Regulation of phosphatidylcholine biosynthesis in apium graveolens

Abstract

When grown in the presence of the sterol biosynthesis inhibitor, paclobutrazol, suspension cultures of Apium graveolens (celery) accumulate substantial amounts of I 4a-methylsterols, at the expense of 4-demethylsterols. These changes have been correlated with reduced synthesis of phosphatidylcholine (PC) via the CDP-base pathway (Roiph & Goad, 1991). It was subsequently proposed that changes in the membrane sterol composition of plant cells may regulate the activity of
CTP: cholinephosphate cytidylyltransferase (CT), the rate-determining enzyme of this pathway (Kinney & Moore, 1989).
In preliminary studies, the membrane-associated form of CT in A.graveolens, was found to exhibit optimal activity at pH 7.7, in the presence of 8.0 mM CTP and 3.5 mM Mg2t Microsomal membrane fractions, in which a large proportion of CT activity
was found to reside, were analysed in terms of lipid composition. The predominant phospholipid in such membranes, PC, constituted approximately 70% of the total phospholipid content. Other, more minor constituents, included phosphatidylethanolamine (PE), phosphatidylglycerol (PU), phosphatidylinositol (P1), phosphatidylserine (PS), and phosphatidic acid (PA). All phospholipids present in A.graveolens were found to be rich in linoleate (18:2) and palmitate (16:0). Lesser amounts of stearate (18:0), oleate (18:1), and a-linolenate (a- 18:3), were also present.
The major phytosterols in microsomes were identified as campesterol, stigmasterol, sitosterol, and isoflicosterol, with trace amounts of cholesterol and 24-methylene cholesterol.
The sterol biosynthesis inhibitors, miconazole, terbinafine, fenpropimorph, and tomatidine, proved to be useflul tools in the manipulation of membrane sterol composition in suspension cultures of A.graveolens. Treatment with these inhibitors caused significant alterations in lipid composition with corresponding changes in the activity of membrane-associated CT. Terbinaflne and fenpropimorph caused a large increase in the stigmasterol/sitosterol ratio of cells with a concomitant stimulation of
CT activity. The latter compound also resulted in the accumulation of various 9$, 19- cyclopropyl sterols. Similarly, the azasterol inhibitor, tomatidine, resulted in an enhancement of CT activity, but with very lift le change in the stigmasterol/sitosterol ratio of cells. Conversely, miconazole resulted in a decline in the stigmasterol/sitosterol
ratio, corresponding to lower membrane-associated CT activity. The latter inhibitor also caused an accumulation of oleoyl residues in the PC fraction of cells, suggesting an inhibition of A 1 2-desaturase activity.
Radiolabelling studies with [3IflS-adenosyl-L-methionine revealed a degree of coordinate regulation between the CDP-base and methyltransferase pathways of PC biosynthesis. Consequently, despite changes in CT activity, levels of phospholipid in
most inhibitor-treated cultures remained relatively constant.
Supplementation of miconazole-treated cultures with free fatty acids partially overcame the cytostatic nature of the azole inhibitor, with a concomitant reactivation of CT. Mono- and diunsaturated fatty acids were found to be the most effective
compounds in this respect. The addition of stigmasterol or sitosterol to miconazoletreated cultures also resulted in partial growth restoration and reactivation of membrane-bound CT.