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The expression of transgenes in the nucleus is an attractive alternative for the expression of recombinant proteins in the green microalga Chlamydomonas reinhardtii. For this purpose, a strong inducible promoter that allows protein accumulation without possible negative effects on cell multiplication and biomass accumulation is desirable. A previous study at our laboratory identified that the CrGPDH3 gene from C. reinhardtii was inducible under NaCl treatments. In this study, we cloned and characterized a 3012 bp sequence upstream of the start codon of the CrGPDH3 gene, including the 285 bp 5' untranslated region. This region was identified as the full-length promoter and named PromA (- 2727 to + 285). Deletion analysis of PromA using GUSPlus as a reporter gene enabled us to identify PromC (- 653 to + 285) as the core promoter, displaying basal expression. A region named RIA1 (- 2727 to - 1672) was suggested to contain the NaCl response elements. Moreover, deletion analysis of RIA1 enabled us to identify a region of 577 bp named RIA3 (- 2727 to - 2150) that, when cloned upstream of PromC, was able to drive the expression of GUSPlus in response to 5 and 100 mM NaCl, and 100 mM KCl, similar to the native CrGPDH3 promoter. These results expand our understanding of the transcriptional mechanism of CrGPDH3 and clearly show that CrGPDH3 promoter and its chimeric forms are highly salt-inducible and can be used as inducible promoters for the overexpression of transgenes in C. reinhardtii.

Cyclooxygenases are responsible for the production of prostaglandin H 2 (PGH 2 ) from arachidonic acid. PGH 2 can be converted into some bioactive prostaglandins, including prostaglandin F 2α (PGF 2α ), a potent chemical messenger used as a biological regulator in the fields of obstetrics and gynecology. The chemical messenger PGF 2α has been industrially produced by chemical synthesis. To develop a biotechnological process, in which PGF 2α can be produced by a microorganism, we transformed an oleaginous fungus, Mortierella alpina 1S-4, rich in triacylglycerol consisting of arachidonic acid using a cyclooxygenase gene from a red alga, Gracilaria vermiculophylla. PGF 2α was accumulated not only in the mycelia of the transformants but also in the extracellular medium. After 12 days of cultivation approximately 860 ng/g and 6421 µg/L of PGF 2α were accumulated in mycelia and the extracellular medium, respectively. The results could facilitate the development of novel fermentative methods for the production of prostanoids using an oleaginous fungus.

The unicellular alga Chlamydomonas ( Chlamydomonas reinhardtii ) exhibits oriented movement responses (phototaxis) to light over more than three log units of intensity. Phototaxis thus depends on the cell's ability to adjust the sensitivity of its photoreceptors to ambient light conditions. In Chlamydomonas , the photoreceptors for phototaxis are the channelrhodopsins (ChR)1 and ChR2; these light-gated cation channels are located in the plasma membrane. Although ChRs are widely used in optogenetic studies, little is known about ChR signaling in algae. We characterized the in vivo phosphorylation of ChR1. Its reversible phosphorylation occurred within seconds as a graded response to changes in the light intensity and ionic composition of the medium and depended on an elevated cytosolic Ca 2+ concentration. Changes in the phototactic sign were accompanied by alterations in the phosphorylation status of ChR1. Furthermore, compared with the wild type, a permanently negative phototactic mutant required higher light intensities to evoke ChR1 phosphorylation. C-terminal truncation of ChR1 disturbed its reversible phosphorylation, whereas it was normal in ChR2-knockout and eyespot-assembly mutants. The identification of phosphosites in regions important for ChR1 function points to their potential regulatory role(s). We propose that multiple ChR1 phosphorylation, regulated via a Ca 2+ -based feedback loop, is an important component in the adaptation of phototactic sensitivity in Chlamydomonas ., (© 2019 American Society of Plant Biologists. All rights reserved.)

Large GTPases of the Dynamin Related Proteins (DRP) family shape lipid bilayers through membrane fission or fusion processes. Despite the highly organized photosynthetic membranes of thylakoids, a single DRP is known to be targeted inside the chloroplast. Fzl from the land plant Arabidopsis thaliana is inserted in the inner envelope and thylakoid membranes to regulate their morphology. Fzl may promote the fusion of thylakoids but this remains to be proven. Moreover, the physiological requirement for fusing thylakoids is currently unknown. Here, we find that the unicellular microalga Chlamydomonas reinhardtii encodes an Fzl ortholog (CrFzl) that is localized in the chloroplast where it is soluble. To explore its function, the CRISPR/Cas9 technology was employed to generate multiple CrFzl knock out strains. Phenotypic analyzes revealed a specific requirement of CrFzl for survival upon light stress. Consistent with this, strong irradiance lead to increased photoinhibition of photosynthesis in mutant cells. Fluorescence and electron microscopy analysis demonstrated that upon exposure to high light, CrFzl mutants show defects in chloroplast morphology but also large cytosolic vacuoles in close contact with the plastid. We further observe that strong irradiance induces an increased recruitment of the DRP to thylakoid membranes. Most importantly, we show that CrFzl is required for the fusion of thylakoids during mating. Together, our results suggest that thylakoids fusion may be necessary for resistance to light stress., Competing Interests: The authors have declared that no competing interests exist.

