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Under high light, oxygenic photosynthetic organisms avoid photodamage by thermally dissipating absorbed energy, which is called nonphotochemical quenching. In green algae, a chlorophyll and carotenoid-binding protein, light-harvesting complex stress-related (LHCSR3), detects excess energy via a pH drop and serves as a quenching site. Using a combined in vivo and in vitro approach, we investigated quenching within LHCSR3 from Chlamydomonas reinhardtii. In vitro two distinct quenching processes, individually controlled by pH and zeaxanthin, were identified within LHCSR3. The pH-dependent quenching was removed within a mutant LHCSR3 that lacks the residues that are protonated to sense the pH drop. Observation of quenching in zeaxanthin-enriched LHCSR3 even at neutral pH demonstrated zeaxanthin-dependent quenching, which also occurs in other light-harvesting complexes. Either pH- or zeaxanthin-dependent quenching prevented the formation of damaging reactive oxygen species, and thus the two quenching processes may together provide different induction and recovery kinetics for photoprotection in a changing environment., Competing Interests: JT, FP, RM, LZ, KB, EJ, SC, MB, GS No competing interests declared, (© 2021, Troiano et al.)

The zea1 mutant of marine microalga Dunaliella tertiolecta accumulates zeaxanthin under normal growth conditions, and its phenotype has been speculated to be related to zeaxanthin epoxidase (ZEP). In this study, we isolated the ZEP gene from both wild-type D. tertiolecta and the mutant. We found that the zea1 mutant has a point mutation of the 1337th nucleotide of the ZEP sequence (a change from guanine to adenine), resulting in a change of glycine to aspartate in a highly conserved region in the catalytic domain. Similar expression levels of ZEP mRNA and protein in both wild-type and zea1 were confirmed by using qRT-PCR and western blot analysis, respectively. Additionally, the enzyme activity analysis of ZEPs in the presence of cofactors showed that the inactivation of ZEP in zea1 was not caused by deficiency in the levels of cofactors. From the predicted three-dimensional ZEP structure of zea1 , we observed a conformational change on the substrate-binding site in the ZEP. A comparative analysis of the ZEP structures suggested that the conformational change induced by a single amino acid mutation might impact the interaction between the substrate and substrate-binding site, resulting in loss of zeaxanthin epoxidase function.

Nuclear-encoded light-harvesting chlorophyll- and carotenoid-binding proteins (LHCPs) are imported into the chloroplast and transported across the stroma to thylakoid membrane assembly sites by the chloroplast signal recognition particle (CpSRP) pathway. The LHCP translocation defect (LTD) protein is essential for the delivery of imported LHCPs to the CpSRP pathway in Arabidopsis. However, the function of the LTD protein in Chlamydomonas reinhardtii has not been investigated. Here, we generated a C. reinhardtii ltd (Crltd) knockout mutant by using CRISPR-Cas9, a new target-specific knockout technology. The Crltd1 mutant showed a low chlorophyll content per cell with an unusual increase in appressed thylakoid membranes and enlarged cytosolic vacuoles. Profiling of thylakoid membrane proteins in the Crltd1 mutant showed a more severe reduction in the levels of photosystem I (PSI) core proteins and absence of functional LHCI compared with those of photosystem II, resulting in a much smaller PSI pool size and diminished chlorophyll antenna size. The lack of CrLTD did not prevent photoautotrophic growth of the cells. These results are substantially different from those for Arabidopsis ltd null mutant, indicating LTD function in LHCP delivery and PSI assembly may not be as stringent in C. reinhardtii as it is in higher plants.

Promoter of the light-inducible protein gene (LIP) of Dunaliella was recently isolated in our laboratory. The aim of this work is to find the light-inducible motif in the Dunaliella LIP promoter and verify its regulatory motif with a Gaussia luciferase reporter gene transformed in Chlamydomonas reinhardtii. 400 bp upstream to the translational start site of the Dunaliella LIP gene was gradually truncated and analyzed for the luciferase expression. Furthermore, this promoter comprising duplicated or triplicated light-responsive motifs was tested for its augmentation of light response. Two putative light-responsive motifs, GT-1 binding motif and sequences over-represented in light-repressed promoters (SORLIP) located in the 200 bp LIP promoter fragment were analyzed for their light responsibility. It is turned out that SORLIP was responsible for the light-inducible activity. With the copy number of SORLIP up to three showed stronger high light response compared with the native LIP promoter fragment. Therefore, we found a light-responsive DNA motif operating in Chlamydomonas and confirm a synthetic promoter including this motif displayed light inducibility in heterologously transformed green algae for the first time. This light-inducible expression system will be applied to various area of algal research including algal biotechnology., (Copyright © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

To better understand the gene expression of the cold-adapted polar diatom, we conducted a survey of the Chaetoceros neogracile transcriptome by cDNA sequencing and expression of interested cDNAs from the Antarctic diatom. A non-normalized cDNA library was constructed from the C. neogracile, and a total of 2,500 cDNAs were sequenced to generate 1,881 high-quality expressed sequence tags (ESTs) (accession numbers EL620615-EL622495). Based on their clustering, we identified 154 unique clusters comprising 342 ESTs. The remaining 1,540 ESTs did not cluster. The number of unique genes identified in the data set is thus estimated to be 1,694. Taking advantage of various tools and databases, putative functions were assigned to 939 (55.4%) of these genes. Of the remaining 540 (31.9%) unknown sequences, 215 (12.7%) appeared to be C. neogracile-specific since they lacked any significant sequence similarity to any sequence available in the public databases. C. neogracile consisted of a relatively high percentage of genes involved in metabolism, genetic information processing, cellular processes, defense or stress resistance, photosynthesis, structure, and signal transduction. From the ESTs, the expression of these putative C. neogracile genes was investigated: fucoxanthin chlorophyll (chl) a,c-binding protein (FCP), ascorbate peroxidase (ASP), and heat-shock protein 90 (HSP90). The abundance of ASP and HSP90 changed substantially in response to different culture conditions, indicating the possible regulation of these genes in C. neogracile.

