Effect of Temperature and Light Intensity on the Polar Lipidome of Endophytic Brown Algae Streblonema corymbiferum and Streblonema sp. In Vitro

The effect of temperature and light intensity on the polar lipidome of endophytic brown algae Streblonema corymbiferum and Streblonema sp. in vitro was investigated. More than 460 molecular species have been identified in four glycoglycerolipids classes, five phosphoglycerolipids classes and one betaine lipid class. The lipids glucuronosyldiacylglycerol and diacylglyceryl-N,N,N-trimethyl-homoserine were found in the algae of the order Ectocarpales for the first time. A decrease in cultivation temperature led to an increase in the unsaturation level in all classes of polar lipids. Thus, at low temperatures, the content of 18:4/18:4 monogalactosyldiacylglycerol (MGDG), 20:5/18:4 digalactosyldiacylglycerol (DGDG), 18:3/16:0 sulfoquinovosyldiacylglycerol (SQDG), 18:3/18:3 and 18:3/18:4 phosphatidylglycerol (PG), 20:4/20:5 and 20:5/20:5 phosphatidylethanolamine (PE), 14:0/20:5, 16:0/20:5 and 20:5/20:5 phosphatidylcholine (PC), 20:5/20:4 phosphatidylhydroxyethylglycine and 18:1/18:2 DGTS increased. At high temperatures, an increase in the content of chloroplast-derived MGDG, DGDG and PG was observed. Both low and high light intensities caused an increase in 20:5/18:3 MGDG and 18:3/16:1 PG. At low light intensity, the content of DGDG with fatty acid (FA) 18:3 increased, and at high light intensity, it was with FA 20:5. The molecular species composition of extraplastid lipids also showed a dependence on light intensity. Thus, the content of PC and PE species with C20-polyunsaturated FA at both sn-positions, 18:1/18:1 DGTS and 16:0/18:1 phosphatidylinositol increased. Low light intensity induced a significant increase in the content of chloroplast-derived 18:1/16:1 phosphatidylethanolamine.


Introduction
Environmental factors, including temperature and light intensity, affect the growth, development and photosynthetic activity of algae. During evolution, algae have developed numerous compensatory mechanisms to smooth out the negative effects of abiotic factors. Lipid metabolism is one of the key tools in the system of adaptation of algae to changing environments [1].
Currently, many studies have been carried out on the effect of temperature on the lipid composition of algae. The main mechanisms of thermal adaptation have been determined, including changes in the degree of unsaturation and carbon chain length of fatty acids (FAs) [2][3][4], the Ñ3/Ñ6 ratio of polyunsaturated fatty acids (PUFAs) [5,6], the content of individual classes of polar lipids as well as the content of neutral lipids [7][8][9][10]. However, separate studies of FA composition and composition of lipid classes do not provide a complete picture of the adaptive reorganization of the lipid matrix of cell membranes. Increasingly popular is the lipidomic approach using HPLC-MS, which makes it possible to determine the molecular composition of all organism lipids. Currently, there are not many works devoted to the study of the effect of temperature on macroalgae lipids, these works mainly describe the composition of molecular species of polar lipids in different seasons [11,12]. However, under natural conditions, in addition to temperature, other environmental factors, such as light, salinity and the concentration of nitrogen and phosphorus, can influence the lipid composition. Recently, the influence of temperature on the molecular composition of glycolipids and betaine lipids of Ulva lactuca and Saccharina japonica cultivated under controlled conditions has been shown, which excludes the influence of other environmental factors [13,14]. Nevertheless, there is no analysis of the molecular species of phospholipids in these works.
The excess or lack of illumination affects, primarily, the activity of photosynthetic processes. The light phases of photosynthesis occur in the thylakoid membranes; therefore, it is expected that the change in the level of illumination is reflected in the FA composition of the lipids that make up these membranes. It is known that reactions to changes in light intensity are species specific, which may be due to different levels of light sensitivity of individual species, as well as differences in the degree of intensity and time of exposure to light [15]. However, a general tendency to increase the content of polar lipids and unsaturated FAs at low light intensity and to increase the content of neutral lipids and saturated FAs (SFA) at high light intensities has been observed in most studies [16,17]. Information about the effect of light on macroalgae lipidome is very limited. Currently, only one study has been carried out at the lipidome level on the example of green macroalgae [18].
The objects of our study are filamentous endophytic algae Streblonema corymbiferum and Streblonema sp. (Ectocarpales, Phaeophyceae), isolated from Eualaria fistulosa (Laminareales, Phaeophyceae). These basiphytes are mainly distributed in the North Pacific and Bering Sea [19]. Endophytes S. corymbiferum and Streblonema sp. are poorly studied and information on their biochemical composition is almost completely absent. These algae have a high growth rate and can be cultivated under controlled conditions in photobioreactors, which makes them convenient objects for research and promising sources of bioactive substances [20]. The aim of this study was to determine the effect of temperature and light intensity on the composition of molecular species of polar lipids in S. corymbiferum and Streblonema sp. and to establish the main mechanisms of thermal and light adaptations in vitro.

