Juan Yang

Jin Liu

Yufang Pan

Eric Maréchal

Alberto Amato

Meijing Liu

Yangmin Gong

Yantao Li

Hanhua Hu
email: hanhuahu@ihb.ac.cn

PDAT regulates PE as transient carbon sink alternative to triacylglycerol in Nannochloropsis

Keywords: Chloroplast endoplasmic reticulum, Nannochloropsis oceanica, Phosphatidylethanolamine, Phospholipid:diacylglycerol acyltransferase, Triacylglycerol, Turnover, Carbon sink

Nannochloropsis oceanica is equipped with more than ten diacylglycerol:acyltransferases (NoDGATs) involved in triacylglycerol (TAG) production, pointing to the puzzling role of the unique phospholipid:diacylglycerol acyltransferase (NoPDAT) it also contains. NoPDAT expression could complement a TAG-deficient yeast phenotype and use a broad range of acyl-donors in vitro. Overexpression and knockdown experiments demonstrated the role of NoPDAT in modulating the transfer of palmitoyl (16:0) and palmitoleyl (16:1) at the sn-3 position of diacylglycerol (DAG), contributing a third of TAG accumulation at the onset of stationary growth phase. No compensatory mechanisms via DGAT pathways could be evidenced, showing that the PDAT pathway is a parallel and independent oilproducing route in N. oceanica. Subcellular localization analysis suggests that NoPDAT resides at the outermost plastid membrane. Besides phosphatidylglycerol (PG) highlighted in enzymatic assays, NoPDAT knockdown led to a two-fold increase in phosphatidylethanolamine (PE), which is attributed to molecular species enriched in 16:0 and 16:1 at the sn-2 position, and suggests the critical role of NoPDAT in the turnover of PE and PG specific molecular species for TAG biosynthesis. Interestingly, accumulation of this specific PE was not only triggered by PDAT lesion but also positively correlated with CO2 availability in the culture, indicating that the specific PE also serves as alternative carbon sink in N. oceanica.

INTRODUCTION

Eustigmatophytes of the Nannochloropsis genus and their close Microchloropsis counterparts are oleaginous microalgae, accumulating the neutral lipid triacylglycerol (TAG), and considered as a promising feedstock for a variety of applications, including alga-based biofuels [START_REF] Vieler | Genome, functional gene annotation, and nuclear transformation of the heterokont oleaginous alga Nannochloropsis oceanica CCMP1779[END_REF]. The de novo synthesis of fatty acids (FA) in Nannochloropsis species generates myristic, palmitic and palmitoleic acids (14:0, 16:0 and 16:1, respectively) within the plastid, which are exported to the cytosol, where they are converted into acyl-CoAs. In the cytosol, 16:0-CoA is elongated into 18:0-CoA (Dolch et al., 2017) prior to a series of desaturations into 18:1 to18:3, elongation into 20:3, and desaturations that ultimately lead to the formation of eicosapentaenoic acid (20:5). In Nannochloropsis oceanica TAG mostly contains 16:0, 16:1, 18:1 and 14:0 [START_REF] Nobusawa | Differently localized lysophosphatidic acid acyltransferases crucial for triacylglycerol biosynthesis in the oleaginous alga Nannochloropsis[END_REF].

Generally, two distinct routes, (i) anacyl-CoA dependent Kennedy pathway and (ii) an acyl-CoA independent pathway, contribute to TAG biosynthesis in many organisms including yeast, land plants, and algae [START_REF] Sorger | Triacylglycerol biosynthesis in yeast[END_REF][START_REF] Chapman | Compartmentation of triacylglycerol accumulation in plants[END_REF][START_REF] Xu | Properties and biotechnological applications of acylcoa:diacylglycerol acyltransferase and phospholipid:diacylglycerol acyltransferase from terrestrial plants and microalgae[END_REF]. On the one hand, the Kennedy pathway starts with glycerol-3phosphate and involves a set of acyltransferases that pull the acyl moiety from acyl-CoAs to the glycerol backbone. N. oceanica contains a single glycerol-3-phosphate acyltransferase (GPAT) transferring acyls to the sn-1 position [START_REF] Nobusawa | Differently localized lysophosphatidic acid acyltransferases crucial for triacylglycerol biosynthesis in the oleaginous alga Nannochloropsis[END_REF].

Then four lysophosphatidic acid acyltransferases (LPAT) add a second acyl at the sn-2 position, using acyl-CoA donors. The function of NoLPAT1, NoLPAT2, NoLPAT3 and NoLPAT4 has been unraveled by the analysis of single-and multiple-gene knockouts [START_REF] Nobusawa | Differently localized lysophosphatidic acid acyltransferases crucial for triacylglycerol biosynthesis in the oleaginous alga Nannochloropsis[END_REF]. NoLPAT1 is mainly involved in the transfer of 16:1 to the sn-2 position of precursors used for phosphatidylcholine (PC) and the betaine lipid (diacylglycerol-N,N,N-trimethylhomoserine, DGTS) biosynthesis. This isoform is not involved into TAG biosynthesis. By contrast, NoLPAT4 transfers 16:0 to the sn-2 position of a pool of phosphatidic acid strictly dedicated to TAG production. NoLPAT2 and NoLPAT3 are involved in both membrane lipid and TAG production. Genetic studies support some functional overlap among NoLPAT1, NoLAPT2 and NoLPAT3 in membrane lipids, but the lpat2;3 double KO produced substantially less phosphatidylethanolamine (PE). In brief, NoLPAT2 and NoLPAT3 are mainly involved in the transfer of 18:1 at the sn-2 position of precursors used for PC, PE and that of 16:0 at the sn-2 position of precursors for TAG [START_REF] Nobusawa | Differently localized lysophosphatidic acid acyltransferases crucial for triacylglycerol biosynthesis in the oleaginous alga Nannochloropsis[END_REF]. Eventually, diacylglycerol (DAG) acyltransferase (DGAT) catalyzes the last and committing step for TAG biosynthesis [START_REF] Chapman | Compartmentation of triacylglycerol accumulation in plants[END_REF]. Many microalgae harbor a high copy number of DGAT-encoding genes, e.g., more than 10 copies in N. oceanica [START_REF] Vieler | Genome, functional gene annotation, and nuclear transformation of the heterokont oleaginous alga Nannochloropsis oceanica CCMP1779[END_REF]Li et al., 2014b). By contrast, land plants typically maintain no more than two copies, e.g., one type I (DGAT1) and one type II (DGAT2) in Arabidopsis [START_REF] Xu | Properties and biotechnological applications of acylcoa:diacylglycerol acyltransferase and phospholipid:diacylglycerol acyltransferase from terrestrial plants and microalgae[END_REF]. Functional characterization of algal DGATs have demonstrated their overlapping yet complementary roles in the spatial and temporal control of TAG biosynthesis with diverse patterns of molecular species (Liu et al., 2016a;Wei et al., 2017a;[START_REF] Xin | Producing designer oils in industrial microalgae by rational modulation of co-evolving type-2 diacylglycerol acyltransferases[END_REF][START_REF] Zienkiewicz | Nannochloropsis, a rich source of diacylglycerol acyltransferases for engineering of triacylglycerol content in different hosts[END_REF][START_REF] Mao | Characterization of type I and type II diacylglycerol acyltransferases from the emerging model alga Chlorella zofingiensis reveals their functional complementarity and engineering potential[END_REF]. Manipulation of DGAT genes via rational engineering has therefore a potential to not only increase TAG level but also tailor FA composition for particular uses (Liu et al., 2016a;[START_REF] Xin | Producing designer oils in industrial microalgae by rational modulation of co-evolving type-2 diacylglycerol acyltransferases[END_REF].

On the other hand, the acyl-CoA independent pathway for TAG biosynthesis is mediated by phospholipid:diacylglycerol acyltransferase (PDAT), transferring an acyl moiety from the sn-2 position of a membrane glycerolipid, usually a phospholipid, to the sn-3 position of DAG (Dahlqvist et al., 2000). In the yeast Saccharomyces cerevisiae, knockout and overexpression of the PDAT-encoding gene LRO1 supported its indispensable contribution to TAG biosynthesis [START_REF] Oelkers | A lecithin cholesterol acyltransferase-like gene mediates diacylglycerol esterification in yeast[END_REF], together with the type II DGAT-encoding gene DGA1 [START_REF] Oelkers | The DGA1 gene determines a second triglyceride synthetic pathway in yeast[END_REF][START_REF] Sandager | Storage lipid synthesis is nonessential in yeast[END_REF].Yet the relative contributions of PDAT and DGA1 are growth stage-dependent: PDAT contributes more at the exponential growth stage while DGA1 contributes more at the stationary stage [START_REF] Oelkers | The DGA1 gene determines a second triglyceride synthetic pathway in yeast[END_REF]. Albeit showing activities on a broad range of polar lipids, S. cerevisiae PDAT prefers the zwitterionic phospholipids PE and PC, particularly the former, as acyl donor for TAG biosynthesis (Dahlqvist et al., 2000;[START_REF] Ghosal | Saccharomyces cerevisiae phospholipid:diacylglycerol acyl transferase (PDAT) devoid of its membrane anchor region is a soluble and active enzyme retaining its substrate specificities[END_REF][START_REF] Feng | Expanding of phospholipid:diacylglycerol acyltransferase (PDAT) from Saccharomyces cerevisiae as multifunctional biocatalyst with broad acyl donor/acceptor selectivity[END_REF]. In contrast to the presence of a single copy of PDAT gene in yeast and algae, many land plants experienced gene expansion events during evolution and contain multiple PDAT copies [START_REF] Pan | Genome-wide analysis of Phospholipid-diacylglycerol acyltransferase (PDAT) genes in plants reveals the eudicot-wide PDAT gene expansion and altered selective pressures acting on the core eudicot PDAT paralogs[END_REF]Falarz et al., 2020). Unlike in S. cerevisiae, neither overexpression nor knockout of PDAT1 in Arabidopsis affected the seed lipid profiles [START_REF] Ståhl | Cloning and functional characterization of a phospholipid:diacylglycerol acyltransferase from Arabidopsis[END_REF][START_REF] Mhaske | Isolation and characterization of an Arabidopsis thaliana knockout line for phospholipid:diacylglycerol transacylase gene (At5g13640)[END_REF]. Nevertheless, in a dgat1 null mutant of Arabidopsis, knockdown of PDAT1 leads to a more severe decrease in seed TAG level, suggesting that PDAT1 and DGAT1 have overlapping activities at least in seeds [START_REF] Zhang | DGAT1 and PDAT1 acyltransferases have overlapping functions in Arabidopsis triacylglycerol biosynthesis and are essential for normal pollen and seed development[END_REF]. By contrast, in actively growing tissues such as developing leaves, PDAT1 functions independently and contributes more than DGAT1 to TAG biosynthesis, by diverting FA from membrane lipids (Fan et al., 2013a(Fan et al., , 2013b)). Arabidopsis PDAT1 has a similar substrate specificity as that of S. cerevisiae PDAT, using PE and PC as acyl donors, as evidenced by the increase in PE and PC in a pdat1 mutant and decrease in PE and PC in a PDAT1-overexpressing line (Fan et al., 2013a(Fan et al., , 2013b)).

While well documented in yeast and land plants, PDAT from algae was less scrutinized. Most detailed biochemical and physiological characterization are restricted to green algae, especially Chlamydomonas reinhardtii [START_REF] Yoon | Phospholipid:diacylglycerol acyltransferase is a multifunctional enzyme involved in membrane lipid turnover and degradation while synthesizing triacylglycerol in the unicellular green microalga Chlamydomonas reinhardtii[END_REF]Liu et al., 2016b).

Differing from yeast or Arabidopsis enzymes, C. reinhardtii PDAT can use a broad range of substrates including phospholipids, glycolipids and DAG as acyl donors. Still, in vitro enzymatic assays highlighted an apparent preference of C. reinhardii PDAT for anionic phospholipids such as phosphatidylinositol (PI), phosphatidylserine (PS) and phosphatidylglycerol (PG) [START_REF] Yoon | Phospholipid:diacylglycerol acyltransferase is a multifunctional enzyme involved in membrane lipid turnover and degradation while synthesizing triacylglycerol in the unicellular green microalga Chlamydomonas reinhardtii[END_REF]. Intriguingly, it is believed that C. reinhardtii PDAT resides in the chloroplast, although the precise localization remains to be experimentally confirmed, and mediates turnover of chloroplast membrane lipids for TAG biosynthesis particularly under non-stress (favorable growth) conditions [START_REF] Yoon | Phospholipid:diacylglycerol acyltransferase is a multifunctional enzyme involved in membrane lipid turnover and degradation while synthesizing triacylglycerol in the unicellular green microalga Chlamydomonas reinhardtii[END_REF]. By contrast, the three type II DGAT genes in C. reinhardtii, CrDGTT1, CrDGTT2 and CrDGTT3, are predominantly active under stress conditions rather than favorable growth conditions (Liu et al., 2016a).

