Elsevier

Phytochemistry

Volume 50, Issue 1, 15 January 1999, Pages 47-51
Phytochemistry

Modulation of phosphatidylcholine biosynthesis in celery by exogenous fatty acids

https://doi.org/10.1016/S0031-9422(98)00488-9Get rights and content

Abstract

The effects of C16 and C18 fatty acids on the synthesis of phosphatidylcholine were studied in Apium graveolens cell suspension cultures and postmitochondrial supernatants. When cells were exposed to exogenous oleic acid, the rate of phosphatidylcholine biosynthesis increased 1.4-fold within 5 min of the addition of the fatty acid to the culture medium. The sensitivity of microsomal CTP:cholinephosphate cytidylyltransferase (EC 2.7.7.15) to saturated and unsaturated fatty acids was monitored through the addition of unesterified fatty acids to postmitochondrial supernatants. The saturated fatty acids, palmitic and stearic, appeared to have little effect on CTP:cholinephosphate cytidylyltransferase activity, whereas exposure to oleic, linoleic and cis-vaccenic acids resulted in significant increases in enzyme activity. Optimal microsomal CTP:cholinephosphate cytidylyl- transferase activities were achieved by the incubation of postmitochondrial supernatants with 500 μM oleate. The exogenous fatty acids were found to be incorporated into microsomal membranes in their unesterified form. Removal of unesterified fatty acids by incubation of microsomal membranes with defatted bovine serum albumin resulted in the reduction of microsomal CTP:cholinephosphate cytidylyltransferase activity; demonstrating that the enzyme requires unesterified unsaturated fatty acids.

Introduction

Phosphatidylcholine (PC) is the major phospholipid in nonplastidial higher plant cell membranes, where it may constitute up to 40 wt% of the membrane lipid profile (Moore, 1982). The functions of PC within these membranes are multiple. For example, PC provides substrates for the synthesis of polyunsaturated fatty acids (Garces, Sarmiento, & Mancha, 1992; Williams, Williams, & Khan, 1992) and triacylglycerols (Stymne, Balfor, Jonsson, Wiberg, & Stobart, 1991) and helps to protect some plants against chilling injury (Kinney, Clarkson, & Loughman, 1987; Cheesbrough, 1989). In addition, in agreement with mammalian studies (Wieder, Haase, Geilen, & Orfanos, 1995; Vance, Houweling, Lee, & Cui, 1996; Cui et al., 1996), PC has also been shown to play a role in the regulation of plant cell division (Parkin, Goad, & Rolph, 1995).

The predominant route of PC biosynthesis in higher plants is via the CDP-base pathway (Moore, 1982). The enzymes involved in this pathway are choline kinase (EC 2.7.1.32), CTP:cholinephosphate cytidylyltransferase (EC 2.7.7.15) and phosphocholine transferase (EC 2.7.8.2). It is generally accepted that the activity of CTP:cholinephosphate cytidylyltransferase (CT) is the rate-limiting step in the production of PC (Weinhold & Feldman, 1995; Shiratori, Houweling, Zha, Tabas, 1995). As in mammalian cells, the CT enzyme of higher plant cells is topodynamically regulated. The membrane-bound enzyme has been found to be the active form in several higher plant species, such as celery and castor bean (Price-Jones & Harwood, 1983; Sauer & Robinson, 1985). However, in pea and maize, the majority of the cellular activity is located in the cytoplasm (Kinney & Moore, 1987; Parkin, Goad, & Rolph, 1993). To date, the regulation of CT activity has been studied in a variety of mammalian, plant and yeast species. Typical regulatory mechanisms include phosphorylation/dephosphorylation cycles, feedback inhibition involving PC and the anionic lipid content of the endoplasmic reticulum (Jamil & Vance, 1990; Cornell, 1991a; Pelech & Vance, 1984). In addition, its activity may be modulated by the presence of C18 unsaturated fatty acids and specific sterol molecules (Terce, Record, Tronchere, Ribbes & Chap, 1992; Parkin, Goad & Rolph, 1995). In mammalian systems, the aforementioned lipids have been shown to influence protein translocation by either enhancing the binding of the CT protein to a binding protein (Weinhold & Feldman, 1995) or by promoting the dephosphorylation of the enzyme (Shiratori et al., 1995), respectively. Nevertheless, the exact mechanisms by which these lipid molecules may regulate CT activity in higher plants remains to be elucidated. In a previous study (Parkin et al., 1995), we demonstrated the ability of oleic acid to promote CT activity in celery (Apium graveolens) cell suspension cultures. The work presented herein extends this study by determining the time-scale for the stimulation of CT activity by unsaturated fatty acids and by partially identifying the mechanism by which these lipid molecules may modulate CT activity.

Section snippets

Results and discussion

In order to ascertain the speed at which celery cell suspension cultures were able to take up exogenous oleic acid from the growth medium, freshly inoculated cultures were exposed to 200 μM oleic acid and the clearance of the fatty acid from the medium monitored with time (data not shown). It was observed that ca. 60% of the exogenous fatty acid had been removed within the first 24 h and 95% had been cleared after 72 h of the incubation period. Hence, it may be concluded that oleic acid had

Cultures

Suspension cultures of celery (A. graveolens cv. New Dwarf White) were grown in Murashige and Skoog medium supplemented with kinetin and 2,4-D according to Haughan, Lenton, and Goad (1988).

Uptake of exogenous oleic acid by suspension cultures

Oleic acid dissolved in EtOH-Tergitol (2:1) was added to freshly inoculated suspension cultures to give a final concentration of 200 μM. Samples of growth media were removed, cells removed by centrifugation and the oleic acid content of the supernatant quantified by GC using pentadecanoic acid as an int.

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    Present address: Department of Biochemistry and Molecular Biology, University of Leeds, Leeds, UK.

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