Effect of Temperature on the Polar Lipidome of Streblonema corymbiferum and Streblonema sp.
The comparative analysis of the content of molecular species of polar lipids, carried out in the present work, showed a distinct dependence of the unsaturation level on temperature. Significant changes were observed both in the composition of the plastid membranes lipids (MGDG, DGDG, SQDG and PG) and in the composition of the structural components of the extraplastid membranes PC, PE and DGTS (Table S1).
The content of PUFA/PUFA and SFA/PUFA MGDG molecular species was maximum at low temperature (5 • C). With increasing temperature, the content of molecules with mo-         At low temperatures, the content of the most unsaturated DGTS molecular species (18:1/18:2) increased, the content of the less unsaturated species (18:1/18:1) was highest at medium temperatures, and the content of the least unsaturated species (16:0/18:1) was highest at high temperatures ( Figure 9). The percentage of unresolved isomers 16:0/20:1 + 18:1/18:0 was higher at low and high temperatures compared to medium temperatures.

Effect of Light Intensity on the Polar Lipidome of Streblonema corymbiferum and Streblonema sp.
Changes in the composition of polar lipid classes under the influence of light intensity were less pronounced and more complex than those under the influence of temperature (Table S2)   There were no significant changes in the SQDG composition depending on the light intensities. At high light intensities, the content of 18:2/16:0 molecular species decreased ( Figure 12).     There were no significant changes in the PHEG composition depending on the light intensity.
At low light intensities, the amount of the most unsaturated DGTS molecular species (18:1/18:2) slightly increased, while at high light intensities the level of the less unsaturated species (18:1/18:1) increased (Figure 17). At low light intensity, the sum of 16:0/20:1 and 18:1/18:0 isomers also increased.  The glycoglycerolipids MGDG, DGDG and SQDG and the phosphoglycerolipids PG, PE, PC, PI and PHEG identified in endophytes are common in brown algae. The high content of PUFA with 18 and 20 carbon atoms is also a characteristic feature of brown algae [21] (pp. 47-64). Glycoglycerolipid GlcADG and betaine lipid DGTS were found in the algae of Ectocarpales for the first time. The presence of GlcADG was previously established in higher plants [22], unicellular algae [23], sea grasses [24] and in some bacteria and fungi [25]. Previously, we identified this lipid in the brown algae Undaria pinnatifida (Laminareales) [26]. It is known that in higher plants GlcADG is synthesized in chloroplasts from UDP-glucuronic acid (UDP-GlcA) and diacylglycerol (DAG) by SQDG synthase (SQD2) [22]. In addition, the authors found that the FA composition of GlcADG and SQDG was similar, which also indicated a common biosynthetic pathway for these lipids. Our results indicate that the molecular species composition of GlcADG is similar to MGDG and DGDG. Thus, two pathways of GlcADG biosynthesis in brown algae can be suggested: (1) synthesis from DAG and UDP-GlcA by SQDG synthase, MGDG can be a possible DAG source; (2) synthesis directly from MGDG by modifying the polar head group. The betaine lipid DGTS is common in green algae, but it is generally rare in brown algae [21] (pp.118-128). Previously, another BL DGTA was found in Ectocarpales algae [27]. The major FA in DGTS composition of endophytes was 18:1. DGTS probably acts as the primary substrate for FA desaturation in the endoplasmic reticulum (ER) [28].

