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(Received for publication, January 25, 1996)
From the We provide evidence that phosphatidic
acid (PtdOH) formed during signaling in plants is metabolized by a
novel pathway. In much of this study,
32Pi-labeled Chlamydomonas cells
were used, and signaling was activated by adding the G-protein
activator mastoparan. Within seconds of activation, large amounts of
[32P]PtdOH were formed, with peak production at about 4 min, when the level was 5-25-fold higher than the control. As the
level of [32P]PtdOH subsequently decreased, an unknown
phospholipid (PLX) increased in radiolabeling; before activation it was
barely detectable. The chromatographic properties of PLX resembled
those of lyso-PtdOH and CMP·PtdOH but on close inspection were found
to be different. PLX was shown to be diacylglycerol pyrophosphate
(DGPP), the product of a newly discovered enzyme, phosphatidate kinase,
whose in vitro activity was described recently (Wissing, J. B., and Behrbohm, H. (1993) Plant Physiol. 102, 1243-1249). The identity of DGPP was established by co-chromatrography
with a standard and by degradation analysis as follows:
[32P]DGPP was deacylated, and the product
(glycerolpyrophosphate, GroPP) was hydrolyzed by mild acid treatment or
pyrophosphatase to produce GroP and Pi as the only
radioactive products. Since DGPP is the pyrophosphate derivative of
PtdOH and is formed as the concentration of PtdOH decreases, we assumed
that PtdOH was converted in vivo to DGPP. This was
confirmed by showing that during a short labeling protocol while the
specific radioactivity of DGPP was increasing, the specific
radioactivity of the 32Pi derived from DGPP as
above was higher than that of [32P]GroP. DGPP was also
formed in suspension cultures of tomato and potato cells, and its
synthesis was activated by mastoparan. Moreover, it was also found in
intact tissues of a number of higher plants, for example, carnation
flower petals, vetch roots, leaves of fig-leaved goosefoot, and common
persicaria and microspores of rape seed. Our results suggest that DGPP
is a common but minor plant lipid that increases in concentration when
signaling is activated. Possible functions of DGPP in phospholpase C
and D signaling cascades are discussed.
Volume 271, Number 26,
Issue of June 28, 1996
pp. 15708-15715
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
,
and
Institute for Molecular Cell Biology,
BioCentrum Amsterdam, University of Amsterdam, Kruislaan 318, NL-1098 SM, Amsterdam, The Netherlands, the ¶ Agrotechnological
Research Institute (ATO-DLO), P. O. Box 17, NL-6700 AA, Wageningen,
The Netherlands, and the 
Inositide Laboratory, The Babraham
Institute, Babraham, CB2 4AT, Cambridge, United Kingdom
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