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First published online April 2, 2004; 10.1104/pp.103.035857 Plant Physiology 134:1708-1717 (2004) © 2004 American Society of Plant Biologists Protein Cryoprotective Activity of a Cytosolic Small Heat Shock Protein That Accumulates Constitutively in Chestnut Stems and Is Up-Regulated by Low and High Temperatures1Departamento de Biotecnología, Escuela Técnica Superior de Ingenieros de Montes, Universidad Politecnica de Madrid, E–28040 Madrid, Spain
Heat shock, and other stresses that cause protein misfolding and aggregation, trigger the accumulation of heat shock proteins (HSPs) in virtually all organisms. Among the HSPs of higher plants, those belonging to the small HSP (sHSP) family remain the least characterized in functional terms. We analyzed the occurrence of sHSPs in vegetative organs of Castanea sativa (sweet chestnut), a temperate woody species that exhibits remarkable freezing tolerance. A constitutive sHSP subject to seasonal periodic changes of abundance was immunodetected in stems. This protein was identified by matrix-assisted laser-desorption ionization time of flight mass spectrometry and internal peptide sequencing as CsHSP17.5, a cytosolic class I sHSP previously described in cotyledons. Expression of the corresponding gene in stems was confirmed through cDNA cloning and reverse transcription-PCR. Stem protein and mRNA profiles indicated that CsHSP17.5 is significantly up-regulated in spring and fall, reaching maximal levels in late summer and, especially, in winter. In addition, cold exposure was found to quickly activate shsp gene expression in both stems and roots of chestnut seedlings kept in growth chambers. Our main finding is that purified CsHSP17.5 is very effective in protecting the cold-labile enzyme lactate dehydrogenase from freeze-induced inactivation (on a molar basis, CsHSP17.5 is about 400 times more effective as cryoprotectant than hen egg-white lysozyme). Consistent with these observations, repeated freezing/thawing did not affect appreciably the chaperone activity of diluted CsHSP17.5 nor its ability to form dodecameric complexes in vitro. Taken together, these results substantiate the hypothesis that sHSPs can play relevant roles in the acquisition of freezing tolerance.
Cold acclimation is a complex process by which the freezing tolerance of certain plants increases after a period of exposure to low nonfreezing temperatures. Because of the enormous agricultural impact of freezing injury, especially in temperate regions, the molecular mechanisms associated with cold acclimation have been the subject of intensive research over the past decades. Studies with Arabidopsis and cold-hardy herbaceous plants, such as winter cereals (Triticum aestivum, Hordeum vulgare), spinach (Spinacia oleracea), oilseed rape (Brassica napus), or cabbage (Brassica oleracea) have led to the identification of numerous genes potentially involved in freezing tolerance (for recent reviews, see Thomashow, 1999  ; Smallwood and Bowles, 2002). Many of these genes encode proteins with known activities, like enzymes for the synthesis of compatible solutes or for the modification of membrane lipids. In other instances, however, the function of the gene products remains unknown. In a few cases the encoded proteins have been shown to contribute functionally to freezing tolerance, such as the stromal polypeptides COR15a (Artus et al., 1996; Steponkus et al., 1998) and WCS19 (NDong et al., 2002). The signal transduction networks involved in cold-regulated gene expression have also been studied in a number of herbaceous species, including Arabidopsis and several crops (Shinozaki et al., 2003).
