Evidence for a Slow-Turnover Form of the
Ca2+-Independent Phosphoenolpyruvate
Carboxylase Kinase in the Aleurone-Endosperm Tissue of Germinating
Barley Seeds1
Lidia Osuna,
Jean-Nöel Pierre,
María-Cruz González,
Rosario Alvarez,
Francisco J. Cejudo,
Cristina Echevarría*, and
Jean Vidal
Departamento de Biología Vegetal, Facultad de
Biología, Universidad de Sevilla, Avenida Reina Mercedes no. 6, 41012 Sevilla, Spain (L.O., R.A., C.E.); Instituto de
Bioquímica Vegetal y Fotosíntesis, Centro de
Investigaciones Científicas "Isla de la Cartuja," Avda
Américo Vespucio s/n, 41092 Sevilla, Spain (M.-C.G., F.J.C.); and Institut de Biotechnologie des Plantes, Unité Associée
Centre National de la Recherche Scientifique, D 1128, Bâtiment 630, Université de Paris-Sud, Centre d
Orsay,
cedex, France (J.-N.P., J.V.)
Phosphoenolpyruvate
carboxylase (PEPC) activity was detected in aleurone-endosperm extracts
of barley (Hordeum vulgare) seeds during germination,
and specific anti-sorghum (Sorghum bicolor) C4 PEPC polyclonal antibodies immunodecorated constitutive
103-kD and inducible 108-kD PEPC polypeptides in western analysis. The 103- and 108-kD polypeptides were radiolabeled in situ after imbibition for up to 1.5 d in 32P-labeled inorganic phosphate. In
vitro phosphorylation by a Ca2+-independent PEPC protein
kinase (PK) in crude extracts enhanced the enzyme's velocity and
decreased its sensitivity to L-malate at suboptimal pH and
[PEP]. Isolated aleurone cell protoplasts contained both
phosphorylated PEPC and a Ca2+-independent PEPC-PK that was
partially purified by affinity chromatography on blue dextran-agarose.
This PK activity was present in dry seeds, and PEPC phosphorylation in
situ during imbibition was not affected by the cytosolic
protein-synthesis inhibitor cycloheximide, by weak acids, or by various
pharmacological reagents that had proven to be effective blockers of
the light signal transduction chain and PEPC phosphorylation in
C4 mesophyll protoplasts. These collective data support the
hypothesis that this Ca2+-independent PEPC-PK was formed
during maturation of barley seeds and that its presumed underlying
signaling elements were no longer operative during germination.
1
This research was supported by grants from the
Dirección General de Investigación Científica y
Técnica (no. PB 92-0675), the Dirección General de Ensenan
za Superior e Investigacion Científica (no. B1097-1205-C02-02),
and the Acción Integrada Hispano-Francesa (no. HF96-0200), and by
the Grupo de Investigación de Fisiología Vegetal from La
Junta de Andalucía. This work was also partially supported by
the Rhone-Poulenc Agro S.A., Torre de la Reina, Sevilla, Spain.
*
Corresponding author; e-mail echeva{at}cica.es; fax
34-95-461-5780.
Plant Physiol. (1999) 119: 511-520
Copyright Clearance Center: 0032-0889/99/119//10
© 1999 American Society of Plant Physiologists