Altogether, the roles of PDAT and DGAT seem evolutionarily conserved, namely, PDAT mediating membrane lipid turnover and functioning under favorable growth conditions, such as in S. cerevisiae and C. reinhardtii or developing leaves of Arabidopsis, while DGAT function is apparently predominant following growth arrest.

The context in which PDAT might operate, i.e. in close relation with the ER or the plastid, is unknown in such clades as Stramenopiles/Heterokonts, whose cells contain plastids originating from a secondary endosymbiosis, bounded by four membranes, with the outermost being related to the endomembrane system. This outermost membrane is known as the chloroplast endoplasmic reticulum (cER) or epiplastid membrane, and its lipid composition has not yet been determined [START_REF] Petroutsos | Evolution of galactoglycerolipid biosynthetic pathways -From cyanobacteria to primary plastids and from primary to secondary plastids[END_REF][START_REF] Flori | Ultrastructure of the periplastidial compartment of the diatom Phaeodactylum tricornutum[END_REF]Dolch et al., 2017).

Unlike C. reinhardtii, the stramenopile alga N. oceanica has been recognized as an industrially important single-cell factory for lipid production because of its fast growth and high lipid content [START_REF] Ma | Lipid production from Nannochloropsis[END_REF]. Genome data [START_REF] Vieler | Genome, functional gene annotation, and nuclear transformation of the heterokont oleaginous alga Nannochloropsis oceanica CCMP1779[END_REF][START_REF] Wang | Nannochloropsis genomes reveal evolution of microalgal oleaginous traits[END_REF] and a variety of forward and reverse genetic tools [START_REF] Wang | Genome editing of model oleaginous microalgae Nannochloropsis spp. by CRISPR/Cas9[END_REF][START_REF] Wei | RNAi-based targeted gene-knockdown in the model oleaginous microalgae Nannochloropsis oceanica[END_REF]Poliner et al., 2018aPoliner et al., , 2018b[START_REF] Poliner | A high-capacity gene stacking toolkit for the oleaginous microalga, Nannochloropsis oceanica CCMP1779[END_REF] are available, providing solid foundation to study lipogenesis. N. oceanica harbors thirteen DGAT genes (two type I referred to as NoDGAT1A and NoDGAT1B, and eleven type II referred to as NoDGAT2A through NoDGAT2K) (Li et al., 2014b) versus six in C. reinhardtii (one type I and five type II) [START_REF] Boyle | Three acyltransferases and nitrogen-responsive regulator are implicated in nitrogen starvation-induced triacylglycerol accumulation in Chlamydomonas[END_REF]. Although both type I and type II N. oceanica DGATs show activities for TAG biosynthesis in vitro, their roles in vivo depend on growth conditions: NoDGAT1A, NoDGAT2A, and NoDGAT2D operate under stress conditions, whereas NoDGAT2C, NoDGAT2J and NoDGAT2K operate more under non-stress conditions (Wei et al., 2017a;[START_REF] Xin | Producing designer oils in industrial microalgae by rational modulation of co-evolving type-2 diacylglycerol acyltransferases[END_REF][START_REF] Xin | Biosynthesis of triacylglycerol molecules with a tailored PUFA profile in industrial microalgae[END_REF]. The differences in the number of functional DGATs between N. oceanica and C. reinhardtii and the presence of DGATs that contribute to non-stress associated TAG biosynthesis in N. oceanica raise the questions whether N. oceanica PDAT is critical, and under what conditions and to what extent it contributes to TAG biosynthesis.

In the present study, we addressed the function of NoPDAT in N. oceanica. NoPDAT resides at the cER, where both plastid and ER lipids are presumed to be present. In vitro assays supported that NoPDAT could use a broad range of glycolipids and phospholipids and suggested an apparent preference for PG as acyl donor. Genetic analysis showed that NoPDAT contributed at least 30% to TAG biosynthesis under nitrogen limited conditions and seemed determinant in the relative proportion of 16:0 and 16:1 at position sn-3 of TAG. In the cellular context, the knockdown of NoPDAT caused an increase, regulated by CO2 availability, of PE with 16:0, 16:1 or 18:1 and of PG with 16:0 and 16:1, at position sn-2 of the lipid glycerol backbone. Our study highlights the role of NoPDAT in mobilizing membrane lipids, particularly PE and PG, in the massive biosynthesis of TAG in N. oceanica and suggests that the saturated/monounsaturated acyl PE can serve as a potential lipid sink under stress with high CO2 level.

RESULTS

Cloning and Bioinformatics Analysis of the PDAT Gene from N. oceanica

The sequenced genome of N. oceanica contains a single PDAT-encoding gene candidate [START_REF] Vieler | Genome, functional gene annotation, and nuclear transformation of the heterokont oleaginous alga Nannochloropsis oceanica CCMP1779[END_REF][START_REF] Wang | Nannochloropsis genomes reveal evolution of microalgal oleaginous traits[END_REF]. We obtained the corresponding full-length cDNA (referred to as NoPDAT), which consists of a 136-bp 5' untranslated region, a 242bp 3' untranslated region, and a 2,835-bp coding sequence interrupted by four introns.

Analysis of the polypeptide sequence of NoPDAT against the conserved domain database of the National Center for Biotechnology Information (NCBI) indicated that this protein belongs to the membrane-bound O-acyltransferase family and contains a phosphatidylcholine-sterol O-acyltransferase domain spanning from 251 to 944 amino acid residues. Transmembrane helix prediction with the TMHMM method [START_REF] Krogh | Predicting transmembrane protein topology with a hidden markov model: Application to complete genomes[END_REF] suggested the presence of a transmembrane domain (spanning 186-205 amino acid residues) at the N-terminus of NoPDAT (Supplemental Figure 1).

To gain insights into the evolutionary relationship between NoPDAT and other homologs from different phyla, we carried out both a Maximum Likelihood (ML) and a Bayesian phylogenetic inference with a 30-sequence dataset containing six stramenopiles, three basidiomycetes and three ascomycetes, five green algae, one streptophyte and nine sequences from monocots and dicots. Although red algae were supposed to lack PDAT [START_REF] Lee | Comparative genomic analyses of transport proteins encoded within the red algae Chondrus crispus, Galdieria sulphuraria, and Cyanidioschyzon merolae[END_REF][START_REF] Mori | Construction of global acyl lipid metabolic map by comparative genomics and subcellular localization analysis in the red alga Cyanidioschyzon merolae[END_REF], we identified and included in the phylogeny one sequence from Porphyra umbilicalis, and two from Porphyridium purpureum. Based on preliminary phylogenetic inferences (see Methods for details), red algal PDATs were used as outgroup to root the tree. Bayesian and ML tree topologies were in perfect accordance on the end branches with higher plants PDAT1 (ML support 100, Bayesian posterior probability 1) and PDAT2 (98, 1), fungi (100, 1), and green algae (60, 0.98) clustering accordingly together in robust individual clades. Interestingly, the Klebsormidium nitens putative PDAT sequence clustered in both inferences in a basal position to the higher plants (100, 1), in accordance with Streptophyta's evolutionary history [START_REF] Hori | Klebsormidium flaccidum genome reveals primary factors for plant terrestrial adaptation[END_REF][START_REF] Wang | Genomes of early-diverging streptophyte algae shed light on plant terrestrialization[END_REF]. Surprisingly, stramenopile PDAT sequences did not cluster together, namely, N. oceanica and Microchloropsis gaditana (formerly known as Nannochloropsis gaditana [START_REF] Fawley | The phylogeny of the genus Nannochloropsis (Monodopsidaceae, Eustigmatophyceae), with descriptions of N. australis sp. nov. and Microchloropsis gen[END_REF] grouped in a sister clade to Phytophtora infestans/Plasmoparahalstedii clade with a weak support (37, 0.74); for diatoms ML did not resolve supraspecific level, while Bayesian analysis robustly (posterior probability= 0.99) clustered diatoms in basal position to the clade grouping plants, fungi, and the other stramenopiles. The polyphyly of stramenopile PDATs may reveal different evolutionary history in diatoms vs eustigmatophyte/oomycetes (Figure 1).

Protein sequence alignment suggested that NoPDAT harbors domains conserved in PDAT polypeptides (Supplemental Figure 1). The analysis of the N-terminal part of NoPDAT using SignalP [START_REF] Nielsen | A Brief history of protein sorting prediction[END_REF] did not show any predicted signal peptide.

The use of HECTAR, a method to predict subcellular targeting in Heterokonts/Stramenopiles [START_REF] Gschloessl | HECTAR: a method to predict subcellular targeting in heterokonts[END_REF], together with other prediction tools such as TargetP and ChloroP [START_REF] Armenteros | Detecting sequence signals in targeting peptides using deep learning[END_REF] or Mitoprot [START_REF] Claros | Computational method to predict mitochondrially imported proteins and their targeting sequences[END_REF] did not support any localization in chloroplast or mitochondrion. The absence of any targeting sequence to chloroplast appears consistent with NoPDAT sequence grouping also with non-photosynthetic Stramenopile sequences such as PDAT from oomycota.

Functional Complementation of NoPDAT in the TAG-Deficient Yeast and in vitro

Assay

To evaluate the function of NoPDAT, the coding sequence was introduced into the TAGdeficient yeast strain H1246 [START_REF] Sandager | Storage lipid synthesis is nonessential in yeast[END_REF] for complementation assay. The H1246 strains expressing the empty vector (EV) and CrDGTT3, a type II DGAT from C. reinhardtii (Liu et al., 2016a), were used as negative and positive controls, respectively. H1246 cells carrying EV gave no detectable level of TAG. By contrast, the expression of NoPDAT, similar to CrDGTT3, restored TAG biosynthesis in H1246 cells, as indicated by the prominent TAG spot resolved by thin-layer chromatography (TLC) (Figure 2A) and green fluorescence following Bodipy 493/503 staining (a specific dye of neutral lipids) (Figure 2B). These results suggested that NoPDAT is functional in transferring acyl moiety from polar lipids to DAG for TAG assembly. They also emphasize that yeast subcellular membranes contain substrates that NoPDAT can possibly utilize.

We developed a non-radiolabeled in vitro assay for acyltransferases using the microsomal protein fraction from yeast cells as a source of crude enzyme [START_REF] Liu | A simple and reproducible nonradiolabeled in vitro assay for recombinant acyltransferases involved in triacylglycerol biosynthesis[END_REF], following the method described in previous studies to measure the activity and substrate specificity of algal DGATs (Liu et al., 2016a;Wei et al., 2017a;[START_REF] Xin | Producing designer oils in industrial microalgae by rational modulation of co-evolving type-2 diacylglycerol acyltransferases[END_REF][START_REF] Mao | Characterization of type I and type II diacylglycerol acyltransferases from the emerging model alga Chlorella zofingiensis reveals their functional complementarity and engineering potential[END_REF]. A total of ten polar lipids were examined, including phosphate-free glycolipids, i.e. monogalactosyldiacylglycerol, MGDG (enriched in 18:3/16:3); digalactosyldiacylglycerol, DGDG (enriched in 18:3/18:3); sulfoquinovosyldiacylglycerol, SQDG (enriched in 18:3/16:0); DGTS (16:1/16:1) and phospholipids, i.e. PC (16:1/18:1); PS (16:1/18:1); phosphatidic acid, PA (16:1/18:1); PE (16:1/18:1); PI (enriched in 18:2/16:0) and PG (racemic mix of 16:1/18:1 and 18:1/16:1).

Among the tested polar lipids, PG led to the highest activity for TAG formation, while others showed either a low (SQDG, MGDG, and DGDG), very low or no apparent activity (Figure 2C). This in vitro analysis confirms that NoPDAT is a glycerolipid acyl transferase, which may accept other substrates than phospholipids, e.g. phosphate-free glycolipids. This enzymatic ability to accept multiple membrane glycerolipids as acyl donors is reminiscent to previous observations made with homologs from C. reinhardtii and S. cerevisiae [START_REF] Yoon | Phospholipid:diacylglycerol acyltransferase is a multifunctional enzyme involved in membrane lipid turnover and degradation while synthesizing triacylglycerol in the unicellular green microalga Chlamydomonas reinhardtii[END_REF][START_REF] Feng | Expanding of phospholipid:diacylglycerol acyltransferase (PDAT) from Saccharomyces cerevisiae as multifunctional biocatalyst with broad acyl donor/acceptor selectivity[END_REF]. Like for the C. reinhardtii enzyme, NoPDAT shows a high in vitro activity with the anionic lipid PG. However, the C. reinhardtii PDAT could also operate on zwitterionic PE or PC substrates in vitro. It must be noted that in N. oceanica, the native plastid PG molecular species are different from that used in this in vitro assay, mainly containing 16:0/16:1, 20:5/16:0 and 20:5/16:1, with a portion of 16:1 being 16:1t a peculiar hexadecenoic acid with a transdesaturation at position Δ3, distinct from palmitoleic acid with a cis-desaturation at position Δ9. The apparent differences observed in NoPDAT activity with the substrates tested in the in vitro assay, and the apparent lack of activity on zwitterionic phospholipids, highlight one of the limits of this enzymatic measurement. Indeed, a lack of in vitro activity might be attributed either to an intrinsic absence of affinity for the considered substrate, a poor availability of the substrate in the microsomal suspension, non-optimal reaction conditions, or a combination of all these hypotheses.

