Plant Molecular Biology 46: 539–548, 2001.
© 2001 Kluwer Academic Publishers. Printed in the Netherlands.
Photoafﬁnity labelling with the cytokinin agonist azido-CPPU of a 34 kDa
peptide of the intracellular pathogenesis-related protein family in the
moss Physcomitrella patens
ere Pagant, Florent Brun and Michel Laloue
Institut National de la Recherche Agronomique, Laboratoire de Biologie Cellulaire, INRA-Versailles-Grignon,
78026 Versailles-Cedex, France (
author for correspondence; e-mail firstname.lastname@example.org)
Received 24 August 2000; accepted in revised form 6 March 2001
Key words: allergen, cytokinin, IPR protein, photoafﬁnity labeling, Physcomitrella patens
As in higher plants, the development of the moss Physcomitrella patens is regulated by environmental signals and
phytohormones. At the protonema level transition from chloronema to caulonema cells is under auxin control.
The formation on second sub-apical caulonema cells of buds that will give rise to the leafy gametophore requires
cytokinins. Using [
H])phenylurea), a photoactivatable cy-
tokinin agonist, we have speciﬁcally photolabelled a soluble 34 kDa protein of P. patens. Urea derivatives were
very efﬁcient competitors of photolabelling while purine-type cytokinins were poor competitors. The protein
UBP34 was puriﬁed by afﬁnity chromatography and the sequences of six internal peptides obtained. A cDNA
encoding UBP34 was cloned by screening a P. patens protonema cDNA library with a probe ampliﬁed by PCR
using degenerate primers designed from the peptide sequences. The UBP34 amino acid sequence shows an average
sequence identity of 42% with both intracellular PR proteins and the BetV1-related family of plant allergens.
Recombinant UBP34 expressed in Escherichia coli was conﬁrmed to bind azidoCPPU.
Abbreviations: azidoCPPU, 1-(2-azido-6-chloropyrid-4-yl)-3-phenylurea; 2,4-D, 2,4-dichlorophenoxyacetic acid;
CPPU, 1-(2-chloropyrid-4-yl)-3-phenylurea; Diuron, (dichloro-3,4-phenyl)-3-dimethyl-1,1-urea; PPP, 3-methyl-
7-(n-pentylamino)pyrazolo[4,3-d]pyrimidine; PU 42, 1-(2,6-dichloropyrid-4-yl)-3-phenylurea; PU 60, 1-(2-
chloropyrid-4-yl-N-oxide)-3-phenylurea; R-MeBA and S-MeBA, 6-(α-methylbenzylamino)purine (R and S
The mechanisms by which cytokinins act at the mole-
cular level still remain largely unknown, despite their
determinant role in plant growth and development
(Faure and Howell, 1999; Kakimoto, 1998).
Numerous cytokinin-binding proteins have been
isolated in various organisms by biochemical meth-
ods (Brault et al., 1997; Kobayashi et al., 2000) but
their physiological functions remain to be established.
Kakimoto (1996) used an activation tagging strategy
to identify cytokinin-independent (cki) mutants. The
CKI1 gene encodes a two-component regulator similar
to the ethylene receptor ETR1 (Bleecker, 1999), and
Kakimoto proposed that CKI1 could be involved in the
cytokinin signalling cascade.
Using the cytokinin agonist azidoCPPU we
have already speciﬁcally photoafﬁnity-labelled a glu-
tathione S-transferase (Gonneau et al., 1998) from
Nicotiana plumbaginifolia and a ﬁbrilin homologue
present in pea thylakoids (Nogué et al., 1996), but the
involvement of these proteins in cytokinin signalling
is still questionable.
We chose the moss Physcomitrella patens as a
model to study the molecular basis of cytokinin action
and more speciﬁcally to identify binding proteins in-