The Papain-like Cysteine Protease HpXBCP3 from Haematococcus pluvialis Involved in the Regulation of Growth, Salt Stress Tolerance and Chlorophyll Synthesis in Microalgae.

The papain-like cysteine proteases (PLCPs), the most important group of cysteine proteases, have been reported to participate in the regulation of growth, senescence, and abiotic stresses in plants. However, the functions of PLCPs and their roles in stress response in microalgae was rarely reported. The responses to different abiotic stresses in Haematococcus pluvialis were often observed, including growth regulation and astaxanthin accumulation. In this study, the cDNA of HpXBCP3 containing 1515 bp open reading frame (ORF) was firstly cloned from H. pluvialis by RT-PCR. The analysis of protein domains and molecular evolution showed that HpXBCP3 was closely related to AtXBCP3 from Arabidopsis. The expression pattern analysis revealed that it significantly responds to NaCl stress in H. pluvialis. Subsequently, transformants expressing HpXBCP3 in Chlamydomonas reinhardtii were obtained and subjected to transcriptomic analysis. Results showed that HpXBCP3 might affect the cell cycle regulation and DNA replication in transgenic Chlamydomonas, resulting in abnormal growth of transformants. Moreover, the expression of HpXBCP3 might increase the sensitivity to NaCl stress by regulating ubiquitin and the expression of WD40 proteins in microalgae. Furthermore, the expression of HpXBCP3 might improve chlorophyll content by up-regulating the expression of NADH-dependent glutamate synthases in C. reinhardtii. This study indicated for the first time that HpXBCP3 was involved in the regulation of cell growth, salt stress response, and chlorophyll synthesis in microalgae. Results in this study might enrich the understanding of PLCPs in microalgae and provide a novel perspective for studying the mechanism of environmental stress responses in H. pluvialis.

Characterization of type-2 diacylglycerol acyltransferases in Haematococcus lacustris reveals their functions and engineering potential in triacylglycerol biosynthesis.

BACKGROUND: Haematococcus lacustris is an ideal source of astaxanthin (AST), which is stored in oil bodies containing esterified AST (EAST) and triacylglycerol (TAG). Diacylglycerol acyltransferases (DGATs) catalyze the last step of acyl-CoA-dependent TAG biosynthesis and are also considered as crucial enzymes involved in EAST biosynthesis in H. lacustris. Previous studies have identified four putative DGAT2-encoding genes in H. lacustris, and only HpDGAT2D allowed the recovery of TAG biosynthesis, but the engineering potential of HpDGAT2s in TAG biosynthesis remains ambiguous. RESULTS: Five putative DGAT2 genes (HpDGAT2A, HpDGAT2B, HpDGAT2C, HpDGAT2D, and HpDGAT2E) were identified in H. lacustris. Transcription analysis showed that the expression levels of the HpDGAT2A, HpDGAT2D, and HpDGAT2E genes markedly increased under high light and nitrogen deficient conditions with distinct patterns, which led to significant TAG and EAST accumulation. Functional complementation demonstrated that HpDGAT2A, HpDGAT2B, HpDGAT2D, and HpDGAT2E had the capacity to restore TAG synthesis in a TAG-deficient yeast strain (H1246) showing a large difference in enzymatic activity. Fatty acid (FA) profile assays revealed that HpDGAT2A, HpDGAT2D, and HpDGAT2E, but not HpDGAT2B, preferred monounsaturated fatty acyl-CoAs (MUFAs) for TAG synthesis in yeast cells, and showed a preference for polyunsaturated fatty acyl-CoAs (PUFAs) based on their feeding strategy. The heterologous expression of HpDGAT2D in Arabidopsis thaliana and Chlamydomonas reinhardtii significantly increased the TAG content and obviously promoted the MUFAs and PUFAs contents. CONCLUSIONS: Our study represents systematic work on the characterization of HpDGAT2s by integrating expression patterns, AST/TAG accumulation, functional complementation, and heterologous expression in yeast, plants, and algae. These results (1) update the gene models of HpDGAT2s, (2) prove the TAG biosynthesis capacity of HpDGAT2s, (3) show the strong preference for MUFAs and PUFAs, and (4) offer target genes to modulate TAG biosynthesis by using genetic engineering methods.