We also tested the preference of NoPDAT for the structure of the DAG acceptor, including seven 1,2-DAG molecular species and one 1,3-DAG, using PG as the acyl donor (Figure 2D). Based on this assay, NoPDAT could operate on a broad spectrum of DAG acceptors, even belonging to the 1,3 series, with a higher apparent activity with 18:1/18:1, 16:0/18:1 and 18:1/16:0. We did not evaluate the main DAG donor from N. oceanica, 16:1/16:0 [START_REF] Nobusawa | Differently localized lysophosphatidic acid acyltransferases crucial for triacylglycerol biosynthesis in the oleaginous alga Nannochloropsis[END_REF], which was not commercially available.

Altogether, this in vitro assay validates the molecular activity of the N. oceanica enzyme as a canonical PDAT, with the potential to use various membrane glycerolipids as acyl donors and various DAG species as acyl acceptors. It must be noted that, due to the constrains of available membrane lipid and DAG substrates, the molecular species used in this in vitro assay differed from the acyl profiles of each lipid class encountered naturally in N. oceanica. The actual substrates used by NoPDAT requires therefore, like for previous studies performed in other organisms, complementary genetic analyses.

Subcellular Localization of NoPDAT at the cER

PDAT proteins are membrane-bound and usually reside at the endoplasmic reticulum (ER) in higher plants and yeast (Dahlqvist et al., 2000;[START_REF] Kim | Endoplasmic reticulum-located PDAT1-2 from castor bean enhances hydroxy fatty acid accumulation in transgenic plants[END_REF]. By contrast, the compartmentalization for green algal PDAT remains ambiguous and lacks experimental evidence [START_REF] Yoon | Phospholipid:diacylglycerol acyltransferase is a multifunctional enzyme involved in membrane lipid turnover and degradation while synthesizing triacylglycerol in the unicellular green microalga Chlamydomonas reinhardtii[END_REF]Liu et al., 2016b). It is worth noticing that in Heterokonts/Stramenopilesthe plastid evolved from a secondary endosymbiosis and is bounded by four membranes (Fussy and Obornik, 2018); the outermost membrane, referred to as cER, is related to the ER and directly connected with the outer membrane of the nuclear envelope [START_REF] Gibbs | The route of entry of cytoplasmically synthesized proteins into chloroplasts of algae possessing chloroplast ER[END_REF][START_REF] Flori | Ultrastructure of the periplastidial compartment of the diatom Phaeodactylum tricornutum[END_REF].

To get insight on the localization of NoPDAT, we fused the NoPDAT coding sequence with the enhanced green fluorescent protein (eGFP) upstream and introduced it into N. oceanica for live cell observation. The eGFP alone was used as control. Laser confocal scanning microscopy analyses showed that in the control transformants the eGFP signal (green) was present in the cytosol, not overlapping with the plastid autofluorescence (PAF, red) (Figure 3A). By contrast, in the NoPDAT-eGFP transformants, the green eGFP signal formed irregular layers surrounding the plastid (red) (Figure 3B), which was further supported by a three-dimensional visualization of eGFP and PAF signals (Figure 3C). We also introduced the NoPDAT-eGFP fusion into Phaeodactylum tricornutum, a Heterokont alga with bigger cells, in which there is experimental demonstration of cERlocalized proteins [START_REF] Hempel | ERADderived preprotein transport across the second outermost plastid membrane of diatoms[END_REF]. Clearly, the eGFP signal (green) surrounded the plastid (red) and nucleus (blue, stained with Hoechst) (Figure 3D), resembling the localization pattern of hDer1-2, a confirmed cER-localized protein [START_REF] Hempel | ERADderived preprotein transport across the second outermost plastid membrane of diatoms[END_REF].

These results suggest that NoPDAT resides at the cER.

Overexpression of NoPDAT in N. oceanica Promotes TAG Accumulation without

Compromised Growth and Suggests a Consumption of 16:0 and 16:1 in Polar Lipids

To investigate the function and biological role of NoPDAT in N. oceanica, we first generated overexpressing lines. The wild-type (WT) strain was used as control. More than ten putative transformants (confirmed by genomic PCR) were screened by quantitative real-time PCR (qPCR). Two lines, PDAT-OE3 and PDAT-OE5 that showed maximal increase in NoPDAT transcripts (>10-fold higher than WT), were selected for subsequent analysis (Figure 4A). Immunoblot analysis using the NoPDAT antibody revealed that NoPDAT protein abundance was higher in the two transformants than in WT (Figure 4B). During a 10-day batch culture period, no difference in the culture optical density (OD730) was observed between the transformants and WT (Figure 4C), suggesting that NoPDAT overexpression had no effect on the growth of N. oceanica.

Accordingly, the nitrate concentration in the culture medium showed no difference between the transformants and WT and was almost consumed within six days (Figure 4D). Fluorescence intensity assay of Nile red-stained algal cells, a rapid and reliable method for relative TAG quantification in algae [START_REF] Ge | Methylcrotonyl-CoA carboxylase regulates triacylglycerol accumulation in the model diatom Phaeodactylum tricornutum[END_REF][START_REF] Li | Transcriptional regulation of microalgae for concurrent lipid overproduction and secretion[END_REF], indicated that N. oceanica accumulated TAG after four days of culture and the transformants had a higher TAG content than WT during the late culture days (days 6-10; Figure 4E). Gas chromatography-mass spectrometry (GC-MS) quantification revealed that NoPDGT overexpression enhanced TAG by ~32%, yet having no consequences on the content in total FA (TFA) (Figure 4F). NoPDAT overexpression had a limited effect on the FA profile of TAG, whereas a slight decrease of 16:0 and 16:1 and increase of 20:5 was noticed in TFA (Figure 4G and 4H). Since the FA profile in TFA accounts for both membrane lipids and TAG, and since the percentage of TAG within this proportion increases, the impact on TFA, even minor, reflects a consumption of 16:0 and 16:1 in polar lipids. Therefore, it can be hypothesized that NoPDAT mainly transfers 16:0 and 16:1 from polar lipids in vivo. We sought to confirm this deduction by analyzing mutants defective in NoPDAT expression.

Knockdown of NoPDAT Attenuates TAG Accumulation and Decreases the

Proportion of TAG with 16:0 and 16:1 at sn-3 Position

We generated NoPDAT knockdown lines of N. oceanica via RNAi-mediated genesilencing. Two out of more than ten putative transformants, PDAT-KD2 and PDAT-KD4 that had the lowest NoPDAT transcript abundance (<10% of that in WT on day 8), were chosen for subsequent experiments (Figure 5A). Similar to the overexpression, NoPDAT knockdown had little impact on the growth of N. oceanica (Figure 5B). Nile red staining and fluorescence intensity assay indicated that the knockdown lines had a TAG content lower than WT, particularly during the late culture days (Figure 5C). GC-MS quantification confirmed the decrease in TAG (reduced by ~32%) caused by NoPDAT knockdown, whereas TFA content showed little change (Figure 5D). Concerning FA profiles, NoPDAT knockdown increased the relative abundance of 16:0 and 16:1 and decreased that of 18:1, 20:4 and C20:5 in TFA; by contrast the FA profile of TAG remained apparently unchanged compared to the WT (Figure 5E and5F). Assuming that TFA reflects FA contained in membrane lipids and TAG, the differences observed in TFA should reflect an impact on membrane glycerolipids. Thus, a knockdown of NoPDAT clearly leads to the accumulation of 16:0 and 16:1 in membrane glycerolipids.

To uncover the NoPDAT knockdown-associated global changes of TAG species in a time-resolved manner, TAG quantification by electrospray ionization mass spectrometry (ESI-MS) analysis was conducted, using the samples of WT and PDAT-KD4 cells from three time points, i.e. day 3, day 5 and day 7. There was no difference in total TAG content between PDAT-KD4 and WT on day 3, a time point before TAG accumulation in N. oceanica cells; by contrast, PDAT-KD4 showed a significantly lower total TAG content than WT on day 5 and day 7 (Figure 6). These results suggest that in N. oceanica, NoPDAT is not involved in the production of a basal level of TAG under favorable growth conditions, but likely acts under nutrient stress, opposite to the function of C. reinhardtii PDAT that contributes more to basal TAG biosynthesis than for TAG accumulation in stationary phase [START_REF] Yoon | Phospholipid:diacylglycerol acyltransferase is a multifunctional enzyme involved in membrane lipid turnover and degradation while synthesizing triacylglycerol in the unicellular green microalga Chlamydomonas reinhardtii[END_REF].

More than 50 TAG molecular species were identified in N. oceanica, shown in Figure 6 and supplemental Data set 1. Among them, seven molecular species, i.e. 52:6 (20:5/16:0/16:1), 50:3 (16:1/18:1/16:1), 50:2 (16:0/16:1/18:1), 48:3 (16:1/16:1/16:1), 48:2 (16:1/16:1/16:0), 48:1 (16:1/16:0/16:0), 46:2 (14:0/16:1/16:1), and 46:1 (16:1/16:0/14:0), accounted for more than 70% of total TAG in both WT and PDAT-KD4 cells on day 3. It is important to notice that PDAT can transfer acyl groups finally recorded at the sn-1 or sn-3 positions of TAG. Over time (e.g., on day 5 and day 7), most of the TAG species followed a similar accumulation pattern and increased considerably in WT and PDAT-KD4. Albeit having no impact on total TAG content in N. oceanica on day 3, NoPDAT knockdown modulated the content of individual TAG species, which were not initially visible when simply considering the TAG FA profile. Focusing on the seven main molecular species, those whose proportion decreased, i.e. (16:1/16:1/16:1), (16:1/16:1/16:0), (14:0/16:1/16:1) and (16:1/16:0/14:0), were more specifically impacted by the knockdown of NoPDAT, highlighting mainly a defect in transfer of 16:0 and 16:1 at position sn-3. At day 3, this trend was also observed for minor TAG species and conversely, the proportion of TAG species containing 20:4 or 20:5 at positions sn-1 or sn-3 increased. At day 5, all TAG species showed a significant decrease in the NoPDAT knockdown lines, consistent with a lowering of the TAG production, and the capacity of NoPDAT to transfer other acyls than 16:0 or 16:1. We sought whether some glycerolipids exhibited a change in their FA that could mirror the decrease in 16:0 and 16:1 in TAG, by a balanced accumulation. 16:0, 16:1 and18:1 PDAT mediates the acyl-CoA independent TAG biosynthesis in which membrane glycerolipids are used as acyl donors (Dahlqvist et al., 2000). We sought whether a specific lipid class accumulated in the NoPDAT knockdown strains compared to WT, based on semi-quantitative TLC analysis of polar lipids, after staining of resolved lipids by iodine. One band clearly accumulated in the KD lines . This band was recovered, subjected to mass spectrometry analysis, and characterized as PE enriched in saturated or monounsatured FA. Henceforth, the TLC spot characterized by such PE molecular species is referred to as "less-unsaturated PE" or LU-PE.

Knockdown of NoPDAT Leads to an Accumulation of PE and PG Enriched in

Quantification via ESI-MS analysis was then conducted for WT and PDAT-KD4 cultures harvested on day 3, day 5, and day 7 under air condition. WT showed a decrease in the overall PE content in the course of cultivation (Figure 7B) and the PE profile was predominantly more-unsaturated, with 20:4/20:4, 20:5/20:4, 20:4/20:3, and20:5/20:5 accounting for over 80% of PE (Figure 7C), consistent with previous observation in Nannochloropsis species [START_REF] Nobusawa | Differently localized lysophosphatidic acid acyltransferases crucial for triacylglycerol biosynthesis in the oleaginous alga Nannochloropsis[END_REF][START_REF] Alboresi | Light remodels lipid biosynthesis in Nannochloropsis gaditana by modulating carbon partitioning between organelles[END_REF]. In PDAT-KD4, the consumption of PE was arrested (Figure 7B). Thus, although NoPDAT had no apparent activity on PE in in vitro enzymatic assay (Figure 2C), PDAT-KD4 accumulated a much higher level of this membrane lipid than the WT (~2.2 fold) regardless of the culture time points (Figure 7B). In PDAT-KD4, the molecular species of PE, which showed an accumulation, were those detected by TLC, i.e. 16:0/16:1 (>100-fold higher than that in WT), followed by 16:1/18:1, 16:1/16:1, 16:0/18:1, 16:0/16:0, 14:0/16:0 and 14:0/16:1 (Figure 7C), corresponding to LU-PE. Considering the sn-2 position in LU-PE molecular species, PE appears therefore as a major donor of 16:0, 16:1 and 18:1, for NoPDAT action.