The molecular aspects of cold acclimation remain largely unexplored in long-lived woody plants. However, it is well established that the capacity of temperate zone woody perennials to cold acclimate is much higher than that of herbaceous species (Weiser, 1970
Among the proteins induced or up-regulated in plants by low temperatures there are heat shock proteins (HSPs). These include members of the HSP70 family in spinach (Neven et al., 1992
In only a few instances low temperatures have been shown to stimulate the accumulation of small HSPs (sHSPs), which are the most diverse and abundant HSPs synthesized by plants (Vierling, 1991
Here we analyze the accumulation patterns of sHSPs in vegetative tissues of both adult chestnuts (Castanea sativa; field conditions) and seedlings kept under controlled conditions. A major constitutive sHSP subject to seasonal periodic changes of abundance was immunodetected in stems. This protein was identified by mass fingerprinting and internal amino acid sequencing as CsHSP17.5, a cytosolic class I sHSP isolated previously from mature chestnut cotyledons (Collada et al., 1997
Detection of sHSPs in Vegetative Organs
To investigate the occurrence of sHSPs in chestnut vegetative organs, 16- to 46-week-old seedlings kept under normal growth conditions were subjected to heat stress (38°C for 4 h), and total proteins were isolated from leaves, stems, and roots. Samples corresponding to equal quantities of protein from both stressed and nonstressed plants were then fractionated by SDS-PAGE and immunoblotted with purified polyclonal antibodies against seed CsHSP17.5 (Collada et al., 1997
Characterization and cDNA Cloning of the Major Stem sHSPs
Two-dimensional western analysis revealed that the 20-kD band detected in stems contained two major cross-reactive components in both seedlings and adult trees. These polypeptides, labeled as St1 and St2 in Figure 2A
, had similar pIs (approximately 6) and overlapped when extracts from seedlings and trees were coelectrophoresed (data not shown; Fig. 2, B and C). Since their N-terminal residues appeared to be blocked (direct sequencing of polyvinylidene difluoride [PVDF]-adsorbed samples), a preparation highly enriched in St1 and St2 was obtained from whole branch slices by selective extraction and size-exclusion HPLC (see "Materials and Methods"). Due to the unusual abundance of condensed tannins and other phenolic compounds in chestnut bark, proteins were extracted under strongly reducing conditions and washed extensively with acetone. Moreover, alkylation was necessary to prevent significant aggregation and to obtain reproducible chromatograms. Both St1 and St2 eluted in a single HPLC peak along with a few minor proteins (Fig. 3A
). To gain information on their primary structure, preparative two-dimensional electrophoresis was carried out followed by endoproteinase Asp-N cleavage of the eluted proteins. The resulting peptidic fragments were then fractionated by reverse-phase HPLC on Nucleosil C-4, with virtually identical chromatograms being obtained for both proteins (Fig. 3C). As shown in Figure 3D, St1 and St2 had matching internal sequences and, furthermore, these agreed at every residue with an internal region of seed CsHSP17.5 spanning amino acids 50 to 77. Such region corresponds to the N-terminal portion of the conserved
Two different approaches were then followed to further characterize St1 and St2. On one hand, both polypeptides were subjected to matrix-assisted laser-desorption ionization time of flight (MALDI-TOF) analysis following two-dimensional electrophoresis and trypsin digestion. The resulting peptide-mass fingerprints were essentially indistinguishable in the range of 0.9 to 3.1 kD, lending further support to the notion that St1 and St2 are modified forms of a single amino acid chain (Table I). The nature of such modification remains to be determined. Moreover, the sharp correspondence between the observed peptide masses and the predicted fingerprint for CsHSP17.5 strongly supports the hypothesis that St1/St2 and the seed sHSP (theoretical pI, 5.95) have identical primary structures, a notion anticipated by internal amino acid sequencing (Fig. 3D). Interestingly, the N-terminal peptides of St1 and St2 were predicted to be acetylated in Ala-2 (see Table I), which would explain the lack of substantial signals upon automatic sequencing. The second approach involved the construction of a chestnut stem cDNA library (4.5 x 107 pfu mL–1) and its screening at moderate stringency with the cDNA for seed CsHSP17.5. Eight independent positive clones were randomly selected and their inserts sequenced. All inserts corresponded to a single nucleotide sequence that showed no mismatches with the Cshsp17.5 seed cDNA, except for the presence of six additional nucleotides at the 5'-untranslated end (accession no. AJ582679). We conclude from these experiments and the reverse transcription (RT)-PCR experiments reported below that CsHSP17.5 is the predominant sHSP in chestnut stems.
