Improving antistress capacity and lipid productivity in the green alga Chlorella pyrenoidosa by adding abscisic acid under salt stress conditions

Microalgae and growth conditions

A FACHB-9 strain of C. pyrenoidosa was acquired from the Freshwater Algae Culture Collection at the Institute of Hydrobiology, Chinese Academy of Science located in Wuhan, China. The algal seeds were introduced into 500 mL of new BG-11 solution (with the addition of 10 g L⁻¹ glucose) and incubated at a temperature of 30 ± 1°C with agitation at 150 rpm for a cultivation period of 8 d. For the subsequent experiments, the algal biomass was obtained through centrifugation at 2,683 ×g and then rinsed three times with deionized water. ABA was obtained from Sigma-Aldrich (Shanghai, China) and was subsequently dissolved in a stock solution of 100 mM ethanol. To subject the algal cells to salt stress, they were suspended again in 300 mL of new BG-11 medium (containing 0.1, 0.21, 0.31, or 0.41 mol L⁻¹ NaCl). The suspension was then cultivated at 150 rpm and an illumination intensity of 150 μE m⁻² s⁻¹. Furthermore, to explore the effect of ABA on resistance and lipid accumulation in algal cells, the cells were individually cultured for 3 d in medium supplemented with the optimal concentration of salt and ABA (0, 100, 150, 200, 250, 280, and 300 μmol L⁻¹). The biomass was then harvested by centrifugation at 2,683 ×g for further analysis. After the results were obtained, the algal cells were subjected to an 8-h treatment with 30 μM JA in the presence of 250 μM ABA + 0.21 mol L⁻¹ NaCl. Subsequently, the impact of JA on cell growth and lipid accumulation in C. pyrenoidosa was assessed. To further validate the mechanism of JA regulation, the culture medium was supplemented with an inhibitor (ibuprofen, IBU) that hinders JA synthesis. The experiment was conducted three times for each treatment.

Measurements of biomass and lipid content

Newly formed algae cells were obtained through centrifugation at a force of 10,000 ×g for 10 min. Subsequently, these cells were promptly frozen in liquid nitrogen for 15 min and then subjected to freeze drying for 24 h until a consistent weight was achieved, after which the cells were weighed.

Total lipids were extracted from freeze-dried algae cells as described in our previous work (Ding et al. 2020). In total, 200–500 mg (w₁) of dehydrated algae cells was pulverized to form a powder, which was subsequently combined with 3 mL of a chloroform-methanol mixture at a ratio of 2 to 1 (v/v). The mixture was agitated on a shaker at a speed of 150 rpm for a duration of 30 min. Then, the mixture was centrifuged at a force of 2,683 ×g for 10 min, and the liquid above the sediment was collected. The extraction process described above was repeated two to three times until the algal residue became white. All of the solvents collected were transferred to a centrifuge tube (w₂) that had been preweighed. The contents were then dried in a vacuum oven at a temperature of 40°C until a constant weight (w₃) was achieved. The percentage of total lipids was determined using the following calculation:

, where V represents the volume of the collected medium (L), while w₅ and w₄ indicate the dry cell weight (g) at times (h), t₂ (biomass harvest) and t₁ (inoculation), respectively.

The lipid weights (g) at time points t₂ and t₁ are referred to as w₇ and w₆, respectively.

Determination of protein, carbohydrate, cell cycle, and chlorophyll a contents

Total protein was measured via a protein assay kit purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). The overall carbohydrate content was determined through trifluoroacetic acid hydrolysis and measured using a glucose assay kit (Comin, Suzhou, China) and assessing the absorbance at 620 nm. The cell cycle was measured using a Cell Cycle and Apoptosis analysis kit (Beyotime, Shanghai, China).

Chlorophyll a (Chl a) was detected by following the steps below. First, 4–8 mL of the suspension solutions of algae cells was centrifuged at a force of 10,000 ×g for 3 min. Next, 0.25 mL of purified water and 0.5 mL of glass spheres (0.05 mm) were added to the Eppendorf tube to lyse the cells through vortexing for 4 min on a vortex shaker. The solution was transferred to a 50 mL trial tube, and 4 mL of 80% ethanol was added for extraction at a temperature of 68°C for 15 min. Following a protocol by Zhang et al. (2018), the mixture was centrifuged at a force of 10,000 ×g for 5 min. Subsequently, the absorbance of the resulting liquid was assessed at wavelengths of 649 and 665 nm.

Determination of ROS, GSH, trehalose, and JA levels

A Reactive Oxygen Species assay kit (DCFH-DA; Beyotime) and GSH assay kit (Beyotime) were used to measure the levels of intracellular ROS and the GSH concentration, respectively, according to the manufacturer’s instructions.

