Lipidome Remodeling and Autophagic Respose in the Arachidonic-Acid-Rich Microalga Lobosphaera incisa Under Nitrogen and Phosphorous Deprivation

Abstract

The green microalga Lobosphaera incisa accumulates triacylglycerols (TAGs) with exceptionally high levels of long-chain polyunsaturated fatty acid (LC-PUFA) arachidonic acid (ARA) under nitrogen (N) deprivation. Phosphorous (P) deprivation induces milder changes in fatty acid composition, cell ultrastructure, and growth performance. We hypothesized that the resource-demanding biosynthesis and sequestration of ARA-rich TAG in lipid droplets (LDs) are associated with the enhancement of catabolic processes, including membrane lipid turnover and autophagic activity. Although this work focuses mainly on N deprivation, a comparative analysis of N and P deprivation responses is included. The results of lipidomic profiling showed a differential impact of N and P deprivation on the reorganization of glycerolipids. The formation of TAG under N deprivation was associated with the enhanced breakdown of chloroplast glycerolipids and the formation of lyso-lipids. N-deprived cells displayed a profound reorganization of cell ultrastructure, including internalization of cellular material into autophagic vacuoles, concomitant with the formation of LDs, while P-deprived cells showed better cellular ultrastructural integrity. The expression of the hallmark autophagy protein ATG8 and the major lipid droplet protein (MLDP) genes were coordinately upregulated, but to different extents under either N or P deprivation. The expression of the Δ5-desaturase gene, involved in the final step of ARA biosynthesis, was coordinated with ATG8 and MLDP, exclusively under N deprivation. Concanamycin A, the inhibitor of vacuolar proteolysis and autophagic flux, suppressed growth and enhanced levels of ATG8 and TAG in N-replete cells. The proportions of ARA in TAG decreased with a concomitant increase in oleic acid under both N-replete and N-deprived conditions. The photosynthetic apparatus’s recovery from N deprivation was impaired in the presence of the inhibitor, along with the delayed LD degradation. The GFP-ATG8 processing assay showed the release of free GFP in N-replete and N-deprived cells, supporting the existence of autophagic flux. This study provides the first insight into the homeostatic role of autophagy in L. incisa and points to a possible metabolic link between autophagy and ARA-rich TAG biosynthesis.

Metabolic response of Botryococcus braunii to high bicarbonate dosages and other conditions: analysis of photosynthetic performance, productivity, and lipidomic profile

“…This is in agreement with our observations of a major abundance of DGDG compared to MGDG species (Fig. 4b, c), and with results published by other authors (Li et al 2014;Han et al 2017;White et al 2019;Kokabi et al 2020).…”

“…The increased abundance of highly unsaturated TAGs (e.g., TAG-50:4/-50:5/-50:6/-52:4/52:5/-52:6/-52:7/-52:8/-52:9) leads us to hypothesize that they may result from membranederived polyunsaturated FA, in agreement with the reduction in glycolipid abundance (Fig. 4b, c), which may be indicative of membrane derived-FA recycling into TAGs (Li et al 2012(Li et al , 2014Kokabi et al 2020).…”

“…From our results, it is not possible to explain the preferential accumulation of PCs over the other GPLs identified, such as the non-nitrogen containing PG. In P-deprived microalgae cells, there are reports of PC and DGDG increase, which may suggest the re-assembly of DGDG from PC, hypothesizing that PC may transiently increase under P deprivation for its reuse in DGDG biosynthesis (Jouhet et al 2003;Kokabi et al 2020). In ND B. braunii cells at D12, we found increased abundance for 71% and 73% of the identified DGTSs (Fig.…”

“…N-deprivation of microalgae triggers membrane lipid turnover and breakdown (Li et al 2012;Simionato et al 2013;Martin et al 2014;Han et al 2017;Kokabi et al 2020) and de novo synthesis of fatty acids (FA) (Li et al 2012;Kokabi et al 2020), to supply precursors for TAG assembly. Lipid turnover and degradation under N deprivation is central to keep membrane homeostasis under stress, especially in chloroplasts, thus explaining the increase of DGDG to MGDG ratio, which corresponds to the major chloroplast lipids (Kobayashi 2016).…”

“…P is a critical nutrient in all domains of life (Dyhrman 2016). The effects of P deprivation have been widely studied in microalgae and cyanobacteria (Martin et al 2011;Cañavate et al 2017;Matich et al 2018;Kokabi et al 2020). The effect of P deprivation in boosting cellular lipid accumulation has rendered contradictory results in microalgae.…”