The green microalgae C. reinhardtii wild-type (WT) strain CC124 (137c) was obtained from the C. reinhardtii resource center (University of Minnesota) and mutants pgrl1 and pgr5 (137c background; Johnson et al., 2014), a kind of gift from Prof. Gilles Peltier (CEA-CNRS- Aix Marseille University, France) and Prof. Michael Hippler (University of Munster, Germany). Cells were grown photoheterotrophically in Tris-acetate phosphate (TAP) medium at 25°C with a photon flux density of 50 μmol photons m^–2 s^–1 in 250 mL conical flask shaken at 120 rpm in an orbital shaker at 25°C. The seed cultures of C. reinhardtii (WT, prgl1, and prg5) were harvested at an optical density (OD) of 0.8 and inoculated at OD of 0.02 for all the cultures. The cultures were cultivated photoheterotrophically at 25°C for 4 days at a continuous light intensity of 50 and 500 μmol photons m^–2 s^–1. Later the cultures were collected after the 3rd day with an OD of 0.8 at 750 nm. Light intensity was measured and adjusted with a light meter (Hansatech).

To calculate the total biomass of cells (WT and mutants pgrl1 and pgr5), 5 mL of culture was collected after the 3rd day of growth in high light and control by centrifugation at 1,000 × g for 10 min at room temperature (RT), and the supernatant was discarded. The excess media was removed by short centrifugation, and cell pellets were lyophilized at −109°C for 12 h. The cell pellet was washed with distilled water and transferred into a pre-weighed 15 mL falcon tube. The total dry weight was later quantified by subtracting the empty tube.

The total ROS was detected with 2,7-dichlorodihydrofluorescein diacetate (H₂DCFDA) (Sigma-Aldrich), a fluorescence dye used to see the ROS in live C. reinhardtii cells. Cells at a 3 million density were collected from all the conditions, and H₂DCFDA staining was performed at 10 μM concentration (Upadhyaya et al., 2020). Further, cells were incubated with dye for 1 h at RT in a continuously rotating shaker under dark. Images were captured using Carl Zeiss NL0 710 Confocal microscope. H₂DCFDA was detected in a 500–530 nm bandpass optical filter with an excitation wavelength of 492 nm and an emission wavelength of 525 nm. Chlorophyll auto-fluorescence was detected using an optical filter of 600 nm. Samples were viewed with a 60× oil immersion lens objective by using the ZEN, 2010 software.

Total ROS produced by C. reinhardtii cells was also quantified by spectrophotometer. Logarithmic-phase cells were centrifuged at 1,500 × g, 20°C for 5 min, resuspended in an equal volume of fresh TAP medium, and then incubated in 5 μM DCFH-DA for 1 h in the dark at 25°C. After dark exposure, the cells were washed twice with TAP to remove excess dye (Devadasu et al., 2019). The fluorescence was measured using a Microplate reader (Tecan M250) at an excitation of 485 nm and an emission of 530 nm. We calculated the total ROS by subtracting the value of fluorescence with dye and without dye.

The cells (3 × 10⁶ cells/ml) were resuspended in 0.1 M phosphate buffer (pH 7.2). Cells were fixed in a 2% Glutaraldehyde (Sigma-Aldrich, St. Louis, United States) solution for 2 h in the dark. The cells were then washed four times in PBS buffer for 1 h each time before being post-fixed in 2% aqueous Osmium Tetraoxide for 3 h, washed six times in deionized distilled water, dehydrated in a series of ethanol solutions (30, 50, 70, and 90%, and three changes of 100% for 10 min each), infiltrated, and embedded in Araldite resin. Ultra-thin sections (60 nm) were cut with a glass ultramicrotome (Leica Ultra cut UCT-GA-D/E-1/100) and placed on copper grids after incubation at 80°C for 72 h for complete polymerization. The sections were stained with uranyl acetate and counterstained with Reynolds lead citrate, with some alterations (Bozzola and Russell, 1998). A Hitachi 7500 (Japan) transmission electron microscope was used to take the images.