Electrogenesis in Plasma Membrane Fraction of Halotolerant Microalga Dunaliella maritima and Effects of N,N'-Dicyclohexylcarbodiimide.

The effects of N,N'-dicyclohexylcarbodiimide (DCCD), non-specific inhibitor of various transport systems functioning in biological membranes, on Na+-transporting P-type ATPase of the green halotolerant microalga Dunaliella maritima were studied in the experiments with vesicular plasma membranes isolated from the alga cells. The effects of DCCD on electrogenic/ion transport function of the enzyme and its ATP hydrolase activity were investigated. Electrogenic/ion transport function of the enzyme was recorded as a Na+-dependent generation of electric potential on the vesicle membranes with the help of the potential-sensitive probe oxonol VI. It was found that unlike many other ion-transporting ATPases, the Na+-ATPase of D. maritima is insensitive to DCCD. This agent did not inhibit either ATP hydrolysis catalyzed by this enzyme or its transport activity. At the same time DCCD affected the ability of the vesicle membranes to maintain electric potential generated by the D. maritima Na+-ATPase. The observed effects can be explained based on the assumption that DCCD interacts with the Na+/H+ antiporter in the plasma membrane of D. maritima.

Functional Characterization of Long-Chain Acyl-CoA Synthetase Gene Family from the Oleaginous Alga Chromochloris zofingiensis.

Long-chain acyl-coenzyme A (CoA) synthetase (LACS) catalyzes the formation of acyl-CoAs from free fatty acids, which is pivotal for lipid metabolism. Here, we confirmed the presence of six CzLACS genes in Chromochloris zofingiensis. Functional complementation and in vitro enzymatic assay indicated that CzLACS2 through CzLACS5 rather than CzLACS1 or CzLACS6 are bona fide LACS enzymes and they have overlapping yet distinct substrate preference. The results of the subcellular colocalization experiment and different expression patterns under three triacylglycerol (TAG)-inducing conditions showed that CzLACS2 through CzLACS4 reside at endoplasmic reticulum (ER) and are involved in TAG biosynthesis, while CzLACS5 resides in peroxisome and participates in fatty acid ß-oxidation. The yeast one-hybrid assay using a library of 50 transcription factors (TFs) constructed in our study identified 12 TFs potentially involved in regulating the expression of CzLACSs. Moreover, heterologous expression of CzLACSs demonstrated their engineering potential for modulating TAG synthesis in yeast and algal cells.

Effects of Excess and Limited Phosphate on Biomass, Lipid and Fatty Acid Contents and the Expression of Four Fatty Acid Desaturase Genes in the Tropical Selenastraceaen Messastrum gracile SE-MC4.

In this study, the effects of limited and excess phosphate on biomass content, oil content, fatty acid profile and the expression of three fatty acid desaturases in Messastrum gracile SE-MC4 were determined. It was found that total biomass (0.67-0.83 g L-1), oil content (30.99-38.08%) and the duration for cells to reach stationary phase (25-27 days) were not considerably affected by phosphate limitation. However, excess phosphate slightly reduced total biomass and oil content to 0.50 g L-1 and 25.36% respectively. The dominant fatty acids in M. gracile, pamitic acid (C16:0) and oleic acid (C18:1) which constitute more than 81% of the total fatty acids remained relatively high and constant across all phosphate concentrations. Reduction of phosphate concentration to 25% and below significantly increased total MUFA, whereas increasing phosphate concentration to ≥ 50% and ≥ 100% significantly increased total SFA and PUFA content respectively. The expression of omega-3 fatty acid desaturase (ω-3 FADi1, ω-3 FADi2) and omega-6 fatty acid desaturase (ω-6 FAD) was increased under phosphate limitation, especially at ≤ 12.5% phosphate, whereas levels of streoyl-ACP desaturase (SAD) transcripts were relatively unchanged across all phosphate concentrations. The first isoform of ω-3 FAD (ω-3 FADi) displayed a binary upregulation under limited (≤ 12.5%) and excess (200%) phosphate. The expression of ω-6 FAD, ω-3 FAD and SAD were inconsistent with the accumulation of oleic acid (C18:1), linoleic acid (C18:2) and alpha-linolenic acid (C18:3), suggesting that these genes may be regulated indirectly by phosphate availability via post-transcriptional or post-translational mechanisms.

