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In winter wheat, low temperature hardening induces a non-specific resistance to snow moulds and other fungi. In a suppressive subtractive hybridization cDNA library between winter wheat unhardened and hardened at 2°C, 17% of 186 sequenced cDNA clones were homologous to the small plant defence related proteins, γ-thionin, γ-purothionin or non-specific lipid transfer proteins (LTPs), based on their nucleotide sequences or deduced from amino acid sequences. Several partial- and full-length cDNA clones homologous to two defensin proteins (γ-thionin, subfamily B-2, γ-purothionin, subfamily I), and non-specific LTPs were isolated. Regulation of defensin transcripts was clearly different from that of the LTPs under controlled environments in the field. The γ-thionin and γ-purothionin transcripts were not expressed in unhardened plants grown at 20°C, strongly upregulated after 1–3 days hardening at 2°C, remained upregulated for 28 days hardening, and disappeared following 1 day of exposure to dehardening conditions at 20°C. The LTPs transcripts were constitutively expressed in plants growing at 20°C, gradually increased to maximum levels following 14–28 days at hardening, and remained upregulated following 1 and 7 days dehardening. In the field, the γ-thionin and γ-purothionin transcripts rapidly increased to maximum levels by mid-November and decreased gradually during the winter until mid-March LTP transcripts were highly expressed in the early autumn, further increased during December–January, then decreased during late winter and early spring. Differences in the pattern and level of expression of these transcripts were evident among cultivars; γ-thionin expression was associated with freezing resistance among the genotypes tested under both controlled environment and field conditions. |
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