For immunofluorescence, cells (3 × 10⁶ cells/ml) were fixed in a solution of 4% paraformaldehyde and 15% sucrose dissolved in phosphate saline buffer (PBS) for 1 h at RT Later, the cells were washed twice with PBS buffer. First, cells were permeabilized by incubation in 0.01% Triton X100 in PBS for 5 min at RT and washed twice in PBS. Next, the samples were transferred to sterile Eppendorf tubes and blocked with a 1% BSA (w/v) in PBS for 1 h. Samples were incubated with anti- ATG8 diluted (1:1000) in PBS buffer, pH = 7.2 containing 1% BSA overnight at 4°C on a rotatory shaker. Cells were then washed twice with PBS for 10 min at 25°C, followed by incubation in a 1:10000 dilution of the fluorescein Dylight 405 labeled goat anti-rabbit secondary antibody (Sigma) in PBS-BSA for 2 h at 25°C. Finally, cells were washed three times with PBS for 5 min. Images were captured with Carl Zeiss NL0 710 Confocal microscope. ATG8 localization was detected with excitation of Dylight 405 nm and emission at 420 nm by analyzing the images with ZEN software.

We studied the impact of high light on non-polar lipid accumulation with confocal microscopy. Cells (3 × 10⁶ cells/ml) were collected after the 3rd-day growth in high light along with control. To stop the mobility of the cells, 5 μL Iodine solution [0.25 g in ethanol (95%)] was mixed and kept for 5 min under dark at RT. The cell suspension was then stained with Nile Red (NR, 1 μg mL^–1 final concentration, Sigma, Sigma-Aldrich) followed by 20 min incubation in the dark. After staining, the samples were placed on a glass slide with a coverslip. Images were captured using Carl Zeiss NL0 710 confocal microscope. A 488 nm scanning laser was used with a 560–615 nm filter to detect neutral lipids. Samples were viewed with a 60× oil immersion lens objective, and the ZEN, 2010 software package was used for image analysis.

The C. reinhardtii cultures were diluted to a density of 3.0 × 10⁶ cells mL^–1 and placed within 96 well microplate wells. To every well, a 5 μL aliquot of 50 μg mL^–1 NR; (Sigma; ready in acetone) was added in 200 μl of equal density cells (3.0 × 10⁶ cells mL^–1), and after a thorough mix, the plate was incubated in the dark for 20 min. The neutral lipids were then measured by visible light at a 485-nm excitation filter and a 595-nm emission filter employing a plate reader (Tecan infinite M200, Magellan). Quantification was achieved by using a standard curve ready with the lipid Triolein (Sigma T4792).

Flow cytometer Calibur (BD Falcon, United States) was use to quantify neutral lipid stain with NileRed. Cells were stained with 1 μg mL^–1 NR (100 μg mL^–1 stock in acetone) in the dark for 30 min before flow cytometry analysis. Measurement of 10,000 cells per sample was analyzed without gating and auto-fluorescence was nullified before measuring the NR fluorescence. The fluorescence reading was obtained at a 488-nm excitation filter and a 545-nm emission. The distribution of the cells stained with NR was expressed as a percent of the total.

C. reinhardtii cells were harvested by centrifugation (1,000 × g for 10 min) after the 3rd day at a cell density of 3.0 × 10⁶. Cell pellets were resuspended in methanol for chlorophyll extractions. The supernatant was used to measure the total chlorophyll (Porra et al., 1989). To calculate total carotenoid cell pellets were resuspended in 80% acetone, and the supernatant was used to measure the total carotenoid. Absorbance was measured at wavelength 480, 663, and 645 nm. Total carotenoid was calculated as = A480 + 0.114 (A663) − 0.638(A645) (Wellburn, 1952). Total carbohydrate was quantified by an Anthrone method (plate reader assay). For quantification of carbohydrate, the cell pellet was resuspended in 50 μL Milli-Q water and 150 μL of Anthrone chemical agent (0.1 g in 100 mL of conc. H₂SO₄ 98%) was added to the 50 μL algal cells. Initially, plates were incubated at 4°C for 10 min and then incubated at 100°C for 20 min. Later on, plates were placed at RT until color development. Plates were measured at 620 nm on an assay plate reader (Tecan M250), and total carbohydrate was calculated (Wase et al., 2019).

