Diatoms are responsible for ∼20% of global primary production and play crucial roles in carbon and silicon biogeochemical cycles (Field et al., 1998). Their fixed carbon is partitioned into either carbohydrates or lipids (Kroth et al., 2008). The lipids of diatoms consist of almost all lipid classes, including both polar lipids (Guschina and Harwood, 2006) and non-polar lipids [free fatty acids, sterols, glycerols and especially triacylglycerols (TAGs)]. TAGs of diatoms have been increasingly studied for their potential as nutritional sources and for biofuel production (Hildebrand et al., 2012; Shahidi and Ambigaipalan, 2018). Fatty acid profiles of diatoms are enriched with medium-chain and very long-chain polyunsaturated fatty acids (PUFAs), namely ω3 fatty acids such as eicosapentaenoic acid (EPA) (Dunstan et al., 1993; Guschina and Harwood, 2006). In aquatic food webs, PUFAs are exclusively synthesized by phytoplankton and cannot be synthesized de novo by metazoans, and therefore must be acquired by non-phytoplanktonic organisms via their diet (Hixson et al., 2015). As such, PUFAs are important determinants of food quality and, consequently, an important indicator for the health and optimal functioning of marine and freshwater food webs (Dalsgaard et al., 2003). The polar lipid fraction of diatoms mainly consists of digalactosyldiacylglycerol (DGDG), monogalactosyldiacylglycerol (MGDG), sulfoquino vosyldiacylglycerol (SQDG), phosphatidylinositol (PI), phosphatidylglycerol (PG), phosphatidylcholine (PC), and minor lipids such as betaine lipids (Stonik and Stonik, 2015).

Global change induces many alterations in marine environments, such as ocean acidification (Gattuso et al., 2015). The molecular, physiological, biochemical and ecological responses of diatoms to ocean acidification have been studied extensively in the last two decades (Gao et al., 2012; Hennon et al., 2015; Petrou et al., 2019; Shi et al., 2019; Li et al., 2021; see also comprehensive reviews by Gao and Campbell, 2014; Bach and Taucher, 2019 and references therein). It has been reported that the lipid contents and fatty acid compositions of diatoms are highly dependent on CO₂ concentrations (Bermúdez et al., 2015; Wang et al., 2017; Abreu et al., 2020). For example, ocean acidification decreased the content of PUFAs in the marine diatom Cylindrotheca fusiformis by ∼3% (Bermúdez et al., 2015). The mechanisms through which CO₂ affects the composition of fatty acids in microalgae are still unclear, however, it has been suggested that elevated CO₂ can enhance the synthesis and accumulation of saturated fatty acids (Sato et al., 2003). This response reduces cell membrane fluidity, which helps the organism to cope with pH reductions and facilitates the regulation of cell homeostasis (Lane and Burris, 1981; Rossoll et al., 2012). More recently, it has been reported that the operation of carbon concentration mechanisms (CCMs) of microalgae are tightly coupled with their lipid metabolisms (Renberg et al., 2010; Abreu et al., 2020) and that the CCMs of diatoms are partially down-regulated under ocean acidification conditions (Trimborn et al., 2009; Wu et al., 2010; Hopkinson et al., 2011; Yang and Gao, 2012). The down-regulations of CCMs led to decreased photorespiration and symptoms of oxidative stress because of an increase in the electron sink constituted by CO₂ fixation (Raven, 2010; Renberg et al., 2010). These changes consequently altered the composition of lipids (such as DGDG, MGDG, and triacylglycerol, given their importance for cell functioning, especially in stress-responses (e.g., oxidative stress) (Bréhélin et al., 2007; Abreu et al., 2020). However, most published studies have analyzed responses of lipid metabolisms in the short term only (i.e., 1–2 weeks), and were thus unable to resolve long-term responses to ocean acidification conditions. Since diatoms are characterized by large population sizes, standing genetic variations and short generation times (Reusch and Boyd, 2013; Collins et al., 2020), they have a high potential to adapt to acidifying oceans, as recently indicated (Li et al., 2017; Zhong et al., 2021).

To address this fundamental knowledge gap, we carried out a ∼400 days selection experiment with the model marine diatom Phaeodactylum tricornutum, and employed a lipidomics approach to investigate long-term responses in the lipid metabolism of diatoms to different emission scenarios associated with ocean acidification.

