With seasonal changes, several proteins accumulate in the endoplasmic reticulum (ER)-enriched fraction in the bark of mulberry tree (Morus bombycis Koidz.). Results of partial amino acid sequence analysis in our previous study suggested that one of these proteins is the ER-localized small heat shock protein (sHSP), designated 20-kD winter-accumulating protein (WAP20). In the present study, molecular and biochemical properties of WAP20 were investigated in detail. The deduced amino acid sequence of the cDNA has the predicted signal sequence to the ER, retention signal to the ER and two consensus regions conserved in sHSPs. Recombinant WAP20 expressed in Escherichia coli also showed typical biochemical features of sHSPs, including the formation of a high-molecular-mass complex between 200 and 300 kD under native conditions, promotion of the renaturation of chemically denaturated citrate synthase and prevention of heat stress-induced aggregation of the enzyme. Transcript levels of WAP20 in the bark tissue were seasonally changed, showing high expression levels from mid-October to mid-December, and the transcript levels were additionally increased and decreased by cold treatment and warm treatment, respectively. WAP20 transcripts were detected abundantly in bark tissue rather than xylem and winter bud tissues during seasonal cold acclimation. The bark tissue specificity of WAP20 accumulation was also observed by exogenous application of phytohormone abscisic acid (ABA) in de-acclimated twigs, whereas WAP20 transcripts were increased in all of these tissues by heat shock treatment at 37 degrees C in summer twigs. The results suggest that ABA may be involved in the expression of the WAP20 gene in bark tissue of the mulberry tree during seasonal cold acclimation.