The 5 mg lyophilized sample was dissolved in 1,000 μL of acetonitrile (ACN), 0.1% formic acid (FA) (v/v), and vortexed for 1 h at 900 rpm at RT. The solution was then centrifuged at 13,000 rpm for 10 min at 4°C. 100 μL of supernatant was collected, and the pellet was dried using a speed vacuum. The pellet was dissolved in 90 μL of acetonitrile 0.1% formic acid (v/v) and spiked with 10 μL of the standard internal mixture (Table 1). The solution was vortexed for 30 min at 750 rpm at RT and speed vacuumed to dry at 40°C. The extracted lipids were derivatized with n-Butanol for 20 min at 60°C, then again speed vacuumed to dry at 60°C for 15 min. The dried pellets were dissolved in 100 μL of ACN 0.1% FA and vortexed for 5 min at 750 rpm on a thermomixer. The samples were then centrifuged at 13,000 rpm for 10 min at RT. The supernatant was taken into HPLC sample vials for further analysis. The 100 μL extracted lipid samples were brought to RT and filled into individual HPLC vials with 200 μL inserts and placed into the autosampler of Nexera X2 UFLC, connected to LC-MS/MS (SHIMADZU 8045) MS system with an ESI source. The data was analyzed by Lab solutions software, and the concentrations were calculated by measuring the area under the curve (AUC) for the internal standards.

Cells were collected by centrifugation. The cell pellet was solubilized in protein extraction buffer (0.1 M DDT, 4% SDS, and 0.1 M Tris, pH- 7.6), and all the samples were kept for heating at 95°C for 5 min. Cell suspensions were centrifuged at 2,810 × g for 10 min, and the supernatant was collected. Protein concentration was quantified using the Bradford method, and 5 μg of proteins were loaded per well. The individual proteins were separated with 15% Bis-Tris gels for diacylglycerol acyltransferases (DGAT2A) and PDAT1 proteins and 12% for ATG8 and Histone3 (H3) proteins. Separated proteins were then transferred to a nitrocellulose membrane (Towbin et al., 1979). Specific primary antibodies were purchased from Agrisera (Sweden) and used at dilutions of DGAT2A (1:2500), PDAT (1:2500), and ATG8 (1:2500). H3 (1:10,000) was used to see equal loading. The secondary antibody, anti-IgG raised from Goat (1:10,000), was used for detection. Immunoblots were obtained by the Chemi-Doc touch imaging system (Bio-Rad) by using Chemiluminescence.

All experiments were conducted in triplicate, and data are obtained as the mean ± standard deviation (SD). Statistical analysis was done using Sigma plot 14.5 software. One-way ANOVA and subsequent Tukey Test were used to analyze the statistical significance (*p < 0.05, ^**p < 0.01, ^***p < 0.001) of the data.

The WT, proton gradient regulation (pgrl)1 and pgr5 (cyclic electron transport dependant mutants) were grown in Tris-acetate phosphate (TAP) medium at light intensities of 50 (as a control) and 500 μmol photon m^–2 s^–1 for 4 days. Under 50 μmol photon m^–2 s^–1 light intensity, all the cultures grew normally (Figure 1). The growth of WT cultures increased (Figure 1A), but pgrl1 and prg5 mutants exhibited slower growth in high light conditions after 2 days (Figures 1A–C). Our results showed that the cell growth pattern of the WT was increased 30%, whereas pgrl1 and pgr5 growth were decreased 21 and 30% compared to the WT under high light (500 μmol photon m^–2 s^–1) after 3rd day and normalized OD data was shown in Supplementary Table 1. Mainly, the pgr5 mutant shows impaired growth, whereas pgrl1 was not affected under the same high light condition (500 μmol photon m^–2 s^–1) after 3rd day of growth (Figures 1B,C). We have studied the effect of light intensity on biomass. The WT shows an increase in biomass yield 75.5% in high light, however, it reduced 64.6 and 43.6% in pgrl1 and pgr5, respectively, after the 3rd day (Figure 1D). However, after the 4th day, biomass is almost the same in both mutants when grown in high light.