Our results show that after 400 days (corresponding to almost 900 generations) of long-term high CO₂ selection, the marine diatom species P. tricornutum revealed significant changes in lipid metabolisms. One of the most apparent findings of the present study is that MGDG, the main glycerolipid in non-plastid membranes, was significantly down-regulated in long-term LC selected cells. Our results are in agreement with the results of a previous short-term (6 h) study with the green algae species Chlamydomonas reinhardtii (Abreu et al., 2020). MGDG is a hexagonal II phase polar membrane lipid, whose role is to give the membrane a high internal lateral pressure among the fatty acyl chains and a pressure on the membrane proteins (Kruijff, 1997). Due to its non-bilayer lipid properties, different ratios of MGDG to other phase properties lipids may affect the lateral pressure on membrane proteins (Kruijff, 1997). For instance, it has been widely reported that a lower ratio of DGDG (a lamellar phase lipid) to MGDG reflects increased sensitivities of microalgae to various abiotic stresses (e.g., low temperature, CO₂ limitation, salt stress, nitrogen starvation) (Du et al., 2018; Liu et al., 2019). Since no significant changes of DGDG were detected in the present study, the down-regulations of MGDG in long-term HC selected cells resulted in an increased ratio of DGDG:MGDG. Therefore, our results suggest that long-term high CO₂ selection may be not stressful for P. tricornutum.

It is also worth noting that the down-regulations of MGDG would result in an increased conductivity of the thylakoid and in an increase of luminal pH, causing the activity of violaxanthin de-epoxidase to decrease (Aronsson et al., 2008). In addition, the down-regulations of MGDG are also likely to decrease the violaxanthin availability from the membrane for violaxanthin de-epoxidase (Schaller et al., 2010). Consequently, such a decrease in de-epoxidase would lead to a low efficiency of the violaxanthin-zeaxanthin interconversion, which is a key process of the xanthophyll cycle, a crucial regulatory component for energy dissipation in diatoms (Miloslavina et al., 2009; Goss and Jakob, 2010; Lepetit et al., 2017). This is evidenced by previous studies, in which diatoms in high CO₂ conditions exhibited higher non-photochemical quenching (NPQ) (i.e., a lower energy dissipation capacity) (e.g., Gao et al., 2012; Wu et al., 2017; Li et al., 2019). These findings were further supported by our recent investigations, in which we found three proteins, namely the precursor protein of zeaxanthin epoxidase (ZEP2), zeaxanthin epoxidase (ZEP1) and violaxanthin de-epoxidase (VDE), were significantly down-regulated in HC selected cells (data not shown). To summarize, our results suggest that the MGDG down-regulation in long-term HC selected P. tricornutum cells is likely an adaptation strategy to cope with the long-term high CO₂ conditions.

PE is one of the major phospholipids (phosphoglycerides) in most algae species (Guschina and Harwood, 2009). Very long chain polyunsaturated fatty acid can be incorporated into PE by acyltransferase reactions of the Kennedy pathway (Sayanova et al., 2017; Li-Beisson et al., 2019). The production of PE by algae was recognized as a response to abiotic stresses, such as light, temperature and nutrient. For instance, it was evidenced that the content of PE significantly increased with increasing temperature (Thompson, 1996). Both dark exposure and phosphate starvation were found to lead to a decrease of PE content in microalgae species (McLarnon-Riches et al., 1998; Khozin-Goldberg and Cohen, 2006). For the response of PE to high CO₂, the degree of unsaturation of PE was reported to decrease in response to high CO₂ by decreasing the contents of C18:3 at the sn-2 position of PE (Sato et al., 2003). Our results indicated that rather than the unsaturation of PE, the concentration of PE was significantly enhanced under long-term high CO₂ selection.

