Jaruswan Warakanont
email: jaruswan.w@ku.th

Yonghua Li-Beisson

Christoph Benning

CrLIP4 Is Involved in Triacylglycerol Degradation in Chlamydomonas reinhardtii Running head: Role of CrLIP4 in Triacylglycerol Degradation in Chlamydomonas

Keywords: proteins, enzymes and metabolism Chlamydomonas reinhardtii, lipid catabolism, SUGAR DEPENDENT1, triacylglycerol lipase, N recovery; TAG remobilization

Degradation of the storage compound triacylglycerol (TAG) is a crucial process in response to environmental stimuli. Failing to respond properly may be detrimental for survival. In microalgae, this process is important for re-growth when conditions become favorable after cells have experienced stresses. Mobilization of TAG is initiated by actions of lipases causing the release of glycerol and free fatty acids, which can be further broken down for energy production or recycled to synthesize membrane lipids. Although key enzymes in the process, TAG lipases remain to be characterized in the model green alga Chlamydomonas reinhardtii.

Here we describe the functional analysis of a putative TAG lipase, i.e. CrLIP4, which shares 44% amino acid identity with the major TAG lipase in Arabidopsis (SUGAR DEPENDENT1 -SDP1). The CrLIP4 transcript level was down regulated during nitrogen deprivation (ND) when TAG accumulates, but was upregulated during nitrogen recovery (NR) when TAG was degraded. Both artificial microRNA knockdown and insertional knockout mutants showed a delay in TAG mobilization during NR. The difference in TAG degradation was more pronounced when the cultures were incubated without acetate in the dark. Furthermore, the crlip4 knock-out mutant over-accumulated TAG during optimal growth conditions. Taken together, the results suggest to us that CrLIP4 likely acts as a TAG lipase and plays a major role in TAG homeostasis in Chlamydomonas.

Introduction

Microalgae are abundant primary producers of the food chain in nature serving as food source for both marine and fresh water animals [START_REF] Guedes | Nutritional value and uses of microalgae in aquaculture[END_REF]. This is due to their high photosynthetic efficiency in converting solar energy to chemical energy. Many algae synthesize and accumulate triacylglycerol (TAG), which has been the basis for many applications such as pharmaceutical products and foods [START_REF] Hasan | Industrial applications of microbial lipases[END_REF], and more recently for biofuels [START_REF] Chisti | Biodiesel from microalgae[END_REF][START_REF] Durrett | Plant triacylglycerols as feedstocks for the production of biofuels[END_REF][START_REF] Li-Beisson | Metabolism of acyl-lipids in Chlamydomonas reinhardtii[END_REF][START_REF] Zienkiewicz | Stress-induced neutral lipid biosynthesis in microalgae -Molecular, cellular and physiological insights[END_REF].

Rapid accumulation of TAG into lipid droplets (LDs) occurs in response to different environmental stresses such as high light intensity, elevated temperature and nutrient deprivation as summarized in [START_REF] Zienkiewicz | Stress-induced neutral lipid biosynthesis in microalgae -Molecular, cellular and physiological insights[END_REF]. Nitrogen (N) deprivation is best investigated and most widely used in a laboratory setting to induce TAG accumulation. Upon the resupply of N when the conditions are favorable, TAG is degraded to support growth and cell division. TAG degradation is carried out by a process called lipolysis. The process starts with sequential hydrolysis of acyl chains from the glycerol backbone of TAG by TAG-, diacylglycerol (DAG)-and monoacylglycerol lipases. The resulting free fatty acids (FFAs) and glycerol are either being recycled for synthesis of membrane lipids or further broken down by β-oxidation in peroxisomes for energy production as summarized in [START_REF] Kong | Lipid catabolism in microalgae[END_REF].

TAG lipase (EC 3.1.1.3) is the first enzyme during lipolysis, and one of the most targeted enzymes in industrial applications. It catalyzes the release of an acyl chain from TAG in an oilwater interface yielding DAG and FFA. The respective proteins often contain an α/β hydrolase fold commonly found in hydrolases [START_REF] Ollis | The alpha/beta hydrolase fold[END_REF]). In addition, the amino acid sequences of these enzymes contain patatin domains with a GXSXG (G: glycine, X: any amino acid, S: serine) motif representing the catalytic site [START_REF] Kelly | Oil is on the agenda: Lipid turnover in higher plants[END_REF]. The reaction is mediated by a catalytic dyad composed of the serine residue serving as nucleophile in the GXSXG motif and an aspartate residue further along the sequence. These features are common in TAG lipases of various biological systems from yeast to human. Biochemical studies of TAG lipases have been proven very difficult, if not impossible.