Effect of Temperature on the Polar Lipidome of Streblonema corymbiferum and Streblonema sp.
The ability of algae to survive changes in environmental temperature indicates the presence of mechanisms to cope with temperature stress. One of the most studied mechanisms of adaptation of organisms to changes in environmental temperature is a change in the degree of unsaturation of membrane lipids, aimed at maintaining the optimal level of membrane fluidity by regulating the activity of desaturases [29]. The obtained results indicate that the degree of contribution of different classes of lipids to the process of adaptation to low and high temperatures is different. In general, the relationship between saturation level and temperature was observed in all classes of polar lipids of endophytes.
Changes in the molecular composition of thylakoid lipids (MGDG, DGDG, SQDG, GlcADG and PG) are of the greatest interest since their function is not only to maintain the membrane structure, but they also play an important role in the photosynthetic process [30]. The FA composition of lipids is important for the stabilization of photosynthetic proteins in the membrane under thermal stress [31]. MGDG and DGDG, the main components of thylakoid membranes, showed a similar reaction in response to temperature changes. The decrease in temperature was accompanied by an increase in the content of MGDG and DGDG molecular species with extremely unsaturated fatty acids: 18:4/18:4 and 20:5/18:4.
Adaptation to low temperatures in the SQDG composition was accompanied by an increase in the content of the most unsaturated molecular species with FA 18:3: 18:3/16:0 and 14:0/18:3. At low temperatures, PG unsaturated species 18:3/18:3 and 18:3/18:4 also accumulated. PUFAs are highly structurally flexible, allowing the lipid to conform to the various forms of numerous photosynthetic proteins. An increase in the content of highly unsaturated molecular species of thylakoid lipids at low temperatures is necessary to maintain the fluidity of thylakoid membranes and, consequently, the efficiency of photosynthesis. In addition, under chilling-induced photoinhibition, the degree of unsaturation of thylakoid membrane lipids affects the rate of photosystem II (PS II) repair, during which the damaged D1 protein is replaced [32][33][34]. High temperature induced an increase in the content of less saturated species of all classes of thylakoid lipids. Thus, the number of MGDG and DGDG molecular species mainly with 18:3, 18:2 and 18:1 FAs at the sn-2 position increased, while the content of species with FA 18:4 decreased. Therefore, thermal adaptation in the composition of galactolipids occurred due to a change in the unsaturation degree of C18 FA. At high temperatures, the level of PG and SQDG molecular species with MUFA and SFA increased. These FAs reduce the fluidity of the membrane bilayer, which, in turn, ensures the efficiency of photosynthetic processes under these conditions. Phosphoglycerolipids PC, PE, PI, PHEG are structural components of extraplastid membranes. PC is the most diverse class of lipids in terms of composition, it is an intermediate in the synthesis of other phosphoglycerolipids. PC is present in the chloroplast outer envelope membrane and can be a FA donor for glycolipid synthesis [35]. It seems that 20:5 FA in the PC and PE composition plays the key role in maintaining the required level of membrane fluidity at low temperatures. With an increase in temperature, an increase in the PC and PE molecular species containing SFA and MUFA, which ensures a decrease in the molecular mobility of the lipid bilayer, should lead to the resistance of cells to high temperatures. The ratio of 20:5 and 20:4 FAs in PHEG, as well as in PC and PE, varied depending on the temperature.
A slight change in the content of DGTS molecular species indicates the secondary participation of this lipid in the processes of temperature adaptation. However, the dependence of the unsaturation degree of this lipid on temperature was also observed. Thus, at low temperatures, the 18:1/18:1 major molecular species desaturates at the sn-2 position to form 18:1/18:2. Under the influence of high temperatures, the activity of desaturases decreased.
It is known that plants are capable of regulating the FA unsaturation level by changing the degree of contribution of the prokaryotic and eukaryotic pathways in lipid synthesis [36]. In plant cells, DAG assembly occurs in both the ER (eukaryotic pathway) and chloroplasts (prokaryotic pathway), while de novo FA synthesis only occurs in chloroplasts. For DAG synthesis in the ER, free FAs are transported from chloroplasts presumably by the FAX 1 protein [37]. Due to the substrate specificity of lysophosphatidic acyltransferases, plastidderived DAGs contain C16 FA at the sn-2 position, while ER-derived DAGs contain C18 FA. DAGs synthesized in the ER can be transported into the chloroplast envelope by the TGD1-5 protein complex and used for the synthesis of thylakoid lipids [38]. Synthesis of C20 FA in algae occurs in the ER, then they can be transported to chloroplasts [39].
MGDG and DGDG S. corymbiferum and Streblonema sp. were predominantly of ER origin, since most molecular species contained C18 and C20 FA at the sn-2 position (96.0% and 98.1% at 15 • C, respectively). A decrease in temperature had almost no effect on the ratio of eukaryotic and prokaryotic galactolipids ( Figure 18). With an increase in temperature, the contribution of the prokaryotic pathway to the biosynthesis of MGDG and DGDG slightly increased. SQDG is the only lipid of S. corymbiferum and Streblonema sp. which is synthesized predominantly in chloroplasts (90.0% and 92.8% at 15 • C, respectively). The change in temperature did not influence the origin of this lipid. At low temperatures, the level of the ER-derived PG increased, which may be due to the limited FA desaturation in chloroplasts. Thus, in chloroplasts, the maximum number of double bonds that can be formed in an FA chain at sn-2 position is one, and in ER it is five. An increase in the content of ER-derived PG at low temperatures was previously described in wheat [36]. At high temperatures, the amount of chloroplast-derived PG increased, mainly through 18:3/16:0 molecular species; however, at the same time, the level of ER-derived PG with 16:0, 18:1 and 18:2 FA also increased. This also indicates the involvement of both biosynthetic pathways in the process of adaptation to heat stress. As a result of the analysis, plastid-derived molecular species of extraplastid lipids, such as PC, PE and DGTS, were found. The presence of such lipids suggests the possibility of DAG transport for their synthesis from chloroplasts. The amount of PC and PE chloroplastderived molecular species was minor (less than 1.5%) and did not change under the influence of temperature, while the amount of DGTS chloroplast-derived molecular species did not exceed 10% of the sum of all molecular species.

