves (2.5-fold) as well as in young leaves (2-fold) after 9 d of treatment (Fig. 5). In both cold-acclimating Solanum species, the lowest changes in SsLTP1 amounts were observed in old leaves. Interestingly, an elevated SsLTP1 level was also observed in drought-stressed S. sogarandinum line 1 and S. tuberosum cv. Irga plants. In S. sogarandinum line 1, a higher SsLTP1 amount (3-fold) was detected in young leaves after 6 d of stress (Fig. 5). Noticeable changes in SsLTP1 abundance were also detected in stems and in well-expanded and old leaves, with maximal levels at the end of the water-deficit period (Fig. 5). In Irga plants, the most pronounced increase in SsLTP1 level was observed in stems, where the protein abundance progressively increased during the water shortage (Fig. 5).
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When plants were exposed for 3 d to 0.15 M NaCl in the nutritive solution, no apparent chlorosis symptom was observed in well-expanded leaves, but some old leaves displayed bleaching (data not shown). In stems of cold-acclimating plants of S. tuberosum cv. Ursus, an increase in SsLTP1 amount was already noticed after 8 h of exposure to NaCl, and the maximal level of accumulation was reached after 3 d of treatment (Fig. 6). After 3 d of exposure to salt, the SsLTP1 abundance in stems was 2.5- and 3.6-fold higher in S. sogarandinum line 2 and in S. tuberosum cv. Ursus, respectively, compared with that observed in control conditions. In leaves of S. sogarandinum line 2 and of S. tuberosum cv. Ursus, a noticeable elevation in SsLTP1 amount was also observed (around 2-fold) (Fig. 6). In S. sogarandinum line 1 and S. tuberosum cv. Irga, an increase in protein abundance occurred only in young leaves and in stems, with maximal levels reached after 3 d of salt exposure (Fig. 6), no noticeable change being observed in well-expanded and old leaves.
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Effect of ABA on SsLTP1 expression
Then the lipid transfer protein abundance was investigated in in vitro grown plantlets of cold-acclimating S. sogarandinum line 2 subjected to an ABA treatment by adding 75 µM to the growth medium. As shown in Fig. 7, no change in the SsLTP1 amount was noticed during the 3 d of exogenous ABA application. This result suggests that expression of the SsLTP1 gene is not regulated through an ABA-dependent pathway.
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Discussion |
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The SsLTP1 gene exhibits an organ-dependent expression pattern
In the present study, the expression at the protein level of a gene encoding a type 1 ns-LTP (SsLTP1) in Solanum species is described during growth and under conditions leading to cellular dehydration. The data reveal that the SsLTP1 protein is present in all aerial plant organs, but with levels varying greatly as a function of organ type. In 2-month-old plants at the flowering stage, a substantial protein level was revealed in open flowers and in stems, a somewhat lower level was noticed in young leaves, and much lower amounts were observed in old leaves. Similar SsLTP1 levels have also been observed in the vegetative organs of 2-week-old plants. SsLTP1 was not detected in roots, in accordance with studies reporting a very low expression of ns-LTP1 genes in roots (Kader, 1996). These data clearly indicate that during growth the SsLTP1 abundance is regulated by factors specific to organ type. In S. tuberosum, one ns-LTP1 gene with 64% sequence identity to SsLTP1 displays a constitutive transcriptional pattern throughout the plant (Horwath et al., 2002), but another one, StnsLTP.2, with higher similarity to SsLTP1 (74% identity) showed an organ-specific expression pattern, with a high level of expression in nodes, internodes, and stolons, and during the tuber life cycle (Horwath et al., 2002). These data are consistent with the western blot data which revealed a high SsLTP1 abundance in stems of S. sogarandinum and of S. tuberosum plants. In Arabidopsis, transcripts of ns-LTP1 genes termed ltp2, ltp1, ltp4, and ltp3 and sharing 49, 47.2, 45, and 42.7% identity with SsLTP1, respectively, were detected in flowers and siliques, but not in roots (Arondel et al., 2000). In addition, only the ltp1 and ltp2 genes displayed noticeable expression in young leaves, but the transcript levels gradually decreased at later stages of leaf development (Arondel et al., 2000). The present study, which delineates the SsLTP1 abundance in a detailed manner, shows that SsLTP1 belongs to the family of ns-LTP genes displaying a pattern of expression strongly regulated as a function of organ type and developmental stage.
