Isolation of chloroplasts from marine microalgae Isochrysis galbana Parke suitable for organelle lipid composition analysis


 Background: Marine microalgae, Isochrysis galbana Parke, is an important diet microalgal species with high nutritional value. Different from other unicellular microalgae, its cell contains two chloroplasts which are the major sites for lipid synthesis. Results: Here, we optimized a chloroplast isolation approach suitable for I. galbana Parke, and evaluated the purity and integrity of the isolated chloroplasts by microscopic observations and biochemical assays. The chloroplast lipids were sequenced by ultrahigh-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). Glycolipids were the main chloroplast lipids, and DGDG, MGDG, and SQDG were the most abundant glyceroglycolipids. DGMG and SQMG were not observed among the chloroplast lipids. In addition, DG was the most abundant neutral lipid. A part of fatty acyl R1/R2 with MGMGs, DGDGs, MGDGs, SQDGs, PEs, and PCs were not found in chloroplasts. The fatty acid proportion of chloroplast lipids were increased, decreased, or remained unchanged compared with the whole-cell. Conclusions: This newly developed isolation approach was a simple and reliable method to isolate chloroplasts with high integrity and purity. Collectively, our findings show that such an isolation approach may be used in studies on many different aspects of chloroplast biology, and offers a useful reference for isolation of chloroplast from other marine microalgae.

facilitates the clari cation of lipid metabolic machinery, which plays a fundamental role in rational engineering of oils and fatty acids for fuel, industrial feedstocks, and nutritional improvement.
Until recently, only a limited number of studies have focused on chloroplast lipid composition and metabolism in land plants. Chloroplasts are particularly rich in monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG), and sulfoquinovosylmonoacylglycerol (SQDG). In higher plant Arabidopsis, galactolipids are essential to support the growth and photosynthesis [7,8]. MGDG, DGDG, SQDG, and phosphatidylglycerol (PG) constitute the thylakoid membranes, which are the integral ingredients of the photosynthetic complexes [5,9]. Phosphatidylcholine (PC), lyso-phosphatidylcholine (lyso-PC), or phosphatidic acid (PA) for chloroplast lipids can be produced in the endoplasmic reticulum (ER) and the chloroplast [10]. In unicellular green alga Chlamydomonas reinhardtii, a model species, the building blocks for triglyceride (TG), and membrane lipids are biosynthesized in the chloroplast. PA and its dephosphorylated product diacylglycerol (DG), are produced in the chloroplast, and they primarily function as precursors for structural lipids of the photosynthetic membrane system [11]. In starved cells, galactolipids are maintained to preserve su cient chloroplast integrity [12]. In general, neutral lipid metabolism of chloroplast has been extensively studied in C. reinhardtii. However, there are no comprehensive studies on the microalgal chloroplast lipid composition.
Marine microalgal biomass plays a critical role in the animal food chain. As a golden-brown agellate marine microalga, Isochrysis galbana Parke is widely adopted as an aquaculture feed for young sh and in bivalve hatcheries due to its high content of polyunsaturated fatty acids (PUFAs) [13]. Previous investigations have shown that the nutritional value of I. galbana to Mollusca larvae is greatly attributed to its unique lipid composition [14][15][16] and that different cultivation temperatures or times can alter the lipid composition of I. galbana. Therefore, many studies have focused on identifying growth conditions that can promote lipid production [17,18]. While the whole-cell lipidomics of I. galbana is widely studied, the lipid synthesis and biosynthesis of chloroplast remain largely unknown.
A major deterrent in studying the lipid composition of the chloroplast is the technical challenge associated with the isolation and puri cation of intact chloroplasts. The currently used techniques employ physical or chemical means to break the cell wall and the plasmalemma [19,20]. The isolation techniques of chloroplast have been con ned to several higher plants and a model alga. For Arabidopsis, a method for chloroplast isolation relies on protoplastation [21]. However, this method is time-consuming and expensive. To resolve this problem, a simple and cost-effective approach has been developed to isolate a large number of intact chloroplasts from Arabidopsis seedlings [22,23]. For marine plant Posidonia oceanica, an optimized protocol has been adapted from terrestrial plants and can be used to isolate chloroplasts from as minimal tissue as possible [24]. For C. reinhardtii mutant, cells are broken by passing them through a 27-gauge syringe needle [15,25]. However, thus far, no universal method for isolation and puri cation of the chloroplast is available due to the differences in the number, size, gravity, thylakoid membrane structure, and lipid composition of the chloroplast of different plant cells. Thus, developing a suitable chloroplast isolation protocol that can be applied to different plant species is essential. The marine microalgae I. galbana Parke, in which one cell contains two chloroplasts, is different from others [26]. The possibility of obtaining intact chloroplasts, which are active in lipid synthesis, from a no cell wall microalga prompted us to attempt the isolation of chloroplasts from I. galbana. There are no established techniques for the isolation of intact chloroplasts from I. galbana, and there are no studies on its chloroplast lipid composition. Therefore, a chloroplast isolation technique for the marine microalgae I. galbana that is suitable for studying the organelle lipid composition is an emerging need.
In the present study, we developed a rapid approach for cellular fractionation of I. galbana, by which intact chloroplasts could be isolated. The chloroplasts puri ed using this approach were mostly intact and contained only minimal contamination. The isolated chloroplasts and their integrity were analyzed by phase-contrast uorescence microscopy (Leica) and transmission electron microscopy (TEM), and their purity was analyzed by enzyme-linked immunosorbent assay (ELISA). The lipid composition of the chloroplasts isolated from I. galbana was identi ed by ultrahigh-performance liquid chromatographytandem mass spectrometry (UHPLC-MS/MS).

