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 coldhardy 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 ex...