The unicellular green alga Haematococcus pluvialis (Volvocales) is known for the ketocarotenoid astaxanthin (3, 3'-dihydroxy-beta, beta-carotene-4, 4'-dione) accumulation, which is induced under unfavorable culture conditions. In this work, we used cDNA microarray analysis to screen differentially expressed genes in H. pluvialis under astaxanthin-inductive culture conditions, such as combination of cell exposure to high irradiance and nutrient deprivation. Among the 965 genes in the cDNA array, there are 144 genes exhibiting differential expression (twofold changes) under these conditions. A significant decrease in the expression of photosynthesis-related genes was shown in astaxanthin-accumulating cells (red cells). Defense- or stress-related genes and signal transduction genes were also induced in the red cells. A comparison of microarray and real-time PCR analysis showed good correlation between the differentially expressed genes by the two methods. Our results indicate that the cDNA microarray approach, as employed in this work, can be relied upon and used to monitor gene expression profiles in H. pluvialis. In addition, the genes that were differentially expressed during astaxanthin induction are suitable candidates for further study and can be used as tools for dissecting the molecular mechanism of this unique pigment accumulation process in the green alga H. pluvialis.

The unicellular green alga Dunaliella salina is an attractive model organism for studying photoacclimation responses and the photosystem II (PSII) damage and repair process in the photosynthetic apparatus. Irradiance during cell growth defines both the photoacclimation and the PSII repair status of the cells. To identify genes specific to these processes, a cDNA library was created from irradiance-stressed D. salina. From the cDNA library, 1112 randomly selected expressed sequence tags (ESTs) were analyzed. Because ESTs constitute the expressed part of the genome, the strategy of randomly sequencing cDNA clones at their 5'-ends allowed us to obtain information about the transcript level of numerous genes in light-stressed D. salina. The results of a BLASTX search performed on the obtained total set of ESTs showed that approximately 1% of the ESTs could be assigned to genes coding for proteins that are known to be up-regulated in response to high-light stress. Specifically, after 48 h of high-light exposure of the cells, an increase in the expression level of antioxidant genes, such as Fe-SOD and APX, was observed, as well as elevated levels of the Cbr transcript, a light-harvesting Chl-protein homolog. Further, the ATP-dependent Clp protease gene was also up-regulated in D. salina cells after 48 h of exposure to high light. The results provide initial insight into the global gene regulation process in response to irradiance.

Microalgae are versatile organisms capable of converting CO2, H2O, and sunlight into fuel and chemicals for domestic and industrial consumption. Thus, genetic modifications of microalgae for enhancing photosynthetic productivity, and biomass and bio-products generation are crucial for both academic and industrial applications. However, targeted mutagenesis in microalgae with CRISPR-Cas9 is limited. Here we report, a one-step transformation of Chlamydomonas reinhardtii by the DNA-free CRISPR-Cas9 method rather than plasmids that encode Cas9 and guide RNAs. Outcome was the sequential CpFTSY and ZEP two-gene knockout and the generation of a strain constitutively producing zeaxanthin and showing improved photosynthetic productivity.

Many microorganisms in Antarctica survive in the cold environment there by producing ice-binding proteins (IBPs) to control the growth of ice around them. An IBP from the Antarctic freshwater microalga, Chloromonas sp., was identified and characterized. The length of the Chloromonas sp. IBP (ChloroIBP) gene was 3.2 kb with 12 exons, and the molecular weight of the protein deduced from the ChloroIBP cDNA was 34.0 kDa. Expression of the ChloroIBP gene was up- and down-regulated by freezing and warming conditions, respectively. Western blot analysis revealed that native ChloroIBP was secreted into the culture medium. This protein has fifteen cysteines and is extensively disulfide bonded as shown by in-gel mobility shifts between oxidizing and reducing conditions. The open-reading frame of ChloroIBP was cloned and over-expressed in Escherichia coli to investigate the IBP’s biochemical characteristics. Recombinant ChloroIBP produced as a fusion protein with thioredoxin was purified by affinity chromatography and formed single ice crystals of a dendritic shape with a thermal hysteresis activity of 0.4±0.02°C at a concentration of 5 mg/ml. In silico structural modeling indicated that the three-dimensional structure of ChloroIBP was that of a right-handed β-helix. Site-directed mutagenesis of ChloroIBP showed that a conserved region of six parallel T-X-T motifs on the β-2 face was the ice-binding region, as predicted from the model. In addition to disulfide bonding, hydrophobic interactions between inward-pointing residues on the β-1 and β-2 faces, in the region of ice-binding motifs, were crucial to maintaining the structural conformation of ice-binding site and the ice-binding activity of ChloroIBP. [ABSTRACT FROM AUTHOR]