Methods
Microalgal culture and sample collection I. galbana Parke was provided by the Marine Biotechnology Laboratory of Ningbo University, China. Seawater (pH 8.2, salinity 22‰) was subjected to ltration using 0.45-μm cellulose acetate membranes, followed by heat sterilization. The culture medium was supplemented with the following nutrients: 100 mg/L KNO 3 , 10 mg/L KH 2 PO 4 , 2.5 mg/L MnSO 4 ·H 2 O, 2.5 mg/L FeSO 4 ·7H 2 O, 10 mg/L EDTA-Na 2 , 6 μg/L vitamin B 1 , and 0.05 μg/L vitamin B 12 . Microalgae were maintained in 5,000 mL conical asks at 20 ± 2°C , with a light intensity of 4,000 Lux (LED) under a 12:12-h light-dark (L:D) photoperiod. Cells were incubated at 4 °C overnight to metabolize the pyrenoid and starch granules in the chloroplasts, followed by sample collection at the late stationary phase. The cells were centrifuged at 6,000×g for 10 min. A blood counting chamber was used to determine the cell density at each time point. All experiments were performed in triplicates, and the results were expressed as the mean ± standard deviation.

Isolation and puri cation of intact chloroplast
Chloroplasts from I. galbana were isolated and puri ed at 4 °C or on ice to protect the integrity of chloroplasts, and the pellet was washed and resuspended gently. First, 100 mg of wet weight microalgae was washed twice with 2 mL of 50 mM HEPES-KOH (pH 7.8) and centrifuged at 800×g for 10 min to remove salts. The chlorophyll concentration of cells was measured according to the study by Arnon [27].
The key step was to gently separate the cell membrane from the plastids, and to this end, the cells were provided with a hypotonic environment by two steps. First, the cells were resuspended in 2 mL of solution A (300 mM sucrose, 50 mM HEPES-Tris, 2 mM EDTA-Tris, 1 mM MgCl 2 , 1% PMSF as a protease inhibitor, pH 7.8) and centrifuged. Second, the cells were subjected to a lower osmotic shock by resuspending the cell pellet in 2 mL of solution B (100 mM sucrose, 50 mM HEPES-Tris, 2 mM EDTA-Tris, 1 mM MgCl 2 , 1% PMSF, pH 7.8) and allowing to stand on ice for 20 min. Subsequently, the cells were successively homogenized at 2,500 r/min for 10 s and 6,800 r/min for 15 s, and this process was repeated 3-4 times, followed by centrifugation at 800×g for 10 min. For effective separation, the supernatant was set aside, and solution B was added to the cell pellet and subjected to homogenization for 3-4 turns.
The supernatant was centrifuged at 1,000×g for 10 min to remove nuclei and intact cells, and the crude chloroplasts were collected by centrifugation at 4,500×g for 10 min. The crude chloroplast pellet was gently resuspended in 1 mL of solution C (300 mM D-sorbitol, 50 mM HEPES-Tris, 10 mM EDTA-Tris, 5 mM MgCl 2 , 1% PMSF, pH 7.8) using a pipette to avoid disrupting the chloroplasts. The resuspended sample was gently loaded onto several Percoll density gradients (top to bottom: 2 mL 10% Percoll, 3 mL 30% Percoll, 3 mL 50% Percoll). All gradient media were prepared in solution C. The Percoll gradients were subjected to centrifugation at 8,000×g for 30 min. The intact chloroplast constituted a band at the 30-50% interface and was collected, diluted with three volumes of solution D (300 mM D-sorbitol, 50 mM HEPES-Tris, 10 mM EDTA-Tris, 5 mM MgCl 2 , pH 7.8), centrifuged at 3,500×g for 10 min twice, and the chlorophyll concentration of the chloroplast was determined. Pellets were stored at -80 °C.

