Mobilisierung von Speicherlipiden in <i>Cucumis sativus</i>- und <i>Arabodopsis thaliana</i>-Keimlingen

Abstract

Germination of oilseeds is characterized by mobilisation of TAGs that are stored in lipid bodies and serve as carbon source. Beside the classical degradation pathway of these storage lipids via the direct action of a triacylglycerol lipase (TGL), an alternative degradation pathway that is dependent on a specific lipid body trilinoleate 13-lipoxygenase (13-LOX) has been found in some plant species. In cucumber seedlings the activity of this specific 13-LOX (CsLbLOX) leads to the formation of ester lipid hydroperoxides (13-HPOD) in the storage lipid fraction. These hydroperoxy fatty acid derivatives where then preferentially cleaved off by a specific TGL and seem to be the substrate for glyoxisomal β-oxidation. As a prerequisite for mobilisation of storage lipids a partial degradation of the phospholipid monolayer of the lipid bodies appears to be necessary. This facilitates access of lipid mobilising enzymes to their substrates inside the lipid bodies. In this context lipid bodies from cucumber cotyledons were isolated and lipid body proteins, enzyme activities as well as storage lipids were analysed. Simultaneously with the detection of LOX activity, the transient accumulation of CsLbLOX, and with the formation of lipid bound 13-HPOD and oxygenated TAG species in lipid bodies during germination, the expression of the patatin-like PLA2 CsPAT and a transient PLA2 activity were observed. Furthermore, CsPAT as well as CsLbLOX could be localised at the lipid body membranes. Analyses of membrane phospholipids of the lipid body showed a decrease in the amount of PC that was accompanied by the accumulation of its degradation product LPC at the same time of germination as PLA2 activity was detectable. Additionally, in vitro experiments with isolated lipid bodies from mature seeds revealed that the formation of 13-HPOD first became visible after incubation with the purified recombinant patatin-like protein. These investigations suggest, that in vivo the degradation of storage lipids is initiated by the activity of CsPAT, which facilitates a decomposition of the lipid body membrane and thereby the substrate for the 13-LOX as well as the TGL becomes accessible.Transgenic expression of CsPAT and CsLbLOX in tobacco seeds did not result in degradation of storage lipids at an early stage or in a definite accumulation of lipid hydroperoxides. An involvement of the transgenic proteins in the wound reaction of tobacco leaves could not be detected as well. Sequencing of lipid body proteins from cucumber and Arabidopsis seedlings did not reveal TGL sequences, which represent possible candidates degrading storage lipids. Heterologous expression of the putative germination specific TGLs AtTGL1 and AtTGL2 from A. thaliana in insect cells and in P. pastoris with sufficient amounts of protein as well as detection of TGL activity of these enzymes was not successful so far. The physiological function of AtTGL1 and AtTGL2 remains unclear.