First published online June 28, 2002; 10.1104/pp.003426
Plant Physiol, August 2002, Vol. 129, pp. 1795-1806
Up-Regulation of Phosphoinositide Metabolism in Tobacco Cells
Constitutively Expressing the Human Type I Inositol Polyphosphate
5-Phosphatase1
Imara Y.
Perera,*
John
Love,2
Ingo
Heilmann,3
William F.
Thompson, and
Wendy F.
Boss
Department of Botany, North Carolina State University, Raleigh,
North Carolina 27695
To evaluate the impact of suppressing inositol
1,4,5-trisphosphate (InsP3) in plants, tobacco
(Nicotiana tabacum) cells were transformed with the
human type I inositol polyphosphate 5-phosphatase (InsP 5-ptase), an
enzyme which specifically hydrolyzes InsP3. The transgenic
cell lines showed a 12- to 25-fold increase in InsP 5-ptase activity in
vitro and a 60% to 80% reduction in basal InsP3 compared
with wild-type cells. Stimulation with Mas-7, a synthetic analog of the
wasp venom peptide mastoparan, resulted in an approximately
2-fold increase in InsP3 in both wild-type and transgenic
cells. However, even with stimulation, InsP3 levels in the
transgenic cells did not reach wild-type basal values, suggesting that
InsP3 signaling is compromised. Analysis of whole-cell lipids indicated that phosphatidylinositol 4,5-bisphosphate
(PtdInsP2), the lipid precursor of InsP3, was
greatly reduced in the transgenic cells. In vitro assays of enzymes
involved in PtdInsP2 metabolism showed that the activity of
the PtdInsP2-hydrolyzing enzyme phospholipase C was not
significantly altered in the transgenic cells. In contrast, the
activity of the plasma membrane PtdInsP 5 kinase was increased by
approximately 3-fold in the transgenic cells. In vivo labeling studies
revealed a greater incorporation of 32P into
PtdInsP2 in the transgenic cells compared with the wild type, indicating that the rate of PtdInsP2 synthesis was
increased. These studies show that the constitutive expression of the
human type I InsP 5-ptase in tobacco cells leads to an up-regulation of
the phosphoinositide pathway and highlight the importance of PtdInsP2 synthesis as a regulatory step in this system.
1
This work was supported in part by the North
Carolina State University-National Aeronautics and Space Administration
Specialized Center of Research and Training (grant no. NAGW-4984), by
the North Carolina Agricultural Research Service (grant to W.F.T. and
W.F.B.), and by the National Aeronautics and Space Administration (grant no. NAG2-1502 to I.Y.P. and W.F.B.).
2
Present address: Department of Plant Sciences,
University of Cambridge, Downing Street, Cambridge CB2 3EA, UK.
3
Present address: Department of Biology, Brookhaven
National Laboratory, 50 Bell Avenue, Upton, NY 11973.
*
Corresponding author; e-mail imara_perera{at}ncsu.edu; fax
919-515-3436.
© 2002 American Society of Plant Physiologists
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