In addition to PE, the majority of the differently regulated PG lipids (seven out of nine) were up-regulated in long-term high CO₂ selected cells (Figure 4). PG is the major phospholipid in chloroplasts, and it contains an uncommon trans-3-hexadecenoic acid (C16:1t), located exclusively at position sn-2 of the glycerol backbone in all eukaryotic photosynthetic organisms (Boudière et al., 2014; Figure 4). PG play an important role in the light harvesting complex trimerization process and in the functioning of the photosystem (Loll et al., 2007). Along with MGDG and PE, PG is also sensitive to various abiotic stressors. It is well recognized that the contents of PG in various algal species, including diatoms, decrease under nutrient starvation (Van Mooy et al., 2009; Abida et al., 2015; Wang et al., 2019), elevated temperatures (Feijão et al., 2017, 2020), and low pH conditions (Vítová et al., 2016). In the present study, we found that ∼80% of the significantly regulated PGs were up-regulated after long-term high CO₂ selection (Figure 4). This may be due to adaptation driven by long-term high CO₂ selection. While the content of PG may decrease after short-term high CO₂ selection, its content may be partially or completely reversed over the long-term high CO₂ selection period. Such an adaptive response has been evidenced in multiple previous studies (Lohbeck et al., 2012; Jin et al., 2020). For instance, the fatty acid and lipid contents of three diatoms partly or entirely recovered following a long-term exposure (∼2 years) to warming conditions (+4°C) (Jin et al., 2020). Thus, our results suggest a high potential of diatoms to adapt their lipid metabolites to long-term high CO₂ selection.

It is known that an increase of SQDG could compensate the absence of PG in plastids (Jouhet et al., 2010; Boudière et al., 2014; Abida et al., 2015), thus the contents of SQDG and PG are expected to be negatively correlated, as shown in our study. Under stressful conditions, such as nutrient starvation, a PG-to-SQDG replacement was considered to be a ubiquitous phenomenon in photosynthetic organisms, enabling the preservation of an anionic lipid environment to the photosystems in the thylakoids (Boudière et al., 2014; Nakamura and Li-Beisson, 2016). Besides a PG-to-SQDG replacement, there was also a PC-to-DGTA replacement occurring in the diatom P. tricornutum, which was also considered to be a strategy to cope with environmental stresses (Abida et al., 2015). Since we observed multiple negative or positive correlations between different lipid metabolites (e.g., positive correlation between PC and PEtOH; negative correlations between MGDG and PC), our results suggest several more lipid remodeling mechanisms that have not been identified yet. We propose that the underlying mechanisms for lipid remodeling in response to various abiotic stressors in diatoms should be further investigated as part of future studies.

It is also recognized that the lipid metabolism of microalgae was regulated by some mineral elements such as silicon and calcium. For instance, lipid production in marine diatoms Chaetoceros gracilis and Thalassiosira pseudonana was reported to increase under silicon limitation conditions (Zendejas et al., 2012; Adams and Bugbee, 2014). Since high CO₂ was reported to affect the biogenic silica cellular contents of diatoms (Xu et al., 2014), such a change induced by high CO₂ is expected to regulate the lipid metabolism as well. Calcium (Ca²⁺), a secondary messenger, plays a crucial role in the signal transduction through the activation of various receptors associated with metabolic in coping with various environmental changes (Chen et al., 2014). Calcium can bind with calmodulin and then affect the energy metabolism by regulating the cyclic electron flow in photophosphorylation and respiratory oxidative phosphorylation in microalgae (Chen et al., 2015). It is known that an appropriate concentration of calcium can stimulate the activity of Acetyl-CoA carboxylase that catalyzes the conversion of acetyl-coenzyme A to malonyl-CoA during the fatty acid biosynthesis (Gorain et al., 2013). However, how would high CO₂ interact with calcium to regulate the lipid modeling of diatoms (through energy metabolism or signal transduction) remains unknown. Hence, we recommend that the underlying mechanisms for lipid remodeling in response to high CO₂ interacting with essential (or non-essential) elements in diatoms are warranted for further investigations.

In conclusion, our observations suggest that long-term high CO₂ selection triggered substantial changes in lipid metabolites of the marine diatom P. tricornutum (Figure 4A). Some lipid metabolites (such as PGs) showed high adaptive potential to the selection. The tightly coupled regulations (positively or negatively correlated) of lipid metabolites reveal that the lipid remodeling is an organismal adaptation strategy of marine diatoms to ongoing ocean acidification. The composition and concentration of lipid are crucial for marine food quality, and their changes can be transferred to high trophic levels (Rossoll et al., 2012; Jin et al., 2015, 2020). Therefore, our results outline the importance of investigating the long-term responses of lipids of primary producers to prolonged ocean acidification conditions, and assessing the ecological consequences.

The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author/s.

PJ and JX conceptualized the study. ZL, HL, JP, PL, QH, YG, and JZ acquired the data. PJ and FL performed the statistical analysis and generated the figures with JW and SO. PJ drafted the manuscript, SO contributed substantially to the editing of the manuscript. All authors discussed and approved the manuscript.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.