Lipidomics of Thalassiosira pseudonana under Phosphorus Stress Reveal Underlying Phospholipid Substitution Dynamics and Novel Diglycosylceramide Substitutes

Abstract

Phytoplankton replace phosphorus-containing lipids (P-lipids) with non-P analogues, boosting growth in P-limited oceans. In the model diatom Thalassiosira pseudonana, the substitution dynamics of lipid headgroups are well described, but those of the individual lipids, differing in fatty acid composition, are unknown. Moreover, the behavior of lipids outside the common headgroup classes and the relationship between lipid substitution and cellular particulate organic P (POP) have yet to be reported. We investigated these through the mass spectrometric lipidomics of P-replete (P+) and P-depleted (P−) T. pseudonana cultures. Nonlipidic POP was depleted rapidly by the initiation of P stress, followed by the cessation of P-lipid biosynthesis and per-cell reductions in the P-lipid levels of successive generations. Minor P-lipid degradative breakdown was observed, releasing P for other processes, but most P-lipids remained intact. This may confer an advantage on efficient heterotrophic lipid consumers in P-limited oceans. Glycerophosphatidylcholine (PC), the predominant P-lipid, was similar in composition to its betaine substitute lipid. During substitution, PC was less abundant per cell and was more highly unsaturated in composition. This may reflect underlying biosynthetic processes or the regulation of membrane biophysical properties subject to lipid substitution. Finally, levels of several diglycosylceramide lipids increased as much as 10-fold under P stress. These represent novel substitute lipids and potential biomarkers for the study of P limitation in situ, contributing to growing evidence highlighting the importance of sphingolipids in phycology. These findings contribute much to our understanding of P-lipid substitution, a powerful and widespread adaptation to P limitation in the oligotrophic ocean.IMPORTANCE Unicellular organisms replace phosphorus (P)-containing membrane lipids with non-P substitutes when P is scarce, allowing greater growth of populations. Previous research with the model diatom species Thalassiosira pseudonana grouped lipids by polar headgroups in their chemical structures. The significance of the research reported here is threefold. (i) We described the individual lipids within the headgroups during P-lipid substitution, revealing the relationships between lipid headgroups and hinting at the underlying biochemical processes. (ii) We measured total cellular P, placing P-lipid substitution in the context of the broader response to P stress and yielding insight into the implications of substitution in the marine environment. (iii) We identified lipids previously unknown in this system, revealing a new type of non-P substitute lipid, which is potentially useful as a biomarker for the investigation of P limitation in the ocean.

A Review of Diatom Lipid Droplets

“…The polar lipid composition of the LD membrane may affect which proteins bind to it [52]. During phosphorous deficiency, phospholipids are replaced by non-phosphorous lipids, such as betaine lipids, sulfolipids and galactolipids [53,54], although it is currently unknown what effect this might have on the LD membrane.…”

Intact polar lipids in the water column of the eastern tropical North Pacific: abundance and structural variety of non-phosphorus lipids

“…The 3G-DAG comprised of C 14 , C 16 and C 18 fatty acids with up to six double bond equivalents is another minor IPL detected in the euphotic zone at all stations except for Station 5. It has been found in some plants (Hölzl and Dörmann, 2007) and some anaerobic gram-positive bacteria (Exterkate and Veerkamp, 1969), which could both be probable sources in the oxic euphotic zone of the ETNP.…”

“…In particular, SQ-DAG in the core OMZ/oxycline contained odd-carbon numbered fatty acids (e.g., C 15:0 / C 16:0 and C 14:0 / C 15:0 ) different from the cyanobacterial SQ-DAG in surface waters (Table S5). Some Gram-positive bacillus and firmicutes biosynthesize 1G, 2G-and SQ-DAG (Hölzl and Dörmann, 2007), and 1G-, 2G-and SQ-DAG in deeply buried Wadden Sea sediments are attributed to anaerobic bacteria (Seidel et al, 2012). However, Gram-positive bacteria are generally not abundant in seawater.…”

“…But even these low levels of oxygen permit the vertical migration of some zooplankton taxa into hypoxic waters (e.g., Seibel, 2011;Wishner et al, 2013). Oxygen depletion stimulates diverse microbial life capable of utilizing alternative electron acceptors for respiration under microaerobic conditions (e.g., Ulloa et al, 2012;Tiano et al, 2014;Carolan et al, 2015; F. Schubotz et al: Intact polar lipids in the water column of the eastern tropical North Pacific Kalvelage et al, 2015). Important prokaryote-mediated processes within OMZs include denitrification and the anaerobic oxidation of ammonium (anammox), which together may account for 30 %-50 % of the total nitrogen loss from the ocean to the atmosphere (Gruber, 2008;Lam and Kuypers, 2011).…”

“…In oxygen-deficient subsurface waters, IPL distributions are more diverse, and other phospholipids such as diacylglycerol phosphatidyl-N-methylethanolamine (PME-DAG), phosphatidyl-N,N-dimethylethanolamine (PDME-DAG) and diphosphatidylglycerol (DPG) increase in abundance; these IPLs occur in a number of bacteria that may inhabit lowoxygen environments (Schubotz et al, 2009;. Diether glycerol phospholipids and glycosidic ceramides with unidentified sources have also been detected (Schubotz et al, 2009;Wakeham et al, 2012), and the latter have been recently observed to be abundant in phosphoruslimited diatoms (Hunter et al, 2018). IPLs that are unique to marine archaea are comprised of glycerol dialkyl glycerol tetraether (GDGT) core lipids with various glycosidic, diglycosidic and mixed phospho-glyco polar head groups (e.g., Schouten et al, 2008;Pitcher et al, 2011;Zhu et al, 2016;Elling et al, 2017).…”