SsLTP1 abundance is increased by environmental conditions leading to cell dehydration
The SsLTP1 level substantially increased in S. sogarandinum and in S. tuberosum plants subjected to drought, salinity, or cold. In the case of low temperature, a higher SsLTP1 abundance was observed in all aerial organs of only S. tuberosum cv. Ursus and S. sogarandinum line 2 phytotron-grown plants (Fig. 4A, B). Interestingly, water shortage led to significant increases in protein level in most aerial organs of the four Solanum lines or cultivars studied. High salinity treatment also resulted in increases in SsLTP1 abundance in all lines and cultivars, but to a lesser extent compared with water deficit. An induction in ns-LTP1 gene expression at the transcriptional level in response to drought stress has been previously reported in the aerial organs of numerous monocotyledon and dicotyledon species (Pearce et al., 1996; Treviño and O'Connell, 1998; Smart et al., 2000; Jung et al., 2003; Wu et al., 2004). Thus, increased ns-LTP abundance has been reported in tobacco leaves during prolonged water deficit (Kawaguchi et al., 2003). In Lycopersicon esculentum, the expression of the SsLTP1-related TSW12 gene is enhanced at the transcript level in stems after 6 h of salt treatment, but not in roots and leaves (Torres-Schumann et al., 1992). In another Solanaceae member, Capsicum annuum, a higher ns-LTP1 transcript abundance was found in leaves of plants treated with NaCl (Jung et al., 2003). In line with these data indicating that ns-LTP1 transcripts are up-regulated in response to various environmental stimuli leading to cell dehydration, the present work reveals that the SsLTP1 protein abundance increases in most aerial organs of Solanum plants during cold, drought, and salt stresses. During low-temperature treatment, a noticeable increase in SsLTP1 abundance was observed only in plants able to acclimatize to cold, no change being observed in the non-acclimating S. sogarandinum line and S. tuberosum cv. Irga. Note that under drought and salt-stress conditions, a noticeable accumulation of the protein was observed in the two groups of Solanum species and lines. These findings led us to conclude that two distinct signals, one related to low temperature and the other to osmotic stress, regulate SsLTP1 expression in response to constraints imposing cell dehydration. The fact that exogenous ABA could not induce the accumulation of SsLTP1 in S. sogarandinum suggests that this phytohormone might not mediate the expression of the gene. Based on the transcriptional regulatory networks participating in the responses to osmotic and cold stresses (Yamaguchi-Shinozaki and Shinozaki, 2006), it appears that several ABA-independent signalling pathways sharing common effectors might regulate SsLTP1 expression in Solanum species under osmotic and cold stresses. The genes involved in cold-acclimation processes are induced through two main independent pathways, one driven by the ICE-DREB1/CBF system (Van Buskirk and Thomashow, 2006; Yamaguchi-Shinozaki and Shinozaki, 2006) and a second one by the HOS9 and HOS10 transcription factors (Zhu, 2004). The CBF/DREB1 transcription factors bind specifically to the cis-DRE/CRT sequence in the promoters of target genes and activate their transcription. Both the DREB1/CBF and HOS9/HOS10 pathways play a pivotal role in the development of freezing tolerance upon low-temperature treatment, but each contributes to only a part of a plant's acclimation capacity (Chinnusamy et al., 2006; Yamaguchi-Shinozaki and Shinozaki, 2006). Thus, SsLTP1 expression under low temperature is likely to be regulated by one of these pathways. In other respects, the elevation in SsLTP1 gene expression upon drought and salinity in the cold non-acclimating Solanum species demonstrates that there is another signalling pathway regulating its expression in conditions imposing cell dehydration. Upon drought and salinity stresses, DREB2 transcription factors have also been found to activate transcription of genes driven by the DRE/CRT sequence, but in a way independent of the CBF/DREB1 factors (Yamaguchi-Shinozaki and Shinozaki, 2006). Interestingly, in A. thaliana, an increased expression of several ns-LTP genes has been reported to be mediated through the DREB1/CBF transcriptional factors under low temperature conditions (Maruyama et al., 2004). These data support the hypothesis that expression of the SsLTP1 gene could be controlled by DRE/CRT cis-elements through DREB1/CBF and DREB2 transcriptional factors under low-temperature and osmotic stress, respectively. Further work is needed to sequence the SsLTP1 promoter to confirm this proposal. The loss of ability to cold acclimate in the S. sogarandinum line 1 may indicate that a signalling element upstream of DREB1/CBF has been inactivated in this line, as in S. tuberosum cv. Irga.