Background: Several abundant proteins have been identified in lipid body of an alkenone-producing marine haptophyte alga Tisochrysis lutea. The gene expression patterns of these proteins were investigated to better understand their roles in alkenone biosynthesis. For this purpose, T. lutea was first cultured in nitrogen-sufficient medium for biomass production and then shifted to nitrogen-deprived medium to induce lipid body formation., Results: There were remarkable increases in the volume of alkenone body (AB) and alkenone content in the alga after they were exposed to nitrogen depletion medium. Relative mRNA levels of the genes coding for the identified proteins V-ATPase subunit V A , V-ATPase subunit V d , hypothetical protein EMIHUDRAFT_465,517, coccolith scale associated protein-1, cycloartenol-c-24-methyltransferase 1-like and SPFH domain-containing protein were investigated over the culture period. RT-PCR data showed that the expression of all these genes except the gene coding for SPFH domain-containing protein was up-regulated during the transition period from nitrogen-sufficient to nitrogen-deficient medium. Among them, the expression of the coccolith scale associated protein-1 gene was up-regulated 50-650 folds. These up-regulations were consistent with the increased alkenone production in nitrogen-deprived medium, suggesting that these proteins are involved in alkenone biosynthesis in T. lutea., Conclusions: Expression analysis of the lipoprotein genes suggests that five out of the six genes are up-regulated and are therefore likely to code for the identified lipoproteins associated with alkenone biosynthesis in T. lutea. These data would help better understand alkenone metabolism and engineer for improved biofuel production in T. lutea.

Motile cilia are microtubule-based organelles that play important roles in most eukaryotes. Although axonemal microtubules are sufficiently stable to withstand their beating motion, it remains unknown how they are stabilized while serving as tracks for axonemal dyneins. To address this question, we have identified two uncharacterized proteins, FAP45 and FAP52, as microtubule inner proteins (MIPs) in Chlamydomonas. These proteins are conserved among eukaryotes with motile cilia. Using cryo-electron tomography (cryo-ET) and high-speed atomic force microscopy (HS-AFM), we show that lack of these proteins leads to a loss of inner protrusions in B-tubules and less stable microtubules. These protrusions are located near the inner junctions of doublet microtubules and lack of both FAP52 and a known inner junction protein FAP20 results in detachment of the B-tubule from the A-tubule, as well as flagellar shortening. These results demonstrate that FAP45 and FAP52 bind to the inside of microtubules and stabilize ciliary axonemes.

Photosynthetic organisms prevent oxidative stress from light energy absorbed in excess through several photoprotective mechanisms. A major component is thermal dissipation of chlorophyll singlet excited states and is called nonphotochemical quenching (NPQ). NPQ is catalyzed in green algae by protein subunits called LHCSRs (Light Harvesting Complex Stress Related), homologous to the Light Harvesting Complexes (LHC), constituting the antenna system of both photosystem I (PSI) and PSII. We investigated the role of LHCSR1 and LHCSR3 in NPQ activation to verify whether these proteins are involved in thermal dissipation of PSI excitation energy, in addition to their well-known effect on PSII. To this aim, we measured the fluorescence emitted at 77 K by whole cells in a quenched or unquenched state, using green fluorescence protein as the internal standard. We show that NPQ activation by high light treatment in Chlamydomonas reinhardtii leads to energy quenching in both PSI and PSII antenna systems. By analyzing quenching properties of mutants affected on the expression of LHCSR1 or LHCSR3 gene products and/or state 1-state 2 transitions or zeaxanthin accumulation, namely, npq4 , stt7 , stt7 npq4 , npq4 lhcsr1 , lhcsr3 -complemented npq4 lhcsr1 and npq1 , we showed that PSI undergoes NPQ through quenching of the associated LHCII antenna. This quenching event is fast-reversible on switching the light off, is mainly related to LHCSR3 activity, and is dependent on thylakoid luminal pH. Moreover, PSI quenching could also be observed in the absence of zeaxanthin or STT7 kinase activity., Competing Interests: The authors declare no conflict of interest.

Two genes of the RACK1 homolog from the photosynthetic dinoflagellate Symbiodinium microadriaticum ssp. microadriaticum (SmicRACK1), termed SmicRACK1A and SmicRACK1B, were found tandemly arrayed and displayed a single synonymous substitution (T/C) encoding threonine. They included two exons of 942 bp each, encoding 313 amino acids with seven WD-40 repeats and two PKC-binding motifs. The protein theoretical mass and pI were 34,200 Da and 5.9, respectively. SmicRACK1 showed maximum identities with RACK1 homologs at the amino acid and nucleotide level, respectively, of 92 and 84% with S. minutum, and phylogenetic analysis revealed clustered related RACK1 sequences from the marine dinoflagellates S. minutum, Heterocapsa triquetra, Karenia brevis, and Alexandrium tamarense. Interestingly, light-dependent regulatory elements were found both within the 282 bp SmicRACK1A promotor sequence, and within an intergenic sequence of 359 nucleotides that separated both genes, which strongly suggest light-related functions. This was further supported by mRNA accumulation analysis, which fluctuated along the light and dark phases of the growth cycle showing maximum specific peaks under either condition. Finally, qRT-PCR analysis revealed differential SmicRACK1 mRNA accumulation with maxima at 6 and 20 d of culture. Our SmicRACK1 characterization suggests roles in active growth and proliferation, as well as light/dark cycle regulation in S. microadriaticum., (© 2018 International Society of Protistologists.)