Characterization of a Type-2 Diacylglycerol Acyltransferase from Haematococcus pluvialis Reveals Possible Allostery of the Recombinant Enzyme.

Haematococcus pluvialis is a green microalga used in the algal biotechnology industry that can accumulate considerable amounts of storage triacylglycerol (TAG) and astaxanthin, which is a high-value carotenoid with strong antioxidant activity, under stress conditions. Diacylglycerol acyltransferase (DGAT) catalyzes the last step of the acyl-CoA-dependent TAG biosynthesis and appears to represent a bottleneck in algal TAG formation. In this study, putative H. pluvialis DGAT2 cDNA (HpDGAT2A, B, D and E) were identified from a transcriptome database and were subjected to sequence-based in silico analyses. The coding sequences of HpDGAT2B, D, and E were then isolated and characterized through heterologous expression in a TAG-deficient Saccharomyces cerevisiae strain H1246. The expression of HpDGAT2D allowed the recovery of TAG biosynthesis in this yeast mutant, and further in vitro enzymatic assays confirmed that the recombinant HpDGAT2D possessed strong DGAT activity. Interestingly, the recombinant HpDGAT2D displayed sigmoidal kinetics in response to increasing acyl-CoA concentrations, which has not been reported in plant or algal DGAT2 in previous studies.

The unusual di-domain structure of Dunaliella salina glycerol-3-phosphate dehydrogenase enables direct conversion of dihydroxyacetone phosphate to glycerol.

Dunaliella has been extensively studied due to its intriguing adaptation to high salinity. Its di-domain glycerol-3-phosphate dehydrogenase (GPDH) isoform is likely to underlie the rapid production of the osmoprotectant glycerol. Here, we report the structure of the chimeric Dunaliella salina GPDH (DsGPDH) protein featuring a phosphoserine phosphatase-like domain fused to the canonical glycerol-3-phosphate (G3P) dehydrogenase domain. Biochemical assays confirm that DsGPDH can convert dihydroxyacetone phosphate (DHAP) directly to glycerol, whereas a separate phosphatase protein is required for this conversion process in most organisms. The structure of DsGPDH in complex with its substrate DHAP and co-factor nicotinamide adenine dinucleotide (NAD) allows the identification of the residues that form the active sites. Furthermore, the structure reveals an intriguing homotetramer form that likely contributes to the rapid biosynthesis of glycerol.

Expression of P-Type ATPases of Marine Green Microalga Dunaliella maritima under Hyperosmotic Salt Shock.

Partial sequences of P-type ATPases were cloned from the marine microalgae Dunaliella maritima, two putative H+-ATPases (DmHA1 and DmHA2) and two putative Ca2+-ATPases (DmCA1 and DmCA2). The probable functions of the cloned proteins were suggested on the basis of their primary structure similarity with the proteins whose functions have been already characterized. The transcriptional response of the cloned D. maritima ATPase genes to a sharp increase in the NaCl concentration in the culture medium (from 100 to 500 mM) was investigated by quantitative RT-PCR. Hyperosmotic salt shock led to a significant increase in the DmHA2 expression and to a slight increase in the DmCA2 expression, whereas the expression of the two other ATPases, DmHA1 and DmCA1, was decreased. These data indicate that the DmHA2 ATPase is involved in maintenance of ion homeostasis in D. maritima cells under hyperosmotic salt shock.

