Plant Molecular Biology 38: 531–538, 1998.
© 1998 Kluwer Academic Publishers. Printed in the Netherlands.
Cloning and characterization of cold-regulated glycine-rich RNA-binding
protein genes from leafy spurge (Euphorbia esula L.) and comparison to
heterologous genomic clones
David P. Horvath and Prudence A. Olson
U.S. Department of Agriculture, Agricultural Research Service, Biosciences Research Laboratory, State University
Station, Fargo, ND 58105-5674, USA
Received 16 December 1997; accepted in revised form 10 April 1998
Key words: cold acclamation, gene regulation, glycine-rich RNA-binding protein
Leafy spurge (Euphorbia esula) is a perennial weed which is capable of acclimating to sub-freezing temperatures.
We have used the differential display technique to identify and clone a cDNA for a cold-regulated gene (cor20)
which hybridizes to mRNAs that accumulate speciﬁcally during the cold acclamation process. The cor20 cDNA
was used to isolate two different genomic clones. Both clones were similar but not identical to each other and the
cDNA. Sequence analysis of the genomic clones indicated that they share considerable homology to a group of
glycine-rich RNA-binding protein genes. Comparison of the promoter region from the three clones (Ccr1 from
Arabidopsis, BnGRP1O from Brassica napus,andGRRBP2 from Euphorbia esula) have identiﬁed at least two
conserved motifs. CAGC is most likely involved in cold regulation and AACCCYAGTTA, is conserved but has
no known function. RNAs which hybridize to cor20 reach maximal expression in less than 2 days after exposure
of the plant to temperatures of 5
C, and remains at high levels in the plant for at least 30 days so long as the
plant is left in the cold. These RNAs drop to control levels within 24 h when the plant is returned to normal
growing temperatures. Transcripts which hybridize to cor20 do not accumulate under conditions of drought or heat
stress. These transcripts are induced in response to low temperatures in roots, stems and leaves, but are expressed
constitutively in tissue culture at control temperatures.
Euphorbia esula, commonly known as leafy spurge, is
a noxious perennial weed that infests 2.5 million acres
of land across the northern United States and Canada
. A primary feature of this plant is the existence of
numerous axillary buds on its stem, crown and roots.
Crown and root buds are capable of cold hardening
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The nucleotide sequence data reported will appear in the EMBL,
GenBank and DDBJ Nucleotide Sequence Databases under the
accession numbers AF03639 (GRRBP1) and AF031933 (GRRBP2).
and provide the plant with active meristems after win-
ter kill of the above-ground portions of the plant .
These subterranean buds are thus responsible for the
perennial nature of leafy spurge. Consequently, under-
standing the mechanisms that control cold hardening
in root and crown buds could aid in developing mea-
sures for controlling leafy spurge and other perennial
weeds with similar survival mechanisms.
The biochemical changes correlated with cold ac-
climation have been extensively studied [13, 20, 22].
Increases in solutes such as carbohydrates and certain
amino acids, changes in the lipid composition of the
cellular membranes and changes in gene expression
have been shown to occur in all plants that have been
studied [13, 20, 22]. However, little is known about
the mechanisms by which plants sense low temper-