Leek seedlings

Elongation of acyl-CoAs by microsomes from etiolated leek seedlings. Phytochemistry 2A, 1187-1192 Mukherjee, K.D., Kiewitt, I. (1984) Changes in fatty acid... 

A methodology was devised to investigate intermembrane transfer of lipids and very long chain fatty acids (VLCFA) in etiolated leek seedlings in vivo (1). 

Extraction, isolation and quantitation techniques were devised to study the penetration of monensin into leek seedlings. As a preliminary step, the complete solubility of monensin in chloroform / methanol ( 2 1, v/v ) was... 

Intermembrane transfer of long chain fatty acids synthesized by etiolated leek seedlings. Phytochemistry 2i, 387-391. 

Intracellular transport of very long chain fatty acids in etiolated leek seedlings. Plant Physiol. Biochem. 26, 173-178. 

Moreau P., Lessire R. and Casagne C. (1984) - In vivo membrane transfer of very long chain fatty acids synthesized by etiolated leek seedlings in Structure Function and Metabolism of plant Lipids, Siegenthaler P.A. et Eichenberger W., eds, Elsevier, Amsterdam, p. 307-310. 

A new system (seven-day-old etiolated leek seedlings) has been employed to study the Intermembrane transfer of lipids and fatty acids in vivo. By vivo pulse-chase experiments, followed by membrane fractionation on linear sucrose gradients, intermembrane transfer events of VLCFA between the light fraction and the heavier ones was demonstrated. 

Since plasma membranes isolated from etiolated leek seedlings are incapable of synthesizing VLCFA,as is the case of the plasma membrane Isolated from leek epidermis, it follows that the VLCFA were transferred to the plasmalemma vivo. 

The results presented here demonstrate, for the first time, the existence of vivo transfers of lipids and fatty acids to the plasma membrane of a higher plant. Hence, etiolated leek seedlings appear to be a good model for the study of the intracellular pathways of lipid and fatty acid transport to the plasmalemma vivo and consequently should provide information that is complementary to what is known about the intracellular transport of proteins. 

Recently (Moreau, 1986 see also Moreau et al., this volume), plasmalatma was purified frcm etiolated leek seedlings by sucrose... 

Schneider F, Lessire R, Bessoule JJ, Juguelin H, Cassagne C. Effect of cerulenin on the synthesis of very-long-chain fatty acids in microsomes from leek seedlings. Biochim Biophys Acta 1993 152 243-252. 

The trans 2-3 enoyl-CoA has been synthesized chemically by K. Levannier and J.P. Lellouche (CEA Saclay). The microsomes, used as enzyme source, were prepared from 7-day old leek seedlings as described earlier (5).The elongation activity was measured using [2- 4c]malonyl-CoA [17 pM] and acyl-CoA [9pM] in the presence of DTT, MgCI2, NADH, NADPH (1) and 60pg of proteins. The fatty acids were extracted and the radioactivity was measured. 

Table 1 Elongation of Cl8 0-CoA and trans 2-3 C20 1-CoA by microsomes from leek seedlings.

Table 2 Trans 2-3 enoyl-CoA elongation by etiolated leek seedling microsomes.

The sensitivity of the leek seedling FAS to these analogs was tested whereas the FAS activity was, as expected inhibited by 90% with 50 pM control cerulenin, the replacement by the Cer 16 analog led to a lesser inhibition (60% at 50 iM). But the most interesting- and promising - result was that cerulenin with 18 carbon atoms had no inhibitory effect on the "de novo" synthesis. 

Moreover, the leek seedling FAS activity was not inhibited by this molecule. On the contrary, this activity seemed to increase as a function of dioxolane-CoA concentration. 

Sturbois B, Moreau P, Maneta-Peyret L, Morre DJ, Cassagne C. Cell-free transfer of phospholipids between the endoplasmic reticulum and the Golgi apparatus of leek seedlings. Biochim Biophys Acta 1994 1189 31-37. 

ISOLATION OF TRANSFER VESICLES ISSUED FROM THE ENDOPLASMIC RETICULUM OF LEEK SEEDLINGS... 

In our laboratory, studies of lipid transfer in leek seedlings in vivo, have already shown the existence of a vesicular process for the transfer of phospholipids and particularly of very long chain fatty acid-containing lipids [6]. This process follows the vesicular endoplasmic reticulum- Golgi apparatus- plasma membrane pathway. Using the cell-free system developed by Morre and coworkers, we have reconstituted in vitro the vesicular transfer of some phospholipids between the ER and the GA. This transfer is ATP and cytosol-dependent, is N Ethyl Maleimide and temperature sensitive and specific for the ER as donor and the GA as acceptor. The phospholipids transferred via an ATP-dependent manner in vitro between the ER and the GA were phosphatidylcholine (PC +79%), phosphatidylethanolamine (PE +67%) and phosphatidylserine (PS +123%) [7]. All those results are in favour of a vesicular transport of phospholipids between the ER and the GA of leek seedlings, and brought us to purify these transition vesicles issued from the ER. 

Bertho P, Moreau P, Morre DJ and Cassagne C. Monensin blocks the transfer of very long chain fatty acid containing lipids to the plasma membrane of leek seedlings. Evidence for lipid sorting based on fatty acyl chain lenght. Biochim.Biophys. Acta 1991 1070 127-134. 

---