Identification and characterization of TAG lipases have only recently made a big leap-forward with the advent of molecular genetics tools, especially the study of gain-or loss-of-function mutants. This has led to the identification of major TAG lipases in Saccharomyces cerevisae (Tgl3p, Tgl4p and Tgl5p) (Athenstaedt andDaum 2003, 2005), in human adipose tissue (ATGL) [START_REF] Jenkins | Identification, cloning, expression, and purification of three novel human calciumindependent phospholipase A2 family members possessing triacylglycerol lipase and acylglycerol transacylase activities[END_REF][START_REF] Villena | Desnutrin, an adipocyte gene encoding a novel patatin domain-containing protein, is induced by fasting and glucocorticoids: ectopic expression of desnutrin increases triglyceride hydrolysis[END_REF][START_REF] Zimmermann | Fat mobilization in adipose tissue is promoted by adipose triglyceride lipase[END_REF]) and in Arabidopsis (the SUGAR DEPENDENT1 (SDP1) [START_REF] Eastmond | SUGAR-DEPENDENT1 encodes a patatin domain triacylglycerol lipase that initiates storage oil breakdown in germinating Arabidopsis seeds[END_REF]) and SDP1-LIKE (SDP1L) [START_REF] Kelly | Seed storage oil mobilization is important but not essential for germination or seedling establishment in Arabidopsis[END_REF]). In Arabidopsis, SDP1 is the major TAG lipase in seeds during post-germination [START_REF] Eastmond | SUGAR-DEPENDENT1 encodes a patatin domain triacylglycerol lipase that initiates storage oil breakdown in germinating Arabidopsis seeds[END_REF]. Loss of a functional gene leads to seedling growth retardation prior to establishment of photosynthetic metabolism. This phenotype can be rescued by sucrose supplementation. Together with its paralogue SDP1L, the two proteins are responsible for 95% of TAG hydrolysis in germinating Arabidopsis seeds [START_REF] Kelly | Seed storage oil mobilization is important but not essential for germination or seedling establishment in Arabidopsis[END_REF]. Recently, two Arabidopsis SDP1 homologues have been characterized in an oleaginous microalga, Lobospaera incisa, and a diatom, Phaeodactylum tricornutum, have been named as LiSDP1 and Tgl1, respectively [START_REF] Barka | Identification of a triacylglycerol lipase in the diatom Phaeodactylum tricornutum[END_REF][START_REF] Siegler | Analysis of the lipid body proteome of the oleaginous alga Lobosphaera incisa[END_REF]. LiSDP1 was able to partially rescue the Arabidopsis sdp1/sdp1l mutant but was not found associated with the LD as SDP1.

In the diatom, down regulation of TGL1 with antisense RNA resulted in a strong increase in TAG content. In addition, recombinant Tgl1 showed esterase activity against a TAG substrate analogue.

Although lipid metabolism in the model microalga Chlamydomonas reinhardtii has recently been subjected to intensive studies [START_REF] Kong | Lipid catabolism in microalgae[END_REF][START_REF] Li-Beisson | Metabolism of acyl-lipids in Chlamydomonas reinhardtii[END_REF][START_REF] Liu | Lipid metabolism in microalgae distinguishes itself[END_REF][START_REF] Merchant | TAG, you're it! Chlamydomonas as a reference organism for understanding algal triacylglycerol accumulation[END_REF], and despite the central importance of TAG lipases, no TAG lipase has yet been identified and characterized in this model green alga. Protein sequence comparison revealed the occurrence of one putative orthologue (CrLIP4) of the Arabidopsis SDP1. Here, we determined that CrLIP4 is a TAG lipase by studying its gene expression and characterizing artificial microRNA repression and insertional disruption mutants. Based on our results we conclude that CrLIP4 plays a key role in TAG degradation.

Results

CrLIP4 is an orthologue of Arabidopsis SDP1

A previous transcriptomic study in Chlamydomonas has identified CrLIP4 as a putative SDP1 orthologue [START_REF] Miller | Changes in transcript abundance in Chlamydomonas reinhardtii following nitrogen deprivation predict diversion of metabolism[END_REF]. Here, we further investigated this putative TAG lipase. Based on amino acid sequence alignment, CrLIP4 shares 44% identity with the Arabidopsis SDP1 protein. To gain more insight into CrLIP4 function, we searched for protein domain in the amino acid sequence and other characterized TAG lipases searching a hidden Markov model (HMM) library (Pfam) through HMMSCAN. All of the tested amino acid sequences contain a patatin domain (PF01734.21) with a GXSXG motif and conserved serine and aspartate residues (Figure 1A). Therefore, we concluded that CrLIP4 belongs to the patatin-like (PTL) protein family. These proteins also contain a DUF3336 domain (PF11815.7). Its function remains to be resolved. As shown in Figure 1A, all TAG lipases included in the analysis except human ATGL contain this protein domain. Therefore, the DUF3336 might not be required or essential for the catalytic reaction of a TAG lipase.

To investigate the evolutionary relationship between these characterized TAG lipases with CrLIP4, phylogenetic reconstruction was carried out. Because the DUF3336 domain is absent in the human ATGL protein, it was excluded from this analysis. As expected, CrLIP4 grouped into the same clade as LiSDP, and was more distantly related to SDP1 and SDP1L, the diatom Tgl1, and the Saccharomyces Tgl4p and Tgl5p, respectively (Figure 1B). Saccharomyces Tgl3p, however, seemed to be separated into another branch. This could be due to the fact that the aspartate residue of the catalytic dyad is not present in its patatin domain [START_REF] Koch | Modifications of the C terminus affect functionality and stability of yeast triacylglycerol lipase Tgl3p[END_REF].

