| dc.description.abstracteng | Microalgae hold great promise with regards to the production of valuable products such as PUFAs and biofuel. They are a highly interesting group of organisms for investigating lipid metabolism and while some insight has been gained from comparison of C. reinhardtii to other well characterized model organisms, it is becoming increasingly clear that substantial diversity exists between algal species. Among them, the terrestrial green microalga L. incisa is unique in its ability to accumulate high levels of ARA and sequester it in neutral lipids within LBs, especially when deprived of nitrogen.
In order to understand the unique mechanisms of sequestering ARA in neutral lipids, LB biogenesis was analyzed on a protein level in L. incisa strain SAG 2468. Following 3 d of nitrogen limitation, a state characterized by TAG and ARA accumulation, a multitude of proteins could be identified in LB isolates by means of LC-MS/MS. Semi-quantitative enrichment analysis through comparison with other cellular fractions was carried out and yielded a number of candidate LB associated proteins. For a subset of these candidates, the subcellular localization was confirmed by heterologous expression in tobacco pollen tubes along with confocal microscopy. Additionally, gene expression was analyzed in L. incisa cultures subjected to nitrogen starvation and subsequently rescued by nitrogen resupply, a time course during which TAG is first accumulated and then remobilized.
The proteins g555, g15430 and g13747 were found to be putative structural components of the lipid storage organelle based on similarities to known algal proteins, strong enrichment in the L. incisa LB fraction and hydrophobicity of the amino acid sequence, respectively.
Furthermore, two putative lipases were investigated in this study, one of them LB-associated. Even though TAG lipase activity could not be established for either of them in this study, they may still play a role in L. incisa LB homeostasis.
An additional lipase candidate, LiSDP1, was demonstrated to hydrolyze TAG when the gene was expressed in an A. thaliana mutant lacking both plant homologs. The protein appears to localize to LBs in tobacco pollen tubes and is postulated to be involved in the degradation of L. incisa LBs during recovery from nitrogen starvation.
Altogether, this study saw the successful isolation and confirmation of LB proteins from L. incisa as well as the identification of a TAG lipase that is most likely involved in storage lipid degradation, thereby contributing to the elucidation of LB biogenesis in this unique microalga. | de |