Galdieria sulphuraria is a unicellular red alga that lives in hot, acidic, toxic metal-rich, volcanic environments, where few other organisms survive. Its genome harbors up to 5% of genes that were most likely acquired through horizontal gene transfer. These genes probably contributed to G.sulphuraria's adaptation to its extreme habitats, resulting in today's polyextremophilic traits. Here, we applied RNA-sequencing to obtain insights into the acclimation of a thermophilic organism towards temperatures below its growth optimum and to study how horizontally acquired genes contribute to cold acclimation. A decrease in growth temperature from 42�C/46�C to 28�C resulted in an upregulation of ribosome biosynthesis, while excreted proteins, probably components of the cell wall, were downregulated. Photosynthesis was suppressed at cold temperatures, and transcript abundances indicated that C-metabolism switched from gluconeogenesis to glycogen degradation. Folate cycle and S-adenosylmethionine cycle (one-carbon metabolism) were transcriptionally upregulated, probably to drive the biosynthesis of betaine. All these cold-induced changes in gene expression were reversible upon return to optimal growth temperature. Numerous genes acquired by horizontal gene transfer displayed temperature-dependent expression changes, indicating that these genes contributed to adaptive evolution in G.sulphuraria., (� The Author(s) 2018. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Carbonic anhydrases (CAs) are a class of zinc-containing metalloenzymes that can reversibly catalyse the hydration reaction of carbon dioxide. Antarctic algae are the most critical component of the Antarctic ecosystem; algae can enter the carbon cycle food chain by fixing carbon dioxide from the air. In this study, the complete open reading frames (ORFs) of CA1 (GenBank ID KY826431), CA2 (GenBank ID KY826432), and CA3 (GenBank ID KY826433), encoding CAs in the Antarctic ice microalga Chlamydomonas. sp. ICE-L, were successfully cloned using reverse transcription-polymerase chain reaction (RT-PCR). In addition, the expression patterns of CAs under blue light, under UV light, and in the dark were determined by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). The CA1, CA2, and CA3 ORFs encode proteins of 376, 430, and 419 amino acids, respectively. Phylogenetic analysis revealed that all amino acid sequences showed high homology with those of C. sp. ICE-L. There are six types of algal CAs; we hypothesised that the CAs studied here are most likely α-CAs. Expression analysis showed that the transcription level of the CAs was influenced by both UV light and blue light. These findings provide additional insight into the molecular mechanisms of CAs and will accelerate the development of CAs for applications in agriculture and environmental governance.

Intraflagellar transport (IFT), which is essential for the formation and function of cilia in most organisms, is the trafficking of IFT trains (i.e. assemblies of IFT particles) that carry cargo within the cilium. Defects in IFT cause several human diseases. IFT trains contain the complexes IFT-A and IFT-B. To dissect the functions of these complexes, we studied a Chlamydomonas mutant that is null for the IFT-A protein IFT140. The mutation had no effect on IFT-B but destabilized IFT-A, preventing flagella assembly. Therefore, IFT-A assembly requires IFT140. Truncated IFT140, which lacks the N-terminal WD repeats of the protein, partially rescued IFT and supported formation of half-length flagella that contained normal levels of IFT-B but greatly reduced amounts of IFT-A. The axonemes of these flagella had normal ultrastructure and, as investigated by SDS-PAGE, normal composition. However, composition of the flagellar 'membrane+matrix' was abnormal. Analysis of the latter fraction by mass spectrometry revealed decreases in small GTPases, lipid-anchored proteins and cell signaling proteins. Thus, IFT-A is specialized for the import of membrane-associated proteins. Abnormal levels of the latter are likely to account for the multiple phenotypes of patients with defects in IFT140.This article has an associated First Person interview with the first author of the paper., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)

The progression of the cell cycle in green algae dividing by multiple fission is, under otherwise unlimited conditions, affected by the growth rate, set by a combination of light intensity and temperature. In this study, we compared the cell cycle characteristics of Desmodesmus quadricauda at 20 °C or 30 °C and upon shifts between these two temperatures. The duration of the cell cycle in cells grown under continuous illumination at 20 °C was more than double that at 30 °C, suggesting that it was set directly by the growth rate. Similarly, the amounts of DNA, RNA, and bulk protein content per cell at 20 °C were approximately double those of cells grown at the higher temperature. For the shift experiments, cells grown at either 20 °C or 30 °C were transferred to darkness to prevent further growth, and then cultivated at the same or the other temperature. Upon transfer to the lower temperature, fewer nuclei and daughter cells were produced, and not all cells were able to finish the cell cycle by division, remaining multinuclear. Correspondingly, cells placed in the dark at the higher temperature divided faster into more daughter cells than the control cells. These differences correlated with shifts in the preceding cyclin-dependent kinase activity, suggesting that cell cycle progression was not related to growth rate or cell biomass but correlated with cyclin-dependent kinase activity.

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., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Total lipids and docosahexaenoic acid (DHA) production by a Chilean isolated thraustochytrid were evaluated under different growth conditions in shake flasks. The analyzed strain was identified as Thraustochytrium striatum according to an 18S rRNA gene sequence analysis. The strain (T. striatum AL16) showed negligible growth in media prepared with artificial seawater at concentrations lower than 50% v/v and pH lower than 5. Maltose and starch were better carbon sources for growth than glucose. DHA content of the biomass grown with maltose (60 g L -1 ) was doubled by increasing the agitation rate from 150 to 250 rpm. The DHA (0.8-6%) and eicosapentaenoic acid (0.2-21%) content in the total lipids varied depending on culture conditions and culture age. Lipid and DHA concentration increased (up to 5 g L -1 and 66 mg L -1 , respectively) by regularly feeding the culture with a concentrated starch solution. Carotenoid accumulation was detected in cells grown with maltose or starch. Contrasting conditions of starch and glucose cultures were selected for comparative proteomics. Total protein extracts were separated by two-dimensional gel electrophoresis; 25 spots were identified using ESI-MS/MS. A protein database (143,006 entries) for proteomic interrogation was generated using de novo assembling of Thraustochytrium sp. LLF1b - MMETSP0199_2 transcriptome; 18 proteins differentially expressed were identified. Three ATP synthases were differentially accumulated in cultures with glucose, whereas malate dehydrogenase was more abundant in cells cultured with starch.

Red algae (Rhodophyta) and land plants belong to the monophyletic clade Archaeplastida, and taxa of both groups are rich producers of terpene secondary metabolites. The terpene carbon skeletons of land plants are made by two types of terpene synthases: typical plant terpene synthases and microbial-type terpene synthases (MTPSLs); however, terpene biosynthesis in red algae is poorly understood. By systematic sequence analysis of seven genomes and 34 transcriptomes of red algae, MTPSL homologs were identified within one genome and two transcriptomes, whereas no homolog of typical plant terpene synthase genes was found. Phylogenetic analysis showed that red algae MTPSLs group with bacterial terpene synthases. Analysis of the genome assembly and characterization of neighboring genes demonstrated red algal MTPSLs to be bona fide red algal genes and not microbial contaminants. MTPSL genes from Porphyridium purpureum and Erythrolobus australicus were characterized via heterologous expression in Escherichia coli and demonstrated to have sesquiterpene synthase activities. We detected a number of volatile sesquiterpenes in the headspace of P. purpureum and E. australicus cultures, most identical to the in vitro products of the respective MTPSLs. Expression of the MTPSL gene in P. purpureum was found to be induced by methyl jasmonate, suggesting a role for this gene in host defense. In summary, this study indicates that the formation of terpene carbon skeletons in red algae is carried out by MTPSLs that are phylogenetically unrelated to typical plant terpene synthases and most likely originated in Rhodophyta via horizontal gene transfer from bacteria., (© 2019 American Society of Plant Biologists. All Rights Reserved.)