SsLTP1 abundance positively correlates with the ability to acclimatize to cold
Under low temperature, either in vitro or in vivo, an increase in SsLTP1 level was observed only in Solanum species or the cultivar able to acclimatize to cold, indicating that an elevated protein level is associated with the first stages of the cold acclimation processes that lead to development of freezing tolerance in Solanum species. Note also that SsLTP1 gene induction by low temperature occurred much faster in vitro than in vivo. An elevation in the SsLTP1 level was already observed in S. sogarandinum in vitro plantlets after 8 h of exposure to low temperature (Fig. 3B). Response mechanisms to cold are likely to be induced faster in small 10-d-old in vitro plantlets than in 2-week-old phytotron-grown plants. The present work is in agreement with those reporting an induction at the transcript level of ns-LTP genes by low temperature in Hordeum vulgare and in Bromus intermis cold-acclimating plants (Dunn et al., 1991; Hughes et al., 1992; Pearce et al., 1996; Wu et al., 2004). The present data provide the first evidence of the induction at the protein level of an ns-LTP1 gene during exposure to low temperature in plants and indicate that SsLTP1 accumulation is associated with the ability to acclimatize to cold. These results strongly support the idea that this type of ns-LTP1 participates in the freezing tolerance in plants. The fact that two S. sogarandinum lines displaying the same genetic background and only differing in their capacity to acclimatize to cold were used gives substantial credence to this hypothesis. In other respects, it is worth mentioning that Arabidopsis plants overexpressing a pepper ns-LTP1 exhibit an increased tolerance to drought and salt treatments in vitro as well as a lower susceptibility to biotic stress in vivo (Jung et al., 2005). Thus, ns-LTPs are very likely to play an essential role in plant responses and adaptation to various environmental constraints.
A putative role of the SsLTP1 in cold acclimation and under osmotic stress
Under control conditions and in a more pronounced manner during low temperature and osmotic constraints, a higher SsLTP1 abundance, more particularly in stems and in young leaves, was observed. In line with the present findings, Horwath et al. (2002) reported a preferential localization of S. tuberosum SsLTP1-related transcript in phloem tissues surrounding nodes in stems and in tubers. Altogether, these data indicate that ns-LTP1s could fulfil an essential role in vascular cells of aerial organs and might be associated with transport of various lipids or hydrophobic molecules needed for proper development and stress acclimation of apical parts and leaves. The function in vivo of ns-LTPs still remains very elusive. Based on the data showing an intracellular localization for SsLTP1, the protein might participate in a symplastic transport process, involved in perception or transduction of abiotic stress signals. Transporting organs are likely to play a fundamental role in the processes leading to freezing tolerance, since a preferential accumulation in stems of another stress-induced protein, an SK3-type dehydrin, during exposure to low temperature of S. sogarandinum has been reported previously (Rorat et al., 2006). The precise subcellular localization of SsLTP1 and the analysis of its lipid-binding capacity will give more insight into the physiological role of the protein during growth and cold acclimation.
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The authors wish to thank Karolina Hofman for helpful assistance in preparing and screening the Solanum sogarandinum cDNA library. This work was supported in part by a Polish Ministry of Science Higher Education grant No. 2/PO6A/023/029.
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