Taken together, these results are consistent with CrLIP4 being an orthologue of Arabidopsis SDP1.

Expression of CrLIP4 is decreased during N deprivation and increased upon N resupply

TAG content can be manipulated easily in Chlamydomonas; i.e., TAG content increases strongly upon N deprivation, and decreases immediately following N resupply [START_REF] Moellering | RNA interference silencing of a major lipid droplet protein affects lipid droplet size in Chlamydomonas reinhardtii[END_REF][START_REF] Siaut | Oil accumulation in the model green alga Chlamydomonas reinhardtii: characterization, variability between common laboratory strains and relationship with starch reserves[END_REF]. In order to see the relationship between the CrLIP4 transcript level and TAG accumulation, we monitored both at different growth conditions; N sufficiency (NS), N deprivation (ND) and N resupply (NR) (Figure 2). Cells were first cultivated in Tris-Acetate-Phosphate (TAP) medium until mid-log phase (NS), then N-deprived for a couple of days (ND), and followed by a 2-d N recovery period (NR) following N resupply. Cells were harvested at each stage for TAG quantification as well as for RNA extraction and quantitative reverse transcription PCR (qRT-PCR) analysis. We confirmed the fluctuation of TAG content in response to changes in the N status of the culture. The CrLIP4 transcript was present at high levels in regular TAP medium (NS). Its abundance dropped strongly to 1.5% of that found in TAP medium (NS) after 20 h of N deprivation. When N was resupplied to the culture, the CrLIP4 transcript level increased slowly to approximately 30% of the level in the original TAP medium after 96 h . Therefore, we concluded that CrLIP4 transcript and TAG levels are inversely correlated.

Repression of the CrLIP4 transcript level delayed TAG mobilization during N recovery

To investigate the in vivo function of CrLIP4, artificial microRNA was employed to repress the transcription of CrLIP4. Two artificial microRNA constructs were generated to target either the 5' or 3' portions of the gene. The linearized plasmid containing the paromomycin resistance gene AphVIII was introduced into the Chlamydomonas dw15.1 genome. Fifteen transformants for each construct were tested for their CrLIP4 expression levels with qRT-PCR. From a total of 30 transformants, three knockdown lines targeting the 3' region exhibited 60% CrLIP4 levels compared to the dw15.1 parental line (PL) (Figure 3A). These three lines named amiRNA1 to -3 were selected for further analysis. The algae were grown in TAP medium until mid-log phase. To induce TAG accumulation the cultures were then transferred to TAP-N medium and incubated for 48 h. Finally, TAG mobilization was initiated by transferring the Ndeprived cultures to TAP medium followed by incubation for another 24 h. During this 24 h period of recovery from N deprivation, the ratio of fatty acids in TAG over total fatty acids in the cell was determined at 0, 12, 16, 20 and 24 h (Figure 3B) to observe relative changes in TAG content. During the first 12 h of N recovery, TAG over total fatty acid ratio of the amiRNA lines were not significantly different from that of the dw15.1 parental line. However, at later time points, 20 and 24 h, the amiRNA lines had a higher TAG over total fatty acid ratio than dw15.1 parental line did. Based on this result we concluded that reduced levels of CrLIP4 led to a delay in TAG degradation consistent with the predicted TAG hydrolytic activity of CrLIP4.

Loss of function Crlip4 insertional mutant alleles

To obtain a clearer picture of CrLIP4 function, we extended our in vivo analysis to insertional mutants that recently became available from the Chlamydomonas mutant library project [START_REF] Li | An Indexed, Mapped Mutant Library Enables Reverse Genetics Studies of Biological Processes in Chlamydomonas reinhardtii[END_REF]. We identified seven putative Crlip4 mutant alleles which could harbor possible insertions either within an intron or 3' UTR of the CrLIP4. Lines with insertions in exons were not available. Among the mutants with an insertion within the second intron, three mutants named Crlip4-1 to -3 were selected for further analysis. According to the information given by the mutant library project, Crlip4-1 and Crlip4-3 only have one insertion junction identified and their flanking sequences are identical (Table S1). However, their internal barcodes are different. In addition, Crlip4-3 seemed to have larger cell diameter (data not shown). This could be due to an additional insertion in its background. For Crlip4-2, both insertion junctions are just a few bases apart. One of the junctions is overlapped with that of Crlip4-1 and -3 (Table S1). Based on this information, we postulated that all three mutants are independent and were chosen for characterization of their phenotype.

We first verified the insertion site. Two sets of PCR reactions were performed to corroborate the insertion sites determined by the developers of the mutant library project (Figure 4A). In the first reaction, primers CLiP-F3 and CLiP-R5 were designed to amplify the genomic region across the insertion site. Only the CC5325 parental line gave rise to a detectable product (Figure 4B). In the second set of analysis, primers oMJ944 and CLiP-R4 were used to amplify DNA from inside the insertion to the flanking genomic region. In this PCR reaction, only the mutants gave rise to detectable PCR products (Figure 4B). The results confirmed the insertion sites for the three mutants.

Because the insertions were in the second intron, we further tested for the presence of CrLIP4 transcripts by RT-PCR. Two positions along the gene were tested (Figure 4A) in parallel to the transcript of the receptor for activated protein kinase C 1 (RACK1) as a control.