To determine the effect of high light on the pigment level associated with C. reinhardtii photosynthetic complex, we have measured the chlorophyll and carotenoid content from standard and high light-grown cells. Chlorophyll content was significantly reduced in pgrl1 and pgr5 mutants, respectively, under high light intensity (Figure 2A). At the same time, we observed twofold increase in carotenoid content in pgrl1 and pgr5 under high light (Figure 2B). It is known that increased carotenoid content acts as a protective mechanism against light stress in the C. reinhardtii (Li et al., 2009). Carotenoids under high light conditions generally tend to increase to protect PSII from photoinhibition. Data shows that an increase in light intensity results in decreased specific growth rate, increased carotenoid, and reduced biomass production of pgrl1 and pgr5. We assume that reduced growth might be due to ROS production. Our results have elicited us to check further studies and detect the ROS in high light stress.

To determine the oxidative stress, cells were stained with 2′,7′-dichlorodihydrofluorescein diacetate (H₂DCFDA). ROS was observed using confocal microscopy as well as quantified by spectrophotometry (Figures 3A,C), where an increase in ROS was observed under the high light condition in WT, prgl1, and pgr5 (Figure 3). However, this increase was predominant in pgr5 (Figures 3A–C). The generation of more ROS in pgr5 could probably explain the reduced growth rate. Further, these results suggest that ROS formation under high light may contribute to autophagy and lipid accumulation. Reports show that ROS may induce multiple metabolic functions like lipid metabolism and autophagy (Shi et al., 2017). High light is one of the factors responsible for ROS formation because of over reduction of the photosynthetic electron transport chain, which might generate ROS that induces autophagy in C. reinhardtii (Pérez-Pérez et al., 2012). Based on the above reports, we have conducted experiments related to autophagy caused by high light.

We have used an anti -ATG8 antibody to examine this protein’s cellular localization in immunofluorescence microscopy (Figure 4A). Autophagy (ATG)8 proteins localize as small red dots in the cytoplasm under the normal light condition in WT, pgrl1, and pgr5 mutant. However, under high light conditions, number of spot significantly increased in pgr5 (40%) (Supplementary Figure 1), which shows activation of autophagy and formation of autophagosomes in close agreement with high accumulation of ATG8 [phosphatidylethanolamine (PE) modified ATG8] detected by western blotting. The autophagy-related protein ATG8-PE was significantly expressed in pgr5 in high light conditions (Figures 4B,C). Upon autophagy activation, ATG8 binds to the autophagosome membrane through phospholipid phosphatidylethanolamine (PE) to form ATG8-PE in a process called lipidation. Hence autophagy may play a significant role in the accumulation of lipids in high light conditions in pgr5. Therefore, in line with autophagy, the accumulation of lipids is lower in WT and pgrl1. However, the ATG8 expression is predominant in pgr5 under high light conditions. These results suggested that induction of ROS due to high light may induce autophagy in pgr5 than WT and pgrl1 (Pérez-Pérez et al., 2012; Perez-Martin et al., 2014). Therefore, it may trigger the lipid metabolism in C. reinhardtii cells.

We have stained the cells with Nile red (NR) for lipid studies, binds explicitly to lipid bodies in the cell, and gives a characteristic yellow-orange fluorescence. Our results clearly show the overall increase of lipid droplets under high light in WT, pgrl1, and pgr5 mutants (Figures 5A,B and Supplementary Figure 2). When we measure the fluorescence intensity from the confocal images, the number of lipid spots is higher in high light-grown cells, but it is more significant in pgr5 (Figure 5C). However, autofluorescence due to chlorophyll was less in pgrl1 and pgr5 than in control, indicating decreased chlorophyll content under high light, even though cells had a significant accumulation of lipid bodies (Figure 5A). Lipid accumulation was observed in all strains under high light conditions (Goold et al., 2016). However, lipid accumulation is more significant in pgr5 (60%) than other strains under high light. We have tested NR fluorescence from fluorescent activated cell sorter (FACS) to assess the lipid accumulation in high light conditions. We obtained twofold higher mean fluorescence intensity from all the strains with high light than normal growth conditions (Figure 5D).

The neutral lipids were semi-quantified by using NR fluorescence from an equal number of cells. NR fluorescence results show that the neutral lipid level was increased with equal cell density. NR fluorescence of C. reinhardtii cells WT, pgrl1, and pgr5 were calculated every 24 h while growing them in light at 50 and 500 μmol photon m^–2 s^–1 (Supplementary Figure 3). After 24 h, cells did not cause any change in total lipid content. However, after 48 h of high light growth, there was an increase in NR fluorescence.

