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First published online July 18, 2002; 10.1104/pp.002360 Plant Physiol, August 2002, Vol. 129, pp. 1664-1673 In Vivo and in Vitro Phosphorylation of Membrane and Soluble Forms of Soybean Nodule Sucrose Synthase1Department of Biochemistry (O.K., G.S., R.C.) and Center for Biotechnology (Y.Z., G.S.), University of Nebraska, Lincoln, Nebraska 68588-0664
Sucrose synthase (SS) is a known phosphoserine
(SerP)-containing enzyme in a variety of plant "sink" organs,
including legume root nodules, where it is phosphorylated
primarily at Ser-11. Using immunofluorescence confocal microscopy, we
documented that part of the total SS (nodulin-100) pool in mature
soybean (Glycine max) nodules is apparently associated
with the plasma membrane in situ, and we report that this association
is very "tight," as evidenced by a variety of chemical and
enzymatic pretreatments of the isolated microsomal fraction. To
investigate the in situ and in planta phosphorylation state of the
membrane (m) and soluble (s) forms of nodule SS, three complementary
approaches were used. First, excised nodules were radiolabeled in situ
with [32P]Pi for subsequent analysis of phosphorylated m-
and s-SS; second, immunopurified s- and m-SS were used as substrate in
"on-bead" assays of phosphorylation by nodule
Ca2+-dependent protein kinase; and third, SS-Ser-11(P)
phosphopeptide-specific antibodies were developed and used. The
collective results provide convincing evidence that microsomal
nodulin-100 is phosphorylated in mature nodules, and that it is
hypophosphorylated relative to s-SS (on an equivalent SS protein basis)
in attached, unstressed nodules. Moreover, the immunological data and
related phosphopeptide mapping analyses indicate that a homologous
N-terminal seryl-phosphorylation domain and site reside in microsomal
nodulin-100. We also observed that mild, short-term inorganic
nitrogen and salt stresses have a significant negative impact on
the content and N-terminal phosphorylation state of nodule m- and s-SS,
with the former being the more sensitive of the two SS forms.
1 This work was supported in part by the U.S. National Science Foundation (grant no. MCB-9727236 to R.C.). This is no. 13,595 in the University of Nebraska Agricultural Research Division journal series. 2 Present address: Department of Computer Science and Engineering, University of Nebraska, Ferguson Hall, Lincoln, NE 68588-0115. * Corresponding author; e-mail RCHOLLET1{at}unl.edu; fax 402-472-7842. © 2002 American Society of Plant Physiologists |
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