Seasonal Changes of Protein and mRNA Abundance To investigate sHSP seasonal accumulation patterns, branch segments were collected from adult trees at monthly intervals and their proteins subjected to western-blot analysis with anti-CsHSP17.5 antibodies. In the representative immunoblot shown in Figure 4A , a cross-reactive 20-kD band is observed in all samples analyzed (January through December 1999). These experiments, along with western-blot analyses of nonstressed seedlings (see Fig. 1A), strongly suggest that CsHSP17.5 is constitutively expressed in chestnut stems. However, its relative abundance appears to vary on a seasonal basis, with the highest levels occurring in winter and late summer (there is approximately a 4-fold difference in signal intensity between January and April). Essentially the same accumulation patterns were observed for 1999 and 2000, supporting the generality of these findings. To test whether the observed changes in protein abundance truly reflected seasonal fluctuations of gene activity, total RNA was isolated from equivalent samples and hybridized at high-stringency with the full-length cDNA for CsHSP17.5 (Fig. 4B). The RNA blots revealed annual oscillations in the amount of hybridizing transcripts, which were basically concurrent with protein data. In fact, after a gradual increase during spring and fall, transcription was found to be maximal in late summer (July and August) and winter. The highest and lowest yearly temperatures at the sample collection site were recorded during these two periods, respectively (Fig. 4C; data for 1999). The presence of mRNA for CsHSP17.5 in the above extracts was verified by RT-PCR (see "Materials and Methods"). Even at low annealing temperatures a single 383-bp band was amplified in all preparations analyzed, the sequence of which was identical to the Cshsp17.5 cDNA (nucleotides 130–512). Taken together, our results are indicative that CsHSP17.5 is constitutively expressed in chestnut stems. Furthermore, protein abundance is subjected to periodic (seasonal) variations, with the highest rates of accumulation being associated with fall and early summer.
Cold Exposure Activates shsp Gene Expression
Many genes induced during cold acclimation are responsive to low temperatures (Thomashow, 1999
Cryoprotective Activity and Cryostability of CsHSP17.5
Molecular chaperone activity has been previously reported for purified CsHSP17.5 (Collada et al., 1997
We also tested the effects of repeated freezing/thawing on both the chaperone activity of purified CsHSP17.5 (prepared at 5 g L–1 in distilled water) and its ability to assemble into homododecameric complexes under nondissociating conditions. For the molecular chaperone activity assays, CsCh1, the major chestnut seed endochitinase (Collada et al., 1992 ), was chemically denatured in 6 M guanidine hydrochloride and then diluted 100-fold into a refolding buffer that was supplemented with or contained no CsHSP17.5 (frozen/thawed up to 10 times before addition to the mixture). CsCh1 was chosen because it is a putative in vivo substrate for CsHSP17.5, since both proteins accumulate abundantly and concomitantly in cotyledonary cells during seed development. As shown in Figure 7A
, the presence of CsHSP17.5 resulted in refolding yields of CsCh1 about 2-fold higher than in control reactions after 120 min of incubation, with no appreciable effect of repeated freezing on chaperone activity. Analogous results were obtained when chemically denatured citrate synthase was used as substrate (data not shown). Figure 7B presents data indicating that the fraction of sHSP molecules able to form dodecameric complexes is not appreciably influenced by repeated freezing/thawing (up to 10 cycles, separated by 1-week storage at –20°C). No variations were found either in the total amount of soluble protein, thus confirming the cryostability of CsHSP17.5 in aqueous solution. In summary, CsHSP17.5 does not appear to be significantly influenced in its conformational stability, aggregation properties, and chaperone activity by alternated exposure to subzero and above-zero temperatures, a condition that plants often endure during winter in temperate climates.
Like other long-lived woody species native to temperate regions, C. sativa exhibits a remarkable freezing tolerance. Here we show that a member of the sHSP family is significantly up-regulated in stem tissues of this species as the winter approaches, reaching the highest abundance during the coldest period of the year (November-March). This protein has been identified by MALDI-TOF mass spectrometry and internal peptide sequencing as CsHSP17.5, a cytosolic class I sHSP previously isolated from mature chestnut cotyledons (Collada et al., 1997 ). Expression of the corresponding gene in stem tissues was confirmed through cDNA cloning and RT-PCR. The accumulation of identical sHSPs in seeds and vegetative organs had been formerly postulated in sunflower (Helianthus annuus) and pea on the basis of electrophoretic mobility (Almoguera et al., 1993; Wehmeyer et al., 1996). Protein and RNA analysis of samples harvested under field conditions (adult trees) suggest that CsHSP17.5 is expressed constitutively in stems (Fig. 4). In agreement with these findings, a single protein band with identical mobility to CsHSP17.5 was consistently immunodetected in stem tissues of nonstressed chestnut seedlings by antibodies raised against the seed sHSP (Fig. 1). The only evidence so far of plant sHSPs constitutively expressed in vegetative organs was obtained in the resurrection plant Craterostigma plantagineum, a model system for molecular studies of desiccation tolerance (Alamillo et al., 1995). Although proteins immunologically related to CsHSP17.5 were not observed in roots or leaves of nonstressed chestnut seedlings, they were induced in both organs after a short exposure to 38°C (Fig. 1). This treatment also stimulated sHSP expression in stems.