Electron microscopy
The modi ed Spurr method [28] was used to prepare the samples for TEM. Brie y, samples were incubated in 2.5% glutaraldehyde in phosphate buffer (0.1 M, pH 7.8) at 4 °C for 48 h, washed with phosphate buffer, and then post-xed with 1% osmium tetroxide (OsO 4 ) for an additional 3 h.
Subsequently, samples were dehydrated in a graded series of ethanol, followed by acetone. The specimens were incubated in a mixture of acetone and nal Spurr resin mixture overnight, followed by incubation at 70 °C for more than 9 h. The samples were sectioned using a LEICA EM UC7 ultratome, and the sections were stained by uranyl acetate and alkaline lead citrate, followed by observation using a Hitachi TEM (Model H-7650) at an acceleration voltage of 80 kV.

ELISA assays
Proteins were extracted according to a modi ed method [29]. Brie y, the samples were ground with liquid nitrogen in a pre-cooled mortar and subsequently sonicated in lysis buffer (8 M urea, 1% PMSF) on ice three times. The remaining debris was removed by centrifugation at 12,000×g at 4 °C for 10 min. The supernatant was collected, diluted with four volumes of cold acetone, and incubated at -20 °C for 24 h, followed by centrifugation at 12,000×g at 4 °C for 10 min. Finally, the obtained pellet was dissolved in phosphate buffer saline (PBS).
According to literature, cytoplasm and mitochondria are common contaminants in chloroplast isolation. The purity of isolated chloroplasts was biochemically assessed using ELISAs to estimate the activities of Phosphoenolpyruvate carboxylase (PEPC) and Cytochrome C oxidase (COX) [24,25,30]. The chloroplastspeci c enzyme, Ribulose bisphosphate carboxylase oxygenase (Rubisco), was used as a positive control. The enzyme activities were estimated using ELISA kits (Meimian Biotechnology, Jiangsu, China), according to the manufacturer's instructions, and the color change was measured spectrophotometrically at 450 nm. Each experiment was conducted with three biological replicates.

Total lipid extraction
The total lipid was obtained from samples using a modi ed Bligh and Dyer method [31]. Brie y, freezedried samples were extracted with chloroform/methanol/water (2:2:0.8, v/v/v) and dried under a gentle stream of N 2 , and the residue was preserved at -20 °C. The nal lipid residue was resuspended in CH 3 OH before further analysis. The above-mentioned procedures were carried out in triplicates. The results were expressed as mean ± standard deviation.

Mass spectrometric conditions
Mass spectrometry was carried out on a Thermo Scienti c TM Q Exactive hybrid quadrupole-Orbitrap mass spectrometer equipped with a HESI-II probe. The instrument was operated according to a data-dependent LC-MS/MS method in positive mode and negative mode, respectively. Data were obtained in a centroid mode from 200 to 2,000 m/z at a resolution of 70 K, and in a high energy collisional dissociation (HCD) MS/MS mode at a resolution of 17.5 K. The automatic gain control (AGC) target was set at 1e 6 for MS and 2e 5 for MS 2 . The capillary voltage was maintained at 3.5 kV, and the capillary temperature was set at 350 °C. The sheath gas was 45 arb, and the aux gas was 10 arb. MS 2 analysis was carried out on the mass spectrometer with different collision energy and ramp of 25, 30 V in a positive ion mode and 20, 24, 28 V in a negative ion mode based on the type of lipids. The instrument was previously calibrated in positive mode and negative mode, respectively.

