Xenobiotics directed against sterol biosynthesis have proved to be useful tools in the determination of which sterol molecules are necessary for successful plant cell growth. However, the exact mode of action by which sterols are able to trigger cell growth remains to be elucidated. Previous studies using the triazole paclobutrazol, demonstrated that in Apium graveolens (cv. New Dwarf White) suspension cultures, sterol and phosphatidylcholine biosynthesis are co-ordinately regulated (C. E. Rolph and L. J. Goad 1991, Physiol. Plant. 83: 605–610). The studies presented herein, were designed to investigate the possible role of phosphatidylcholine in the stimulation of plant cell growth.
Sterol biosynthesis, and hence cell growth, was inhibited by the use of the azole xenobiotic miconazole. Treatment of the cultures with miconazole lead to compositional changes in the free sterol content of the cells. For example, 30 μM miconazole treatment led to a reduction in the stigmasterol/sitosterol ratios from 1.53 to 1.24. In contrast, the phospholipid content of the cells remained relatively unchanged with phosphatidylcholine accounting for approximately 25% of the total phospholipids present in both control and miconazole-treated cells. The cytostatic effect of miconazole could be partially counteracted by supplementation of the growth medium with the phytosterol stigmasterol and/or the unsaturated fatty acids oleate and linoleate.
The activity of CTP:cholinephosphate cytidylyltransferase (EC 2.7.7.15), a rate-limiting enzyme in phosphatidylcholine biosynthesis, was significantly reduced in cells whose growth had been arrested by miconazole treatment. In miconazole-treated cultures whose growth had been partially restored by supplementation with either specific sterols or unesterified fatty acids, the activity of this key enzyme was increased. In the case of stigmasterol, oleate and linoleate supplementation, the microsomal activity was found to be similar to that exhibited by control cultures.
From these studies, it may be concluded that certain phytosterols and unsaturated fatty acids play key roles with respect to phosphatidylcholine biosynthesis and that phosphatidylcholine biosynthesis via the CDP-base pathway is an important pre- and/or co-requisite for successful culture growth.