In contrast, there is an increase in signal at 750 nm. Overall, high light-grown cells exhibit a relative two-fold increase in neutral lipid content over 3–4 days. However, this accumulation of neutral lipids is predominantly more in the pgr5 mutant when compared to other strains. Therefore, our results indicate that an increase in light intensity facilitates the production of neutral lipids in C. reinhardtii. Further, we have performed electron microscopic studies to determine whether high light induces lipid accumulation in the cells.

To see the clear lipid vacuoles in the cell, we have carried out transmission electron microscopy (TEM). It reveals that pgrl1 and pgr5 exhibit a large cytoplasmic vesicle under high light-grown cells (Figures 6A–C and Supplementary Figure 4). It showed that the membrane structure is aberrant under high light, especially the pgrl1 and pgr5 mutants. The majority of the thylakoid membranes were loosen in high light-grown cells. While in the WT cells, the thylakoid membranes are arranged as layers (stacked) in the chloroplast, with some membrane appressed. Recently we reported that photosynthetic efficiency is reduced under the high light condition in WT, pgrl1, and pgr5 mutants, which may be due to a change in chloroplast structure (Yadav et al., 2020). Such phenotype raised the question that mutant strains are defective in generating the functional membrane under high light conditions. The earlier report emphasized that autophagic bodies accumulate in plant cell vacuoles under Concanamycin A treatment since vacuolar hydrolase cannot act (Thompson et al., 2005). However, in our case, under high light conditions, we have observed a high degree of vacuolization and an increase of vacuole size in mutants (Figures 6A,B and Supplementary Figure 4). It was also being reported that vacuole lytic function is needed to synthesize TAG and lipid bodies in C. reinhardtii cells when subjected to light stress (Couso et al., 2018).

Carbohydrate is a polysaccharide, and it is the storage form of sugars and starch in plants/algae. We quantified the carbohydrate content in WT, pgrl1, and pgr5 strains in normal and high light conditions. The carbohydrate content of WT and pgrl1 was increased significantly under the high light condition. The carbohydrate content was cross-checked with a starch deficient mutant (sta6) under normal light and high light. It shows about 8 μg of carbohydrate/10⁶ cells in standard and high light conditions (Figure 7) as measured with the anthrone reagent, which estimates starch and soluble sugars. Overall, our reports indicate that carbohydrate content has increased under the high light condition in WT and pgrl1. However, it reduced in pgr5 mutant under high light. Microalgae change their metabolism and convert excess energy into an energy-rich product such as lipid, starch, carbohydrate, or proteins under unfavorable conditions (Heredia-Arroyo et al., 2010). However, pgr5 may have an altered NADPH and ATP ratio during photosynthesis, switching metabolic pathways that lead to lipid accumulation. These results indicate that high light induces the lipid pathways due to more energy equivalents in the pgr5 mutant. Further, we have tested the protein/enzyme of the lipid pathway from all the conditions.

We have focused on the enzymes involved in the triacylglycerol (TAG) synthesis known as the Kennedy pathway. This pathway is mainly catalyzed by the acylation of diacylglycerols (DAGs) by diacylglycerol acyltransferases (DGAT), in which acyl-CoA is a substrate to form TAG. Acylation of diacylglycerols (DAGs) by phospholipid: diacylglycerol acyltransferase 1 (PDAT1) enzyme is the acyl-CoA independent pathway in the ER. To determine the involvement of PDAT and DGAT in high light response in C. reinhardtii and their protein level regulation was calculated (Figures 8A–C). The PDAT and DGAT were transiently upregulated in response to high light conditions. Protein expression was increased after the 3rd day, specifically in pgr5 expression of PDAT compared to pgrl1 (Figure 8A). Our results confirm an increase in TAG in pgrl1 and pgr5 due to the enhanced content of PDAT and DGAT enzymes.