Since in vitro assays support the capacity of NoPDAT to use other acyl donors, we examined other lipid classes. In line with the strong in vitro activity of NoPDAT on PG (Figure 2C), the PDAT-KD4 line was marked by a ~80% higher level of this lipid class compared to the WT, at all time points (Figure 7B). A total of eight PG species were detected, and like for PE, those harboring a 16:0 or 16:1 at position sn-2 accumulated in the PDAT-KD4 line (Figure 7D). However, different from PE that tended to increase in PDAT-KD4 line, PG decreased gradually in both WT and PDAT-KD4 cells as the culture persisted.

Minor quantitative variations were noticeable for phospholipids such as PC or PI (Figure 7B), consistent with in vitro tests (Figure 2C). However, contrary to the results obtained with plastid glycolipids in in vitro assays, MGDG or DGDG levels changed a little in the PDAT-KD4 line compared to the WT (Figure 7B). As for the FA profiles of MGDG, DGDG or PC, a slight enrichment in molecular species measured in the PDAT-KD4 line concerned 16:0 or 16:1 at the sn-2 position, which could be related to a utilization by NoPDAT (Supplemental Figure 2). These findings suggest that NoPDAT may have poor activity on PC, PI, MGDG or DGDG in in vivo conditions, either by a lack of specificity or due to a location of the enzyme with limited access to these lipids.

Interestingly, in the course of cultivation, the WT strain showed some variations in the PC profile, such as a decrease in molecular species with a 16:1 at the sn-2 position, balanced by an increase of molecular species enriched in 18:1 or 18:2, but these changes were unaffected by the NoPDAT knockdown, and are therefore unrelated to this enzyme.

DGTS relative quantity was evaluated based on the staining intensity on TLC plate,

showing no moderate change except for day 3 in the PDAT-KD4 line (Supplemental Figure 3).

Eventually, plasmenylethanolamine (PME) was detected in trace amount in N. oceanica and accumulated considerably (~15 fold) following NoPDAT knockdown, showing that this unusual phospholipid, structurally close to PE, could also serve as acyl donor for NoPDAT (Figure 7E).

Transcriptional Response of Lipid Metabolic Genes to NoPDAT Knockdown

To investigate the effect of NoPDAT knockdown on lipid metabolism network at the transcriptional level, reverse-transcription qPCR (RT-qPCR) was conducted for 55 lipid metabolic genes in WT and PDAT-KD4 (Figure 8 and supplemental Data set 2). The pyruvate dehydrogenase (PDH) that catalyzes the production of acetyl-CoA, the enzymes involved in de novo fatty acid biosynthesis such as acetyl-CoA carboxylase (ACC), malonyl-CoA:ACP transacylase (MCAT), ketoacyl-ACP synthase III and I (KASIII/KASI), ketoacyl-ACP reductase (KAR), hydroxyacyl-ACP dehydrase (HAD), and enoyl-ACP reductase (ENR), acyl-ACP desaturase (AAD) that is responsible for the formation of 16:1 ∆7 -ACP and 18:1 ∆9 -ACP, and long-chain acyl-CoA synthetase 2 (LACS2) that catalyzes the production of acyl-CoA pool were down-regulated at the transcriptional level in response to NoPDAT knockdown. Since NoPDAT knockdown attenuates TAG biosynthesis (Figure 5D and Figure 6), it is likely that the cellular demand for FAs is lower, giving rise to a transcriptional down-regulation feedback on FA biosynthesis (Figure 8). In line with this, PDAT-KD4 had a slightly lower TFA content than WT (Figure 5D).

The ∆6-fatty acid desaturase (∆6 FAD) that introduces a double bond between carbons 6 and 7 on C18 acyls and fatty acid elongase 2 (FAE2) that catalyzes the formation of C20 acyls from C18 acyls were also down-regulated in the NoPDAT knockdown line (Figure 8). This may partially explain that the knockdown strains had lower percentage of very long-chain fatty acids 20:4 and 20:5 (Figure 5E).

When considering enzymes involved in the Kennedy pathway, the expression of GPAT, LPAT1, LPAT2, LPAT3, LPAT4 and of DGATs was little impacted. This indicates that a lack of NoPDAT does not trigger any compensatory mechanism via the acyl-CoA dependent pathway, consistent with the observed impact of overexpression and knockdown on TAG levels. The PDAT pathway appears therefore independent from the Kennedy pathway.

Finally, although PE, PG and PME increased considerably (Figure 7), no up-regulation of their biosynthesis at the transcriptional was observed in response to NoPDAT knockdown (Figure 8). Altogether, the analysis of the transcription of lipid metabolism genes suggests an orchestrated control of FA production, but no clear reprograming of genes involved in membrane lipid or TAG assembly. The phenotype of NoPDAT knockdown line observed at the level of lipid classes is therefore mainly attributable to a regulation of the metabolic network, and not to a long term genetic reprogramming. Enriched in 16:0, 16:1 and18:1 The accumulation of LU-PE was observed in NoPDAT knockdown cultures aerated with air and was visible by TLC analysis (Figure 7A-left), however, it was absent in PDAT-KD4 line grown at static condition (Figure 7A-right). For the WT, the LU-PE band was not visible even with the aeration of 2% CO2 until day 7 (Supplemental Figure 4A), let alone the static (Supplemental Figure 3) and air conditions (Figure 7A-left). Accordingly, PDAT-KD4 aerated with 2% CO2 accumulated more LU-PE than air condition as shown on TLC plate (Figure 7A-right).

CO2 Availability Affects the NoPDAT Knockdown-Associated Accumulation of PE

To gain the detailed changes in response to CO2 availability, quantification of glycerolipids in PDAT-KD4 via ESI-MS was performed. Compared to cultures aerated with air, static cultures showed a higher level of glycolipids (MGDG and DGDG) but a lower level of phospholipids (PG, PE, PC and PI); Cultures aerated with 2% CO2, on the other hand, was comparable to cultures aerated with air in the level of MGDG, DGDG and PG, yet having a higher level of PE and PI and a lower level of PC (Figure 7B).

Unlike those PE enriched in 20:4/20:4, 20:5/20:4 and 20:4/20:3 that had the highest level in cultures aerated with air, LU-PE species were all correlated with the CO2 availability and their levels followed the order of static cultures <cultures aerated with air< cultures aerated with 2% CO2 (Figure 7C). Similarly, the PG enriched in 16:0, 16:1 and 18:1 in PDAT-KD4 were positively correlated with CO2 availability (Figure 7D). PME species were also increased by CO2 availability: the higher CO2 supply, the higher level of PME (Figure 7E). In addition, the level of some more-unsaturated MGDG and DGDG species was lower in cultures aerated with air than that in static cultures (Supplemental Figure 2). Under static condition, LU-PE maintained a rather low level in PDAT-KD4 line on day 5, though it was higher than that on day 5 in WT aerated with air. Moreover, LU-PE content of PDAT-KD4 under static condition was comparable to that on day 3 and less than a half of that on day 7 in WT aerated with air, and it was less than 10% and 5% of that of PDAT-KD4 aerated with air and 2% CO2 respectively.

PDAT mRNA level increased in the course of cultivating and maintained the highest from day 5 to 7 at air condition in WT (Supplemental Figure 4B), and immunoblotting analysis showed that increasing CO2 supply (from air to 2% and 5% CO2) in the culture enhanced PDAT expression (Supplemental Figure 4C). It was evident that accumulation of LU-PE in WT cells either at day 7 or with elevated CO2 supply was not attributed to the down-regulated PDAT, showing that LU-PE may act as carbon sink under stress with high CO2 level. In fact, aeration with 5% CO2 led to a visible LU-PE band not only in WT but also in PDAT overexpression strains (Supplemental Figure 4A).

Although TAG species responded differentially to CO2 availability in PDAT-KD4, the major more-unsaturated TAG species such as 52:6 (20:5/16:0/16:1), 52:5 (20:4/16:1/16:0) and 52:4 (20:3/16:0/16:1) were correlated with CO2 supply and their levels followed the order of static cultures <cultures aerated with air< cultures aerated with 2% CO2 (Figure 6). These data suggest under high CO2 supply conditions, acyldonors with 16:0 and 16:1 are likely partitioned preferentially to PE (maybe also PG) while acyl-donors with 20:4 and 20:5 are partitioned preferentially to TAG, with LU-PE and TAG serving as carbon sinks.

LU-PE Accumulation is Independent from Genes Involved in PE Homeostasis

The analysis of the NoPDAT knockdown line suggests that this enzyme is responsible for the specific consumption of PE with 16:0 and 16:1 at position sn-2 of molecular species, collectively called LU-PE, which then represent a minor proportion of PE molecular species. We wondered whether the molecular specificity of other enzymes involved in the production or catabolism of PE could also control LU-PE. We performed overexpression and/or knockdown of a series of genes (Figure 9), including those encoding a putative plant-type DAG:CDP-alcohol (choline/ethanolamine) phosphotransferase (CPT/EPT), a bifunctional enzyme responsible for the last biosynthetic step of PE and PC in the Kennedy pathway, a base-exchange-type phosphatidylserine synthase (PSS) that converts PE into PS, a phosphatidylethanolamine N-methyltransferase (PEMT) and a phospholipid N-methyltransferase (PLMT) that catalyze the methylation of PE to form PC, and phosphoethanolamine N-methyltransferase (PEAMT) that converts phosphoethanolamine to phosphocholine for subsequent PC synthesis [START_REF] Han | Metabolic remodeling of membrane glycerolipids in the microalga Nannochloropsis oceanica under nitrogen deprivation[END_REF][START_REF] Li-Beisson | The lipid biochemistry of eukaryotic algae[END_REF]Zienkiewicz et al., 2020). One-dimension TLC was used to screen phenotypes, based on the detection of the specific LU-PE band, used as readout. Overexpressing of each of the two CPT/EPT genes did not induce any accumulation of LU-PE in N. oceanica (Supplemental Figure 5). The turnover of LU-PE species is therefore unlikely counterbalanced by an increase in PE production. Likewise, overexpression of PSS or knockdown of PSS, PEMT, PLMT or PEAMT did not lead to any LU-PE accumulation (Supplemental Figure 5). Altogether, these results indicated that LU-PE accumulation in N. oceanica is not linked to the genes involved in PE homeostasis, and that NoPDAT is solely responsible for the observed profile change.

DISCUSSION

NoPDAT is a Canonical Glycerolipid:Diacylglycerol Acyltransferase, with a Broad Spectrum of Possible Acyl-Donors, Using Mainly PG and PE in vivo N. oceanica is able to accumulate high levels of TAG, probably due to the presence of a great number of acyltransferases involved in TAG biosynthesis, namely, thirteen DGATs and one PDAT [START_REF] Vieler | Genome, functional gene annotation, and nuclear transformation of the heterokont oleaginous alga Nannochloropsis oceanica CCMP1779[END_REF][START_REF] Wang | Nannochloropsis genomes reveal evolution of microalgal oleaginous traits[END_REF]. Only three out of the six DGATs and the PDAT from C. reinhardtii have been shown to restore TAG synthesis in TAG-deficient yeast [START_REF] Yoon | Phospholipid:diacylglycerol acyltransferase is a multifunctional enzyme involved in membrane lipid turnover and degradation while synthesizing triacylglycerol in the unicellular green microalga Chlamydomonas reinhardtii[END_REF][START_REF] Hung | Functional study of diacylglycerol acyltransferase type 2 family in Chlamydomonas reinhardtii[END_REF]Liu et al., 2016a). By contrast, in addition to the PDAT (Figure 2), six DGATs from N. oceanica have been proven to enable TAG synthesis in the same species (Wei et al., 2017a;[START_REF] Xin | Producing designer oils in industrial microalgae by rational modulation of co-evolving type-2 diacylglycerol acyltransferases[END_REF][START_REF] Xin | Biosynthesis of triacylglycerol molecules with a tailored PUFA profile in industrial microalgae[END_REF]. C. reinhardtii PDAT showed a lower in vitro TAG-synthesizing activity than DGATs [START_REF] Yoon | Phospholipid:diacylglycerol acyltransferase is a multifunctional enzyme involved in membrane lipid turnover and degradation while synthesizing triacylglycerol in the unicellular green microalga Chlamydomonas reinhardtii[END_REF]Liu et al., 2016a). N. oceanica PDAT, on the other hand, seemed more active than DGATs in in vitro TAG synthesis (Figure 2; Wei et al., 2017a;[START_REF] Xin | Producing designer oils in industrial microalgae by rational modulation of co-evolving type-2 diacylglycerol acyltransferases[END_REF].