The RT-PCRs were carried out for 25, 30 and 35 cycles. Both RT-PCR reactions showed that all three mutants were not be able to amplify any product even after 35 cycles, while the CC5325 parental line started to show a band after 30 cycles (Figure 4C). The RACK1 reactions all produced products ensuring the quality of the RNA/cDNA preparations. Based on these results we concluded that these three Crlip4 mutants are true loss of function mutants.

Crlip4 mutants are impaired in TAG breakdown following N resupply and accumulated TAG during normal growth Artificial microRNA repression of CrLIP4 led to a delay in TAG degradation and we expected that the insertional mutant had a more pronounced effect. Therefore, we determined the TAG fatty acid over total fatty acid ratio in cells cultivated under four different growth conditions as shown in Figure 5A: NS, ND, NR using TAP medium (NRT) and NR using minimal medium (NRM). Moreover, N resupply was done in the dark to force the utilization of TAG for the production of energy and cell building blocks. During the NS phase of the experiment, three cultures for each line were grown in TAP medium under normal growth conditions in the light until mid-log phase was achieved. The cells were then transferred to TAP-N medium and incubated for 48 h in the light to achieve N deprivation (ND). Finally, to induce maximum TAG mobilization, ND cultures were divided and half of the cells were resuspended following centrifugation in regular TAP medium (NRT) or minimal medium (MM, NRM) and incubated for 24 h in the dark. During the normal growth in Nreplete medium in the light (NS), the Crlip4 mutants showed significantly higher ratio of TAG over total fatty acids than the CC5325 parental line (Figure 5B). During the TAG accumulation phase (i.e. following N deprivation), the Crlip4 mutants also had an increased TAG over total fatty acid ratio compared to the CC5325 parental line (Figure 5B). However, this difference was only statistically different for one of the mutant lines. When the cultures were subsequently incubated in N-replete media, the Crlip4 mutants were strongly delayed in TAG degradation especially in MM as indicated by their significantly higher TAG over total fatty acid ratio after 24 h compared to the respective parental line (Figure 5B). Taken together this result for the Crlip4 insertional lines along with that for the lines with artificial microRNA repression of CrLIP4, strongly indicated that CrLIP4 is involved in degradation of TAG during N resupply.

Moreover, we showed that removal of CrLIP4 increased oil content in general and especially following N starvation providing a promising engineering strategy for increasing the TAG content in the algal biomass.

Fatty acid compositional changes in the Crlip4 mutants To test the effect of CrLIP4 ablation on the fatty acid profile, TAG acyl groups and total fatty acids were analyzed. During normal N-replete growth conditions (NS), TAG acyl groups and total fatty acid profiles of the mutants were similar to those of the CC5325 parental line (Figure S1). In cultures during later stages of the experiment, saturated (16:0 and 18:0; number of carbons : number of double bonds, with position of double bonds indicated counting from the carboxyl end) were lower and unsaturated fatty acids (especially 18:1 ∆9 ) were more abundant in TAG of the mutants compared to those of the CC5325 parental line. (Figure S1 and Figure 5C). The strongest differences were observed in cells following N resupply in MM medium (NRM) (Figure 5C). The reverse effect was observed for the total fatty acid profile (Figure S1 and Figure 5D). One interpretation of this result might be that CrLIP4 has a preference for a subfraction of TAG molecular species.

However, other changes in lipid metabolism due to the loss of CrLIP4 could cause this phenotype.

Discussion

TAG lipases play important functions in maintaining lipid homeostasis, energy balance, and physiological activities in living organisms. Here we identified and characterized a TAG lipase in the model green alga Chlamydomonas. The phenotype of both amiRNA repression and insertional disruption mutants suggested that CrLIP4 plays a role in TAG degradation particularly during recovery from N deprivation when TAG is abundant and needs to be rapidly degraded. CrLIP4 transcription is most abundant during normal growth conditions when the N supply is sufficient, but decreases following N deprivation when TAG is synthesized and TAG degradation should slow down. While the abundance of the CrLIP4 transcript does not necessarily reflect the abundance or activity of the CrLIP4 protein, its presence under normal growth conditions highlights the importance of TAG homeostasis during active growth. Aside from providing insights into TAG homeostasis under different conditions, this study of CrLIP4 and its role in lipid degradation in microalgae is providing an avenue towards the engineering of TAG content of interest to various industries, not the least biodiesel production.

CrLIP4 transcript level is conversely related to TAG accumulation

In this study we observed an inverse correlation between CrLIP4 transcript and TAG level similar to what was initially observed by [START_REF] Miller | Changes in transcript abundance in Chlamydomonas reinhardtii following nitrogen deprivation predict diversion of metabolism[END_REF] and corroborated in other transcriptomic studies [START_REF] Boyle | Three acyltransferases and nitrogen-responsive regulator are implicated in nitrogen starvationinduced triacylglycerol accumulation in Chlamydomonas[END_REF][START_REF] Lv | Transcriptome analysis of Chlamydomonas reinhardtii during the process of lipid accumulation[END_REF][START_REF] Tsai | Recovery from N Deprivation Is a Transcriptionally and Functionally Distinct State in Chlamydomonas[END_REF][START_REF] Tsai | The protein Compromised Hydrolysis of Triacylglycerols 7 (CHT7) acts as a repressor of cellular quiescence in Chlamydomonas[END_REF].