The trehalose concentration was quantified by using a trehalose content kit (Comin). Plant JA enzyme-linked immunoassay kits (Mlbio Biotechnology, Shanghai, China) were used to measure the levels of endogenous JA. To summarize, 0.1 g of collected algal cells was placed in a mortar and suspended in 1 mL of 10 mM PBS (pH = 7.4) before being crushed using liquid nitrogen. Afterward, the mixture was subjected to centrifugation at a force of 2,683 ×g for a duration of 20 min at a temperature of 4°C, and the concentration of the above liquid was measured at a wavelength of 450 nm. JA contents can be obtained according to the following standard curves:

Y represents the level of JA, while X denotes the absorbance at OD450 nm.

Transcriptome analysis

Statistical analysis

For each of the parameters tested, a minimum of three parallel experiments were carried out, and the data were presented as the mean ± standard deviation. SPSS version 19.0 (SPSS Inc., Chicago, IL, USA) was utilized for performing the statistical analyses. A significance level of less than 0.05 was considered to indicate statistical significance for all the analyses.

ABA enhanced lipid productivity in Chlorella pyrenoidosa under salt stress

In this work, the biomass concentration decreased with increasing salt concentration at the same culture time. The control group (Fig. 1A) exhibited the highest biomass concentration (0. 91 g L⁻¹), which indicates that biomass production was inhibited in the NaCl stress groups. This inhibition might be attributed to hyperosmotic stress, ion toxicity, and secondary stresses, such as oxidative damage caused by high salinity. Ji et al. (2018) reported that under salt stress, the cell growth rate of Scenedesmus obliquus XJ002 decreased due to damage to the photosystem II reaction center and oxygen evolution complex and inhibiting electron transport to the reaction center.

Fig. 1

Biomass concentration and lipid production under varying levels of NaCl (A & B) and abscisic acid (ABA) with the optimized NaCl concentration (C & D) in Chlorella pyrenoidosa. DW, dry weight. The error bars indicate the standard deviations from three independent samples. * and ** indicate statistical significance compared with the control at p < 0.05 and p < 0.01, respectively.

The lipid content versus different NaCl concentrations is shown in Fig. 1B, which shows an upward trend followed by a decrease as the salt concentration increases. The NaCl treatment group had a lipid content of 38.31%, which was 1.63 times greater than that of the control group (23.45%). These results suggest that salt stress can induce lipid accumulation in microalgal cells. Furthermore, according to the data presented in Table 1, the highest lipid production rate (31.23 mg L⁻¹ d⁻¹) was attained in the 0.21 M NaCl experimental group, surpassing that of the control group (20.91 mg L⁻¹ d⁻¹) by 1.49 times. Thus, 0.21 M NaCl was the optimal concentration for salt stress-induced lipid biosynthesis.

Table 1

Effects of NaCl on the biomass concentration, biomass productivity, lipid content, and lipid productivity of Chlorella pyrenoidosa

Algal proliferation is impacted by ionic toxicity, severe osmotic stress, and oxidative damage induced by salinity stress. However, ABA has been reported to promote cell growth and improve the fatty acid quality of microalgae (Norlina et al. 2020). In this work, different concentrations of ABA were added to the test groups to explore the mechanism by which ABA activates the resistance of microalgae to high salt stress, Fig. 1C shows the change in biomass concentration versus ABA concentration. The biomass in these groups treated with salt stress + ABA (150, 200, 250, and 280 μM) increased by 4.29, 18.29, 30.49, and 12.2%, respectively, compared with that in the test group (only salt stress). Taken together, these findings show that after ABA addition, algal cell growth could be promoted under salt stress conditions.

Fig. 1D shows the lipid contents in the groups subjected to NaCl stress (0.21 M) + ABA. Compared to those in the control group, the lipid contents in the treatment groups increased by 2.33, 7.52, and 9.51% at ABA concentrations of 150, 200, and 250 μM, respectively. Wu et al. also discovered that ABA plays crucial roles in lipid accumulation in Chlorella (Wu et al. 2018). Furthermore, the group treated with 250 μM ABA demonstrated the highest lipid productivity (45.35 mg L⁻¹ d⁻¹), which was 1.45 and 2.17 times greater than that of the NaCl treatment and control groups, respectively (Tables 1 & 2). Therefore, lipid productivity in microalgae can be improved by salt stress + ABA, and treatment with 0.21 M NaCl + 250 μM ABA was optimal for algal cell growth and lipid accumulation and was selected for subsequent experiments.