While DGATs use acyl-CoAs, PDATs transfer acyl groups already esterified to a lipid donor. Since the early characterization utilized PC for investigating in vitro PDAT activities, the enzyme was named as phospholipid:diacylglycerol acyltransferase (Dahlqvist et al., 2000). In fact, PDATs can utilize not only phospholipids but many other substrates as acyl donors, yet the activity and substrate preference are dependent on the PDAT sources (Dahlqvist et al., 2000;[START_REF] Yoon | Phospholipid:diacylglycerol acyltransferase is a multifunctional enzyme involved in membrane lipid turnover and degradation while synthesizing triacylglycerol in the unicellular green microalga Chlamydomonas reinhardtii[END_REF][START_REF] Feng | Expanding of phospholipid:diacylglycerol acyltransferase (PDAT) from Saccharomyces cerevisiae as multifunctional biocatalyst with broad acyl donor/acceptor selectivity[END_REF]. Both S. cerevisiae and Arabidopsis PDATs preferentially transfer the acyl at the sn-2 position of PC and PE to the sn-3 position of DAG to form TAG (Dahlqvist et al., 2000;[START_REF] Ghosal | Saccharomyces cerevisiae phospholipid:diacylglycerol acyl transferase (PDAT) devoid of its membrane anchor region is a soluble and active enzyme retaining its substrate specificities[END_REF]Fan et al., 2013aFan et al., , 2013b;;[START_REF] Feng | Expanding of phospholipid:diacylglycerol acyltransferase (PDAT) from Saccharomyces cerevisiae as multifunctional biocatalyst with broad acyl donor/acceptor selectivity[END_REF]. Concerning C. reinhardtii, the in vitro analysis of CrPDAT highlighted distinct levels of activity and substrate preference, when using various truncated forms of the enzyme, supporting that PDAT conformation, insertion into a membrane, and exposure of hydrophobic residues were critical for interaction with potential substrates. Thus, CrPDAT substrate preference evaluated with a truncated form, highlighted a higher activity with anionicacyl-donors, such as PG, and a lower activity using zwitterionic lipids such as PE and PC [START_REF] Yoon | Phospholipid:diacylglycerol acyltransferase is a multifunctional enzyme involved in membrane lipid turnover and degradation while synthesizing triacylglycerol in the unicellular green microalga Chlamydomonas reinhardtii[END_REF]. When evaluating MGDG, DGDG or SQDG as possible acyl donors, only MGDG could be used as substrate for CrPDAT, but this in vitro activity could be only detected with a fulllength version of the enzyme [START_REF] Yoon | Phospholipid:diacylglycerol acyltransferase is a multifunctional enzyme involved in membrane lipid turnover and degradation while synthesizing triacylglycerol in the unicellular green microalga Chlamydomonas reinhardtii[END_REF]. Here, N. oceanica PDAT had no detectable activity on PE or PC, but exhibited an apparent in vitro preference for PG, and to some extent MGDG, DGDG or SQDG, reminiscent to the results obtained with the C. reinhardtii enzyme (Figure 2C). It must be noticed that the lipidic substrates, obtained from commercial suppliers, contained molecular species differing from those occurring naturally in N. oceanica. On the acceptor side, NoPDAT was efficient on a broad series of DAG molecular species, suggesting that this enzyme could potentially function with a variety of substrates.

As the analyses of overexpression and knockdown lines supported that LU-PE, harboring 16:0, 16:1 or 18:1 at position sn-2, were natural acyl-donors for NoPDAT (Figure 7C), we tested additional PE species, but none of them proved to be utilized by the enzyme in the in vitro conditions (Supplemental Figure 6). Similarly, N. oceanica PDAT exhibits no observed in vitro activity on PC regardless of the acyl compositions (Supplemental Figure 6). Noteworthy, when PG was used as the acyl donor, N. oceanica PDAT showed a consistent preference for molecular species harboring 16:0 and 16:1 at the sn-2 position in in vitro conditions (Supplemental Figure 6). Considering all in vitro and in vivo results, we thus demonstrated that NoPDAT is a canonical glycerolipid:diacylglycerol acyltransferase, which can accept a broad range of acyl-donors with 16:0, 16:1 and to some extent 18:1 at position sn-2, transferred to DAG.

In vivo, main substrates appear to be PE and PG.

The substrate variation of PDATs may also arise from their diversification during evolution. Green and red algae (Chlorophyta and Rhodophyta) originated from a primary endosymbiosis, whereas Stramenopile/Heterokonts emerged from a secondary endosymbiosis, in which eukaryotic hosts engulf and retain a red alga [START_REF] Mcfadden | Primary and secondary endosymbiosis and the origin of plastids[END_REF][START_REF] Yoon | The single, ancient origin of chromist plastids[END_REF][START_REF] Maréchal | Primary endosymbiosis: emergence of the primary chloroplast and the chromatophore, two independent events[END_REF]Fussy and Obornik, 2018). Although both N. oceanica and P. tricornutum belong to Heterokont algae, the balance between secondary host vs endosymbiont genetic importance differed over the course of speciation. In N. oceanica, the symbiont nucleus likely dominated over the host nucleus, whereas in P. trocornutum the symbiont nucleus was probably discarded [START_REF] Guo | Genome assembly of Nannochloropsis oceanica provides evidence of host nucleus overthrow by the symbiont nucleus during speciation[END_REF]. It is possible that N. oceanica and P. tricornutum PDATs were inherited from the symbiont and host nuclei, respectively. Consistent with this hypothesis, the PDAT sequences from Eustigmatophytes and Bacillariophyceae did not group together in reconstructed phylogenetic tree (Figure 1). It has been proposed that PDATs evolution could be driven by different selection constraints and that positive selection may play critical roles in PDAT function (Falarz et al., 2020). Certain amino acids under positive selection within the 'lid' region and adjacent to the catalytic serine (designated by the red arrows; Supplemental Figure 1) are probably involved in this diversification of PDAT structure and function. More functional data need to be gathered in the Heterokont/Stramenopile phylum to investigate this hypothesis.

N. oceanica PDAT Contributes to Membrane Lipid Turnover during Stress-Induced

TAG Biosynthesis

In the batch cultures performed in the present work, wild-type N. oceanica showed a rapid growth within the first four days and then the algal growth slowed down, indicative of the onset of nutrient limitation stress (Figure 4C and Figure 5B). Consistently, the nitrate content monitored in the culture medium was drastically consumed within the first four days (Figure 4D) and thereafter TAG began to accumulate (Figure 4E and Figure 5C). Neither NoPDAT overexpression nor NoPDAT knockdown caused any important change in TAG content of N. oceanica before day 4, thus considered as the favorable growth (nutrient replete) condition with a basal level of synthesized TAG (Figures 456).

By contrast, after four days of cultivation, when the algal cells were exposed to nutrients' limitation, a striking increase of TAG was observed in NoPDAT overexpressing lines, and an opposite trend was observed in NoPDAT knockdown lines (Figures 456). These results suggest a significant contribution of NoPDAT to TAG production at the onset of the stationary phase. In line with this deduction, in the WT background, the NoPDAT transcripts showed a regular increase on day 5 and day 8 compared to day 2 (Figure 4A and Figure 5A). In this context, NoPDAT may function as a TAG contributor in N. oceanica under stress conditions, but not under favorable fast-growing conditions, differing from C. reinhardtii or S. cerevisiae in which PDAT contributes mainly to TAG accumulation under favorable growth conditions [START_REF] Oelkers | The DGA1 gene determines a second triglyceride synthetic pathway in yeast[END_REF][START_REF] Yoon | Phospholipid:diacylglycerol acyltransferase is a multifunctional enzyme involved in membrane lipid turnover and degradation while synthesizing triacylglycerol in the unicellular green microalga Chlamydomonas reinhardtii[END_REF].

Nevertheless, the analysis of knockdown lines showed that even in early cultivation stages, the acyl-donors of PDAT, i.e.LU-PE, accumulated. In addition, we observed a decrease of several TAG species caused by NoPDAT knockdown on day 3, though the total TAG level showed little change (Figure 6). Thus, we cannot rule out the possibility that NoPDAT operates on a basal level and contributes to non-stress associated TAG synthesis as well, but not detectable compared to the bulk produced via the Kennedy pathway, as several NoDGAT genes have been demonstrated to contribute mainly to TAG biosynthesis in N. oceanica under non-stress conditions (Wei et al., 2017a;[START_REF] Xin | Producing designer oils in industrial microalgae by rational modulation of co-evolving type-2 diacylglycerol acyltransferases[END_REF][START_REF] Xin | Biosynthesis of triacylglycerol molecules with a tailored PUFA profile in industrial microalgae[END_REF]. Moreover, NoPDAT, resembling CrPDAT that harbors a conserved lipase motif and can hydrolase various lipids (Supplemental Figure 1; [START_REF] Yoon | Phospholipid:diacylglycerol acyltransferase is a multifunctional enzyme involved in membrane lipid turnover and degradation while synthesizing triacylglycerol in the unicellular green microalga Chlamydomonas reinhardtii[END_REF], may not only transfer acyl moieties from membrane lipids to DAG for TAG biosynthesis but act only as a lipase, as suggested by the increase of many membrane lipids on day 3 in response to NoPDAT knockdown (Figure 7B).

Parallel and Possibly Independent TAG Productions via NoPDAT and NoDGAT

Pathways

Of the three N. oceanica DGAT genes that function mainly under stress conditions, knockdown of NoDGAT1A, NoDGAT2A and NoDGAT2D leads to TAG decrease by 25%, 26% and 47%, respectively (Wei et al., 2017a;[START_REF] Xin | Producing designer oils in industrial microalgae by rational modulation of co-evolving type-2 diacylglycerol acyltransferases[END_REF]. In the present study, NoPDAT knockdown caused ~38% decrease in TAG level (Figure 5D). Thus, although these experiments were not strictly conducted under the same conditions and the gene knockdown efficiencies may vary, NoPDAT likely makes a comparable contribution to stress-induced TAG accumulation as each of the above-mentioned NoDGATs. It is worth noting that, if summing up the TAG decrease ratios caused by silencing each of the four acyltransferase genes in N. oceanica, the value exceeds 100%. In this context, these acyltansferases may have overlapping roles. The interactions between enzymes involved in TAG synthesis including DGAT and PDAT have been reported in mammals [START_REF] Man | Colocalization of SCD1 and DGAT2: implying preference for endogenous monounsaturated fatty acids in triglyceride synthesis[END_REF][START_REF] Jin | Diacylglycerol acyltransferase-2 (DGAT2) and monoacylglycerol acyltransferase-2 (MGAT2) interact to promote triacylglycerol synthesis[END_REF], land plants [START_REF] Lee | Interaction of DGAT1 and PDAT1 to enhance TAG assembly in Arabidopsis[END_REF]Xu et al., 2019a) and algae [START_REF] Xu | Characterization of type-2 diacylglycerol acyltransferases in the green microalga Chromochloris zofingiensis[END_REF]. In Arabidopsis, "oil-producing metabolons" were recently characterized, containing either DGAT1 or DGAT2 and PDAT, with accesses to separate substrate pools [START_REF] Regmi | Oil-Producing metabolons containing DGAT1 use separate substrate pools from those containing DGAT2 or PDAT[END_REF]. It would be important to evaluate whether similar metabolons might occur in such a species with so many oil-producing enzymes. In our study, we noticed little impact on the expression of genes coding for NoGPAT, NoLPATs and NoDGATs in response to a knockdown of NoPDAT (Figure 8). Only a down-tuning of FA synthetic genes seemed to occur.

Altogether, NoPDAT is likely to operate as a parallel and independent oil-producing route, possibly dispensable to feed an actual form of carbon storage, and more important as an actor of membrane lipid turnover [START_REF] Yoon | Phospholipid:diacylglycerol acyltransferase is a multifunctional enzyme involved in membrane lipid turnover and degradation while synthesizing triacylglycerol in the unicellular green microalga Chlamydomonas reinhardtii[END_REF]Fan et al., 2013bFan et al., , 2014)). The occurrence of such PDAT-mediated membrane lipid turnover in N. oceanica is further supported by several lines of evidence. First, as the culture time lasted (from favorable growth to nutrient limitation stress), NoPDAT showed a steady increase in its transcript level (Figure 4A, Figure 5A and Supplemental Figure 4B), accompanied by the decrease of many membrane lipids (Figure 7B). Second, following NoPDAT knockdown, membrane lipids particularly PG and PE exhibited a considerable increase regardless of the culture time points examined (Figure 7B). The increase of PG particularly PG with 16:0, 16:1 and 18:1 at the sn-2 position caused by NoPDAT knockdown is in agreement with the in vitro enzymatic assay results that NoPDAT exhibited a stronger activity on these PG species (Figure 7D; Supplemental Figure 6). The impact on PE level is even more evident.

