An osmotin-like cryoprotective protein from the bittersweet nightshade Solanum dulcamara

Cold acclimation in plants is a polygenic phenomenon involving increased ex pression of several genes. The gene products participate either directly or indirectly towards increasing cold tolerance. Evidence of proteins having a direct effect on cold tolerance is emerging but limited. With isolated pr otoplasts from warm-grown kale (Brassica oleracea) as a model system, we te sted protein fractions from winter bittersweet nightshade, Solanum dulcamar a, stems for the presence of proteins that have a cryoprotective effect. Pu rification of one such fraction resulted in isolation of a 25 kDa protein. N-terminal Edman degradation amino acid sequence analysis showed that it ha s high homology to osmotin and osmotin-like proteins. When added to warm-gr own protoplasts, it increased the cryosurvival of frozen-thawed protoplasts by 24% over untreated or BSA-treated controls at -8 degreesC. A cDNA libra ry which was made in November from stems and leaves of S. dulcamara was suc cessfully screened for the corresponding cDNA clone. The deduced amino acid sequence indicated that the protein consists of 206 amino acid residues in cluding a N-terminal signal sequence and a putative C-terminal propeptide. The mature protein, without the N-terminal signal sequence, was expressed i n Escherichia coli. The partially purified protein in the supernatant fract ion of the culture medium had cryoprotective activity.