Lipid Turnover in Plants and Yeast, A Study of Storage Lipid Synthesis and Lipid-Based Signalling in Plants and Yeast

Sammanfattning: In this thesis, lipid biosynthesis is discussed focussing on the genes and enzymes involved in the final step of storage lipid synthesis, on the enzyme phospholipase A2, and on cell membrane lipid homeostasis. The investigation of lipid metabolism revealed that in Saccharomyces cerevisiae, there are four genes that are essential for the final step of storage lipid biosynthesis; ARE1, ARE2, DGA1, and LRO1. A yeast strain disrupted in the four genes is viable and has no apparent growth defect under normal conditions. It is devoid of both storage lipids and lipid bodies. We therefore suggest that the presence of storage lipids and lipid bodies in yeast is non-essential in contrast to previous suggestions where both are considered a prerequisite for vegetative growth. It was shown that yeast cells disrupted in storage lipid biosynthesis has a higher de novo fatty acid biosynthesis during the early vegetative growth phase to compensate for the loss of storage lipids while the rate of fatty acid biosynthesis is down-regulated at stationary growth phase compared to the wild type strain. The overall acyl group composition as well as the phospholipid composition was however altered by the disruption of storage lipid biosynthesis. Our data further revealed that acyl-CoA:diacylglycerol acyltransferase encoded by DGA1 is responsible for the major part of the triacylglycerol biosynthesis and a significant contribution is also given by phospholipid:diacylglycerol acyltransferase, encoded by LRO1. ARE1 and to a minor extent ARE2 exhibit acyl-CoA:diacylglycerol acyltransferase activity apart from their steryl ester biosynthesis activity and under normal growth conditions ARE1 mainly functions as an acyl-CoA:diacylglycerol acyltransferase. The investigation of lipid turnover in plants demonstrated that plasma membranes isolated from spinach leaves contain a phospholipase A2 activity. Characterisation of the phospholipase A2 activity showed an absolute requirement for calcium ions, with full activation at 10 µM calcium. Further characterisation revealed that spinach plasma membranes contain a phospholipase A2 activity, not previously characterised in plants. Cold acclimation of spinach and rye plants resulted in a 2-fold activation of plasma membrane phospholipase A2. Other phospholipase activities, i.e. phospholipase D implied in cold stress, remained unaffected. These results suggest a role for phospholipase A2 in cold stress in plants. To investigate the physiological role(s) of phospholipase A2 in cells, a genetic approach to find genes encoding the enzyme in yeast was undertaken. A screening for genes encoding a membrane-associated phospholipase A2 in yeast knockout mutants revealed four candidate genes. Sequence lipase motif, disruption and overexpression results, and activity analyses strongly suggest that the open reading frame YDL109c, encodes a novel microsomal phospholipase A2 in S. cerevisiae.