NoPDAT Location at the cER Highlights the Presence of Both PG and PE in the

Outermost Membrane of the Plastid

There are two possible origins for CDP-diacylglycerol-glycerol-3-phosphate 3phosphatidyltransferase (or phosphatidylglycerolphosphate synthase, PGPS) producing PG. One is prokaryotic, at the origin of the plastid PGPS in Archeplastida, and the other is eukaryotic, for PGPS in the mitochondrion [START_REF] Luévano-Martínez | The chimeric origin of the cardiolipin biosynthetic pathway in the Eukarya domain[END_REF]. The situation in Heterokont has not been established. Based on sequence similarity search, both types are encountered. We could identify one sequence in N. oceanica (Nanoce1779|7997, in the CCMP1779 genome) related to prokaryotic PGPS, harboring both signal peptide and chloroplastic transit sequences together with an ASAFAP motif, indicating that a prokaryotic-like PGPS is likely to synthesize PG in the plastid. Homologues were also detected in M. gaditana (EKU22790 in CCMP526) and P. tricornutum (Phatr3_J8663).

We could also identify one sequence related to eukaryotic PGPS in M. gaditana (EKU20337), P. tricornutum (Phatr3_J47576) and a C-terminal fragment in N. oceanica (Nanoce1779|6370). A mitochondrial transit peptide is predicted using MitoprotII for Phatr3_J47576 and EKU20337, which suggests that these eukaryotic-type PGPS produce PG in the mitochondrion. Eventually, PG might also be exported to the endomembrane system, via inter-organellar transport [START_REF] Vance | Phospholipid synthesis and transport in mammalian cells[END_REF], via a protein machinery which remains to be characterized. In N. oceanica profile we cannot unambiguously assess which of the detected PG molecular species is mitochondrial and/or plastidial or even in the ER. Although there is no information available about the distribution of PG in N. oceanica, the predominance of C16 acyls over C18 acyls in PG sn-2 position (Figure 7D) suggests that N. oceanica synthesizes the bulk of its PG in the chloroplast. Considering the subcellular localization of NoPDAT at the cER (Figure 3), it could possibly access PG from both the ER (low abundance) and the plastid outer envelope. NoPDAT may not use PG species located at the thylakoid membrane or the chloroplast inner envelope per se, unless this lipid is translocated to the cER, which needs to be assumed to interpret the knockdown phenotype.

PE represents a major non-bilayer phospholipid widely distributed in the extrachloroplastic membranes of Archaeplastida. It is believed that PE biosynthesis involves mainly two metabolic pathways, i.e., an ER-localized CDP-ethanolamine pathway (de novo biosynthetic pathway) and a mitochondrial PS decarboxylation pathway, with the former being the major one [START_REF] Gibellini | The Kennedy pathway-De novo synthesis of phosphatidylethanolamine and phosphatidylcholine[END_REF]. In N. oceanica, PE is present predominantly in the form of molecular species with no less than four double bonds (Figure 7C; Li et al., 2014b;[START_REF] Han | Metabolic remodeling of membrane glycerolipids in the microalga Nannochloropsis oceanica under nitrogen deprivation[END_REF][START_REF] Meng | Identification of the role of polar glycerolipids in lipid metabolism and their acyl attribution for TAG accumulation in Nannochloropsis oceanica[END_REF]. Based on our analysis of genes involved in the biosynthesis or conversion of PE, the produced LU-PE was mainly consumed by cER NoPDAT to form TAG and otherwise accumulated, and could not be converted to PE enriched in 20:3, 20:4 and 20:5. The cER thus appears as a major membrane lipid remodeling site, during long nutrient stress, marked by a consumption of PE and PG molecular species.

LU-PE serves as an alternative carbon reservoir in N. oceanica under stress

Since LU-PE species are the acyl-donors for NoPDAT, knockdown of NoPDAT could result in LU-PE accumulation, which is boosted by elevated CO2 supply. It is suggested that LU-PE may serve as an alternative yet essential carbon reservoir for photosynthetically assimilated carbon. The large pool of LU-PE is produced in response to nutrient limitation when TAG biosynthesis is impaired or under high CO2 conditions, which indicated a potential protection mechanism in cells under stress. The protective role of LU-PE is further supported as there's no difference between the growth rate of WT and NoPDAT knockdown lines under stress, despite lower TAG content in the latter.

By contrast, knockdown of PDAT and knockout of plastid galactoglycerolipid degradation 1 gene (pgd1) in C. reinhardtii decreased cellular TAG content and their growth rates in the mid-exponential growth phase and stationary phase under stress, respectively [START_REF] Li | A galactoglycerolipid lipase is required for triacylglycerol accumulation and survival following nitrogen deprivation in Chlamydomonas reinhardtii[END_REF][START_REF] Yoon | Phospholipid:diacylglycerol acyltransferase is a multifunctional enzyme involved in membrane lipid turnover and degradation while synthesizing triacylglycerol in the unicellular green microalga Chlamydomonas reinhardtii[END_REF], supporting a role of TAG in protecting algal cells against oxidative damage. In NoPDAT knockdown lines, this protective role appears to be replaced by accumulation of LU-PE as an alternative electron and carbon sink against overreduction of photosynthetic electron transport chain and possibility production of reactive oxygen species under stress.

Considering that PE enriched in 20:3, 20:4 and 20:5 showed no increase while LU-PE rose drastically (Figure 7C), the fatty acid elongases and desaturases [START_REF] Han | Metabolic remodeling of membrane glycerolipids in the microalga Nannochloropsis oceanica under nitrogen deprivation[END_REF]Poliner et al., 2018a) may not function on the acyl groups of those LU-PE species in N. oceanica (Figure 9). Due to the greatly different acyl-chain length and unsaturation of LU-PE, if the vastly increased LU-PE species are transported to the biologically functional two-layer membranes, the membrane structure and function would be changed.

To avoid such a drastic change, LU-PE species may be stored on the monolayer of lipid droplets, which generally occur in algal cytosol under stress conditions to accommodate TAG [START_REF] Wang | Algal lipid bodies: Stress induction, purification, and biochemical characterization in wild-type and starchless Chlamydomonas reinhardtii[END_REF]Vieler et al., 2012a;[START_REF] Wang | Proteomics analysis of lipid droplets from the oleaginous alga Chromochloris zofingiensis reveals novel proteins for lipid metabolism[END_REF]. More researches are to be carried out to identify the location and function of LU-PE in cells in the future.

CONCLUSIONS

The remarkably high content of TAG in N. oceanica has been attributed to the strongly redundant equipment of NoDGATs. This is absolutely plausible, since some are more expressed and active in favorable growth conditions and others during severe nutrient limitations. Based on these assumptions, the function of NoPDAT might appear as dispensable. Interestingly, in the NoPDAT knockdown lines, no compensatory mechanisms via the Kennedy pathway could be observed, neither by metabolic regulation, which could fill up the cell with oil to the same level as the WT, nor via transcriptional reprogramming, by overexpression of GPAT, LPATs or DGATs. All results support that the PDAT pathway is a parallel and independent oil-producing pathway. This role in coupling membrane glycerolipid breakdown at the periphery of the plastid, mainly via PG and PE, with TAG production, is likely one of the features determining the remarkable capacity of Nannochloropsis species to accumulate such high levels of TAG.

It may be part of the mechanisms adjusting membrane lipids with the cellular physiological status, here during a long nutrient stress. In particular, the specific saturated/monounsaturated acyl PE, accumulating under stress, is a substitute for TAG to respond quickly and directly to the excessive carbon flux. The activation of the PDAT pathway now needs to be evaluated in various physiological and environmental contexts to route the specific PE to TAG effectively. Such feature should be also useful for future strategies aiming at further improving the production of TAG by genetic engineering of Nannochloropis and related species.

METHODS

Algal Strains

Nannochloropsis oceanica (MBIC10090) and Phaeodactylum tricornutum (CCMP2561) were obtained from the Marine Biotechnology Institute Culture Collection (Iwate, Japan) and the culture collection of the Provasoli-Guillard National Center for Culture of Marine Phytoplankton (Maine, USA) respectively. Algal cultures were grown axenically in artificial seawater enriched with f/2 nutrients (nitrate concentration was reduced to 500 μM) [START_REF] Guillard | Culture of phytoplankton for feeding marine invertebrates[END_REF] at 22 °C under continuous illumination of 120 μmol photons m -2 s -1 . Transformants were grown in medium with the presence of 1 (for N. oceanica) or 40 μg/ml (for P. tricornutum) zeocin. To obtain the correct full-length cDNA of N. oceanica PDAT (NoPDAT), three primer pairs were designed based on the three annotated gene sequences and three DNA fragments amplified from the cDNA template were obtained. The obtained long fragment not only included one of the short fragments but also is overlapped with the 3' end of the other. The full-length cDNA of this gene was further verified by 3' and 5'-RACE. The latest annotation information in JGI Nannochloropsis oceanica CCMP1779 v2.0 on the Protein ID 640709 issues its full-length, which is the same with that obtained in our experiment (Supplemental Figure 7). The full-length coding regions of NoPDAT were amplified by using N. oceanica cDNA as template with the primer pair 11464-fw and 1202-rv. Total RNA was extracted with TRIzol reagent (Invitrogen) from exponentially growing cells homogenized by grinding in liquid nitrogen. DNA was removed with gDNA Eraser and first-strand cDNA was synthesized by PrimeScript RT reagent Kit (TaKaRa) according to the manufacturer's instructions.

Identification of Putative

All the primers are shown in Supplementary Data set 3.

For phylogenetic analyses, a final dataset composed of 30 sequences (see supplementary method) was aligned using MUSCLE (Edgar, 2004) then curated using BMGE (Block Mapping and Gathering with Entropy) software [START_REF] Criscuolo | BMGE (Block Mapping and Gathering with[END_REF] to select the phylogenetic informative regions. The [START_REF] Le | An improved general amino acid replacement matrix[END_REF] substitution model for Maximum Likelihood (ML) phylogenetic inference method was chosen among the 56 models implemented in MEGA X [START_REF] Kumar | MEGA X: Molecular evolutionary genetics analysis across computing platforms[END_REF] (Molecular Evolutionary Genetics Analyses) software as the model best fitting the dataset. A Maximum Likelihood phylogenetic analysis was hence performed on the curated alignment with LG+G+I model and 5000 bootstrap pseudo-replicates (details of the analyses are presented in supplementary method). A Bayesian phylogenetic inference was carried out as well. Two independent Metropolis-coupling Markov Chain Monte Carlo analyses were performed using the MrBayes3.2 [START_REF] Ronquist | MrBayes 3.2: efficient bayesian phylogenetic inference and model choice across a large model space[END_REF] software (details in the supplementary method).

NoPDAT Expression in Yeast

The open reading frame (ORF) of NoPDAT was cloned into the yeast expression vector pYES2-CT (Invitrogen). The resulting vectors pYES2-NoPDAT was confirmed by restriction digestion and sequencing. The recombinant pYES2-NoPDAT plasmid was then transformed into the Saccharomyces cerevisiae TAG-deficient quadruple mutant strain H1246 (dga1, lro1, are1, and are2) by using S.c. EasyComp Transformation Kit (Invitrogen). Transformants were selected on solid plates of SC-uracil medium (Teknova, Hollister, CA, USA). H1246 cells carrying the empty plasmid pYES2-CT (EV control) and pYES2-CrDGTT3 (a type 2 DGAT gene from Chlamydomonas reinhardtii) were from Liu et al. (2016a). Yeast cells were grown in SC-uracil medium with 2% raffinose for 24 hours. Cells were harvested by centrifugation at 1,500 × g for 5 min and resuspended in SC-uracil medium containing 2% galactose with an initial OD600 of 0.4 for induction. After the induction of 2 days, total lipids from yeast cells were extracted as described by [START_REF] Yoon | Phospholipid:diacylglycerol acyltransferase is a multifunctional enzyme involved in membrane lipid turnover and degradation while synthesizing triacylglycerol in the unicellular green microalga Chlamydomonas reinhardtii[END_REF] and neutral lipids were separated on a Silica gel 60 thin layer chromatography (TLC) plate (EMD Chemicals, Merck, Darmstadt, Germany) using a mixture of hexane/tert-butylmethyl ether/acetic acid (80/20/2, by volume) as the mobile phase. Lipids were detected by spraying the TLC plate with 10% CuSO4 in 8% phosphoric acid, followed by charring at 180°C for 3 min. BODIPY 493/503 (Invitrogen), a neutral lipid-specific fluorescent dye, was used for the staining of yeast cells.