Liquid chromatography/mass spectrometry (LC/MS) was used to analyze the fatty acids transesterification and the resultant fatty acid methyl esters (FAMEs) from control strains WT, and mutants pgrl1 and pgr5 under normal (50 μmol photons m^–2 s^–1) and high light (500 μmol photons m^–2 s^–1) cells (Table 1). Different compositions of fatty acids are observed in the high light grown C. reinhardtii. There was an increase in C16:1, C16:2, C16:3, C18:0, C18:1, and C18:3 in pgrl1 and pgr5 mutant under high light conditions. We recently observed increasing total fatty acids in severe Fe deficiency from C. reinhardtii (Devadasu et al., 2019; Devadasu and Subramanyam, 2021). In our case also the total fatty acid content is significantly increased in pgrl1 and pgr5 mutant strains. Overall saturated fatty acid increases from 6 to 10%, 13 to 18%, and 12 to 37% in WT, pgrl1, and pgr5 mutant, respectively, and monounsaturated fatty acid increases from 5 to 15%, 10 to 20%, and 12% to 38% in WT, pgrl1, and pgr5 mutant, respectively. However, there are also changes in total polyunsaturated fatty acids from 6 to 23%, 11 to 16%, and 17 to 27%. This result implies that high light increases SFA and MUFA in WT, pgrl1, and pgr5 mutant strains, but specifically, the SFA and MUFA increased significantly in pgr5.

To examine the fatty acid formed under high light, we studied the changes in neutral lipid, especially TAG and membrane lipid content and composition. We prepared neutral lipid extract from equal dry weights of WT, pgrl1, and pgr5 cells under 50 and 500 μmol photon m^–2 s^–1. Further, the extracted lipids were separated on TLC plates from the 3rd day of culture. This was stained with iodine, and results revealed that accumulation of more TAG was observed in pgr5 compared to pgrl1 and WT (Supplementary Figure 5). To quantify the TAG, the TLC bands were recovered, and FAMES quantifies TAG content through of GC (Figure 9). TAG concentration in WT and pgrl1 increased from 9 to 18%; however, pgr5 significantly increased from 11 to 34% (Figure 9D). The fatty acid composition of TAG bands, 16:0, was raised in all strains under high light. However, the level of 18:0 was increased significantly in pgrl1 and pgr5 mutants. Further, 18:1ω9c majorly increased in pgr5 mutant under high light (Figures 9A,D). The accumulation of a significant amount of TAG in pgrl1 and pgr5 under high light may suggest the role of membrane lipid in TAG synthesis (Fan et al., 2011).

To assess all the membrane lipids of C. reinhardtii, TLC is carried out. In WT, we observed not much change in chloroplast-specific lipid monogalactosyldiacylglycerol (MGDG), but chloroplast lipid digalactosyldiacylglycerol (DGDG) was marginally increased. However, in pgr5 mutant, MGDG and DGDG content reduced significantly under high light conditions (Supplementary Figure 5). Further GC quantification confirmed that the MGDG and DGDG were significantly decreased in high light (Figures 9B,D). The fatty acid composition shows that DGDG 18:1 and 18:1ω9c increased in pgrl1 mutant while decreased in pgr5 mutant; however, not much change was observed in WT (Figures 9C,D). Further, the MGDG is enriched with C16:0, C18:1 ω9c, C18:3, and C18:0. Not much change was seen in the fatty acid composition of MGDG in WT and pgrl1 but significantly decreased in pgr5 under high light (Figures 9B,D). We assume that the reduced amounts of MGDG and DGDG are metabolized to TAG synthesis in pgr5, and a similar report was also observed from other studies (Li et al., 2008; Devadasu and Subramanyam, 2021). The TLC and the GC data of membrane lipids support that the electron microscopic data that the stacks of thylakoids disturbed because of change in membrane lipids. Thus fatty acids from the chloroplasts and other intracellular membrane systems may have converted into TAG.

Light is an essential factor in the production of biomass. Half of the microalgae species’ dry weight is carbon and lipid (Zhu, 2015). This study demonstrates that high light intensity (500 μmol photons m^–2 s^–1) enhanced the growth rate, promoted biomass and lipid accumulation of C. reinhardtii compared to regular light intensities in WT. However, growth and biomass were reduced in mutant (Figures 1B–D). PGRL1 and PGR5 proteins are essential in the acclimation process to high light. Thus, the growth curves show that the absence of these leads to less biomass and growth (Figure 1). In addition, the light stress response has been previously studied and shows the importance of light intensity in producing biomass and fatty acids in microalgae (Nzayisenga et al., 2020).

