Freezing tolerance in plants develops through acclimation to cold by growth at low, above-freezing temperatures. Wheat is one of the most freezing-tolerant plants among major crop species and the wide range of freezing tolerance among wheat cultivars makes it an excellent model for investigation of the genetic basis of cold tolerance. Large numbers of genes are known to have altered levels of expression during the period of cold acclimation and there is keen interest in deciphering the signaling and regulatory pathways that control the changes in gene expression associated with acquired freezing tolerance. A 5740 feature cDNA amplicon microarray that was enriched for signal transduction and regulatory genes was constructed to compare changes in gene expression in a highly cold-tolerant winter wheat cultivar CDC Clair and a less tolerant spring cultivar, Quantum. Changes in gene expression over a time course of 14 days detected over 450 genes that were regulated by cold treatment and were differentially regulated between spring and winter cultivars, of these 130 are signaling or regulatory gene candidates, including: transcription factors, protein kinases, ubiquitin ligases and GTP, RNA and calcium binding proteins. Dynamic changes in transcript levels were seen at all periods of cold acclimation in both cultivars. There was an initial burst of gene activity detectable during the first day of CA, during which 90% of all genes with increases in transcript levels became clearly detectable and early expression differential between the two cultivars became more disparate with each successive period of cold acclimation.

Over the last 10 years considerable progress has been made in the immunological and biochemical characterization of oviduct-specific glycoproteins. It is now well established that a subclass of these secretory products, designated as oviductins, associate with the zona pellucida of the ovulated oocyte and with the early embryo. Recent reports on the cloning of cDNAs of oviductins from various species, including that of golden hamster (Mesocricetus auratus) oviductin by our laboratory, allowed us to compare their deduced amino acid sequences with those of other proteins. Optimal alignment analysis showed that oviductins contain regions of significant similarity with catalytically inactive mammalian members of the bacterial and microfilarial chitinase protein family. Most importantly, a close examination of the hamster and human deduced amino acid sequences revealed that both glycoproteins possess contiguous Ser/Thr rich repeated units, clustered in their carboxy-terminal portions. These mucin-type motifs are similar in the hamster and human glycoprotein, although hamster oviductin contains more of these complete units. This striking feature might indicate that these molecules play a similar role to mucin-type glycoproteins, e.g., in protecting the oocyte and early embryo against attacks from their environment. W e propose a model whereby oviductins are targeted to the oocyte via the interaction of their chitinase-like domains with specific oligosaccharide moieties of the zona pellucida. Once localized to this structure, oviductin molecules would act as a protective shield around the oocyte and early embryo by virtue of their densely glycosylated mucin-type domains. o 1995 Wiley-Liss, Inc.

Oviductins are a family of glycoproteins, synthesized and released by oviductal secretory cells, which bind to the zona pellucida of the oocyte after ovulation. Hamster oviductin migrates as diffuse species of 160-350 kDa during SDS/PAGE under reducing as well as non-reducing conditions. In this report, we describe the one-step purification of hamster oviductin using either immuno- or lectin-affinity chromatography. Probing with specific lectins showed that the glycoprotein contains terminal alpha-D-GalNAc, and either terminal alpha-D-NeuAc or non-terminal beta-D-(GlcNAc)2 residues, but fails to react with concanavalin A and Ulex Europeus A-1 lectins which are specific for branched alpha-D-mannose and alpha-L-fucose moieties respectively. Intraovarian oocytes do not contain this glycoprotein and we demonstrate here that the immunoaffinity-purified oviductin readily binds to their zonae pellucidae in vitro, thus mimicking the in vivo phenomenon. Two major immunologically related forms of hamster oviductin (named alpha and beta) were characterized using one- and two-dimensional gel electrophoresis. The alpha-form (160-210 kDa) has an acidic pI of 3.5-4.5 and the beta-form (approx. 210-350 kDa) is localized at the cathodic site in the isoelectric focusing dimension; in between these two major forms lies a smear of minor-charge isomers. Peptide mapping of both major forms with papain and Staphylococcus aureus V8 protease yielded fragments of identical size. Moreover, the two forms share the same N-terminal sequence which display no significant homology with other reported proteins. Treatment with trifluoromethanesulphonic acid showed that a protein with the size and pI of the alpha-form can be generated from the beta-form. Both the alpha- and beta-forms are sulphated on O-linked oligosaccharide side chains but are not phosphorylated. Collectively, these results suggest that the hamster oviductin polymorphism observed in two-dimensional PAGE is a consequence of different glycosylation patterns and not the polypeptide chain itself. Hamster oviductin is mostly O-glycosylated and contains a few N-linked oligosaccharide side chains (approx. 10 kDa). We propose that hamster oviductin is a mucin-type glycoprotein which might act as a protective secretion influencing the first steps of the reproductive process necessary for the normal triggering of fertilization and early embryonic development.