Because the CrLIP4 transcript level sharply fell during N deprivation, it was expected that following N resupply, the CrLIP4 transcript level would recover rapidly to the same level as during normal growth conditions. However, following N resupply, the level of CrLIP4 increased slowly and had not reached the original levels by the end of the experiment. This result was similar as observed in transcriptomics data from [START_REF] Tsai | Recovery from N Deprivation Is a Transcriptionally and Functionally Distinct State in Chlamydomonas[END_REF]. It seems possible that transcription of CrLIP4 requires more time than we allowed in this experiment to fully recover its original level. Another possibility is that expression of CrLIP4 is dependent on the stage of the culture as shown by [START_REF] Lv | Transcriptome analysis of Chlamydomonas reinhardtii during the process of lipid accumulation[END_REF], which could mean that the level of CrLIP4 transcript will not recover to the same level until the culture is diluted and allowed to grow to mid-log phase again.

CrLIP4 is involved in TAG degradation during N recovery

The in vivo functions of CrLIP4 was addressed in two independent experiments using artificial microRNA repression of the gene and loss-of-function insertional mutants. Data from both sets of experiments were consistent with a role of CrLIP4 in degrading TAG during N recovery. In the artificial microRNA lines, delayed TAG degradation was not observed until later time points (Figure 3B). This could be due to the fact that the level of CrLIP4 transcript was still more than 50% of the parental line giving rise to substantial levels of the CrLIP4 protein during N recovery. Furthermore, during this experiment TAG mobilization might have coincided with de novo TAG synthesis due to the incubation in the light as discussed below. Ultimately, the actual level of a metabolic end product or in this case intermediate such as TAG is determined by its concurrent synthesis and degradation.

During the analysis of the loss-of-function mutants, the highest difference in the ratio of TAG over total fatty acids between Crlip4 mutants and the parental line was observed when the cultures were kept in the dark after N was resupplied (NRM) (Figure 5B). We assumed that this condition allowed us to examine TAG degradation only without interference from concurrent TAG synthesis. Under this condition, because no photosynthesis occurred to provide reductant and ATP for TAG biosynthesis nor acetate was provided to serve as precursor for TAG biosynthesis in the dark, only minimal de novo fatty acid synthesis and therefore TAG assembly should occur at a limited level. Nevertheless, we observed the TAG mobilization defect in the mutants under all conditions tested, albeit to different extent, suggesting that CrLIP4 plays a key role in TAG degradation during recovery from N degradation.

Substrate specificity of CrLIP4 can be inferred from fatty acid compositional changes

Lipases have a diverse range of substrate specificities. Saccharomyces Tgl3p hydrolyzes a wide range of TAG substrates [START_REF] Athenstaedt | YMR313c/TGL3 encodes a novel triacylglycerol lipase located in lipid particles of Saccharomyces cerevisiae[END_REF], while Tgl4p and Tgl5p are more specific for TAGs with 14:0 and 16:0, and 26:0 fatty acids, respectively [START_REF] Athenstaedt | Tgl4p and Tgl5p, two triacylglycerol lipases of the yeast Saccharomyces cerevisiae are localized to lipid particles[END_REF]. The recombinant Arabidopsis SDP1 protein was able to use a broad range of TAG substrates [START_REF] Eastmond | SUGAR-DEPENDENT1 encodes a patatin domain triacylglycerol lipase that initiates storage oil breakdown in germinating Arabidopsis seeds[END_REF]. Fatty acid in the TAG fraction of Crlip4 mutants following N resupply showed reduced 16:0 and increased 18:1 ∆9 fatty acids compared the PL. Based on this result one might infer that CrLIP4 prefers 18:1 ∆9 . However, the real substrate specificity of an enzyme may not be able to determine only from the composition of fatty acids in the mutants. This is because the phenotype could be a result of pleiotropy. In addition, substrate specificity should be inferred from an in vitro assay by using recombinant protein as the TAG lipases mentioned above. Nonetheless, lipase assay alone may not indicate the true substrate in the biological system as in the case of Arabidopsis PLIP1, 2 and 3 [START_REF] Wang | A Plastid Phosphatidylglycerol Lipase Contributes to the Export of Acyl Groups from Plastids for Seed Oil Biosynthesis[END_REF][START_REF] Wang | Two abscisic acid-responsive plastid lipase genes involved in jasmonic acid biosynthesis in Arabidopsis thaliana[END_REF]. Therefore, for CrLIP4, more study needs to be carried out to determine the substrate preference.