On the other hand, when microalgal cells were grown in high light, PSII undergoes severe damage. Hence photosynthetic electron transport chain induces the ROS (Li et al., 2009). Antioxidants usually accumulate in cells like carotenoids to repair the damage caused by ROS (Perez-Martin et al., 2014). Similarly, we also observed in our results that the total carotenoid content increased (Figure 2B). Interestingly, the ROS generation was much higher in pgr5 because the lack of this gene led to acceptor side limitations as PGR5 is involved in protecting PSI. Thus, PSI’s acceptor side is limited in pgr5. Because of acceptor side limitation, one could expect more ROS generation at the PSI acceptor side (Tiwari et al., 2016), suggesting that lipid synthesis could serve as a receptor to excess electrons to acclimatize to the abiotic stress. As already reported, oxidative stress causes lipid accumulation under nitrogen stress in microalgae (Yilancioglu et al., 2014).

Transmission electron microscopy (TEM) images of C. reinhardtii showed a pronounced increase in vacuoles’ size under high light intensity, particularly in mutant strains (Figure 6). Accumulated autophagy bodies can play an essential role in lipid metabolism. A similar report was observed in nutrient limitations like nitrogen limiting conditions in C. reinhardtii (Couso et al., 2016). It has been reported that in stress conditions (like oxidative stress, rapamycin stress, and ER stress), the ATG8 gene was expressed in C. reinhardtii (Perez-Martin et al., 2014). In agreement with this hypothesis, we reported that significant expression of ATG8-PE protein was observed under high light conditions by immunofluorescence microscopy. It indicates that ATG8 is a pivotal protein to induce the autophagy process in C. reinhardtii (Figure 4). An abundance of ATG8-PE protein was increased in mutants due to high light, especially in pgr5. It seems an increase in stromal redox poise, which induces autophagy, thereby leading to triacylglycerol (TAG) accumulation. High light stress leads to ROS formation, which activates the autophagy mechanism. Increased ROS was seen in high light conditions; however, this was more prevalent in pgr5 mutant when grown in high light, indicating that high light increases oxidative stress and autophagy, which accompanies increased lipid content in the cell.

The autophagy of C. reinhardtii contains numerous autophagy-related proteins. Among various ATG proteins, ATG8-PE protein is essential for forming autophagosomes (Pérez-Pérez et al., 2016). In this study, we measured the expression level of ATG8-PE protein to evaluate the autophagy role in WT, pgrl1, and pgr5 (Figure 4). ATG8 appeared as a faint band in the WT and mutants (pgrl1 and pgr5) grown under optimal conditions (50 μmol photons m^–2 s^–1), and it was increased when cells were exposed to high light. However, lipidated form ATG8-PE was detected in the pgr5 mutant. Further, TEM confirmed several lytic vacuoles and small vesicles inside the vacuoles in high light treated cells (Figure 6). Under high light vacuoles size was markedly increased abundantly. This supports our hypothesis that light stress causes ROS production, especially in the pgr5 mutant. In turn, ROS induces autophagy, as reported earlier, leading to an increase in lipid production. Confocal and fluorescent activated cell sorter (FACS) data also suggested an increase in lipid droplet formation under high light in pgr5 mutant (Figures 5A,D). Continuous light exposure leads to the accumulation of biomass and the cellular over reduction and formation of ROS, which induces autophagy and lipids in microalgae (Shi et al., 2017). Our previous results indicate that PSI and PSII were damaged when cells were grown in high light (Nama et al., 2019; Yadav et al., 2020). Based on this result, we interpret that lipid accumulation in mutant strains could be explained by PSII or PSI being over-reduced, leading to high ROS production. These increased ROS levels act as an autophagy inducer (Heredia-Martinez et al., 2018).