A cysteine protease belonging to peptidase C1A superfamily from the eukaryotic, symbiotic dinoflagellate, Symbiodinium sp. strain KB8, was characterized. The protease was purified to near homogeneity (566-fold) by (NH4)2SO4 fractionation, ultrafiltration, and column chromatography using a fluorescent peptide, butyloxycarbonyl-Val-Leu-Lys-4-methylcoumaryl-7-amide (Boc-VLK-MCA), as a substrate for assay purposes. The enzyme was termed VLKP (VLK protease), and its activity was strongly inhibited by cysteine protease inhibitors and activated by reducing agents. Based on the results for the amino acid sequence determined by liquid chromatography-coupled tandem mass spectrometry, a cDNA encoding VLKP was synthesized. VLKP was classified into the peptidase C1A superfamily of cysteine proteases (C1AP). The predicted amino acid sequence of VLKP indicated a tandem array of highly conserved precursors of C1AP with a molecular mass of approximately 71 kDa. The results of gel-filtration chromatography and SDS-PAGE suggested that VLKP exists as a monomer of 31-32 kDa, indicating that the tandem array is likely divided into two mass-equivalent halves that undergo equivalent posttranslational modifications. The VLKP precursor contains an inhibitor prodomain that might become activated after acidic autoprocessing at approximately pH 4. Both purified and recombinant VLKPs had a similar substrate specificity and kinetic parameters for common C1AP substrates. Most C1APs reside in acidic organelles such as the vacuole and lysosomes, and indeed VLKP was most active at pH 4.5. Since VLKP exhibited maximum activity during the late logarithmic growth phase, these attributes suggest that, VLKP is involved in the metabolism of proteins in acidic organelles., Competing Interests: The authors have declared that no competing interests exist.

Small heat shock proteins (sHSPs) are found in all three domains of life (Bacteria, Archaea, and Eukarya) and play a critical role in protecting organisms from a range of environmental stresses. However, little is known about their physiological functions in red algae. Therefore, we characterized the sHSPs (PysHSPs) in the red macroalga Pyropia yezoensis, which inhabits the upper intertidal zone where it experiences fluctuating stressful environmental conditions on a daily and seasonal basis, and examined their expression profiles at different developmental stages and under varying environmental conditions. We identified five PysHSPs (PysHSP18.8, 19.1, 19.2, 19.5, and 25.8). Real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis showed that expression of the genes PysHSP18.8, PysHSP19.5, and PysHSP25.8 was repressed at all the developmental stages under normal conditions, whereas PysHSP19.1 and PysHSP19.2 were overexpressed in mature gametophytes and sporophytes. Exposure of the gametophytes to high temperature, oxidative stress, or copper significantly increased the mRNA transcript levels of all the five genes, while exogenous application of the ethylene precursor 1-aminocylopropane-1-carboxylic acid (ACC) significantly increased the expression levels of PysHSP19.2, PysHSP19.5, and PysHSP25.8. These findings will help to further our understanding of the role of PysHSP genes and provide clues about how Pyropia species can adapt to the stressful conditions encountered in the upper intertidal zone during their life cycle.

HD-Zip proteins are unique to plants, and contain a homeodomain closely linked to a leucine zipper motif, which are involved in dimerization and DNA binding. Based on homology in the HD-Zip domain, gene structure and the presence of additional motifs, HD-Zips are divided into four families, HD-Zip I⁻IV. Phylogenetic analysis of HD-Zip genes using transcriptomic and genomic datasets from a wide range of plant species indicate that the HD-Zip protein class was already present in green algae. Later, HD-Zips experienced multiple duplication events that promoted neo- and sub-functionalizations. HD-Zip proteins are known to control key developmental and environmental responses, and a growing body of evidence indicates a strict link between members of the HD-Zip II and III families and the auxin machineries. Interactions of HD-Zip proteins with other hormones such as brassinolide and cytokinin have also been described. More recent data indicate that members of different HD-Zip families are directly involved in the regulation of abscisic acid (ABA) homeostasis and signaling. Considering the fundamental role of specific HD-Zip proteins in the control of key developmental pathways and in the cross-talk between auxin and cytokinin, a relevant role of these factors in adjusting plant growth and development to changing environment is emerging.

