Protein Cryoprotective Activity of a Cytosolic Small Heat Shock Protein That Accumulates Constitutively in Chestnut Stems and Is Up-Regulated by Low and High Temperatures1

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). This is probably a consequence of having longer life cycles and generation times, and also of the more extreme conditions endured by their aerial parts in winter. Woody plants seem to attain freezing tolerance through various stages, which are sequentially activated by daylength shortening and increasingly lower temperatures (Weiser, 1970; Sakai and Larcher, 1987). Very recently, evidence has been obtained in hybrid poplar (Populus tremula ? P. tremuloides) that these environmental cues trigger cold acclimation through distinct pathways (Welling et al., 2002). In herbaceous plants, low temperature is the primary signal responsible for inducing this process (Thomashow, 1999).
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; Anderson et al., 1994) and soybean (Glycine max L. Merr.; Caban? et al., 1993), and also HSP90 isoforms in rice (Oryza sativa; Pareek et al., 1995) and oilseed rape (Krishna et al., 1995). Homologous cold-responsive HSPs have been described in other organisms, such as Drosophila melanogaster (Burton et al., 1988) or mice (Mus musculus; Matz et al., 1995), conveying the generality of this response. Since HSP70 and HSP90 exhibit molecular chaperone activity, a protective role against freeze-induced protein denaturation has been hypothesized (e.g. Guy et al., 1998).
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; Waters et al., 1996; van Montfort et al., 2001). This was first described by van Berkel et al. (1994) in cold-stored potato (Solanum tuberosum) tubers. Chilling-induced sHSP synthesis has also been observed in tomato (Lycopersicon esculentum L. cv Daniella) fruits, but only following heat treatment (Sabehat et al., 1998). More recently, Ukaji et al. (1999) reported that accumulation of WAP20, an endoplasmic reticulum-localized sHSP, is associated with cold acclimation in Morus bombycis (mulberry tree). Winter-specific accumulation of sHSPs has also been observed in Acer platanoides, Sambucus nigra, and Aristolochia macrophylla (Lubaretz and zur Nieden, 2002). The possible relationship of these sHSPs with protective mechanisms against low temperature stress is still a matter of speculation. Many experiments have shown that sHSPs have molecular chaperone activity (van Montfort et al., 2001). Besides, members of this protein family can enhance stress tolerance in a variety of cell systems (e.g. Lavoie et al., 1993; Yeh et al., 1997; Soto et al., 1999). Direct evidence for a chaperone function in cellular thermotolerance has been recently obtained for a cyanobacterial sHSP (Giese and Vierling, 2002). Other studies have reported that sHSPs have stabilizing effects on model membranes formed of synthetic and cyanobacterial lipids, suggesting a role for these proteins in preserving membrane integrity during thermal fluctuations (T?r?k et al., 2001; Tsvetkova et al., 2002). While these properties might contribute to cell survival under freezing stress, more studies are obviously needed to understand the hypothetical role of sHSPs in relation to cold acclimation.
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). The main finding is that this protein, the expression of which is maximal in winter and quickly responds to cold exposure (4?C), shows significant protein cryoprotective activity in vitro. Besides suggesting a novel function for this protein family, our results substantiate the notion that sHSPs may play relevant roles in the acquisition of freezing tolerance. A similar function has been postulated for high-molecular mass chaperones of the HSP70 family (Guy et al., 1998; Sung et al., 2001).
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