The TSA Chlorella was previously isolated from extreme saline-alkali soil on the Songnen Plain, Heilongjiang Province, China (Wang et al., 2011), which is rich in different salt types, including NaCl and NaHCO₃, and grown in liquid Bold’s basal medium (BBM, Bold and Wynne, 1984). The culture conditions were 23°C under a 16-h light/8-h dark photoperiod. The illumination intensity was 40-mmol photons m⁻¹ s⁻¹. The TSA Chlorella cells were maintained in solid BBM, and the sub-culturing and rapid propagation of TSA Chlorella was cultured in liquid BBM. A full-length cDNA yeast library of TSA Chlorella was constructed (Qiao et al., 2018). A sequence screened from the 1-M NaCl-treated TSA Chlorella library had close similarity levels to sequences of other species’ PAP genes. Accordingly, it was named ChPAP.

The full-length ChPAP sequence was analyzed using BlastX and ORFfinder on the NCBI Web site.¹ GeneDoc 3.0 software was used to align the sequences of the ChPAP protein and other species. The maximum-likelihood-based phylogenetic tree was constructed using MEGA 5.1 software. The transmembrane domains in the ChPAP sequence were predicted using the TMHMM server v. 2.0.²

The ChPAP cDNA fragment harboring the open reading frame was amplified from TSA Chlorella using PCR with the ChPAP-forward (5'-GGATCCATGTTGCACGCGATGGTGG-3'; BamHI restriction site) and -reverse (5'-GTCGACTCAAACAGGCACCATGCTGC-3'; KpnI restriction site) primers and Phanta Max Super-Fidelity DNA Polymerase (Vazyme Biotech Co., Ltd., Nanjing, China). For yeast transformation, ChPAP was ligated into the pYES2 vector (Invitrogen, United States) digested with BamHI and NotI restriction enzymes to construct the plasmid pYES2-ChPAP, which was transferred into InVSC1 yeast cells using the PEG/LiAC method (Gietz et al., 1992). First, to test the tolerance of transgenic yeast under different stress conditions, yeast cells containing, independently, the pYES2-ChPAP and empty pYES2 vectors were cultivated in liquid SD-Uracil (pH 5.8) medium at 30°C for 2days. The concentration of the transgenic yeast cells was adjusted to an OD₆₀₀ value of 0.5 and then diluted to 10⁻¹, 10⁻², 10⁻³, and 10⁻⁴ with sterile H₂O. A total of 4.5μl of each dilution series was placed into a solid yeast (1% yeast extract, 2% peptone, and 2% galactose) medium supplemented independently with 0.8-M NaCl, 1.0-M NaCl, and 1.6-M sorbitol. The empty pYES2 vector in yeast cells was used as a control, and the growth of yeast cells at 30°C was observed and photographed for 5–10days.

To determine the subcellular localization of ChPAP, the expression plasmid pYES2-ChPAP-EGFP was constructed. The ChPAP full-length sequence with restriction sites was cloned using PCR with EGFP-specific forward GFP-F (5'-GGATCCATGTTGCACGCGATGGTGGAC-3') and reverse GFP-R (5'-GGTACCCCAACAGGCACCATGCTGCTTGC-3') primers. The PCR product was ligated into the pEGFP plasmid (Clontech) digested with the BamH1 and KpnI sites. The ChPAP-pEGFP fusion fragment was digested at the BamHI and NotI sites in the pEGFP plasmid to construct the empty pYES2 vector.

The pYES2-ChPAP-EGFP and pYES2-EGFP plasmids were transferred independently into yeast cells. The transgenic yeast cells were pre-cultured in liquid medium containing 1% yeast extract, 2% peptone, and 2% glucose at 30°C for 2days. Afterward, they were washed three times to remove the remaining glucose. The EGFP and ChPAP-EGFP plasmids in yeast cells were induced to express in liquid yeast (1% yeast extract, 2% peptone, and 2% galactose) medium at 30°C for 6h, and then, ChPAP-EGFP yeast cells were incubated at 30°C with 20-μm FM4-64 dye for 3h. The remaining dye was removed by washing three times with sterile H₂O before samples were observed. The fluorescence was detected using laser-scanning confocal imaging system (Olympus Fluoview, FV500). The EGFP and FM4-64 signals were excited at 488nm and 543nm, respectively.

To investigate the ChPAP transcript levels under high salinity and drought stresses, the TSA Chlorella samples were grown on medium supplemented independently with 200-mm NaCl and 300-mm sorbitol. The samples were collected at 0, 3, 6, 12, 24, and 48h and then ground with a mortar and pestle in liquid nitrogen for RNA isolation.

The total RNA of TSA Chlorella was extracted using RNAiso Plus reagent (TaKaRa, Japan). The ChPAP-specific forward (5'-ATGGGCCTCAAGGAAGAC-3') and reverse (5'-TCAAGCGTACTTCGCCTTCAG-3') primers were used to amplify the cDNA probes using a PCR Digoxigenin Probe Synthesis kit (Roche, Switzerland). The northern blot analysis was performed in accordance with a previously published protocol (Qiao et al., 2018).