Cold acclimation is characterized in trees by increased cold tolerance in fall, reaching a maximum in winter and then decreasing rapidly in spring (Weiser, 1970
The precise reasons why CsHSP17.5 is constitutively expressed in chestnut stems are not yet known, and the same is true for its periodic changes of abundance. It is now well established that sHSPs have a high capacity to bind nonnative polypeptides, and current models propose that they cooperate with other cell chaperones to promote proper protein folding and assembly (van Montfort et al., 2001
Some stresses characteristic of winter can also augment the cellular requirements for chaperones. Low temperature directly affects the stability and solubility properties of many globular proteins, making thermodynamically favorable the exposure of nonpolar side chains to the aqueous solvent (Pace, 1990
In summary, the biochemical data and the expression patterns reported here for CsHSP17.5 are strongly supportive of a protective role in vivo at periods when thermal extremes often occur. On the other hand, the constitutive expression observed in stems also suggests a role in normal cell maintenance. Virtually identical sHSP expression patterns have been observed in a preliminary study of phylogenetically distant woody species, and the characterization of a Cedrus atlantica polypeptide is now under way (R. Casado, I. Allona, C. Collada, C. Aragoncillo, and L. Gomez, unpublished data). The ability of purified CsHSP17.5 to act as cryoprotectant in vitro has not been reported before for any member of this protein family. Such activity may help explain its strong up-regulation in association with the acquisition of cold hardiness in adult trees, and also its cold-responsiveness in seedlings. Nonetheless, the protection of cold-labile proteins is only a working model within the chaperone frame. Cell membranes are thought to be the primary site of freezing injury, and some sHSPs have been shown to stabilize the liquid-crystalline state of model bilayers (Török et al., 2001
Plant Material and Stress Conditions
Stem material (2-year-old branch internodes) and mature seeds of Castanea sativa Mill. were harvested from adult trees growing in Zarzalejo, Madrid (4°11' W, 40°35° N). Chestnut seedlings were kept in growth chambers as previously described (Soto et al., 1999
Stem material was ground in liquid nitrogen to the consistency of flour. This powder was extracted (10:1, v/w) with 62.5 mM Tris-HCl, pH 6.8, 5% mercaptoethanol, 0.5% SDS, 1 mM phenylmethylsulfonyl fluoride for 60 min at 22°C. After centrifugation at 30,000g for 30 min, proteins in the supernatants were precipitated with 12% (w/v) TCA, washed three times with 80% acetone in water, and then analyzed by SDS-PAGE. For subsequent protein fractionation and purification, the extracted proteins (dried acetone pellets) were first suspended in 0.1 M Tris-HCl, pH 8.6, 2 mM EDTA, 2 mM DTT, 6 M guanidine hydrochloride and then carboxymethylated with 0.2 M iodoacetic acid for 30 min at 37°C. This step reduced significantly protein aggregation caused by phenolic compounds. The carboxymethylated proteins were dialyzed against water, lyophilized, and fractionated by size-exclusion HPLC on a Spherogel TSK-G column (3 x 215 mm) eluted with 50 mM ammonium acetate, pH 5.5, 0.1% SDS. The appropriate HPLC fractions were further fractionated by two-dimensional isoelectrofocusing (IEF) x SDS-PAGE on a Bio-Rad Miniprotein II system (Bio-Rad Laboratories, Hercules, CA). Seed CsHSP17.5 was purified to homogeneity as previously described (Collada et al., 1997