Table 2

Effects of abscisic acid (ABA) on the biomass concentration, biomass productivity, lipid content, and lipid productivity of Chlorella pyrenoidosa under NaCl treatment

ABA restored cell growth under salt stress

In addition to the lipid content, the levels of Chl a, carbohydrates, proteins, and cell cycle components were measured. As shown in Fig. 2A, the Chl a levels were significantly lower in all groups treated with NaCl than in the control group. However, adding ABA to the microalgal cells not only restored the Chl a content but also surpassed that of the control group. These changes were consistent with the trends in cell growth (Fig. 1A).

Fig. 2

The level of chlorophyll a (A), flow cytometric study of Chlorella pyrenoidosa’s cell cycle (B), protein (C), and carbohydrate content (D) under various circumstances. ABA, abscisic acid; DW, dry weight. The error bars indicate the standard deviations from three independent samples. * and ** indicate statistical significance compared with the control at p < 0.05 and p < 0.01, respectively.

The S phase of the cell cycle was suppressed in the presence of salt stress, but this suppression was reduced upon the addition of ABA. Consequently, the proportion of cells in the S phase was comparable to that in the control group, as shown in Table 3 and Fig. 2B. Based on the aforementioned results, it can be concluded that the presence of salt stress hindered the process of cell division and growth, resulting in a reduction in Chl a levels. However, this inhibition was mitigated by the application of ABA, which leading to an increase in the growth and lipid biosynthesis of algal cells.

Table 3

Changes of cell cycle in different treatment groups

As shown in Fig. 2C, the protein levels in the NaCl treatment groups were lower than those in the control group. However, in the ABA-treated groups, there was a noticeable increase in protein levels, with the maximum protein content increasing to 27.33%. Under NaCl stress conditions, the cellular carbohydrate content decreased from 33.65 to 21.13% with increasing ABA concentration (Fig. 2D). This result indicated that the metabolic flow of carbon was transferred to lipid and protein biosynthesis under salt stress + ABA conditions, possibly due to the influence of ABA on carbon reallocation (Yang et al. 2023).

ABA-mediated regulation of cellular metabolism under salt stress

Summary of the transcriptome sequencing and annotation data

To gain deeper knowledge on the metabolic reaction caused by ABA, the transcriptome of C. pyrenoidosa was analyzed under salt stress + ABA. Three parallel samples (labeled C01, C02, and C03) were collected from the treatment group treated with 0.21 M NaCl + 250 μM ABA, and the 0.21 M NaCl-treated group served as the control (labeled B01, B02, and B03). Supplementary Tables S1 and S2 show the GO categorization results based on the gene annotations. The DEGs were evenly distributed across the GO categories of biological process, cellular component, and molecular function. Supplementary Tables S3 and S4 show the functional annotations of the DEGs in the KEGG pathway when ABA was added. ABA induced notable alterations in the expression levels of genes linked to sugars, fats, proteins, and cell communication. These findings further indicate that ABA promoted growth and intracellular lipid production under salt stress conditions (Fig. 1). However, additional investigations are needed to further explore the mechanism by which ABA regulates the growth and lipid accumulation of C. pyrenoidosa.

To confirm the accuracy of upregulation and downregulation of gene expression in the lipid synthesis pathway identified by transcriptome sequencing, the upregulated genes g8893, g6886, g6008, and g2851 related to lipid synthesis and downregulated genes g5675 and g1137 related to lipid catabolic metabolism in this pathway were selected for fluorescence quantitative PCR verification. g4994 and g378, which are related to the synthesis and metabolism of JA, were also verified (Fig. 3). The results of fluorescence quantitative PCR were consistent with the transcriptome results, which further confirmed that the regulation of genes in the lipid synthesis pathway by ABA is conducive to the accumulation of lipid, and confirmed that the JA signaling pathway is involved in the regulation of the intracellular lipid anabolism by ABA.

Fig. 3

The relative expression of different genes. ABA, abscisic acid; ALDH, aldehyde dehydrogenase; 1-AGPAT, aldehyde dehydrogenase; CDP, phosphatidate cytidylyltransferase; accA, phosphatidate cytidylyltransferase; ALDH2, aldehyde dehydrogenase-2; ADH, alcohol dehydrogenase; DAD1, DAD1-phospholipase A1; MYB, v-myb avian myeloblastosis viraloncogene homolog transcription factors. The error bars indicate the standard deviations from three independent samples. * and ** indicate statistical significance compared with the control at p < 0.05 and p < 0.01, respectively, paired t test.