It should be noted that 16:0 and 18:1 ∆9 in the total fatty acid pool seem to increase, while 16:4 ∆4,7,10,13 and 18:3 ∆9,12,15 seem to decrease in the mutants (Fig. 5D). This result was not unexpected because 16:0 and 18:1 ∆9 are the major fatty acids in TAG [START_REF] Zäuner | A cytochrome b5-containing plastid-located fatty acid desaturase from Chlamydomonas reinhardtii[END_REF] which contribute almost 40% of total fatty acids in the mutants. On the other hand, 16:4 ∆4,7,10,13 and 18:3 ∆9,12,15 are major fatty acids found in membrane lipids [START_REF] Giroud | Lipids of Chlamydomonas reinhardtii. Analysis of Molecular Species and Intracellular Site(s) of Biosynthesis[END_REF], which were present in a lower ratio compared to the parental line simply refection the difference in relative TAG content. A similar result was observed for the Tgl1 TAG lipase inactivation in a diatom mutant [START_REF] Barka | Identification of a triacylglycerol lipase in the diatom Phaeodactylum tricornutum[END_REF].

The role of CrLIP4 in actively growing cells

Chlamydomonas accumulates small amount of TAG during normal growth conditions. This could be due to the fact that flux of carbon in the form of acyl groups goes toward other pathways such as membrane lipids under these conditions. [START_REF] Deng | Effect of the expression and knockdown of citrate synthase gene on carbon flux during triacylglycerol biosynthesis by green algae Chlamydomonas reinhardtii[END_REF] also reported that in Chlamydomonas citrate synthase (CrCIS) diverts flux of acetyl-CoA from TAG biosynthesis to the tricarboxylic acid cycle. In plants, during vegetative growth, fluxes of DAG and phosphatidylcholine go toward membrane lipids more than to TAG synthesis [START_REF] Bates | The significance of different diacylgycerol synthesis pathways on plant oil composition and bioengineering[END_REF]. Based on analyses of transcript levels of genes involved in TAG metabolism during normal growth condition, Chlamydomonas synthesizes and degrades TAG concomitantly [START_REF] Boyle | Three acyltransferases and nitrogen-responsive regulator are implicated in nitrogen starvationinduced triacylglycerol accumulation in Chlamydomonas[END_REF][START_REF] Lv | Transcriptome analysis of Chlamydomonas reinhardtii during the process of lipid accumulation[END_REF][START_REF] Miller | Changes in transcript abundance in Chlamydomonas reinhardtii following nitrogen deprivation predict diversion of metabolism[END_REF][START_REF] Tsai | Recovery from N Deprivation Is a Transcriptionally and Functionally Distinct State in Chlamydomonas[END_REF]). This concurrent synthesis and break down results in a low TAG concentration during optimal growth. Once the breakdown of TAG is inhibited as is in the case of the Crlip4 mutants, higher TAG was detected (Fig. 5B). Hence, CrLIP4 plays an important role in TAG and lipid homeostasis during normal growth as well as during the TAG mobilization phase, when energy and acyl-chains are needed to support growth and membrane synthesis. Therefore, TAG degradation in microalgae, i.e. CrLIP4, provides a potential engineering target to shift the balance towards accumulation of TAG in growing cells of microalgae.

Materials and methods

Chlamydomonas strains and growth conditions

The Chlamydomonas cell wall-deficient strain dw15.1 (cw15, nit1, mt + ) kindly provided by Arthur Grossman (Carnegie Institute for Science, Stanford University) was used for analysis of transcript levels and served as the parental strain for artificial microRNA studies. The Crlip4 insertional mutant strains LMJ.RY0402.162064, LMJ.RY0402.162453 and LMJ.RY0402.216596 were obtained from the Chlamydomonas Resource Center [START_REF] Li | An Indexed, Mapped Mutant Library Enables Reverse Genetics Studies of Biological Processes in Chlamydomonas reinhardtii[END_REF]. The background strain CC-4533 (cw15, mt -) was used as a parental strain in experiments of the Crlip4 insertional mutants. Unless otherwise specified, the algal cultures were grown in Tris-Acetate-Phosphate (TAP) medium [START_REF] Gorman | Cytochrome f and plastocyanin: their sequence in the photosynthetic electron transport chain of Chlamydomonas reinhardi[END_REF] under continuous light at 80-100 μmol m -2 sec -1 and at 22°C. For N deprivation, the cells were collected by centrifugation at 3,000 g, washed twice and resuspended in TAP-N. For N resupply, the cell pellets were collected similarly but resuspend in either TAP or minimal medium (MM) (TAP medium adjusted pH with HCl instead of acetic acid) and incubated either under the light or in the dark, as indicated in the results part.

Phylogenetic tree reconstruction

The conserved domains, GXSXG motif and catalytic residues were predicted by HMMER [START_REF] Finn | HMMER web server: 2015 update[END_REF]. Amino acid sequences alignment was carried out with MUSCLE [START_REF] Edgar | MUSCLE: a multiple sequence alignment method with reduced time and space complexity[END_REF]). The phylogenetic tree was reconstructed by the Maximum Likelihood method based on the JTT matrix-based model [START_REF] Jones | The rapid generation of mutation data matrices from protein sequences[END_REF]) using MEGA7 [START_REF] Kumar | MEGA7: Molecular Evolutionary Genetics Analysis Version 7.0 for Bigger Datasets[END_REF]). Test of phylogeny was carried out with the Bootstrap method using 1000 replicates [START_REF] Felsenstein | Confidence limits on phylogenies: an approach using the bootstrap[END_REF].