Following IEF x SDS-PAGE, proteins were electrotransferred onto PVDF membranes (Immobilon-P, Millipore, Bedford, MA) on a Bio-Rad Mini Trans-Blot cell. Selected protein spots were subjected to in situ digestion for 5 h at 37°C with endoproteinase Asp-N (Roche Diagnostics, Indianapolis) in 50 mM sodium phosphate, pH 8.0. The resulting peptidic fragments were eluted with 80% (v/v) formic acid in water and separated by reverse-phase HPLC on a Nucleosil C-4 column (4.6 x 250 mm) using a two-step gradient of 0.1% trifluoroacetic acid in acetonitrile (0%–70% acetonitrile in 70 min; 70%–100% acetonitrile in 10 min; flow rate 1 mL/min). The purified peptides were sequenced by standard methods using an Applied Biosystems (Foster City, CA) gas phase 470A sequenator. For MALDI-TOF analyses, peptide-mass fingerprints were obtained as in Pineiro et al. (2001)
A cDNA library was constructed in Lamda Uni-ZAP XR using chestnut stem poly(A+) RNA and the ZAP-cDNA Gigapack III Gold Cloning kit (Stratagene, La Jolla, CA). The library was enriched in 2,000- to 400-bp fragments by gel filtration on Sepharose CL-2B (Amersham Biosciences, Piscataway, NJ). Approximately 105 clones were screened using as probe the full-length cDNA for seed CsHSP17.5 (Soto et al., 1999
Nylon membranes (Magna, MSI, Minnetonka, MN) were prehybridized for 2 h at 42°C in 50% formamide, 5x SSPE (1x SSPE is 0.15 M NaCl, 10 mM sodium phosphate, pH 7, 1 mM EDTA), 5x Denhardt's solution, 0.2% (w/v) SDS, 10% dextran sulfate with denatured salmon sperm DNA at 0.1 g L–1, and then hybridized for an additional 16 h in the same solution with 32P-labeled probe (see below). After hybridization, membranes were washed at room temperature twice in 2x SSC (1x is 0.15 M NaCl, 15 mM sodium citrate, pH 7), 0.1% SDS for 15 min, twice in 1x SSC, 0.1% SDS for 15 min, and twice in 0.1x SSC, 0.1% SDS for 15 min (moderate stringency). The 660-bp EcoRI-SalI fragment from the Cshsp17.5 cDNA was gel-purified and labeled with [
PVDF-adsorbed proteins were probed with purified polyclonal antibodies to CsHSP17.5 prepared as in Collada et al. (1997)
The cryoprotective activity of CsHSP17.5 (purified as in Collada et al., 1997
Purified CsCh1, the major chestnut seed endochitinase (Collada et al., 1992 Sequence data from this article have been deposited with the EMBL/GenBank data libraries under accession number AJ582679.
We thank J.M. Malpica for his help with statistical treatments, J. Jordano for helpful comments, and J. Varela for N-terminal protein sequencing. We also thank E. Camafeita and A. Marina for MALDI-TOF analyses. P.N. was the recipient of a predoctoral fellowship from Ministerio de Asuntos Exteriores of Spain, and A.S. was the recipient of a predoctoral F.P.I. fellowship from Ministerio de Educación, Cultura y Deportes of Spain. Received November 7, 2003; returned for revision January 13, 2004; accepted January 13, 2004.
1 This work was supported by Plan Nacional de Biotecnología, Ministerio de Ciencia y Tecnología, Spain (grant no. BIO99–0931) and by Comunidad Autónoma de Madrid (grant no. 07M/0047/2000). 
2 Present address: Facultad de Ciencias, Centro de Biología Molecular Severo Ochoa, Universidad Autónoma de Madrid, E–28049 Madrid, Spain.
3 Present address: Departamento de Silvicultura, Facultad de Ciencias Forestales, Universidad de Chile, Santiago, Chile.
4 Present address: Centro de Investigación Forestal-CIFOR, Instituto Nacional de Investigaciones Agrarias, Ministerio de Agricultura, Pesca y Alimentación, E–28040 Madrid, Spain. Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.103.035857. * Corresponding author; e-mail lgomez{at}montes.upm.es; fax 34–91–3366387.
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ABSTRACT
RESULTS
-crystallin domain present in all sHSPs. 
, sHSP; 
, STI; 
, HEWL; 
, ConA. The CP50 values were determined as described in "Materials and Methods". The results shown are the average of four independent experiments. SE of the CP50 values were ±10.4% for sHSP, ±9.7% for STI, ±11.8% for HEWL, and ±14.5% for ConA.