Data processing
The raw data acquired from LC/MS runs were analyzed on the Thermo Xcalibur TM system. Lipidsearch software version 4.1 (Thermo Scienti c TM ) was used to perform peak identi cation, lipid identi cation, peak extraction, peak alignment, peak area, and intensity. According to characteristic fragment ions and fragmentation pathways, glycolipids, phospholipids (PLs), betaine lipids, and non-polar lipids could be determined [32][33][34][35][36][37][38][39]. Identi cation of each lipid class and the acyl chains were further con rmed by MS/MS in positive or negative ion mode, as previously described. The semi-quantitative determination of each lipid was based on the lipid standards (Avanti Polar Lipids Inc., Alabaster, AL) area [32,40].

Chloroplast yield
The chlorophyll concentration of the whole-cell and chloroplast were determined. The average yield of intact chloroplasts was 10±1% based on the chlorophyll recovery (Additional le 1: Table S1).

Chloroplast characterization and integrity
Chloroplast debris was accumulated at the 10-30% percoll gradient interface, the intact chloroplasts constituted a band at the 30-50% interface, and intact cells were found at the bottom of the tube (Fig. 1). The microscopic pictures for each fraction are shown in Fig. 2 and Fig. 3. Fig. 3a and Fig. 3b show that there were no intact cells in the puri ed chloroplasts. Under a phasecontrast microscope, the intact chloroplasts were green and surrounded by a bright halo due to the presence of the chloroplast envelope, and had an integrity of >85% (Fig. 3d). When observing the intact cells and isolated chloroplasts using uorescence microscopy, the isolated chloroplasts emitted red light and were smaller than intact cells (Fig. 3e and Fig. 3f). Examination of cell ultrastructure showed that puri ed chloroplasts retained their shape, and were intact ( Fig. 3g and Fig. 3h).
Chloroplast purity PEPC and COX activities in the cell homogenates, crude chloroplasts, and puri ed chloroplasts were determined, and the data is shown in Table 1. The differential centrifugation could reduce 54% cytoplasm and 45% mitochondrial contamination, and a discontinuous density gradient centrifugation could further reduce 41% cytoplasm and 51% mitochondrial contamination. This method resulted in only low activities of PEPC and COX, con rming that there was no signi cant organelle contamination in the isolated chloroplasts.

