Stabilisation of plant membranes during freezing by
specific proteins
D.K. Hincha, B. Neukamm, F. Sieg and J.M. Schmitt,
Pflanzenphysiologie, Freie Universität Berlin, Germany
We have developed in vitro-assays using isolated spinach
thylakoid membranes, to assess the cryoprotective activity
of proteins. When thylakoids from nonacclimated plants are
frozen and thawed under damaging conditions, they rupture
and collapse. Collapsed thylakoid membranes can be
distinguished from intact membranes by a volumetric
hematocrit centrifugation assay and by immunological
determination of the loss of the lumenal protein
plastocyanin. We have isolated a cryoprotective protein
(cryoprotectin) from cold acclimated cabbage (Brassica
oleracea) leaves. Analysis by SDS-PAGE and silver staining
revealed a single polypeptide of 7 kD in the most active
fractions. Gel filtration chromatography confirmed that the
cryoprotective activity is associated with a protein of 7
kD under nondenaturing conditions. This protein could be
isolated only from cold acclimated leaves, but not from
plants grown under nonacclimating conditions. N-terminal
sequencing and comparison with sequence data banks revealed
a high degree of sequence identity with several plant lipid
transfer proteins, especially with the family of WAX
proteins from Brassica. An isolated WAX protein from the
cuticle of cabbage leaves had lipid transfer activity for
fluorescently labeled phosphatidylcholine, but no
cryoprotective activity. Conversely, cryoprotectin showed
no lipid transfer activity. It can be concluded that these
structurally very similar proteins are functionally
different in vitro and may also play distinct roles in
plant metabolism.
Abstract 1998