In microalgae, lipid and carbohydrate are the primary energy storage forms and share the common carbon precursors for biosynthesis. Carbon partitioning is essential for the development of biofuels and chemicals. Most carbohydrates are stored as starch and sugars in microalgae. In several algal species, carbohydrate and lipid accumulate, while in some species, carbohydrate level decreases, and TAG increases, suggesting that lipids may be synthesized from carbohydrate degradation. Our results showed that both lipid and carbohydrate content increased in WT and pgrl1. However, lipid accumulation precedes carbohydrate accumulation (Figures 5, 7), consistent with the previous studies in the microalga, Pseudochlorococcum, under nitrogen stress conditions (Li et al., 2011). However, the carbohydrate content was reduced in pgr5 mutant under high light growth, explaining that carbohydrate degradation would have been converted to lipids in pgr5. Therefore more lipid accumulation was observed. Complex metabolic regulations in the cell may control the accumulation of lipid droplets. The pgr5 prevents the proton gradient and PSI dependant CET; therefore, the altered NADPH and ATP ratio may switch the metabolic pathways during the photosynthesis process. Some of the metabolic alterations would have converted to lipids, which is necessary to acclimatize to the high light in pgr5. Carbohydrate is the dominant sink for carbon storage in C. reinhardtii mutant, which lacks cell walls, so lipid synthesis occurs only when the carbon supply exceeds carbohydrate synthesis (Fan et al., 2012). Under saturating light, the C. reinhardtii culture induces lipid droplets formation without disrupting growth, while N starvation leads to significantly lower level (Goold et al., 2016).

In our case, a significant accumulation of TAG was observed in high light conditions and the accumulation of TAG is more in pgr5 (Figure 5). In, Arabidopsis thaliana and C. reinhardtii TAG synthesis originated from multiple types of acyltransferases. The final step of TAG biosynthesis was catalyzed by diacylglycerol acyltransferases (DGAT) and phospholipid diacylglycerol acyltransferase (PDAT) both involved in seed oil accumulation (Zhang et al., 2009). In the present study, we find the regulation of PDAT and DGAT at the protein level. Both the enzymes are accumulated under high light intensity, supporting these enzymes in the synthesis of TAG (Figure 8). Thus, the accumulation of TAG would be either through DGAT or PDAT routes.

The fatty acid composition also plays a significant role in biofuel production. The results of total fatty acids show that the composition and content of fatty acids were different in WT, pgrl1, and pgr5 under normal and high light. The pgrl1 and pgr5 have a higher percentage of SFA and MUFA under high light, as shown in Table 1. Palmitic (C16:0), stearic acid (C18:0), oleic acid content (C18:1), and palmitoleic (C16:1) increased in WT, pgrl1, and pgr5 under high light. This result implies high light stress conditions increase SFA and MUFA content in WT, pgrl1, and pgr5 mutant because the excess fatty acids could provide energy sink to the cells. Overall total fatty acid content was enhanced in pgr5 mutant compared to WT and pgrl1. We also observed an increase in the individual TAG band, especially the oleic acid (18:1 ω9c) (Figure 9A). Usually, oleic acid is a source of carbon that requires a functional β-oxidation cycle in which oleic acid is reduced to acetyl-CoA then to sugars or synthesis of other fatty acids or lipids (Graham, 2008).

Interestingly, the thin layer chromatography results show that the membrane lipids have been decreased, which indicates that the degradation of these lipids could have converted to TAG, especially in pgrl1 and pgr5 (Supplementary Figure 4). Also, the decreased fatty acids from MGDG and DGDG could have converted to TAG (Figure 9). Similar reports were observed suggesting the recycling of membrane lipids, monogalactosyldiacylglycerol (MGDG), and digalactosyldiacylglycerol (DGDG) into TAG accumulation in C. reinhardtii (Abida et al., 2015; Devadasu and Subramanyam, 2021). Therefore, we can interpret that fatty acids are dissociated from glycolipids, which are involved in the Kennedy pathway to synthesize the TAGs through the DGAT enzyme (Yoon et al., 2012).

Our results provided complete evidence that the two mutants, pgrl1 and pgr5, of C. reinhardtii, accumulate fatty acid and induced autophagy under high light conditions. The correlation between stress and TAG accumulation in photosynthesis of algae has been known for a century. Under high light conditions, these mutant’s fatty acid compositions may be an important study for biotechnological applications. It suggests that one possible reason for the increase in lipid content may be the unbalanced redox state of the cells, leading to the generation of ROS. Therefore, the increased ROS levels in pgr5 cells could play a dual role as signals to activate lipid biosynthesis and autophagy inducers to start recycling cellular components to fuel lipid production in this mutant (Tran et al., 2019). Therefore, pgrl1 and pgr5 of C. reinhardtii would be appropriate for producing high yield lipids (Lipids/TAG). Hence, our study could offer an essential value: microalgae-based lipid production can be promoted by applying various feedstocks to biodiesel and animal feed.