Background: The green algae Chlamydomonas reinhardtii and Volvox carteri are important models for studying light perception and response, expressing many different photoreceptors. More than 10 opsins were reported in C. reinhardtii, yet only two-the channelrhodopsins-were functionally characterized. Characterization of new opsins would help to understand the green algae photobiology and to develop new tools for optogenetics., Results: Here we report the characterization of a novel opsin family from these green algae: light-inhibited guanylyl cyclases regulated through a two-component-like phosphoryl transfer, called "two-component cyclase opsins" (2c-Cyclops). We prove the existence of such opsins in C. reinhardtii and V. carteri and show that they have cytosolic N- and C-termini, implying an eight-transmembrane helix structure. We also demonstrate that cGMP production is both light-inhibited and ATP-dependent. The cyclase activity of Cr2c-Cyclop1 is kept functional by the ongoing phosphorylation and phosphoryl transfer from the histidine kinase to the response regulator in the dark, proven by mutagenesis. Absorption of a photon inhibits the cyclase activity, most likely by inhibiting the phosphoryl transfer. Overexpression of Vc2c-Cyclop1 protein in V. carteri leads to significantly increased cGMP levels, demonstrating guanylyl cyclase activity of Vc2c-Cyclop1 in vivo. Live cell imaging of YFP-tagged Vc2c-Cyclop1 in V. carteri revealed a development-dependent, layer-like structure at the immediate periphery of the nucleus and intense spots in the cell periphery., Conclusions: Cr2c-Cyclop1 and Vc2c-Cyclop1 are light-inhibited and ATP-dependent guanylyl cyclases with an unusual eight-transmembrane helix structure of the type I opsin domain which we propose to classify as type Ib, in contrast to the 7 TM type Ia opsins. Overexpression of Vc2c-Cyclop1 protein in V. carteri led to a significant increase of cGMP, demonstrating enzyme functionality in the organism of origin. Fluorescent live cell imaging revealed that Vc2c-Cyclop1 is located in the periphery of the nucleus and in confined areas at the cell periphery.

Red algae are the oldest identifiable multicellular eukaryotes, with a fossil record dating back more than a billion years. During that time two major rhodophyte lineages, bangiophytes and florideophytes, have evolved varied levels of morphological complexity. These two groups are distinguished, in part, by different patterns of multicellular development, with florideophytes exhibiting a far greater diversity of morphologies. Interestingly, during their long evolutionary history, there is no record of a rhodophyte achieving the kinds of cellular and tissue-specific differentiation present in other multicellular algal lineages. To date, the genetic underpinnings of unique aspects of red algal development are largely unexplored; however, they must reflect the complements and patterns of expression of key regulatory genes. Here we report comparative evolutionary and gene expression analyses of core subunits of the SWI/SNF chromatin-remodeling complex, which is implicated in cell differentiation and developmental regulation in more well studied multicellular groups. Our results suggest that a single, canonical SWI/SNF complex was present in the rhodophyte ancestor, with gene duplications and evolutionary diversification of SWI/SNF subunits accompanying the evolution of multicellularity in the common ancestor of bangiophytes and florideophytes. Differences in how SWI/SNF chromatin remodeling evolved subsequently, in particular gene losses and more rapid divergence of SWI3 and SNF5 in bangiophytes, could help to explain why they exhibit a more limited range of morphological complexity than their florideophyte cousins., (© 2018 Phycological Society of America.)

The marine diatom Phaeodactylum tricornutum is attractive for basic and applied diatom research. We isolated putative endogenous gene promoters derived from genes that are highly expressed in P. tricornutum: the fucoxanthin chlorophyll a/c-binding protein (FCP) C gene, the vacuolar ATP synthase 16-kDa proteolipid subunit (V-ATPase C) gene, the clumping factor A gene and the solute carrier family 34 member 2 gene. Five putative promoter regions were isolated, linked to an antibiotic resistance gene (Sh ble) and transformed into P. tricornutum. Using quantitative RT-PCR, the promoter activities in the transformants were analyzed and compared to that of the diatom endogenous gene promoter, the FCP A gene promoter which has been used for the transformation of P. tricornutum. Among the five isolated potential promoters, the activity of the V-ATPase C gene promoter was approximately 2.73 times higher than that of the FCP A gene promoter. The V-ATPase C gene promoter drove the expression of Sh ble mRNA transcripts under both light and dark conditions at the stationary phase. These results suggest that the V-ATPase C gene promoter is a novel tool for the genetic engineering of P. tricornutum., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Microalgae contribute significantly to carbon fixation on Earth. Global warming influences their physiology and growth rates. To understand algal short-term acclimation and adaptation to changes in ambient temperature, it is essential to identify and characterize the molecular components that sense small temperature changes as well as the downstream signaling networks and physiological responses. Here, we used the green biflagellate alga Chlamydomonas reinhardtii as a model system in which to study responses to temperature. We report that an RNA recognition motif (RRM)-containing RNA-binding protein, Musashi, occurs in 25 putative splice variants. These variants bear one, two, and three RRM domains or even lack RRM domains. The most abundant Musashi variant, 12, with a molecular mass of 60 kD, interacts with two clock-relevant members of RNA metabolism, the subunit C3 of the RNA-binding protein CHLAMY1 and the 5'-3' exoribonuclease XRN1. These proteins are able to integrate temperature information by up- or down-regulation of their protein levels in cells grown at low (18°C) or high (28°C) temperature. We further show that the 60-kD Musashi variants with three RRM domains can bind to (UG) 7 repeat-containing RNAs and are up-regulated in cells grown at a higher temperature during early night. Intriguingly, the 60-kD Musashi variant 12, as well as C3 and XRN1, confer thermal acclimation to C. reinhardtii , as shown with mutant lines. Our data suggest that these three proteins of the RNA metabolism machinery are key members of the thermal signaling network in C. reinhardtii ., (© 2018 American Society of Plant Biologists. All rights reserved.)

Photozipper (PZ) is a light-activatable basic leucine zipper (bZIP) protein composed of a bZIP domain and a light-oxygen-voltage-sensing domain of aureochrome-1. Blue light induces dimerization and subsequently increases the affinity of PZ for the target DNA sequence. We prepared site-directed PZ mutants in which Asn131 (N131) in the basic region was substituted with Ala and Gln. N131 mutants showed spectroscopic and dimerization properties almost identical to those of wild-type PZ and an increase in helical content in the presence of the target sequence. Quantitative analyses by an electrophoretic mobility shift assay and quartz crystal microbalance (QCM) measurements demonstrated that the half-maximal effective concentrations of N131 mutants to bind to the target sequence were significantly higher than those of PZ. QCM data also revealed that N131 substitutions accelerated the dissociation without affecting the association, suggesting that a base-specific interaction of N131 occurred after the association between PZ and DNA. Activation of PZ by illumination decreased both the standard errors and the unstable period of QCM data. Optical control of transcription factors will provide new knowledge of the recognition of the target sequence.