Effect of Light Intensity on the Polar Lipidome of Streblonema corymbiferum and Streblonema sp.
Light is a critical environmental factor affecting algae metabolism. Due to the lack of solar energy, the efficiency of photosynthesis can decrease, which causes the development of algae to slow down [40]. Light stimulates photosynthesis, but with excessive light intensity, an imbalance between the absorbed energy and the ability to use it can occur, resulting in oxidative stress, accompanied by the destruction of chloroplasts [41]. To maintain photosynthetic activity at an optimal level and protect photosystems, various adaptation mechanisms have been developed in algae. Modifications in the lipid composition of endophytes that occur with changes in light intensity are probably also adaptive responses. At low light intensity, an increase in the content of the most unsaturated MGDG species, containing predominantly 20:5 and 18:4 FA, may be necessary to increase membrane fluidity and, consequently, the rate of photosystem electron transport [42]. Previously, it was shown that the accumulation of 20:5 n-3 in MGDG under low light conditions in U. pinnatifida is accompanied by an increase in the chlorophyll content, which together leads to an increase in photosynthetic activity [43]. In addition, FA desaturation of plastid lipids induces a change in lipid-protein interactions, which, in turn, affects the self-assembly of active chlorophyll-protein complexes [44]. At low light intensity, the amount of DGDG containing 18:3 FA at the sn-1 position also increased. At high light intensity, an increase in some highly unsaturated molecular species of MGDG and DGDG may be related to both the structural and protective function of these lipids. It is known that MGDG is involved in the xanthophyll cycle, a key mechanism for protecting the photosynthetic apparatus of plants from damage caused by excessive light exposure. The presence of highly unsaturated non-bilayer MGDG is a necessary condition for carotenoid solubilization and activation of violaxanthin de-epoxidase, which catalyzes the formation of the photoprotector zeaxanthin [45]. Previously, it was shown that PSII repair is inhibited in DGDG-deficient mutants under high light conditions compared to the wild type [46]. It is likely that highly unsaturated DGDG (20:5/18:4 and 20:5/18:3) may be necessary to maintain the rate of resynthesis of the D1 protein of PSII. An increase in the unsaturation level of galactolipids may also be associated with the PUFA ability to neutralize reactive oxygen species and thus perform a protective function [47]. At low and high illuminations, the content of the 18:3/16:1 molecular species increased in the PG composition. We suggest that 16:1 FA at the sn-2 position is a 16:1∆3-trans PG-specific FA, inducing an increase in the light-harvesting complex II (LHCII) trimerization [48]. Under low levels of illumination, this may be necessary to increase the light-harvesting ability of PSII. The increase in the number of LHCII trimers at high light intensity is probably associated with a photoprotective function.
Changes in light intensity also affected the composition of extraplastid lipids. Low light intensity induced an increase in the content of the 18:1/16:1 PE molecular species, indicating increased transport of DAG from chloroplasts to the ER for the synthesis of this lipid. This was especially noticeable in Streblonema sp., where the content of this species was 0.3% at the maximum level of illumination and 26.3% in the absence of light. It is also interesting that in other classes of polar lipids, the 18:1/16:1 species occur in trace amounts.
At high light intensity, an increase in the content of molecular species with C20 PUFA in PC and PE was noted, which primarily indicates an increase in FA synthesis and can also be associated with the antioxidant function of PUFA.
In DGTS, with increasing light intensity, the amount of 18:1 FA increases (approximately from 60 to 80% of all DGTS acyl groups in both endophytes), and the amount of the 18:1/18:1 DGTS increases from 40 to 60-70%. This most likely indicates an increase in de novo FA synthesis and confirms the role of DGTS as a substrate for the primary FA extraplastid desaturation. The amount of 18:1 FA and the 16:0/18:1 species also increases in another extraplastid lipid PI.

