Plant Molecular Biology 41: 233–243, 1999.
© 1999 Kluwer Academic Publishers. Printed in the Netherlands.
High expression level of a gene coding for a chloroplastic amino acid
selective channel protein is correlated to cold acclimation in cereals
, Maria Grossi, Nicola Pecchioni, Giampiero Val
e and Luigi Cattivelli
Istituto Sperimentale per la Cerealicoltura, Via S. Protaso 302, 29017 Fiorenzuola d’Arda (PC), Italy (
Received 14 October 1998; accepted in revised form 4 August 1999
Key words: 5
-RACE, cold resistance, cold-regulated gene, differential display, pore protein
A cold-regulated gene (cor tmc-ap3) coding for a putative chloroplastic amino acid selective channel protein was
isolated from cold-treated barley leaves combining the differential display and the 5
-RACE techniques. Cor tmc-
ap3 is expressed at low level under normal growing temperature, and its expression is strongly enhanced after
cold treatment. A positive correlation between the expression of cor tmc-ap3 and frost tolerance was found both
among barley cultivars and among cereal species. The COR TMC-AP3 protein was expressed in vitro, puriﬁed
and used to raise a polyclonal antibody. Western analysis showed that the cor tmc-ap3 gene product is localized to
the chloroplastic outer envelope fraction, supporting its putative function. The frost-resistant winter cultivar Onice
accumulated COR TMC-AP3 more rapidly and at a higher level than the frost-susceptible spring cultivar Gitane.
After 28 days of cold acclimation the winter cultivar had about 2-fold more protein than the spring genotype.
All these results suggest that an increased amount of a chloroplastic amino acid selective channel protein could
be required for cold acclimation in cereals. Hypotheses about the role of COR TMC-AP3 during the hardening
process are discussed.
Most plants growing in temperate climates develop
frost tolerance when exposed to low but non-freezing
temperatures. This process, known as cold accli-
mation or hardening, involves physiological as well
as molecular changes (Cattivelli and Bartles, 1992).
Manycold-regulated(COR) genesfrom differentplant
species have been cloned after the demonstration of
altered gene expression during the cold acclimation
process (Cattivelli and Bartles, 1990). The role of
many COR genes is still unknown, although for some
of them a putative function based on peculiar features
and/or homologies with other known genes has been
The accumulation of some COR proteins has been
related to the protection of cells from frost damage.
The nucleotide sequence data reported will appear in the
EMBL, GenBank and DDBJ Nucleotide Sequence Databases under
the accession number AJ011921 (cor tmc-ap3).
A number of COR gene products show the character-
istics of the dehydrin class of LEA proteins (Houde
et al., 1992; Welin et al., 1994; Grossi et al., 1995).
These proteins, which also accumulate during the late
stages of seed development, are believed to have a
role in cold acclimation so as to prevent dehydra-
tion of plant cells during extracellular ice formation
(Wolfraim et al., 1993), although it has also been
suggested that they may have a role in membrane pro-
tection (Danyluk et al., 1998). Recently, Hincha et al.
(1997)havefoundthatclass I β-1,3-glucanasepuriﬁed
from tobacco protects thylakoids against freeze-thaw
injury in vitro. They also report that a β-1,3-glucanase
isoform accumulated in spinach and cabbage during
cold hardening under natural conditions, suggesting
a cryo-protective role for this protein during cold
Other COR genes are thought to be involved in
the signal transduction pathway or to interact with
RNAs. Two related COR genes which show strong