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We report here the identification and characterization of a new leaf-specific light-stimulated gene induced during cold acclimation of wheat. Sequence analysis revealed that the gene encodes a protein of 19 kDa with a pI of 8.8. This is a novel protein with a particular charge distribution. The C-terminal half has a high propensity to form an alpha-helix and contains all the acidic amino acids with a net negative charge of -7. On the other hand, the N-terminal half is rich in proline, lysine and arginine with a net positive charge of +10. These properties are commonly found in several transcription factors. The protein is also rich in alanine (21%), is hydrophilic but not boiling soluble in contrast to other alanine-rich proteins. During low temperature exposure, the corresponding mRNA accumulates rapidly in the leaf and remains at a constant level in two tolerant cultivars used. However, in a less tolerant cultivar, the mRNA level declines despite maintaining the plants at 4 degrees C. Southern blot analysis indicates that the differential expression in the less tolerant genotype is not due to a different genomic organization or gene copy number. The mRNA was specifically localized in leaf tissues and increased several-fold during the greening at 4 degrees C. Furthermore, this gene is not induced in callus cultures acclimated in the absence or presence of light. This suggests that the full expression of this gene is dependent on organized leaf tissue. The expression of this gene was not affected by ABA, drought, heat shock, salinity, wounding or anaerobiosis, demonstrating that it is specifically induced by low temperature. The Wcs19 mRNA is preferentially expressed in tolerant Gramineae species.

Low temperature is one of the primary stresses limiting the growth and productivity of wheat (Triticum aestivum L.) and rye (Secale cereale L.). Winter cereals low-temperature-acclimate when exposed to temperatures colder than 10°C. However, they gradually lose their ability to tolerate below-freezing temperatures when they are maintained for long periods of time in the optimum range for low-temperature acclimation. The overwinter decline in low-temperature response has been attributed to an inability of cereals to maintain low-temperature-tolerance genes in an up-regulated state once vernalization saturation has been achieved. In the present study, the low-temperature-induced Wcs120 gene family was used to investigate the relationship between low-temperature gene expression and vernalization response at the molecular level in wheat and rye. The level and duration of gene expression determined the degree of low-temperature tolerance, and the vernalization genes were identified as the key factor responsible for the duration of expression of low-temperature-induced genes. Spring-habit cultivars that did not have a vernalization response were unable to maintain low-temperature-induced genes in an up-regulated condition when exposed to 4°C. Consequently, they were unable to achieve the same levels of low-temperature tolerance as winter-habit cultivars. A close association between the point of vernalization saturation and the start of a decline in the Wcs120 gene-family mRNA level and protein accumulation in plants maintained at 4°C indicated that vernalization genes have a regulatory influence over low-temperature gene expression in winter cereals.

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Low-temperature (LT) induced genes of the Wcs120 family in wheat (Triticum aestivum) were mapped to specific chromosome arms using Western and Southern blot analysis on the ditelocentric series in the cultivar Chinese Spring (CS). Identified genes were located on the long arms of the homoeologous group 6 chromosomes of all 3 genomes (A, B, and D) of hexaploid wheat. Related species carrying either the A, D, or AB genomes were also examined using Southern and Western analysis with the Wcs120 probe and the WCS120 antibody. All closely related species carrying one or more of the genomes of hexaploid wheat produced a 50 kDa protein that was identified by the antibody, and a Wcs120 homoeologue was detected by Southern analysis in all species. In the absence of chromosome arm 6DL in hexaploid CS wheat no 50 kDa protein was produced and the high-intensity Wcs120 band was missing, indicating 6DL as the location of Wcs120 but suggesting silencing of the Wcs120 homoeologue in the A genome. Levels of proteins that cross-reacted with the Wcs120 antibody and degrees of cold tolerance were also investigated in the Chinese Spring/Cheyenne (CS/CNN) chromosome substitution series. CNN chromosome 5A increased the cold tolerance of CS wheat. Densitometry scanning of Western blots to determine protein levels showed that the group 5 chromosome 5A had a regulatory effect on the expression of the Wcs120 gene family located on the group 6 chromosomes of all three hexaploid wheat genomes.