Cold acclimation of winter wheat (Triticum aestivum L.) seedlings induces accumulation in the apoplast of taTLPs that are similar to thaumatin-like proteins (TLPs), which are pathogenesis-related proteins. We characterized a cDNA of WAS-3a encoding the major isoform of taTLPs from winter wheat cells and showed that WAS-3a transcripts were markedly increased by treatment with ABA and by treatment with elicitors (chitosan, beta-glucan and cell wall fractions of Fusarium oxysporum and Microdochium nivale) in wheat cells. To analyse the function of WAS-3a, a highly efficient expression system using wheat cells was established, and a large amount of recombinant WAS-3a protein (rWAS-3a) was obtained with near homogeneity. Antifungal assays using various fungi grown on agar plates revealed that rWAS-3a inhibits hyphal growth of pink snow mould, Microdochium nivale, at a low temperature. The results suggest that cold-induced taTLPs that accumulate in the apoplast contribute to snow mould resistance of winter wheat.

Cortical parenchyma cells of mulberry (Morus bombycis Koidz.) trees acquire extremely high freezing tolerance in winter as a result of seasonal cold acclimation. The amount of total proteins in endoplasmic reticulum (ER)-enriched fractions isolated from these cells increased in parallel with the process of cold acclimation. Protein compositions in the ER-enriched fraction also changed seasonally, with a prominent accumulation of 20-kD (WAP20) and 27-kD (WAP27) proteins in winter. The N-terminal amino acid sequence of WAP20 exhibited homology to ER-localized small heat-shock proteins (smHSPs), whereas that of WAP27 did not exhibit homology to any known proteins. Like other smHSPs, WAP20 formed a complex of high molecular mass in native-polyacrylamide gel electrophoresis. Furthermore, not only WAP20 but also 21-kD proteins reacted with antibodies against WAP20. Fractionation of the crude microsomes by isopycnic sucrose-gradient centrifugation revealed that both WAP27 and WAP20 were distributed on a density corresponding to the fractions with higher activity of ER marker enzyme, suggesting localization of these proteins in the ER. When ERenriched fractions were treated with trypsin in the absence of detergent, WAP20 and WAP27 were undigested, suggesting localization of these proteins inside the ER vesicle. The accumulation of a large quantity of smHSPs in the ER in winter as a result of seasonal cold acclimation indicates that these proteins may play a significant role in the acquisition of freezing tolerance in cortical parenchyma cells of mulberry trees.

Plants require substantial resistance against freezing and pathogens for overwintering. These two traits are acquired through cold acclimation. In contrast to freezing tolerance, molecular basis of disease resistance acquired through cold acclimation is poorly understood. Recent studies have suggested that pathogenesis-related (PR) proteins that are secreted into the apoplast during cold acclimation are responsible for the disease resistance. Interestingly, some of the cold-induced PR proteins display both antifungal and antifreeze activities, suggesting a dual function in protecting plants from overwintering stresses. The signaling pathway for cold-induced disease resistance is currently unknown but can be independent of pathogeninduced defense mechanisms.

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Seasonal evaluation of total soluble protein fractions extracted from cortical parenchyma cells of mulberry ( Morus bombycis Koidz.) tree identified a predominant 18 kDa protein that was directly correlated to periods of cold acclimation. The 18 kDa protein, designated as WAP18 ( w inter a ccumulating 18 kDa p roteins) increased from September to December and then gradually decreased until June. The maximum levels of WAP18 were detected in mid-winter, which corresponds to the maximum freeze tolerance in cortical parenchyma cells of mulberry tree. Two-dimensional gel electrophoresis confirmed that WAP18 consists of at least three proteins that range between an isoelectric point of 5.0 and 6.0. All three proteins reacted with anti-WAP18 antibodies, thereby suggesting that they represent individual isoforms. Furthermore, N-terminal amino acid sequence analysis demonstrated that all three proteins contain high sequence similarity to each other and high homology to pathogenesis-related (PR) ----10/Bet v 1 protein families. The purified WAP18 exhibited in vitro cryoprotective activity for the freeze labile L -lactate dehydrogenase (LDH) enzyme. These results suggest that WAP18 may function in the freezing tolerance mechanism of cortical parenchyma cells of mulberry tree during winter.