Yeast Microsome Preparation and in vitro PDAT Activity Assay

The transformant cells of H1246 bearing pYES2-NoPDAT were grown in SC-uracil medium with 2% galactose for 12 h at 30°C. Cells were then harvested and washed twice with ice-cold distilled water. The cell pellets were resuspended in cell lysis buffer (containing 5% glycerol, 20 mM Tris-HCl (pH 8.0), 0.3 M ammonium sulfate, 10 mM MgCl2, 1 mM EDTA, 1 mM DTT, 1× EDTA-free protease inhibitor cocktail set X (Calbiochem), 1 mM PMSF) to an OD600 of approximately 100 and lysed by passing twice through a French pressure cell (Spectronics Instruments, Rochester, NY, USA) at an internal pressure of 15,000 PSI. Cell debris was removed from the suspension by centrifugation at 10,000 × g for 10 min at 4°C and the supernatant was centrifuged further at 100,000 × g for 2 h at 4°C. The resulting microsomal membrane pellets were resuspended in microsome storage buffer (50 mM Tris-HCl, pH 7.5, 10% glycerol) to give a protein concentration of 10 µg µL -1 for immediate use or stored at -80 °C.

The in vitro PDAT assay was conducted according to our previously described procedures (Liu et al., 2016a). The polar lipids tested included 1,2-dipalmitoyl-sn-

glycero-3-O-4'-[N,N,N-trimethyl(d9)]-homoserine (DGTS), 1-palmitoyl-2-oleoyl-sn- glycero-3-phosphocholine (PC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (PE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (PG), 1-palmitoyl-2-
oleoyl-sn-glycero-3-phospho-L-serine (PS), digalactosyldiacylglycerol (DGDG, plant sourced), phosphatidylinositol (PI, soy sourced), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphate (PA), monogalactosyldiacylglycerol (MGDG, plant sourced), sulphoquinovosyldiglyceride (SQDG, plant sourced), and the diacylglycerols (DAGs) were 16:0/16:0, 16:1/16:1, 18:1/16:0, 16:0/18:1, 18:1/18:1, 1,3-18:1/18:1, 18:2/18:2, and 18:3n3/18:3n3. 16:1/16:1, 18:2/18:2, and 18:3n3/18:3n3 DAGs, and SQDG were purchased from Larodan Fine Chemicals (Malmo, Sweden), whereas 18:1/16:0 DAG was prepared by partial digestion of 18:1/16:0/18:1 triacylglycerol (TAG) (Larodan Fine Chemicals) with Rhizopus arrhizus lipase (Sigma-Aldrich) and recovery of DAG. All other lipids were purchased from Avanti Polar Lipids (Alabaster, AL, USA).

GFP Fusion and Overxpression Constructs

For the localization analysis of NoPDAT, two constructs were generated to express Cterminal enhanced green fluorescent protein (eGFP) fusion proteins in N. oceanica and P. tricornutum cells respectively. NoPDAT ORF was amplified by PCR using primers nopdat_pP-EcoRI-fw and nopdat-KpnI-rv and inserted into the Kpn I-EcoR I sites (upstream of the eGFP sequence) of the pPhaT1-eGFP vector [START_REF] Zhang | High-efficiency nuclear transformation of the diatom Phaeodactylum tricornutum by electroporation[END_REF], to generate the pPhaT1-NoPDAT-eGFP vector. Plasmid pPha-NoVCPp containing the endogenous bidirectional N. oceanica violaxanthin/chlorophyll a-binding protein 2 (NoVCP2) promoter and NOVCP1 3'UTR (terminator) as well as sh ble resistance gene was constructed according to [START_REF] Moog | In vivo localization studies in the stramenopile alga Nannochloropsis oceanica[END_REF] and has been described in our previous study [START_REF] Chen | High efficiency transformation by electroporation of the freshwater alga Nannochloropsis limnetica[END_REF]. NoPDAT ORF was amplified using primer pair nopdat_KpnI-fw and nopdat-XbaI-rv and ligated into the pPha-NoVCPp (between the Kpn I and Xba I sites), to obtain NoPDAT overexpression vector pPha-NoVCPp-NoPDAT. To generate the pPha-NoVCPp-NoPDAT-eGFP vector, eGFP was cloned and inserted downstream of the NoVCP2 promoter of pPha-NoVCPp [START_REF] Chen | High efficiency transformation by electroporation of the freshwater alga Nannochloropsis limnetica[END_REF], then the PCR product amplified by primers nopdat_KpnI-fw and nopdat-KpnI-rv was digested and cloned into the Kpn I site of the vector pPha-NoVCPp-eGFP. Inserts of all constructs were sequenced to confirm that no mutations were present. Hpa I linearized pPhaT1-NoPDAT-eGFP was introduced into wild-type P. tricornutum, and Hpa I linearized pPha-NoVCPp-NoPDAT and pPha-NoVCPp-NoPDAT-eGFP were respectively introduced into wild-type N. oceanica following our previous protocol [START_REF] Zhang | High-efficiency nuclear transformation of the diatom Phaeodactylum tricornutum by electroporation[END_REF]Li et al., 2014a). The transformants were screened by PCR for the presence of transgene using specific primers. GFP positive cells were observed using a Leica TCS SP8 laser scanning confocal microscope. Chlorophyll autofluorescence and GFP fluorescence were excited at 488 nm, and were detected with 630-690 nm and 500-550 nm, respectively. Fluorescence of the nucleus stained by Hoechst 33342 was detected at 425-475 nm emission by 405 nm excitation. NoPDAT overexpression in N. oceanica was confirmed by quantitative real-time PCR (qPCR) and western blot analysis.

For the overexpression of choline phosphotransferase (CPT, Protein ID 9914), ethanolamine phosphotransferase (EPT, Protein ID 8504) and phosphatidylserine synthase (PSS Protein ID 6823) in N. oceanica, their ORFs were amplified using primer pairs nocpt_KpnI-fw and nocpt-XbaI-rv, noept_KpnI-fw and noept-XbaI-rv, and nopss_KpnI-fw and nopss-XbaI-rv respectively, then ligated into the pPha-NoVCPp vector. Before being transformed into wild-type N. oceanica, the overexpression vectors were linearized with Hpa I (EPT-OE and PSS-OE vectors) or ScaI (CPT-OE vector) and then purified with DNA purification kit according to our previous method (Li et al., 2014a).

Quantitative Real-Time PCR and Western Blot Analysis

Total RNA was extracted from algal cells at different growth phases and cDNA was prepared as mentioned above. Relative gene expression was measured using the SsoAdvanced Universal SYBR Green Supermix (Bio-Rad) and aLightCycler 480 Real-Time PCR System (Roche). Primers used for real-time PCR are shown in Supplementary Data set 3. Histone H4 served as the internal control gene [START_REF] Siaut | Molecular toolbox for studying diatom biology in Phaeodactylum tricornutum[END_REF]. Technical triplicates were performed for duplicate cultures. The mean values of relative mRNA quantities were calculated according to the 2 -ΔΔCt -method.

For the western blot analysis, total protein was extracted from liquid nitrogen-frozen algal cells in RIPA lysis buffer containing 1 mM phenylmethanesulfonyl fluoride (Beyotime Biotechnology, China) by ultrasonication for 10 min at 10% intensity (5 s duration, 5 s intervals, on ice using Scientz JY96-IIN Sonifier). Extracts were centrifuged and protein concentration in the supernatant was determined using a BCA protein assay kit (Beyotime Biotechnology, China). After denaturation, total protein was separated by 10% SDS-PAGE gel and then transferred to polyvinylidene fluoride membrane. The membranes were incubated with anti-NoPDAT antibody followed by HRP-conjugated secondary antibody and the blots were developed with Immobilon Western Chemiluminescent HRP Substrate (Millipore, USA). Images of the blots were obtained using an ImageQuant LAS 4000 mini apparatus (GE Healthcare Life Science). Polyclonal antibody of NoPDAT was generated by Genscript Corporation (Nanjing, China) against a synthetic peptide CPHLEKETGTFSSEE.

Growth and Nile Red Staining

Growth was determined by measuring the optical density (OD) at 750 nm and the initial inoculation density is OD750=0.05. Nitrate concentration in the culture was determined using spectrophotometry at 220 nm [START_REF] Collos | An optical method for the rapid measurement of micromolar concentrations of nitrate in marine phytoplankton cultures[END_REF]. The relative abundance of the algal neutral lipid was estimated by fluorescence spectroscopy (Perkin-Elmer, LS 55) with the fluorescent dye Nile red (Sigma-Aldrich) using procedures described before [START_REF] Ge | Methylcrotonyl-CoA carboxylase regulates triacylglycerol accumulation in the model diatom Phaeodactylum tricornutum[END_REF] with some modification. Briefly, a total of 0.5 OD cell suspensions were harvested by centrifugation, and 500 μl of 70% ethanol was added to resuspend cell pellets. The resuspended solution was subject to incubation at 4°C for 10 min, and were subsequently incubated at 37°C for 30 min after addition of 2.5 ml of ddH2O and 30 μl of 100 μg/ml Nile red solution. Fluorescent emission was measured at 572 nm with an excitation wavelength of 531 nm. Background fluorescence for this filter set was subtracted prior to the addition of Nile red.

Total Lipid and TAG Content Analysis of N. oceanica

Total lipids of algal cells were extracted in a chloroform/methanol (1:1, v/v) solvent system [START_REF] Bligh | A rapid method of lipid extraction and purification[END_REF] and were further separated by TLC on Silica gel plates 60 using hexane/diethyl ether/acetic acid (70:30:1, v/v/v) as developing solvent [START_REF] Reiser | Isolation of mutants of Acinetobacter calcoaceticus deficient in wax ester synthesis and complementation of one mutation with a gene encoding a fatty acyl-coenzyme A reductase[END_REF]. TAGs were identified by staining with iodine vapor and recovered with methanol from the plates. Triolein was used as the standard and was purchased from Sigma-Aldrich. Total lipids and TAGs were transesterified with 1 M sulfuric acid in methanol at 100°C for 1 h. Fatty acid methyl esters from the transesterification of total lipids and TAGs were analyzed by gas chromatography (TRACE GC; Thermo Scientific) and quantified using methylheptadecanoate (C17:0) as the internal standard following our previously described procedures [START_REF] Ge | Methylcrotonyl-CoA carboxylase regulates triacylglycerol accumulation in the model diatom Phaeodactylum tricornutum[END_REF].

Construction of RNAi Strains

To silence NoPDAT in N. oceanica, a hairpin construct was built using the primer pairs nopdat535_fw and nopdat975_rv1 for 'long' fragment (LF), and nopdat535_fw and nopdat761_rv2 for 'short' fragment (SF) following our previous methods [START_REF] Wei | RNAi-based targeted gene-knockdown in the model oleaginous microalgae Nannochloropsis oceanica[END_REF]. After linearization with Sca I, the hairpin construct driven by the native β-tubulin promoter was introduced into wild-type N. oceanica according to the procedure described by Li et al. (2014a). The transformants were screened by checking the integration of the sh ble gene with the primers ble_fw and ble_rv.

To construct RNAi expression vector for PSS, phosphatidylethanolamine N-methyltransferase (PEMT), phospholipid N-methyltransferase (PLMT) and phosphoethanolamine N-methyltransferase (PEAMT), primer pairs pss344_fw and pss739_rv1, pemt229_fw and pemt630_rv1, plmt102_fw and plmt524_rv1, peamt652_fw and peamt1051_rv1 were used for LF amplification, and primer pairs pss344_fw and pss554_rv2, pemt229_fw andpemt429_rv2, plmt102_fw andplmt314_rv2, peamt652_fw and peamt871_rv2 were used for SF amplification. Prior to transformation to wild-type N. oceanica, the four RNAi vectors were linearized using Sca I (PEMT, PLMT and PEAMT-RNAi vector) or Kpn I (PSS-RNAi vector).