RNA isolation and quantitative reverse transcription PCR (qRT-PCR)

Cell pellets were harvested from 10-15 ml Chlamydomonas culture as described above. The pellets were flash frozen in liquid N2 and stored at -80°C until use. Total RNA isolation from the frozen cell pellets was carried out with RNeasy Plant Mini Kit (QIAGEN). The total RNA was treated with RNase-Free DNase Set (QIAGEN). cDNA synthesis was performed with either RETROscript® Reverse Transcription Kit (Ambion) or QuantiTect Reverse Transcription Kit (QIAGEN). The PCR reactions were prepared with Applied Biosystems® SYBR® Green PCR Master Mix (ThermoFisher SCIENTIFIC). Primers Lip4-jw2-fwd and Lip4-jw2-rev were used to amplify the CrLIP4 transcript. Sequences of these primers, and also all others used in this study, are given in Table S2. The reference gene used for

Chlamydomonas samples was RACK1. The qRT-PCR reactions were carried out using the Applied Biosystem 7500 Fast real-time PCR system. The qRT-PCR data were calculated based on the 2 -∆∆C T method as previously described [START_REF] Livak | Analysis of relative gene expression data using realtime quantitative PCR and the 2(-Delta Delta C(T)) Method[END_REF].

Generation of artificial microRNA knockdown lines

Target sequences of artificial microRNA were determined through WMD3-Web MicroRNA Designer (http://wmd3.weigelworld.org/cgi-bin/webapp.cgi?page=Home;project=stdwmd).

The Designer page of WMD3 was used for primer design. Target sequence and primers can be found in Table S2. Oligonucleotides were then inserted into the pChlamiRNA3int vector according to [START_REF] Molnar | Highly specific gene silencing by artificial microRNAs in the unicellular alga Chlamydomonas reinhardtii[END_REF]. The plasmid was linearized with KpnI restriction enzyme and transformed into Chlamydomonas dw15.1 using the glass bead method [START_REF] Kindle | High-frequency nuclear transformation of Chlamydomonas reinhardtii[END_REF]) with minor modifications; Cells were grown in liquid TAP medium and the use of polyethyleneglycol (PEG) was omitted. Transformants were selected on agar-solidified TAP medium containing 10 µg/ml paromomycin. Single colonies were picked and tested for the CrLIP4 transcript level by qRT-PCR as described above. Lipid analysis was performed as described below.

Genotyping of Crlip4 insertional mutants

PCR was used to test the genotype of the Crlip4 mutants. Three single colonies were tested for each mutant. Genomic DNA was extracted with Chelex-100 (Sigma-Aldrich) as previously described [START_REF] Cao | Chlamydomonas (Chlorophyceae) colony PCR[END_REF]. PCR across the predicted insertion site was performed with CLiP-F3 and CLiP-R5 primers. While PCR from the insert to the flanking sequence was performed with oMJ944 and CLiP-R4 primers. The PCR was carried out with KOD DNA polymerase (MERCK) for 35 cycles.

Reverse transcription PCR for Crlip4 insertional mutants

RNA extraction was performed as previously described [START_REF] Nguyen | The green microalga Chlamydomonas reinhardtii has a single omega-3 fatty acid desaturase that localizes to the chloroplast and impacts both plastidic and extraplastidic membrane lipids[END_REF]. DNase treatment was carried out with TURBO™ DNase (Thermo Fisher SCIENTIFIC). The treated RNA was then purified with NucleoSpin® RNA Clean-up (MACHEREY-NAGEL). cDNA was synthesized with SuperScript™III First-Strand Synthesis System (Thermo Fisher SCIENTIFIC). PCR was performed at two locations with CrLIP4-RT-F1 and CrLIP4-RT-R1, and CrLIP4-RT-F4 and CrLIP4-R4 primers. The PCRs were conducted at 25, 30 and 35 cycles.

In addition, RACK1 was amplified with LP-F and LP-R primers for 25 cycles as an internal standard.

Lipid analysis

Four to 15 ml of Chlamydomonas cultures were harvested and stored at -80°C as described above. Total lipid was extracted from the frozen cell pellets using the method from [START_REF] Bligh | A rapid method of total lipid extraction and purification[END_REF]. The extracted lipids were dried under an N2 stream and stored at -20°C. TAG was separated from total lipids (30 µl from 200 µl resuspended in chloroform) with thin layer chromatography (TLC) using a solvent system for neutral lipids as described in [START_REF] Warakanont | Chloroplast lipid transfer processes in Chlamydomonas reinhardtii involving a TRIGALACTOSYLDIACYLGLYCEROL 2 (TGD2) orthologue[END_REF]. TAG bands were isolated from the TLC plate and being converted to fatty acid methyl esters (FAMEs) as described in [START_REF] Benning | Isolation and genetic complementation of a sulfolipid-deficient mutant of Rhodobacter sphaeroides[END_REF] with some modifications. Generally, after incubation with 1 ml of 1 N methanolic HCl (Sigma-Aldrich) at 80°C for 30 min, 1 ml of 0.9% NaCl (w/v) was added for phase separation. FAMEs were extracted into 1 ml hexane which was then dried and resuspend in 50-100 µl hexane for quantification with gas chromatography using DB-23 column (Agilent Technology) as described in [START_REF] Zäuner | A cytochrome b5-containing plastid-located fatty acid desaturase from Chlamydomonas reinhardtii[END_REF]. For all experiments the instrument was HP6890 coupled with a flame ionization detector (Agilent Technology), except GCMS-QP2020 (Shimadzu) was used for the lipid analysis of Crlip4 mutants. Cells were first cultivated under N sufficiency in TAP medium, then deprived for N for a number of days and followed by N recovery in TAP medium again. The transcript of CrLIP4 was normalized to that of the receptor for activated kinase C 1 (RACK1). This experiment was carried out with Chlamydomonas dw15-1 as a parental line (PL). Noted that the culture in all conditions was incubated under the light. The error bars represent standard deviations calculated from three biological replicates. RACK1 is used as a control.