Lipid composition of the whole-cell and isolated chloroplast
In the present study, we analyzed the contents of glycolipids, PLs, sphingolipids, betaine lipids, and acylglycerols in the whole-cell and chloroplast of I. galbana Parke. Figure 4 lists the relative amounts of lipid classes in total lipid and the relative amounts of major lipid subclasses in total lipid.
Glycolipids were the main lipid components in the chloroplast and accounted for 55.7% of the total lipid.
As the most abundant glyceroglycolipid in the chloroplast, DGDG, MGDG, and SQDG made up approximately 27.0%, 18.4%, and 8.2% of the total lipid, respectively, which were much higher than those in the whole-cell. The fatty acyl R1/R2 were mostly occupied by 16 Table S2). The percentage of trigalactosyldiacylglycerol (TGDG) in the chloroplast was 1.2%, which was nearly the same as that in the whole-cell. Remarkably, monogalactosylmonoglyceride (MGMG) constituted 9.5% of glycolipids in the whole-cell, while it only accounted for 0.7% in the chloroplast. Besides, the fatty acid chains of MGMGs with 16:2 were present in the whole-cell but absent in the chloroplast. Compared to the whole-cell, digalactosylmonoglyceride (DGMG) and sulfoquinovosylmonoacylglycerol (SQMG) were nearly undetectable in the chloroplast. Therefore, our data suggested that these two types of lipids were not components of the chloroplast lipids.
Based on our experimental results, the proportion of acylglycerols was second only to glycolipids, which were abundant in both the whole-cell (25.1%) and the chloroplast (28.9%) of I. galbana. Microscopic observations con rmed that chloroplast contained lipid droplets (LD), which were conserved neutral lipid storage organelles (Fig. 5). The proportion of DG in total chloroplast lipid was 20.4%, which was more than that in the whole-cell. Meanwhile, the proportions of monoglyceride (MG) and TG in chloroplast were less than those in the whole-cell.
The proportion of PLs in the total lipid of chloroplast was only slightly more than that in the whole-cell.
The proportion of PG in PL was greater compared with the other four PLs in the whole-cell and chloroplast, and the second most abundant PL was PC. The chloroplast exhibited a slight increase in PG and a slight decrease in PC compared with the whole-cell. Other PLs in the chloroplast were detected at lower amounts. PA content of the chloroplast was slightly higher than that of the whole-cell, while phosphatidylinositol (PI) and phosphatidylethanolamine (PE) showed the opposite trends compared with We also found sphingolipids, which made up 8.3% in the whole-cell and 10.4% in the chloroplast. There was a lot of cerebroside and a small amount of ceramide in sphingolipids. The proportion of betaine lipids was low, which was nearly the same between the whole-cell and chloroplast. Moreover, 14:0 lyso-DGCC (lyso-diacylglycerylcarboxyhydroxymethylcholine) and 16:0 lyso-DGCC were not found in chloroplast compared with the whole-cell.
The PC composition was deeply modi ed in the chloroplast, with a dramatic elevation in 18:1 and 19:1 fatty acids and a remarkable reduction in 16:0, 18:2, and 20:4 fatty acids (Fig. 6g). In addition, in PGs of the chloroplast, the proportion of 18:1 fatty acid was substantially increased, and the proportions of other fatty acids were slightly reduced (Fig. 6h). In contrast, the proportions of DGDG, MGDG, SQDG, and glycosphingolipid (GerGl) in the chloroplast were essentially similar to those in the whole-cell.

