Plant Molecular Biology 42: 923–936, 2000.
© 2000 Kluwer Academic Publishers. Printed in the Netherlands.
Characterization of a small GTP-binding protein of the rab 5 family in
Mesembryanthemum crystallinum with increased level of expression during
early salt stress
, Karin Schiene
and Karl-Josef Dietz
Department of Physiology and Biochemistry of Plants,
Genetics Department, Faculty of Biology, Uni-
versity of Bielefeld, P.O. Box 100131, 33501 Bielefeld, Germany (
author for correspondence; e-mail:
email@example.com; fax: +49 521 106 6039)
Received 13 July 1999; accepted in revised form 17 February 2000
Key words: Mesembryanthemum crystallinum, small GTPase, salt stress
A cDNA encoding a member of the Ypt/Rab family of small GTP-binding proteins was cloned from the facultative
CAM plant Mesembryanthemum crystallinum. Mcrab5b includes an open reading frame of 201 amino acids. The
deduced amino acid sequence shows 91% similarity to LjRAB5b isolated from Lotus japonicus. The amino acid
sequence of McRAB5b provides interesting features suggesting that McRAB5b and its homologue from Lotus
japonicus represent a new subclass of Ypt/Rab proteins. The fact that proteins like McRAB5b and LjRAB5b
were only found in plants and not in yeast or vertebrates suggests that they have plant-speciﬁc functions. The
expression of Mcrab5b as investigated by northern blot hybridization and RT-PCR was stimulated under salt stress.
After heterologous expression in Escherichia coli an antibody was raised against recombinant McRAB5b protein.
Western blot analysis revealed that McRAB5b was bound to membranes. It is present in a monomeric and a dimeric
form in vitro and in vivo. In vitro only the monomeric protein exhibits a binding capacity for radiolabelled GTP,
while the dimer is unable to do so, indicating that the activity may be regulated by monomer/dimer transition.
Abbreviations: GAP, GTPase-activating protein; GDI, guanine nucleotide dissociation inhibitor; GEF, guanine
nucleotide exchange factors; SGP, small GTP-binding protein
Mesembryanthemum crystallinum L. (common ice
plant; Aizoaceae, Caryophyllales) is a facultativehalo-
phyte that shifts from C3 photosynthesis to crassu-
lacean acid metabolism (CAM) in response to envi-
ronmental stress or as part of its developmental pro-
gramme. The plant tolerates high salt concentrationsin
the soil and has an efﬁcient mechanism of salt seques-
tration into the vacuole and into the epidermal bladder
cells. In addition to the physiological and genetic
changes, stress conditions also induce considerable
The nucleotide sequence data reported will appear in the EMBL,
GenBank and DDBJ Nucleotide Sequence Databases under the
accession number AJ006255.
changes in plant morphology (Adams et al., 1998).
Due to its metabolic versatility and small genome size
(390 Mb), M. crystallinum has become a model plant
for physiological and genetic studies of salt tolerance.
The adaptation to salt stress involves changes in
enzyme activities and gene expression. Salt-dependent
up-regulation of gene expression is observed, for
example, for several V-ATPase subunits (Dietz and
Arbinger, 1996; Löw et al., 1996; Tsiantis et al.,
1996), for phosphoenolpyruvate carboxylase (Cush-
man et al., 1989), for pyruvate orthophosphate
dikinase (Fisslthaler et al., 1995) and for a ser-
ine/threonine kinase (Baur et al., 1994). Many other
genes have been found to respond to salt stress but yet
little is known about signalling events involved in the