The bifunctional identification of both lycopene ß- and ε-cyclases from the lutein-rich Dunaliella bardawil.

The halophilic green alga Dunaliella bardawil FACHB-847 is rich in lutein and α-carotene, which has great potential for carotenoid production in open ponds. In this study, genes encoding lycopene ß- and ε-cyclases (DbLcyB and DbLcyE) from D. bardawil FACHB-847 were functionally identified by genetic complementation in E. coli. The bifunctional DbLcyB not only catalyzed the formation of both mono- and bi-cyclic ß-rings with a major ß-cyclase activity, but also possessed a weak ε-cyclase activity. In contrast, DbLcyE preferred to convert lycopene into monocyclic δ-carotene, and possessed a weak ß-monocyclase activity. Lutein and α-carotene were the prominent carotenoids in D. bardawil FACHB-847, which was in agreement with the result of genetic complementation of co-expression of DbLcyB and DbLcyE in E. coli with α-carotene as the prominent product. The bifunctional DbLcyB and DbLcyE may contribute to the high accumulation of α-carotene in D. bardawil FACHB-847. Interestingly, the accumulation of lutein in D. bardawil FACHB-847 was more sensitive to salt stress, while the accumulation of ß-carotene in D. salina CCAP 19/18 was induced by salt stress. In brief, the production of different carotenoid compositions from these two Dunaliella species can be induced by different growth conditions.

Gene Expression Analysis of Zeaxanthin Epoxidase from the Marine Microalga Dunaliella tertiolecta in Response to Light/Dark Cycle and Salinity.

Zeaxanthin is an important pigment in the photo-protection mechanism of microalgae. However, zeaxanthin epoxidase, an enzyme involved in the accumulation and conversion of zeaxanthin, has not been extensively studied in microalgae. In this work, we report the expression pattern of zeaxanthin epoxidase in Dunaliella tertiolecta (DtZEP) at different light and diverse salinity conditions. To confirm the responsiveness to light conditions, the ZEP expression pattern was investigated in photoperiodic (16 h of light and 8 h of dark) and continuous (24 h of light and 0 h of dark) light conditions. mRNA expression levels in photoperiodic conditions fluctuated along with the light/dark cycle, whereas those in continuous light remained unchanged. In varying salinity conditions, the highest mRNA and protein levels were detected in cells cultured in 1.5 M NaCl, and ZEP expression levels in cells shifted from 0.6 M NaCl to 1.5 M NaCl increased gradually. These results show that mRNA expression of DtZEP responds rapidly to the light/dark cycle or increased salinity, whereas changes in protein synthesis do not occur within a short period. Taken together, we show that DtZEP gene expression responds rapidly to light irradiation and hyperosmotic stress. In addition, ZEP expression patterns in light or salinity conditions are similar to those of higher plants, even though the habitat of D. tertiolecta is different.

Characterization and diverse evolution patterns of glycerol-3-phosphate dehydrogenase family genes in Dunaliella salina.

The glycerol-3-phosphate dehydrogenase (GPD) gene family plays a major role in glycerol synthesis and adaptation to abiotic stresses. Few studies on GPD family genes from the halotolerant algae Dunaliella salina are available. In this study, seven DsaGPD genes were identified by mining D. salina sequencing data. Among them, DsaGPD5 contained the canonical NAD+-GPD protein domain, called si-GPD. In comparison, DsaGPD1-4 not only contained the canonical NAD+-GPD domain but also a unique domain, the haloacid dehalogenase (HAD)-like superfamily domain, in their N-terminal region, called bi-GPD. DsaGPD6, 7 contained the FAD+-GPD domain. In the transient expression system, DsaGPD1, 3, 4 were found in the cytosol of Arabidopsis thaliana protoplast, DsaGPD2, 5 in the chloroplast, and DsaGPD6, 7 in the mitochondria. MEME analysis showed that six conserved motifs were present in both si-GPDs and bi-GPDs, whereas seven highly conserved motifs were only present in bi-GPDs. The quantitative real-time PCR results showed significant induction of the DsaGPD genes under abiotic stresses, indicating their tolerance-related role in D. salina. DsaGPD2 and DsaGPD5 may be the osmoregulator form and glyceride form in the chloroplast, respectively. The evolutionary forces acting on si-GPDs and bi-GPDs were different in the same organism: bi-GPDs were under purifying selection, while si-GPDs were mainly under positive selection. Furthermore, evolution of the N_HAD domain and C_GPD domain in bi-GPDs is highly correlated. In summary, this study characterizes DsaGPD gene family members and provides useful information for elucidating the salt tolerance mechanism in D. salina.

