Cloning and characterization of a thermal hysteresis (antifreeze) protein with DNA-binding activity from winter bittersweet nightshade, Solanum dulcamara

The gene for a thermal hysteresis (antifreeze) protein (sthp-64) from the bittersweet nightshade, Solanum dulcamara, was cloned and characterized. An expression cDNA library prepared from November S. dulcamara was screened using a polyclonal antibody generated against a previously purified 67 kDa thermal hysteresis protein, and positive clones were identified and sequenced. The full-length thermal hysteresis protein gene was cloned into an Escherichia coli expression vector and expressed as a fusion protein. The putative thermal hysteresis protein (STHP-64) contains two conserved regions 56 and 57 amino acids in length which have the C-X-4-C-X(2)2-23-H-X-1-H zinc finger motif which is present in WRKY proteins, a family of transcription factors which play a role in regulating expression of pathogenesis-related proteins in plants. Additional features of transcription factors, such as an acidic domain between the two zinc-fingers and a glutamine-rich region upstream of the first zinc-finger are also present in STHP-64. A DNA binding assay showed that the expressed STHP-64 fusion protein has specific DNA-binding ability. A unique feature of STHP-64 is that the C-terminus contains 10 consecutive 13-mer repeats. Such repeats are a common feature of animal antifreeze proteins. The expressed STHP-64 fusion protein had low levels of thermal hysteresis activity, but this activity was considerably increased by addition of citrate, which is known as an enhancer of certain insect antifreeze proteins. Northern blots demonstrated that the STHP-64 transcript was not present in leaves until November and December, suggesting that cold acclimation induces STHP-64 production.