Effects of ABA on the JA, ROS, GSH, and total antioxidant capacity levels of Chlorella pyrenoidosa under salt stress

To examine whether exogenous ABA influences the ROS and JA signaling pathways during cell growth and lipid formation in microalgae under salt stress conditions, the intracellular ROS, trehalose, GSH, JA, and total antioxidant capacity (T-AOC) levels were measured.

JA is generally considered an oxidizing lipid signaling molecule in plants and microalgae (Yang et al. 2023). More than that, Pretorius et al. have demonstrated that JA was involved in the synthesis of volatile lipids in plants under biotic stress (Pretorius et al. 2021). As shown in Fig. 4A, the concentration of JA increased in the salt stress + ABA treatment group. On the eighth day, the peak JA concentration (928.06 pmol L⁻¹) was 1.10- and 1.19-fold greater than that in the salt stress and control groups, respectively. Therefore, treatment with salt stress + ABA promoted intracellular JA accumulation. As shown in Fig. 1D, under salt stress + ABA conditions, the intracellular lipid content of the microalgae increased, and the change was consistent with that of the JA level; in addition, the maximal lipid content was observed on the eighth day. Therefore, it can be deduced that the intracellular JA concentration in algae was positively correlated with the lipid content. Combined with the upregulation of JA synthesis-related genes in the ABA-supplemented group described above, it can be inferred that ABA regulates intracellular lipid synthesis by affecting JA synthesis.

Fig. 4

Effect of different conditions on the jasmonic acid (JA) content (A), reactive oxygen species (ROS) levels (B), total antioxidant capacity (T-AOC) levels (C), trehalose content (D), and glutathione (GSH) levels (E) of Chlorella pyrenoidosa. NaCl + ABA, NaCl stress combined with abscisic acid treatment; FW, fresh weight. The error bars indicate the standard deviations from three independent samples. * and ** indicate significant difference compared with the control at p < 0.05 and p < 0.01, respectively.

ROS serve as typical second messengers in response to various stress conditions and are linked to the cellular regulation of lipid biosynthesis in microalgae (Kou et al. 2020, Eibl and Schneemann 2022, Zhao et al. 2023). GSH is a major antioxidant in plants and microalgae, which combines with free radicals through its thiol group to produce easily metabolized acids, thereby accelerating the excretion of free radicals and reducing oxidative damage to cells (Tamaki et al. 2021). Trehalose, besides being a source of carbon and energy, serves as a protective agent against abiotic stresses such as heat, salt, drought, and oxidation (Reina-Bueno et al. 2012). As a stress-responsive metabolite, trehalose significantly protects intracellular macromolecules like proteins, nucleic acids, and cell membranes, thereby enhancing stress resistance—a function discovered in microalgae (Colina et al. 2020). Furthermore, trehalose acts as a signaling molecule, participating in specific metabolic regulatory pathways including ABA signaling, sugar signaling, and plant photosynthesis (Islam et al. 2019).

As shown in Fig. 4B, ROS levels increased by 46.38% compared to the control on the 8th day under salt osmotic stress, while ROS levels in the ABA treatment group decreased to 86.41% of the salt osmotic stress group. However, while ROS levels gradually increased, T-AOC levels in algal cells also increased and reached the highest level on the sixth day, when the ABA added group was 1.21 times that of the control group (Fig. 4C). Fig. 4D & E indicate that GSH and trehalose levels in the ABA treatment group reached their highest, at 4.31 μmol g⁻¹ and 16.58 μg g⁻¹, respectively, increasing by 71.71 and 71.64% compared to the salt osmotic stress group at the same time. Previous studies have reported that salt osmotic stress induces the production of substantial ROS in plant and microalgae cells (Ben Rejeb et al. 2014). To counteract oxidative damage, the activities of intracellular SOD, POD, glutathione reductase, and catalase, as well as non-enzymatic components such as GSH, trehalose, ascorbic acid, and α-tocopherol, generally increase (Sakhno et al. 2019).

The increase in GSH and trehalose content after ABA addition might be due to ABA regulation of the cellular antioxidant system, which aligns with findings in plants where certain plant hormones enhance antioxidant and stress resistance capabilities, thereby reducing intracellular oxidative levels and mitigating oxidative damage (Stirk and van Staden 2020, Kusvuran 2021). Anjum et al. demonstrated that enhancing glutathione and related enzyme functions and altering their levels might effectively improve plant stress resistance (Anjum et al. 2012). Guajardo et al. found that exogenous ABA could increase the intracellular antioxidant level of algae to counteract dehydration stress (Guajardo et al. 2016). Yoshida et al. conducted similar studies in Chlamydomonas reinhardtii, showing that adding ABA to stressed algal cells increased the activity of antioxidant enzymes, enhancing oxidative stress tolerance (Yoshida et al. 2003). Therefore, it is concluded that ABA caused an obvious effect on the cellular antioxidant system.