Rotary substates of mitochondrial ATP synthase reveal the basis of flexible F1-Fo coupling.

F1Fo-adenosine triphosphate (ATP) synthases make the energy of the proton-motive force available for energy-consuming processes in the cell. We determined the single-particle cryo-electron microscopy structure of active dimeric ATP synthase from mitochondria of Polytomella sp. at a resolution of 2.7 to 2.8 angstroms. Separation of 13 well-defined rotary substates by three-dimensional classification provides a detailed picture of the molecular motions that accompany c-ring rotation and result in ATP synthesis. Crucially, the F1 head rotates along with the central stalk and c-ring rotor for the first ~30° of each 120° primary rotary step to facilitate flexible coupling of the stoichiometrically mismatched F1 and Fo subcomplexes. Flexibility is mediated primarily by the interdomain hinge of the conserved OSCP subunit. A conserved metal ion in the proton access channel may synchronize c-ring protonation with rotation.

Effects of triethylamine on the expression patterns of two G3PDHs and lipid accumulation in Dunaliella tertiolecta.

Glycerol-3-phosphate (G3P) is the important precursors for triacylglycerol synthesis, while glycerol-3-phosphate dehydrogenase (GPDH) determines the formation of G3P. In this study, two GDPH genes, Dtgdp1 and Dtgdp2 were isolated and identified from Dunaliella tertiolecta. The full-length Dtgdp1 and Dtgdp2 CDS were 2016 bp and 2094 bp, which encoded two putative protein sequences of 671 and 697 amino acids with predicted molecular weights of 73.64 kDa and 76.73 kDa, respectively. DtGDP1 and DtGDP2 both had a close relationship with those of algal and higher plants. DtGDP1 shared two conserved superfamily (A1 and A2) and four signature motifs (I-IV), and the DtGDP2 showed six signature domains (from motif I to VI) and DAO_C conserved family. Our previous work showed that the triethylamine intervention could greatly increase the triacylglycerol content (up to 80%) of D. tertiolecta. This study aims to investigate the effect of triethylamine on GPDH expression. Results showed that, when treated by triethylamine at 100 ppm and 150 ppm, the expression levels of Dtgdp1 and Dtgpd2 were increased to 5.121- and 56.964-fold compared with the control, respectively. Triethylamine seemed to enhance lipid metabolic flow by inducing the expressions of Dtgdp1 and Dtgdp2 to increase the lipid content, which provides a new insight into the desired pathway of lipid synthesis in algae through genetic engineering.

Subunit Asa3 ensures the attachment of the peripheral stalk to the membrane sector of the dimeric ATP synthase of Polytomella sp.

The mitochondrial ATP synthase of Polytomella exhibits a peripheral stalk and a dimerization domain built by the Asa subunits, unique to chlorophycean algae. The topology of these subunits has been extensively studied. Here we explored the interactions of subunit Asa3 using Far Western blotting and subcomplex reconstitution, and found it associates with Asa1 and Asa8. We also identified the novel interactions Asa1-Asa2 and Asa1-Asa7. In silico analyses of Asa3 revealed that it adopts a HEAT repeat-like structure that points to its location within the enzyme based on the available 3D-map of the algal ATP synthase. We suggest that subunit Asa3 is instrumental in securing the attachment of the peripheral stalk to the membrane sector, thus stabilizing the dimeric mitochondrial ATP synthase.