Background: The marine alga Ulva compressa is the dominant species in coastal areas receiving effluents from copper mines. The alga can accumulate high amounts of copper and possesses a strong antioxidant system. Here, we performed short-term transcriptomic analyses using total RNA of the alga cultivated with 10 μM of copper for 0, 3, 6, 12 and 24 h by RNA-seq., Results: De novo transcriptomes were assembled using the Trinity software, putative proteins were annotated and classified using Blast2GO. Differentially expressed transcripts were identified using edgeR. Transcript levels were compared by paired times 0 vs 3, 0 vs 6, 0 vs 12 and 0 vs 24 h at an FDR < 0.01 and Log2 Fold Change > 2. Up-regulated transcripts encode proteins belonging to photosystem II (PSII), Light Harvesting II Complex (LHCII), PSI and LHCI, proteins involved in assembly and repair of PSII, and assembly and protection of PSI. In addition, transcripts encoding enzymes leading to β-carotene synthesis and enzymes belonging to the Calvin-Benson cycle were also increased. We further analyzed photosynthesis and carotenoid levels in the alga cultivated with 10 μM of copper for 0 to 24 h. Photosynthesis was increased from 3 to 24 h as well as the level of total carotenoids. The increase in transcripts encoding enzymes of the Calvin-Benson cycle suggests that C assimilation may also be increased., Conclusions: Thus, U. compressa displays a short-term response to copper stress enhancing the expression of genes encoding proteins involved in photosynthesis, enzymes involved carotenoids synthesis, as well as those belonging to the Calvin-Benson cycle, which may result in an increase in C assimilation.

Diacylglycerol acyltransferase (DGAT) is a rate-limiting enzyme in the synthesis of triacylglycerol (TAG), the most important form of energy storage in plants. Some residues have previously been proven to be crucial for DGAT1 activity. In this study, we used site-directed mutagenesis of the CeDGAT1 gene from Chlorella ellipsoidea to alter 16 amino acids to investigate effects on DGAT1 function. Of the 16 residues (L482R, E542R, Y553A, G577R, R579D, Y582R, R596D, H603D, H609D, A624R, F629R, S632A, W650R, A651R, Q658H, and P660R), we newly identified 5 (L482, R579, H603, A651, and P660) as being essential for DGAT1 function and 7 (E542, G577, R596, H609, A624, S632, and Q658) that significantly affect DGAT1 function to different degrees, as revealed by heterologous expression of the mutants in yeast strain INVSc1. Importantly, compared with CeDGAT1 , expression of the mutant CeDGAT1Y553A significantly increased the total fatty acid and TAG contents of INVSc1. Comparison among CeDGAT1Y553A, GmDGAT1Y341A, AtDGAT1Y364A, BnDGAT1Y347A, and BoDGAT1Y352A, in which tyrosine at the position corresponding to the 553rd residue in CeDGAT1 is changed into alanine, indicated that the impact of changing Y to A at position 553 is specific for CeDGAT1. Overall, the results provide novel insight into the structure and function of DGAT1, as well as a mutant gene with high potential for lipid improvement in microalgae and plants.

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.

Sulfated fucans, often denoted as fucoidans, are highly variable cell wall polysaccharides of brown algae, which possess a wide range of bioactive properties with potential pharmaceutical applications. Due to their complex architecture, the structures of algal fucans have until now only been partly determined. Enzymes capable of hydrolyzing sulfated fucans may allow specific release of defined bioactive oligosaccharides and may serve as a tool for structural elucidation of algal walls. Currently, such enzymes include only a few hydrolases belonging to the glycoside hydrolase family 107 (GH107), and little is known about their mechanistics and the substrates they degrade. In this study, we report the identification and recombinant production of three novel GH107 family proteins derived from a marine metagenome. Activity screening against a large substrate collection showed that all three enzymes degraded sulfated fucans from brown algae in the order Fucales. This is in accordance with a hydrolytic activity against α-1,4-fucosidic linkages in sulfated fucans as reported for previous GH107 members. Also, the activity screening gave new indications about the structural differences in brown algal cell walls. Finally, sequence analyses allowed identification of the proposed catalytic residues of the GH107 family. The findings presented here form a new basis for understanding the GH107 family of enzymes and investigating the complex sulfated fucans from brown algae. DATABASE: The assembled metagenome and raw sequence data is available at EMBL-EBI (Study number: PRJEB28480). Sequences of the GH107 fucanases (Fp273, Fp277, and Fp279) have been deposited in GenBank under accessions MH755451-MH755453., (© 2018 Federation of European Biochemical Societies.)

HAP2 is a class II gamete fusogen in many eukaryotic kingdoms. A crystal structure of Chlamydomonas HAP2 shows a trimeric fusion state. Domains D1, D2.1 and D2.2 line the 3-fold axis; D3 and a stem pack against the outer surface. Surprisingly, hydrogen-deuterium exchange shows that surfaces of D1, D2.2 and D3 closest to the 3-fold axis are more dynamic than exposed surfaces. Three fusion helices in the fusion loops of each monomer expose hydrophobic residues at the trimer apex that are splayed from the 3-fold axis, leaving a solvent-filled cavity between the fusion loops in each monomer. At the base of the two fusion loops, Arg185 docks in a carbonyl cage. Comparisons to other structures, dynamics, and the greater effect on Chlamydomonas gamete fusion of mutation of axis-proximal than axis-distal fusion helices suggest that the apical portion of each monomer could tilt toward the 3-fold axis with merger of the fusion helices into a common fusion surface., Competing Interests: JF, XD, JP, JZ, CL, RI, JE, WS, TS No competing interests declared, (© 2018, Feng et al.)

