Plant Physiol, April 2002, Vol. 128, pp. 1368-1378
Pyruvate,Orthophosphate Dikinase in Leaves and Chloroplasts of
C3 Plants Undergoes Light-/Dark-Induced Reversible
Phosphorylation1
Chris J.
Chastain,*
Jason P.
Fries,
Julie A.
Vogel,
Christa L.
Randklev,
Adam P.
Vossen,2
Sharon K.
Dittmer,
Erin
E.
Watkins,
Lucas J.
Fiedler,
Sarah A.
Wacker,
Katherine C.
Meinhover,
Gautam
Sarath, and
Raymond
Chollet
Department of Biology, Minnesota State University, Moorhead,
Minnesota 56563 (C.J.C., J.P.F., J.A.V., C.L.R., A.P.V., S.K.D.,
E.E.W., L.J.F., S.A.W., K.C.M.); and Department of Biochemistry, George
W. Beadle Center, University of Nebraska, Lincoln, Nebraska 68588-0664
(G.S., R.C.)
Pyruvate,orthophosphate (Pi) dikinase (PPDK) is best
recognized as a chloroplastic C4 cycle enzyme. As one of
the key regulatory foci for controlling flux through this
photosynthetic pathway, it is strictly and reversibly regulated by
light. This light/dark modulation is mediated by reversible
phosphorylation of a conserved threonine residue in the active-site
domain by the PPDK regulatory protein (RP), a bifunctional protein
kinase/phosphatase. PPDK is also present in C3 plants,
although it has no known photosynthetic function. Nevertheless, in this
report we show that C3 PPDK in leaves of several
angiosperms and in isolated intact spinach (Spinacia oleracea) chloroplasts undergoes light-/dark-induced changes in phosphorylation state in a manner similar to C4 dikinase.
In addition, the kinetics of this process closely resemble the
reversible C4 process, with light-induced dephosphorylation
occurring rapidly (
15 min) and dark-induced phosphorylation occurring
much more slowly (
30-60 min). In intact spinach chloroplasts,
light-induced dephosphorylation of C3 PPDK was shown to be
dependent on exogenous Pi and photosystem II activity but independent
of electron transfer from photosystem I. These in organello results
implicate a role for stromal pools of Pi and adenylates in regulating
the reversible phosphorylation of C3-PPDK. Last, we used an
in vitro RP assay to directly demonstrate ADP-dependent PPDK
phosphorylation in desalted leaf extracts of the C3 plants
Vicia faba and rice (Oryza sativa). We
conclude that an RP-like activity mediates the light/dark modulation of
PPDK phosphorylation state in C3 leaves and chloroplasts and likely represents the ancestral isoform of this unusual and key
C4 pathway regulatory "converter" enzyme.
1
This work was supported in part by the U.S.
National Science Foundation (grant nos. RUI-0094497 to C.J.C. and
MCB-9727236 to R.C.) and by the Center for Biotechnology, University
of Nebraska, Lincoln, funded through the Nebraska Research Initiative
(to G.S.).
2
Present address: University of Minnesota Medical
School, 420 Delaware Street, S.E., Minneapolis, MN 55455-0310.
*
Corresponding author; e-mail chastain{at}mhd1.mnstate.edu; fax
218-236-2168.
© 2002 American Society of Plant Physiologists