The influence of JA on cellular proliferation and the production of lipids are altered in the presence of salt stress and ABA

The role of JA signaling in cell proliferation, lipid accumulation, and metabolism was further confirmed through adding exogenous JA and a JA inhibitor (IBU) to the salt stress + ABA treatment group.

JA levels were measured in the different treatment groups. As shown in Fig. 5A, with the addition of exogenous JA, the intracellular JA concentration gradually increased in the salt stress + ABA treatment group; however, the addition of IBU (as an inhibitor) significantly decreased the intracellular JA concentration. Simultaneously, as illustrated in Table 4, the introduction of JA from an external source significantly promoted the development and synthesis of lipids in C. pyrenoidosa when exposed to salt stress + ABA. Conversely, the opposite effect was observed when IBU was added. Specifically, compared with those in the other groups, the biomass concentration, lipid content, and lipid productivity in the IBU-treated group were substantially lower. Therefore, it can be inferred that the intracellular level of JA is closely related to the growth and lipid synthesis of algal cells. As previously mentioned, adding ABA under conditions of salt stress resulted in an increase in intracellular JA levels, leading to the promotion of algal cell growth and the accumulation of lipids. Taken together, these findings indicate that ABA effects the growth and lipid storage of algal cells exposed to salt stress through intracellular JA signaling molecules.

Fig. 5

Effect of different conditions on the jasmonic acid (JA) content (A), reactive oxygen species (ROS) levels (B), total antioxidant capacity (T-AOC) levels (C), trehalose content (D), and glutathione (GSH) levels (E) of Chlorella pyrenoidosa. NaCl + ABA, NaCl stress combined with abscisic acid treatment; NaCl + ABA + JA, NaCl stress combined with ABA and JA treatment; NaCl + ABA + IBU, NaCl stress combined with ABA and ibuprofen treatment; FW, fresh weight. The error bars indicate the standard deviations from three independent samples. * indicates significant difference compared with the control at p < 0.05.

Table 4

Effect of different treatments on the biomass concentration, biomass productivity, lipid content, and lipid productivity of Chlorella pyrenoidosa

In addition, the effects of JA or IBU addition on the levels of intracellular ROS, trehalose, GSH, and T-AOC in the salt stress + ABA treatment group were analyzed. Under salt stress + ABA conditions, exogenous JA significantly inhibited the formation of ROS (Fig. 5B) but increased the T-AOC, trehalose, and GSH levels (Fig. 5C–E). In contrast, the ROS levels in the IBU-supplemented group obviously increased compared with those in the JA-supplemented group, and the T-AOC, trehalose and GSH levels markedly decreased. Therefore, the extracellular hormone ABA triggers the cell’s antioxidant system under salt stress by regulating intracellular JA levels, thereby mitigating oxidative stress in algal cells. Thus, the intracellular level of JA is closely related to the levels of antioxidants and antioxidant enzymes in algal cells.

Table 5 shows the biodiesel quality and fatty acid profiles of C. pyrenoidosa under the different treatments. Compared to the control, ABA additions under salt stress conditions resulted in an increase in the cetane number and a decrease in the degree of unsaturation, iodine value, long-chain saturated factor, and cold filter plugging point. Under salt stress + ABA, the results indicated an improvement in the combustion quality, oxidation stability, and low-temperature fluidity of C. pyrenoidosa biodiesel.

Table 5

Effects of different treatments on the fatty acid profiles and biodiesel quality of Chlorella pyrenoidosa

Although salt stress conditions promote lipid accumulation in algal cells, the oxidative stress induced by salt stress generally inhibits the growth of algal cells, resulting in low lipid productivity. In this work, we found that exogenous ABA could activate the intracellular JA signaling pathway in algal cells and increase antioxidant levels, relieving the damage caused by salt stress-induced cellular oxidative stress. As a result, algal cell growth recovered, and lipid productivity significantly increased due to the upregulation of the genes related to lipid biosynthesis (Fig. 6).

Fig. 6

A possible mechanism by which jasmonic acid (JA) signaling regulates lipid accumulation in Chlorella pyrenoidosa under salt stress + abscisic acid (ABA) condition. DAG, diacylglycerol; FA, fatty acid; GSH, glutathione; LBs, lipid bodys; ROS, reactive oxygen species; TAG, triacylglycerol.