Ethylene favors carposporogenesis in the red seaweed Grateloupia imbricata. Analyses of cystocarp development in vitro in thalli treated with ethylene suggest an interconnection between polyamine and ethylene biosynthesis pathways. Yet, little is known about molecular mechanisms underlying carposporogenesis. Here, we used droplet digital PCR to analyze genes encoding enzymes related to polyamine (Spermidine [Spd] synthase) and ethylene (ACC synthase) synthesis; a pivotal compound of both pathways (S-adenosyl methionine synthase, SAMS); the gene that encodes amine oxidase, which is involved in polyamine degradation, and a candidate gene involved in seaweed reproduction (ornithine decarboxylase, ODC). In addition, we analyzed genes encoding proteins related to stress and reactive oxygen species, ascorbate peroxidase (APX), cytochrome P450 and WD 40. We characterized gene expression in fertilized and fertile thalli from G. imbricata that were exposed to ethylene for 15 min at two time points after treatment (1 and 7 d). The differential gene expression of SAMS, Spd synthase, ACC synthase, and cytochrome P450 was related to disclosure and development of cystocarps in fertilized thalli that transitioned from having no visible cystocarps at 1 d to developing cystocarps at 7 d. Likewise, cytochrome P450 was associated with cystocarp disclosure and maturation. In addition, amine oxidase and APX were involved in fine-tuning polyamine and reactive oxygen species during carposporogenesis, respectively, whereas WD 40 did so in relation to ethylene signaling. Expression of the candidate gene ODC was increased when cystocarps were not visible (fertilized thalli, 1d), as previously described. This analysis suggests developmental stage-specific roles for these genes during carposporogenesis., (© 2018 Phycological Society of America.)

Recently developed transgenic techniques to explore and exploit the metabolic potential of microalgae present several drawbacks associated with the delivery of exogenous DNA into the cells and its subsequent integration at random sites within the genome. Here, we report a highly efficient multiplex genome-editing method in the diatom Phaeodactylum tricornutum, relying on the biolistic delivery of CRISPR-Cas9 ribonucleoproteins coupled with the identification of two endogenous counter-selectable markers, PtUMPS and PtAPT. First, we demonstrate the functionality of RNP delivery by positively selecting the disruption of each of these genes. Then, we illustrate the potential of the approach for multiplexing by generating double-gene knock-out strains, with 65% to 100% efficiency, using RNPs targeting one of these markers and PtAureo1a, a photoreceptor-encoding gene. Finally, we created triple knock-out strains in one step by delivering six RNP complexes into Phaeodactylum cells. This approach could readily be applied to other hard-to-transfect organisms of biotechnological interest.

Saccharina japonica is one of the most important marine crops in China, Japan and Korea. Candidate genes associated with blade length and blade width have not yet been reported. Here, based on SLAF-seq, the 7627 resulting SNP loci were selected for genetic linkage mapping to 31 linkage groups with an average spacing of 0.69 cM, and QTL analyses were performed to map the blade length and blade width phenotypes of S. japonica. In total, 12 QTLs contributing to blade length and 10 to width were detected. Some QTL intervals were detected for both blade length and width. Additive alleles for increasing blade length and width in S. japonica came from both parents. After the QTL interval regions were comparatively mapped to the current reference genome of S. japonica (MEHQ00000000), 14 Tic20 (translocon on the inner envelope membrane of chloroplast) genes and three peptidase genes were identified. RT-qPCR analysis showed that the transcription levels of four Tic20 genes were different not only in the two parent sporophytes but also at different cultivation times within one parent. The SNP markers closely associated with blade length and width could be used to improve the selection efficiency of S. japonica breeding.

The B race of a green microalga Botryococcus braunii Kützing produces triterpene hydrocarbons that is a promising source for biofuel. In this algal race, precursors of triterpene hydrocarbons are provided from the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway. The terminal enzyme of this pathway, 4-hydroxy-3-methylbut-2-enyl diphosphate reductase (HDR) is regarded as one of the key enzymes that affect yields of products in terpene biosynthesis. In order to better understand the MEP pathway of the alga, cDNA and genomic clones of HDR were obtained from B. braunii Showa strain. B. braunii HDR (BbHDR) is encoded on a single copy gene including a 1509-bp open reading frame that was intervened by 6 introns. The exon-intron structure of BbHDR genes did not show clear relation to phylogeny, while its amino acid sequence reflected phyla and classes well. BbHDR sequence was distinctive from that of the HDR protein from Escherichia coli in the residues involved in hydrogen-bond network that surrounds substrate. Introduction of BbHDR cDNA into an E. coli HDR deficient mutant resulted in recovery of its auxotrophy. BbHDR expression level was upregulated from the onset of liquid culture to the 24th day after inoculation with a 2.5-fold increase and retained its level in the subsequent period.

The impact of cadmium on the diatom Nitzschia palea (Kützing) W. Smith 1856 was studied by examining the relation between valve deformities and response through biological processes and genetic expression. Cultures of N. palea were exposed to two Cd treatments (C 1  = 2.4 ± 0.6 and C 2  = 42.6 ± 4.2 µg Cd/L) along with a control (C 0  = 0 µg Cd/L) for 28 days. Cadmium bioaccumulation, diatoms growth, photosynthetic efficiencies, valve deformities and genetic expression were investigated during the course of the experiment. Cadmium exposure had significant effects on bioaccumulation, growth, valve deformities and genetic expression. Maximal effects for all studied endpoints were recorded after 7 days of exposure for the C 2 treatment, which corresponded to the sampling time and condition with maximum cadmium bioaccumulation. Abnormal raphe formations (deviation from its lateral position) were significantly more abundant in the C 2 treatment compared to the control. Molecular responses were related to cadmium level based on the number of genes impacted, intensity of the response and the frequency of observations. The expression of genes involved in the regulation of mitochondrial metabolism, photosynthesis, oxidative stress and silica metabolism was affected by cadmium exposure.

