Plant Molecular Biology 34: 897–911, 1997.
1997 Kluwer Academic Publishers. Printed in Belgium.
Cloning and characterization of the calreticulin gene from
Ricinus communis L.
Sean J. Coughlan
, Craig Hastings and Ron Winfrey Jr.
Trait and Technology Development Department, Pioneer-Hi-Bred International, P.O. 1004, 7300 NW 62ND
Avenue, Johnston, IA 50131-1004, USA (
author for correspondence)
Received 17 October 1996; accepted in revised form 1 May 1997
Key words: calreticulin, gene characterization, Ricinus communis L.
endosperm cDNA library with antisera raised to the total lumenal fraction of puriﬁed plant endoplasmic reticulum.
The calcium-bindingproperties ofthe recombinant protein were characterized and shown to be essentially identical
to those reported for the mammalian calreticulin. Calcium overlays and immune blot analysis conﬁrmed the
endoplasmic lumenal identity of this reticuloplasmin. Probing protein blots of endoplasmic reticulum subfractions
with radio-iodinatedcalreticulin showed speciﬁc associations with various polypeptides including one identiﬁed as
the abundant reticuloplasmin protein disulﬁde isomerase.
Characterization of the corresponding genomic clones revealed that calreticulin is encoded by a single gene of
3 kb in castor. The full genomic sequence reveals the presence of 12 introns, 12 translated exons, and one exon
containing the last three amino acids of the translated sequenceand the 3
-untranslatedregion of the gene. Northern
blot analysis of RNA isolated from variousorgan tissues showed a basal constitutivelevel of expression throughout
the plant, but more abundant mRNA being detected in tissues active in secretion. This was conﬁrmed by analysis
of transgenic tobacco plants containing 1.8 kb of 5
-untranslated genomic sequence fused to the
reporter gene (GUS) showed a more localized pattern of expression. Activity being localized to the vasculature
(phloem, root hairs and root tip) in vegetativetissue, and being strongly expressed in the ﬂoral organsincluding the
developing and germinating seed.
The endoplasmic reticulum is one of the most impor-
tant organelles of the higher-plant cell, comprising as
it does the initial part of the protein secretory pathway
. It is also the site of the majority of fatty acid mod-
iﬁcation and triacylglycerol biosynthesis , and is an
important intracellular store of reversibly bound calci-
tion . In developing seed storage tissue, the endo-
plasmic reticulum is the primary site of seed storage
protein synthesis, folding and assembly . These
Thenucleotidesequencedatareportedwillappear in theEMBL,
GenBank and DDBJ Nucleotide Sequence Databases under the
accession numbers 74764 (castor calreticulin promoter
74752 (castor calreticulin cDNA).
processesrequiresthe coordinatedaction of an arrayof
chaperones, both soluble and membrane-bound .
The soluble ER lumenal proteins collectively known
numerous plant homologues have been cloned includ-
ing BIP , PDI , GRP94 , and calreticulin
been implicated in the mechanism of protein assembly
, and protein disulﬁde bond formation. Calretic-
ulin, in contrast, has been considered to be the primary
calcium-bindingprotein of the endoplasmicreticulum,
has recently been presentedthat this protein might also
have a role in protein folding and/or assembly [20, 38,
41] as well as signal transducing roles based on its
calcium-binding properties [9, 49].