Discussion
The marine algae of I. galbana differ from other marine unicellular microalgae in their lack of the cell wall. Due to this difference in structure, chloroplast isolation techniques need to be speci cally adapted for I. galbana, and here, we report a new isolation approach. SDS, frequently presented in isolation buffers to break up the cell wall, was not added to the isolation buffer in this study. Referred to the disruption method for animal cells [41], our improved method used two-step hypotonic buffers, which was better compared with simplex lysis buffers. Buffer A contained 300 mM sucrose, and buffer B contained only 100 mM sucrose. The two-step hypotonic buffers allow the microalgae cells to gradually detach from the plasma membrane, and the amount of buffers added needs to be strictly controlled. However, as the plasma membrane of I. galbana was extremely hard, separation could further be achieved by homogenization to save time. Meanwhile, the two-step hypotonic buffers provided conditions for the next homogenization. A rapid isolation method of chloroplast consisting of the twostep hypotonic buffers and homogenization could save time and increase the yield.
In particular, the multiple homogenization steps were a key feature of the isolation procedure and ensured a high yield of intact chloroplasts. During homogenization, quartz sand mixture (0.1mm and 0.4mm) was added into the tube to break the cells of I. galbana. The homogenization needs to be thorough, and several short-time homogenizations were used until very little sediment was observed after centrifugation at 800 × g for 10 min. The supernatant was examined using phase-contrast uorescence microscopy to ensure the absence of intact cells. The speed of homogenization is also vital for intact chloroplast isolation, and we adopted the intermittent low-speed and high-speed method. With this modi cation, we could not only isolate the intact chloroplasts but could also avoid thylakoid membrane fragmentation that is usually caused by mechanical heat generated during continuous homogenization. Additionally, continuous low-speed homogenization increases the isolation time substantially, which is not conducive to the separation and protection of intact chloroplasts. Therefore, our method of successive low and highspeed homogenization in this experiment mitigates these issues. The homogenization condition presented here are optimal for I. galbana Parke, and we demonstrated that the osmotic shock and homogenization play a fundamental role for effective cell lysis, organelle separation, and the nal isolation of intact chloroplasts.
After homogenization, intact chloroplasts were puri ed from damaged chloroplasts and intact cells by a Percoll density gradient centrifugation, and two-step gradients were employed for most experiments. Before gradients were added, the tube was rinsed with 1% BSA. Then the gradients were added slowly along the tube wall to form distinct interfaces. The gradient centrifugation step allowed for the puri cation of intact, photosynthetically active chloroplasts from I. galbana Parke.
Microscopy and ELISAs were used to estimate the integrity and purity of the isolated chloroplast. ELISAs is a powerful method for measuring the enzymatic activity and is often applied in biochemical assays not only for animal cells but also for plants and algae [42,43]. ELISAs are rapid, sensitive, speci c, and precise [44], therefore, we used it to quantify enzymatic activity. To avoid the inactivation of the enzymes, the samples should go through the entire experimental process, and the sampling process should be synchronous. Cell homogenate, rather than cell fractionation, was selected to determine 100% of individual enzymatic activity. PEPC and COX are enzymes unique to the cytoplasm and mitochondria, respectively, and chloroplasts do not contain these enzymes [25]. Microscopic images and the Rubisco activity data con rmed the integrity of the isolated chloroplast. The results of the PEPC and COX activities in the cell homogenates, crude chloroplasts, and puri ed chloroplasts ensured the purity of the isolated chloroplast, indicating that they were suitable for chloroplast lipid composition analysis.
To fully understand the chloroplast lipid composition in I. galbana Parke, we chose the late stationary phase of microalgae for analysis because it is the optimal period with the most lipid accumulation [45]. The chloroplast lipid composition of I. galbana Parke was qualitatively similar to that of other photosynthetic plants. The results of the cellular and chloroplast lipid composition in I. galbana Parke indicated that chloroplasts were the major sites for fatty acid synthesis and lipid biosynthesis. The results of glycolipids were consistent with those reported for chloroplast from land plants like Arabidopsis [46,47]. One important exception was that the content of DGDG was higher compared with the MGDG in this alga. This was probably a consequence of different plant species or cultivation conditions as depending on the plant species, changes in growth conditions can affect the amounts of DGDG and MGMG [12,48].
According to previous reports, acylglycerols are present in minor amounts in Vicia faba [49] and in signi cant amounts in Mimosa [50]. Our results indicate similar acylglycerols levels in I. galbana Parke and Mimosa. In eukaryotes, lipid droplets serve as the primary depot for energy and neutral lipid storage [51,52], and consistent with other literature, we also observed lipid droplets in the chloroplasts of I. galbana Parke (Fig. 5) [53,54]. I. galbana chloroplast lipid droplets contained substantial quantities of storage lipids, and DGs were used for the synthesis of glycolipids and PGs.
In plants and algae, galactolipid, sulfolipid, and PGs are synthesized by DAG, which is derived from the eukaryotic pathway or eukaryotic and prokaryotic pathways [55,56]. I. galbana chloroplast lipids had high amounts of unsaturated fatty acids, and were mostly occupied with 14:0, 16:1, 18:3, 18:4, and 18:5 in DGDGs, 14:0, 16:1, 18:1, 18:3, 18:4, and 18:5 in MGMGs, and 14:0, 16:0, 18:1, 18:3, and 18:4 in SQDGs. In PGs, 16:0 and 18:1 were the main fatty acids. The position of these fatty acids in the glycerol backbone was different. Fatty acyl residues of DGDGs and SQDGs were mainly with 18 carbon atoms at the sn-2 position of the glycerol backbone. In I. galbana, DGDG and SQDG were mostly derived from the eukaryotic pathway. The other two photosynthetic membrane lipids, fatty acyl residues with 16 and 18 carbon atoms, were both distributed at the sn-2 position. Thus, in I. galbana Parke, the MGMG and PG were produced by both the prokaryotic and eukaryotic pathways. Activity is expressed as U·per L of chlorophyll in three independent replicates. Cell homogenate was obtained by homogenization. Crude chloroplasts were obtained by differential centrifugation. Puri ed chloroplasts were obtained by differential centrifugation followed by puri cation using discontinuous density gradient centrifugation. Figure 1 Typical Percoll gradient after centrifugation, and the percentages of Percoll in buffer for the various layers. a: broken chloroplasts, b: intact chloroplasts, c: intact cells.