TAG Molecular Composition and Content

TAG molecular composition and content were analyzed as described previously (Hu et al., 2014;[START_REF] Ge | Methylcrotonyl-CoA carboxylase regulates triacylglycerol accumulation in the model diatom Phaeodactylum tricornutum[END_REF]. In brief, TAGs recovered from TLC plates as mentioned above were redissolved in chloroform/methanol (1:1, v/v). The TAG samples were dried and dissolved in isopropanol, and then were analyzed by a Shimadzu LC-20AD HPLC system coupled with an AB 4000 Q trap tandem MS system (Applied Biosystems, USA) using APCI in positive mode. A ZORBAX Eclipse Plus phenyl-hexyl column (4.6 mm × 250 mm i. d., 5μm particle size, Agilent Technologies, USA) was used for the separation, and mobile phase was consisted of acetonitrile (solvent A) and 0.5% ammonia-methanol (w/v) (solvent B) in gradient elution mode. For the elution, 50% of solution B was used during first 7.5 min. Then a gradient from 50% to 72% of solution B was supplied during next 0.5 min with an isocratic hold at 72% for additional 5 min, then 72% to 50% in 0.1 min, followed by an isocratic hold at 50% for 17 min. The injection volume was 10 μL, with flow rate = 0.8 mL/min. The heated interface temperature, curtain gas pressure and collision gas were set to 450 °C, 30 psi and medium respectively. The EMS (enhanced MS) -EPI (Enhanced Product Ion) scan mode was applied for qualitative analysis, and the MRM (multiple reaction monitoring) -EPI mode was used for quantification. The scan rate was 1000 μ/s, and the ion source gas 1 pressure was set at 45 psi. The declustering potential and the collision energy were set at 84 and 35±15 V, respectively. Scan range of the EMS was set at m/z 700-1000, and the EPI at m/z 100-1000.

Qualitative and Quantitative Analysis of Polar Lipids

Total lipid extracts and polar lipid standards were subjected to TLC as above in a solvent system consisting of chloroform/methanol/acetic acid/distilled water (75:13:9:3 v/v/v/v).

The standards of MGDG, PE, PG, PC, PS, PI purchased from Avanti Polar Lipids (Alabama, USA) were used to identify the classes of polar lipids on the plates. Furthermore, each band on the plates was scraped off respectively, and the silica gel powder was extracted with methanol and dried, then redissolved in chloroform/methanol (1:1, v/v). The redissolved single lipids were identified using shotgun MS approach according to [START_REF] Wei | Rapid profiling and quantification of phospholipid molecular species in human plasma based on chemical derivatization coupled with electrospray ionization tandem mass spectrometry[END_REF]. In addition, total lipids extracted from wild-type and NoPDAT knockdown strains were also analyzed using shotgun MS method directly. For the shotgun MS method, an Shimadzu LC-20AD HPLC system and an AB 4000 Q trap tandem MS system (Applied Biosystems, USA) with an electrospray ionization (ESI) source in positive-ion mode were used. The ionization voltages were set to 5 kV. The ion source temperature (TEM), curtain gas (CUR) and ion source gas 1 (GS1) were set to 350 °C, 10 psi and 16 psi, respectively.The flow rate of the sample injected into the ESI source was set to 20 μL/min.The declustering potential (DP) of PC (+PIS 184), PE (+NLS 141), PS (+NLS 185), PG (+NLS 189), PA (+NLS 115), PI (+NLS 277), MGDG (+NLS 179), and DGDG (+NLS 341) were set to 166.8, 139.9, 127.7, 64.6, 71.3, 120.4, 86.3, and 73 V, respectively, and the collision energy (CE) was set to 47.7, 35.3 , 38.8, 28.5, 25.7, 32.6, 20.2, and 23.7 V respectively. The MS scan was from 400 to 1000 m/z. PC (12:0/12:0), PE (12:0/12:0), PS (14:0/14:0), PG (14:0/14:0), PA (14:0/14:0) and PI (8:0/8:0) were used as internal standards for quantification of phospholipids (PLs). . The effect of NoPDAT knockdown on lipid metabolism network in N. oceanica. Four time points were selected for qPCR. Genes were considered to be significantly differentially expressed if: the expression level fold change between NoPDAT knockdown line (PDAT-KD4) and wild-type (WT) was no less than 2-fold with P < 0.01 for at least one time point or no less than 1.5-fold with P < 0.01 for at least two time points. The genes in blue and red were down-and up-regulated, respectively. Abbreviations: AAD (acyl-ACP desaturase); ACC (acetyl-CoA carboxylase); ADHAPR (acyl/alkyl-dihydroxyacetone

  PDAT Encoding Gene in N. oceanica and Phylogenetic Analyses Since the annotation of PDAT in JGI Nannochloropsis oceanica CCMP1779 v1.0 (Protein ID 11464 and Protein ID 1202) and v2.0 (Protein ID 530913) are different, and the transcript of Protein ID 530913 overlaps partial transcripts of Protein ID 11464 and Protein ID 1202. Protein ID 530913 contains all the conserved regions ('lid' region, salt bridge and catalytic S-D-H triad) of PDAT (Falarz et al., 2020), while Protein ID 11464 contains only the PDAT 'lid' region and Protein ID 1202 contains incomplete catalytic triad (Asp and His).

Figure 1 .

 1 Figure1. Bayesian and Maximum Likelihood (ML) phylogenetic analyses of PDATs from different phyla. The Basien tree only is represented here and is drawn to scale, with branch lengths measured in the number of substitutions per site. The posterior probability (PP) from the Bayesian analysis and the bootstrap value from the ML analysis are reported on the nodes. The nodes where PP equals 1.0 and ML support equals 100 were removed. ---= node not present in ML. Rhodophyte sequences (red) were used as outgroup to root both Bayesian and ML trees.

Figure 2 .

 2 Figure 2. Characterization of NoPDAT activity. (A, B) Functional complementation of NoPDAT in TAG-deficient S. cerevisiae H1246 cells as determined by TLC analysis of lipid extracts (A) and Bodipy staining (B) of NoPDAT-expressing H1246 cells. EV (empty vector) and CrDGTT3(Liu et al., 2016) were used as negative and positive controls, respectively. (C, D) Substrate preference of NoPDAT for polar lipids (C) and DAGs (D). DAG (18:1/18:1) was used as the acyl acceptor in (C) and PG was used as the acyl donor in (D). Data represent mean ± SD (n=3).

Figure 3 .Figure 4 .Figure 5 .

 345 Figure 3. Subcellular localization of NoPDAT in N. oceanica and P. tricornutum cells. (A, B) N. oceanica cells transformed with the empty vector (eGFP, A) and NoPDAT-eGFP (B). (C) A 3D visualization of PAF+GFP in (B). (D) P. tricornutum cells transformed with NoPDAT-eGFP. BF, bright field; PAF, plastid autofluorescence; H, Hoechst. Red indicates the plastid autofluorescence (PAF), green indicates the GFP signal, while blue indicates the staining of nucleus with Hoechst (H). Bar, 4 μm.

Figure 7 .

 7 Figure 7. Effect of NoPDAT knockdown on polar lipids of N. oceanica. (A) TLC analysis of lipid extracts from wild-type (WT) and NoPDAT knockdown lines (PDAT-KD2 and PDAT-KD4) aerated with air (left) and PDAT-KD4 under different CO 2 availability conditions (right). (B) Content of polar lipids. (C-E) Content of PE species (C), PG species (D) and PME species (E). Static, static culture; Air, culture aerated with air; CO 2 , culture aerated with 2% CO 2 . Data represent mean ± SD (n=3). MU-PE: "more-unsaturated PE", PE enriched in 20:3, 20:4 and 20:5 fatty acids; LU-PE: "less-unsaturated PE", PE enriched in saturated or monounsaturated fatty acids; PME: plasmenylethanolamine.

Figure 9 .

 9 Figure9. A hypothesized working model illustrating the role of NoPDAT in lipid metabolism in N. Figure9. A hypothesized working model illustrating the role of NoPDAT in lipid metabolism in N. oceanica. Down-regulated genes and decreased metabolite by PDAT knockdown (KD) are illustrated in blue, and up-regulated gene, increased metabolites and carbon flux by PDAT KD in red. Knockdown (KD) and overexpression (OE) of genes involved in PE homeostasis that were carried out in the study are also shown in the model. PGA (3-phosphoglyceric acid); G3P (Glycerol 3phosphate); DHAP (dihydroxyacetone phosphate); CDP-Etn (cytidine diphosphate-ethanolamine); P-Etn (phosphoethanolamine); P-Cho (phosphocholine); P-MMEtn (phospho-monomethylethanolamine);CPT (cholinephosphotranferase); EPT (ethanolaminephosphotranferase); PSS (phosphatidylserine synthase); PEMT (phosphatidylethanolamine N-methyltransferase); PLMT (phospholipid N-methyltransferase); PEAMT (phosphoethanolamine methyltransferase); LU-PE: "less-unsaturated PE", PE enriched in saturated or monounsaturated fatty acids; PME: plasmenylethanolamine.

Figure 9 . 3 .Supplemental Figure 4 .

 934 Figure9. A hypothesized working model illustrating the role of NoPDAT in lipid metabolism in N. Figure9. A hypothesized working model illustrating the role of NoPDAT in lipid metabolism in N. oceanica. Down-regulated genes and decreased metabolite by PDAT knockdown (KD) are illustrated in blue, and up-regulated gene, increased metabolites and carbon flux by PDAT KD in red. Knockdown (KD) and overexpression (OE) of genes involved in PE homeostasis that were carried out in the study are also shown in the model. PGA (3-phosphoglyceric acid); G3P (Glycerol 3phosphate); DHAP (dihydroxyacetone phosphate); CDP-Etn (cytidine diphosphate-ethanolamine); P-Etn (phosphoethanolamine); P-Cho (phosphocholine); P-MMEtn (phospho-monomethylethanolamine);CPT (cholinephosphotranferase); EPT (ethanolaminephosphotranferase); PSS (phosphatidylserine synthase); PEMT (phosphatidylethanolamine N-methyltransferase); PLMT (phospholipid N-methyltransferase); PEAMT (phosphoethanolamine methyltransferase); LU-PE: "less-unsaturated PE", PE enriched in saturated or monounsaturated fatty acids; PME: plasmenylethanolamine.

ACKNOWLEDGMENTS

This work was supported by the National Natural Science Foundation of China (41976119, 91751117). A.A., E.M., H.H. and Y.G. were supported by a CEA-CAS bilateral program. J.L. was supported by the National Key R&D Program of China (2018YFA0902500). A.A. and E.M. were supported by the French National Research Agency (GRAL Labex ANR-10-LABEX-04, and EUR CBS ANR-17-EURE-0003).

MGDG and DGDG were quantified by external standard method using PE. The fatty acid compositions of PLs were analyzed by EPI scanning in negative-ion mode using manual tuning. Data acquisition and processing were performed using AB SCIEX Analyst 1.5 Software (Applied Biosystems, Foster City, CA, USA).

Statistical Analysis

Statistical analysis was performed using the SPSS Statistics 20.0 software. Data were expressed as mean ± SD (standard deviation) based on three independent biological replicates. Two-tailed Student's t tests (t test) were performed to determine statistical significance, and the threshold was set at 0.05 or 0.01.

Supplemental Data

The following materials are available in the online version of this article. Supplemental Figure 2. Content of MGDG, DGDG and PC species in wild-type (WT) and the NoPDAT knockdown line (PDAT-KD4) of Nannochloropsis oceanica. Static, static culture; Air, culture aerated with air; CO 2 , culture aerated with 2% CO 2 . Data represent mean ± SD (n=3).

Supplemental

2 0 : 5 / 2 0 : 5 2 0 : 5 / 1 6 : 1 2 0 : 5 / 1 6 : 0 2 0 : 5 / 1 4 : 0 2 0 : 4 / 1 6 : 0 2 0 : 4 / 1 4 : 0 1 6 : 1 / 1 6 : 0 1 6 : 0 / 1 6 : 0 0 1 2 3 DGDG species (nmol mg -1

)

WT day 3 PDAT-KD4 day 3 WT day 5 PDAT-KD4 day 5 WT day 7 PDAT-KD4 day 7 PDAT-KD4 day 5 Static PDAT-KD4 day 5 CO 2 Air 2 0 : 5 / 1 8 : 2 2 0 : 5 / 1 8 : 1 2 0 : 5 / 1 6 : 1 2 0 : 5 / 1 8 : 0 1 8 : 3 / 1 8 : 2 2 0 : 4 / 1 6 : 0 1 8 : 1 / 1 8 : 2 1 8 : 1 / 1 8 : 1 2 0 : 5 / 1 4 : 0 1 6 : 1 / 1 8 : 3 1 6 : 1 / 1 8 : 2 1 6 : 0 / 1 8 : 2 1 6 : 0 / 1 8 : 1 1 6 : 2 / 1 6 : 1 1 6 : 1 / 1 6 : 1 1 6 : 0 / 1 6 : 1

PG PE PC

1 6 : 0 / 1 8 : 1 1 8 : 1 / 1 8 : 1 1 6 : 0 / 1 6 : 0 1 8 : 2 / 1 8 : 2 1 6 : 0 / 2 0 : 4 1 6 : 0 / 1 8 : 1 1 6 : 0 / 1 6 : 0 1 6 : 1 / 1 6 : 1 2 0 : 4 / 2 0 : 4 1 6 : 0 / 1 8 : 1 1 6 : 0 / 1 6 : 0 1 6 : 1 / 1 6 : 1 1 6 : 0 / 1 8 : 2