ISSN 1021-4437, Russian Journal of Plant Physiology, 2008, Vol. 55, No. 2, pp. 193–200. © Pleiades Publishing, Ltd., 2008.
Original Russian Text © E.O. Fedina, F.G. Karimova, I.A. Tarchevsky, I.Yu. Toropygin, V.A. Khripach, 2008, published in Fiziologiya Rastenii, 2008, Vol. 55, No. 2, pp. 210–218.
Phosphorylation is an effective and widely spread
means for modulation of enzyme and protein activities.
Protein phosphorylation usually occurs on serine, thre-
onine, histidine, and tyrosine residues . Tyrosine
phosphorylation in plants is investigated to lesser extent
than phosphorylation on serine and threonine residues.
Nevertheless, substantial evidence has accumulated on
the involvement of this phosphorylation in the regula-
tion of plant metabolism. Recent studies have demon-
strated signiﬁcant contribution of tyrosine phosphory-
lation in the regulation of cell metabolism .
Comparatively scarce studies were devoted to phos-
phorylation–dephosphorylation of enzymes involved in
photosynthetic assimilation of carbon dioxide. It was
found that some Calvin cycle enzymes could be phos-
phorylated on serine and threonine . There is only
one report showing that small subunits of Rubisco can
be phosphorylated on serine and tyrosine residues.
Dephosphorylation of these amino acids resulted in dis-
sociation of small and large Rubisco subunits and
diminished the Rubisco activity .
Activities of Rubisco and other Calvin cycle
enzymes can be controlled through phytohormone-
induced changes in their phosphorylation. Brassino-
steroids are plant cell metabolites that are structurally
similar to animal steroid hormones. One of the most
active brassinosteroids is 24-epibrassinolide (EPB). It
induces a large range of cell responses including plant
growth, seed germination, and nitrogen ﬁxation; it also
improves plant resistance to cold, pathogens, and salt
stress [5, 6]. The mutants defective in EPB metabolism
are characterized by dwarf traits, low biomass, and low
fruit yield . Modern studies aim primarily at elucida-
tion of molecular mechanisms of EPB signaling. These
works revealed EPB receptors in the plasma mem-
branes and their phosphorylation on Ser/Thr residues
. Other pathways can be also implicated in brassi-
nosteroid signaling . The kinase responsible for
phosphorylation of nuclear proteins was identiﬁed ,
and a new class of transcription factors involved in
Effect of Epibrassinolide on Tyrosine Phosphorylation
of the Calvin Cycle Enzymes
E. O. Fedina
, F. G. Karimova
, I. A. Tarchevsky
, I. Yu. Toropygin
, and V. A. Khripach
Kazan Institute of Biochemistry and Biophysics, Kazan Research Center, Russian Academy of Sciences,
Kazan, a/ya 30, Tatarstan, 420111 Russia;
fax: 7 (843) 297-7347; e-mail: email@example.com
Orekhovich Institute of Biomedical Chemistry, Russian Academy of Medical Sciences, Moscow, Russia
Institute of Bioorganic Chemistry, National Academy of Sciences of Belarus, Minsk, Belarus
Received March 23, 2007
—Two-dimensional electrophoresis was used to separate proteins from crude extracts of pea (
L.) leaves, and thus isolated proteins were subjected to Western blot analysis with monoclonal antibod-
ies against PY20 phosphotyrosine polypeptides. This analysis revealed 44 polypeptides phosphorylated on
tyrosine residues. Phosphorylation of some of these proteins was changed under the action of epibrassinolide.
Some of these polypeptides were identiﬁed by means of MALDI-TOF MS analysis. The results indicate that
eight of these proteins belong to the Calvin cycle enzymes, namely, the isoforms of Rubisco large and small
subunits, fructose-1,6-phosphate aldolases 1 and 2, and the precursor of
-subunit of Rubisco-binding protein.
The observed changes in phosphorylation of these proteins may partly explain the effects of brassinosteroids
on photosynthesis. The tyrosine phosphorylation sites were identiﬁed
for the fragments of polypeptides
Key words: Pisum sativum - tyrosine phosphorylation - epibrassinolide - Rubisco - Rubisco-binding protein -
MALDI-TOF MS analysis
: 2D electrophoresis—two-dimensional electro-
1-propanesulfonate; DTT—dithiothreitol; EPB—24-epibrassino-
lide; IEF—isoelectric focusing; MALDI-TOF MS—matrix-
assisted laser desorption/ionization time-of-ﬂight mass spectrom-
etry; PMSF—phenylmethylsulfonyl ﬂuoride; PVDF mem-
branes—polyvinyldiﬂuoride membranes; TBST—Tris buffered
saline supplemented with Tween-20; TEMED—tetramethylethyl-
enediamine; TFA—triﬂuoroacetic acid.