The ChPAP nucleic acid sequence contained an open reading frame of 1,002 nucleotides that translated into 333 amino acids. The ChPAP amino acid sequence shared close similarities with the previously reported PAP sequences of other species, such as 70% similarity with C. variabilis and 53% similarity with Micractinium conductrix. A comparison of the ChPAP domains with those in PAPs of other species revealed the presence of conserved domains 1, 2, and 3 (Figure 1), which contained two arginine, one histidine, and two histidine residues, respectively. These results were consistent with previously reported PAP2 protein structures (Hemrika et al., 1997; Neuwald, 1997; Stukey and Carman, 1997). The phylogenetic analysis showed that ChPAP was closely related to the PAP of C. variabilis (XP_005848979.1). The TSA Chlorella firstly clustered with unicellular microalgae, and then clustered with microbes, animals, and plants (Figure 2). Thus, ChPAP2 is the PAP gene of TSA Chlorella.

The five serial dilutions of transgenic yeast cells were spotted into a solid yeast medium. The growth of yeast cells harboring the empty pYES2 vector was similar to that of the ChPAP2 transgenic yeast in medium containing 1% yeast extract, 2% peptone, and 2% glucose without any stress. The ChPAP2 transgenic yeast grew better than the controls in the presence of 0.8-M NaCl, 1-M NaCl, or 1.6-M sorbitol (Figure 3). Thus, the expression of ChPAP2 in yeast cells improved the tolerance to NaCl and sorbitol, which indicated that ChPAP2 functions as lipid signaling molecule during abiotic stress (Munnik et al., 1996). PAH1 encoded PAP1 was regulated to express under Zn deficiency and enhanced the activity of PAP enzyme (Soto-Cardalda et al., 2012). In addition, Arabidopsis PAP2 was found that was involved with ABA signaling and regulating the stomatal movements (Paradis et al., 2011). However, there was no relevant report on the investigation of PAP2 under abiotic stress.

A northern blot analysis was used to detect ChPAP2 expression patterns. Total RNA was used to analyze the effects of high salinity and drought stresses on ChPAP2 expression. The TSA Chlorella cells were exposed independently to NaCl and sorbitol for 0, 3, 6, 12, 24, and 48h. The ChPAP2 mRNA expression dramatically increased with the 200-mm NaCl treatment from 6 to 48h compared with the control (0h), indicating that ChPAP2 was upregulated (Figure 4A). Thus, in yeast, the increased ChPAP2 expression level may increase NaCl resistance. The PA and DGPP levels also increase under hyperosmotic stress conditions (Munnik et al., 2000). The presence of NaCl in liquid media not only leads to high salinity stress, but also to hyperosmotic stress in plants. We hypothesized that the ChPAP2 expression level may be upregulated, allowing it to catalyze the excess DGPP into PA.

In this study, a sorbitol solution was used as the drought agent (Bocchini et al., 2018; Lu et al., 2019). The ChPAP2 expression levels did not differ significantly compared with those of the control after 3–6h of exposure to 300-mm sorbitol. The ChPAP2 mRNA level began to rise at 12h, and the level was the most significantly different from that of the control at 48h after treatment (Figure 4B). In Saccharomyces cerevisiae, PAP accumulates during exposure to hyperosmotic and dehydration stresses (Munnik et al., 2000). Thus, the upregulated ChPAP2 expression may enhance drought tolerance.

The deduced ChPAP2 amino acid sequence was predicted to contain six transmembrane domains (Figure 5A). To determine its subcellular localization, GFP was fused to the C-terminus of ChPAP2 (ChPAP2-GFP). The green fluorescence of the GFP protein alone was almost evenly distributed throughout the yeast cells (Figures 5B1,2). FM4-64 stained the vacuole membrane, and its localization signal was consistent with the vacuolar membrane signal (red fluorescence area; Figure 5B4). Thus, the ChPAP2 protein appeared to localize on the plasma membrane and in the lumen of vacuoles in yeast cells (Figures 5B3,5). The results suggested that ChPAP2 might play important roles in transporting lipids through the plasma membrane and in catalyzing DGPP into PA in the vacuoles.

ChPAP2, as a catalytic enzyme, participates in lipid metabolism. ChPAP2 localized on the plasma membrane may function in maintaining cell membrane stability and the ion balance of the cytoplasm in response to abiotic stresses. The ChPAP2 protein also is localized in the lumen of vacuoles, where it may be involved with lipid translocation and DGPP catalysis to form PA. Therefore, we hypothesized that ChPAP2 participates in catalyzing DGPP in the lumen of vacuoles.

The PAP protein may function as a signaling molecule in planta under stress conditions, and its levels accumulate during hyperosmotic and dehydration stresses (Munnik et al., 1996). Therefore, the PAP enzyme may play a role in regulating specific cellular DGPP and PA pools under stress conditions (Oshiro et al., 2003).

In summary, the ChPAP in TSA Chlorella was upregulated expression in treated with high salinity and drought, and the ChPAP in yeasts could tolerate high salinity and drought stresses. Its protein was localized at the plasma membrane and in the lumen of vacuoles. The ChPAP might translocate excess NaCl and sorbitol from plasma membrane and then segregate them into vacuole to regulate ion balance in the cytoplasm. As a consequence, the PAP as a novel transporter can enhance high salinity tolerance and accumulate excess high salinity. These characteristics make ChPAP for the bioremediation of saline-alkali soil.