Algae material
S. corymbiferum. and Streblonema sp. were isolated from a culture of gametophyte clones of Eualaria fistulosa (Laminareales, Phaeophyceae) (Bering Island). Basiphyte (host algae) thalli fragments containing endophytes were placed in Petri dishes with sterilized (ultraviolet, 0.2 µm filtration, boiling) seawater enriched with ES medium [49] and kept at a temperature of 15 ± 1 • C, a medium light intensity of 10 µmol photons m −2 s −1 and a photoperiod of 12 h light:12 h dark for spore release. After 2 days, the basiphyte fragments were removed. The filaments of endophytes were removed from culture and grown separately in sterilized (ultraviolet, 0.2 µm filtration, boiling) natural seawater fertilized with an ES medium [49]. The cultures were illuminated with cool light fluorescence lamps (Phillips 39 W) that provided a medium light intensity of 25-30 µmol photons m −2 s −1 . The photoperiod was 12 h of light and 12 h of dark.

Temperature Treatment
Samples were grown in 200 mL flasks with sterile seawater enriched with ES medium [49] for three weeks at temperatures of 5, 10, 15, 20 and 25 • C, a medium light intensity of 200 µmol photons m −2 s −1 and a photoperiod of 12 h light:12 h dark. Three samples of each endophyte species were cultivated in each temperature regime.

Light Treatments
Samples were grown in 200 mL flasks with sterile seawater enriched with ES medium [49] for three weeks at light intensities of 0, 5, 12, 20, 50, 100, 150 and 200 µmol photons m −2 s −1 , temperature of 14-16 • C and a photoperiod of 12 h light:12 h dark. Three samples of each endophyte species were cultivated in each light regime.