Figure 1 .

 1 Figure 1. Protein domain and phylogenetic analysis. (A) A schematic diagram showing the positions of protein domains. The DUF3336 (PF11815.7) and Patatin (PF01734.21) domains, GXSXG motif, serine and aspartate residues were predicted by HMMER (Finn et al 2015) of characterized TAG lipases and CrLIP4. The length is drawn to scale from N-to C-terminus. (B) Phylogenetic analysis of CrLIP4 homologues. The percentage of trees in which the associated taxa clustered together is shown next to the branches. The tree was drawn to scale, with branch lengths measured in the number of substitutions per site.

Figure 2 .

 2 Figure 2. CrLIP4 transcript level and TAG content during the three N status.

Figure 3 .

 3 Figure 3. Artificial microRNA knockdown of CrLIP4. (A) Relative expression of CrLIP4. (B) TAG concentration during N resupply of artificial microRNA knockdown lines 1 to 3 (amiRNA-1 to -3). Transcript levels of CrLIP4 from 3 amiRNAs were analyzed with RACK1 as an internal control. The amiRNA lines were tested for their TAG content after 0, 12, 16, 20 and 24 h of N resupply under the light. The error bars represent standard deviations calculated from three biological replicates. Statistical analysis was carried out with two-tailed student t-test based on whether the variances are equal or not. * indicates statistically difference between amiRNA and dw15.1 PL with p-value ≤ 0.05. ns indicates no statistically different between amiRNA and dw15.1 PL at p-value ≤ 0.05.

Figure 4 .

 4 Figure 4. CrLIP4 gene model and genotyping of Crlip4 knockout mutants.

Figure 5 .

 5 Figure 5. TAG phenotype of Crlip4 mutants. (A) Schematic drawing shows growing conditions of Chlamydomonas. The cultures were grown in TAP medium until mid-log phase was achieved. They were transferred to TAP-N for 48 h and to either TAP or MM for another 24 h under the dark. N sufficient (NS), N deprivation (ND), N resupply with TAP (NRT) and N resupply with MM (NRM) samples were taken as indicated. (B) TAG/total fatty acids during NS, ND, NRT and NRM. (C) Fatty acid composition of TAG. (D) Total fatty acids of cultures from NRM condition. The error bars represent standard deviations calculated from three biological replicates. Statistical analysis was carried out with student t-test (one-tailed in B and two-tailed in C and D). The type of the t-test was chosen based on the equality of variances. * and ** indicate statistical difference with p-value ≤ 0.05 and 0.01, respectively. ns indicates no statistical difference at p-value ≤ 0.05.

Figure S1 Fatty

 S1 Figure S1 Fatty acid composition of TAG and total fatty acids of four N status Fatty acid compositions of TAG (left panel) and total fatty acid (right panel) fractions from NS (first row), ND (second row), NRT (third row) and NRM (fourth row) of WT and Crlip4 mutants were shown. The error bars represent standard deviations calculated from three biological replicates. Statistical analysis between each Crlip4 mutant and WT was carried out with two-tailed student t-test based on whether the variances are equal or not. * and ** indicate statistical difference with p-value ≤ 0.05 and 0.01, respectively. ns indicates no statistical difference at p-value ≤ 0.05.

Funding

This work was supported by grants to JW from the Thailand Research Fund (MRG6080038) and Faculty of Science, Kasetsart University (PRF-PII 60 P2 60), to YLB from MUsCA. CB was supported by the Division of Chemical Sciences, Geosciences and Biosciences, Office of Basic Energy Sciences of the United States Department of Energy (Grant DE-FG02-91ER20021) and MSU AgBioResearch. JW was supported during initial stages of this work by a Royal Thai Government Scholarship and later by a Junior Research Fellowship Program from the Embassy of France in Thailand.

Disclosures:

The authors declare no conflicts of interest.

Acknowledgments:

The authors thank Bertrand Légeret on advice for lipid analysis, Saowapa Chaipitak for advices on statistical analysis, and Fred Beisson and Kittisak Yokthongwattana for helpful discussions.

We also appreciate Anchalee Sirikhachornkit and Supachai Vuttipongchaikij for providing laboratory resource and facility.

Supplemental information:

Supplemental Table S1. Information of Crlip4 insertional mutant alleles Supplemental Table S2. Sequences of primers and target sequences used for different purposes as indicated.