Thesis presented October 28, 2024

Abstract:
Phosphate (Pi) starvation is a frequent abiotic stress encountered by plants in nature and significantly affecting plant growth and crop yield. To adapt to Pi starvation, plants activate various mechanisms to ensure their survival, including increasing Pi uptake from soil and remobilizing intracellular phosphorus reserve. Notably, during Pi starvation, phospholipids (PL) located at extra-plastidial membranes are partially degraded to release Pi, while plastidial digalactosyldiacylglycerol (DGDG), a non-phosphorous plastid-synthesized lipid, is transferred to extra-plastidial organelles to substitute PL in order to maintain their integrities and functions. Previous studies have revealed that the DAG backbone of newly formed DGDG originated from extra-plastidial PC, indicating that the partially degraded PC is recycled to synthesize DGDG. However, we uncovered that the total lipid content drastically decreased under Pi deprivation. Given that, we hypothesized a potential association between lipid remodeling and ß-oxidation, a lipid degradation pathway occurring in peroxisomes. It prompted us to further investigate if the ß-oxidation pathway would be important in recycling free fatty acids derived from the partially degraded PL under Pi deprivation and, more generally, if peroxisomes would be involved in lipid remodeling during Pi starvation. We attempt to purify peroxisome from Arabidopsis calli grown under both Pi-enriched and Pi-deprived conditions using an adapted isolation protocol from previous studies, to refine our understanding of peroxisomal lipid composition in Arabidopsis thaliana and to study its lipidomic and proteomic responses to Pi starvation. Using this optimized protocol, we enrich peroxisomes with satisfactory yields for analyses under both standard and stress conditions, and de-enrich other organelles. Our results highlight the major phospholipid composition of peroxisomal membranes, similar across various species. We also identify proteins related to oxidative stress response and nitrogen metabolism that exhibit significant differences between normal growth conditions and Pi deficiency. However, low purity of the isolated peroxisomes is insufficient to conclusively assess the lipidomic and proteomic differences in response to Pi deprivation. In order to elucidate the role of ß-oxidation process in lipid remodeling in response to Pi starvation, we selected two enzymes involved in ß-oxidation pathway, PXA1 and MFP2, and employed multiple approaches, such as gene expression profiling, growth phenotype assessment, lipidomic analyses, and subcellular imaging, onto the wild type and mutant strains deficient in these two genes under Pi-replete and Pi-deplete conditions. Our results show that ß-oxidation pathway is not involved in the metabolic processes of lipid remodeling in response to Pi starvation in A. thaliana cell cultures. Interestingly, a resistant phenotype of pxa1 calli under Pi-starved conditions is observed. The phenotypic behavior of pxa1 plants in response to Pi starvation requires further investigations. Moreover, an abnormal mitochondrial morphology, that remain to be investigated, is observed in these two ß-oxidation mutants, particularly under standard conditions. Additional studies remains to be conducted to assess the redox status of these two mutant calli under both conditions. Overall, we revealed that peroxisomal ß-oxidation pathway does not play a major role in the metabolic flow of phospholipid degradation in response to Pi starvation, and characterized notable cellular responses in ß-oxidation mutants.

Keywords:
phosphate starvation, peroxisome, membrane lipid remodeling, ß-oxidation, Arabidopsis thaliana