Lipid Extraction
Samples of endophytes were collected on filter paper, dried, weighed and transferred to glass tube. Lipids were extracted using the mixture of chloroform: methanol (1:1). The organic phase was collected in pear-shaped flasks using filter paper. The biomass was re-extracted six times. The final organic phase was dried by rotary evaporator, transferred to glass vials, dried, weighed and stored in chloroform at −20 • C. Quantitative analyses of molecular species and their identification using a fragmentation pattern were performed on a Shimadzu LCMS-8060 triple-quadrupole mass spectrometer (Kyoto, Japan) with electrospray ionization (ESI) ion source. The temperature of the interface, heat block and desolvation line was 300, 400 and 250 • C, respectively. The flow rates of drying gas (N 2 ), nebulizer gas (N 2 ) and heating gas (zero air) were 10 L min −1 , 3 L min −1 and 10 L min −1 , respectively. The negative ion mode was applied for quantitative analysis of PI and the positive mode was applied for analyzing others lipid classes. Previously described fragmentation reactions were used for sn-position of acyl chains determination in all polar lipid classes [56][57][58][59][60], except GlcADG and PHEG. For the identification of MGDG, DGDG and PC, [M + Li] + ions were used, in this case 5mM LiOH in methanol with 0.02 mL/min flow was added postcolumn. Mass spectrometer parameters (precursor and fragment ions, collision energy and type of registered fragmentation reaction) for qualitative and quantitative analyses of each class of polar lipids are given in Table S3. MS/MS spectra and fragmentation reactions are shown in Figure S2.

Statistical Analysis
All statistical analyses were performed using Microsoft Excel (Microsoft, Redmond, WA, USA). All values were presented as mean ± standard deviation for triplicate. The data were assessed statistically by one-way ANOVA and Tukey HSD test for a posteriori comparisons. A probability level of p < 0.05 was considered significant.

Conclusions
The obtained results indicate that changes in temperature affected the composition of all classes of lipids to varying degrees. At low temperatures, the content of extremely unsaturated (possible for the particular polar lipid class) molecular species of lipids (MGDG and DGDG with 18:4 and 20:5, SQDG with 18:3, PG with 18:3 and 18:4, PC, PE and PHEG with 20:5, PI and DGTS with 18:2) increased. At high temperatures, the amount of these molecular species decreased, while the content of other (less unsaturated) species increased. Such modifications are necessary to maintain the fluidity of cell membranes at an optimal level. Temperature had the least significant effect on the composition of PI and PHEG.
With changes in illumination, the modifications of the plastid lipid composition are mainly aimed at maintaining the function of the photosynthetic apparatus. Light intensity had a significant effect on the composition of plastid membrane lipids, with the exception of SQDG. Both low and high light intensities induced an increase of 18:3/16:1 PG amount. With increasing light intensity, the amount of C20-PUFAs (especially 20:5) in MGDG and DGDG increased, which is associated with the role of these lipids in the photosynthetic apparatus of chloroplasts. The change in light intensity had little effect on the SQDG molecular composition; apparently, this lipid is not involved in light adaptation. Changes in the extraplastid lipid composition are probably the result of increased de novo FA synthesis, which leads to the accumulation of 18:1 FA in PI and DGTS and the end products of FA synthesis in brown algae-C20-PUFA in PC and PE.
The study of the contribution of pro-and eukaryotic pathways of synthesis revealed an increase in the level of plastid-derived molecular species in MGDG, DGDG and PG at high cultivation temperatures. This additional mechanism allows us to regulate the level of unsaturated lipids, which is presumably related to the peculiarities of desaturation in ER and plastids. A significant increase in plastid-derived PE 18:1/16:1 under low light conditions indicates the presence of a DAG transport pathway from chloroplasts. In general, the high level of unsaturation of most classes of lipids under optimal conditions indicates the cold and light resistance of endophytes. On the other hand, this may indicate an increased sensitivity to high temperatures [61].
It is also worth noting that despite the close relationship of the two species of endophytes, the quantitative composition of some molecular types of lipids and adaptation mechanisms differed in them.
Thus, the analysis of the polar lipidome, including the separation of isobaric molecular species and the determination of the sn-position of acyl chains, made it possible to obtain a complete picture of the adaptive changes that occur in brown endophytic algae during cultivation under various temperature and illumination conditions. Supplementary Materials: The following supporting information can be downloaded at https: //www.mdpi.com/article/10.3390/md20070428/s1.  Table S3: MS parameters for polar lipid molecular species quantification and identification. Figure S1: Identification of the endophytic algae Streblonema corymbiferum and Streblonema sp. Figure

Conflicts of Interest:
The authors declare no conflict of interest.