The critical and ubiquitous enzyme adenylate kinase (ADK) catalyzes the nucleotide phosphoryl exchange reaction: 2ADP ↔ ATP + AMP. The ADK3 in the chloroplasts of the green alga Chlamydomonas reinhardtii, bears an unusual C-terminal extension that is similar to the C-terminal end of the intrinsically disordered protein CP12. In this study, we report that this enzyme, when oxidized but not when reduced, is able to interact with the chloroplast glyceraldehyde-3-phosphate dehydrogenase (GAPDH) forming a stable complex as shown by native electrophoresis and mass spectrometry. In this bienzyme complex, the activity of ADK3 is unchanged while the NADPH-dependent activity of GAPDH is significantly inhibited. Moreover ADK3, like CP12, can protect GAPDH against thermal inactivation and aggregation. The ADK3-GAPDH bienzyme complex is unable to recruit phosphoribulokinase (PRK), in contrast with the ternary complex formed between GAPDH-CP12 and PRK. The interaction between ADK3 and GAPDH might be a mechanism to regulate the crucial ATP: NADPH ratio within chloroplasts to optimize the Calvin-Benson cycle during rapid fluctuation in environmental resources., Enzymes: Adenylate kinase (EC 2.7.4.3), glyceraldehyde-3-phosphate dehydrogenase (GAPDH, EC 1.2.1.13), phosphoribulokinase (PRK, EC 2.7.1.19)., (© 2018 Federation of European Biochemical Societies.)

The proposal that the respiratory complexes can associate with each other in larger structures named supercomplexes (SC) is generally accepted. In the last decades most of the data about this association came from studies in yeasts, mammals and plants, and information is scarce in other lineages. Here we studied the supramolecular association of the F 1 F O -ATP synthase (complex V) and the respiratory complexes I, III and IV of the colorless alga Polytomella sp. with an approach that involves solubilization using mild detergents, n-dodecyl-β-D-maltoside (DDM) or digitonin, followed by separation of native protein complexes by electrophoresis (BN-PAGE), after which we identified oligomeric forms of complex V (mainly V 2 and V 4 ) and different respiratory supercomplexes (I/IV 6 , I/III 4 , I/IV). In addition, purification/reconstitution of the supercomplexes by anion exchange chromatography was also performed. The data show that these complexes have the ability to strongly associate with each other and form DDM-stable macromolecular structures. The stable V 4 ATPase oligomer was observed by electron-microscopy and the association of the respiratory complexes in the so-called "respirasome" was able to perform in-vitro oxygen consumption., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Epigenetic factors such as histone modifications play integral roles in plant development and stress response, yet their implications in algae remain poorly understood. In the industrial oleaginous microalgae Nannochloropsis spp., the lack of an efficient methodology for chromatin immunoprecipitation (ChIP), which determines the specific genomic location of various histone modifications, has hindered probing the epigenetic basis of their photosynthetic carbon conversion and storage as oil. Here, a detailed ChIP protocol was developed for Nannochloropsis oceanica, which represents a reliable approach for the analysis of histone modifications, chromatin state, and transcription factor-binding sites at the epigenetic level. Using ChIP-qPCR, genes related to photosynthetic carbon fixation in this microalga were systematically assessed. Furthermore, a ChIP-Seq protocol was established and optimized, which generated a genome-wide profile of histone modification events, using histone mark H3K9Ac as an example. These results are the first step for appreciation of the chromatin landscape in industrial oleaginous microalgae and for epigenetics-based microalgal feedstock development., (© 2018 Phycological Society of America.)

The Rhodophyta Sheathia arcuata is exclusively distributed in freshwater, constituting an important component in freshwater flora. This study presents the first transcriptome profiling of freshwater Rhodophyta taxa. A total of 161,483 assembled transcripts were identified, annotated and classified into different biological categories and pathways based on BLAST against diverse databases. Different gene expression patterns were caused principally by different irradiances considering the similar water conditions of the sampling site when the specimens were collected. Comparison results of gene expression levels under different irradiances revealed that photosynthesis-related pathways significantly up-regulated under the weak light. Molecular responses for improved photosynthetic activity include the transcripts corresponding to antenna proteins (LHCA1 and LHCA4), photosynthetic apparatus proteins (PSBU, PETB, PETC, PETH and beta and gamma subunits of ATPase) and metabolic enzymes in the carbon fixation. Along with photosynthesis, other metabolic activities were also regulated to optimize the growing and development of S. arcuata under appropriate sunlight. Protein-protein interactive networks revealed the most responsive up-expressed transcripts were ribosomal proteins. The de-novo transcriptome assembly of S. arcuata provides a foundation for further investigation on the molecular mechanism of photosynthesis and environmental adaption for freshwater Rhodophyta., Competing Interests: The authors have declared that no competing interests exist.

PCP and acpPC are the two major antennae proteins that bind pigments in peridinin-containing dinoflagellates. The relationship between antennae proteins and cellular pigments at molecular level is still poorly understood. Here we identified and characterized the two antennae protein genes in dinoflagellate Prorocentrum donghaiense under different light conditions. The mature PCP protein was 32 kDa, while acpPC was a polyprotein each of 19 kDa. Both genes showed higher expression under low light than under high light, suggesting their possible role in a low light adaptation mechanism. The two genes showed differential diel expression rhythm, with PCP being more highly expressed in the dark than in the light period and acpPC the other way around. HPLC analysis of cellular pigments indicated a diel change of chlorophyll c2, but invariability of other pigments. A stable peridinin: chlorophyll a pigment ratio was detected under different light intensities and over the diel cycle, although the diadinoxanthin:chlorophyll a ratio increased significantly with light intensity. The results suggest that 1) PCP and acpPC genes are functionally distinct, 2) PCP and acpPC can function under low light as an adaptive mechanism in P. donghaiense, 3). the ratios of diadinoxanthin:chlorophyll a and peridinin: chlorophyll a can potentially be used as an indicator of algal photophysiological status and a pigment signature respectively under different light conditions in P. donghaiense., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The content of intrinsically disordered protein (IDP) is related to organism complexity, evolution, and regulation. In the Plantae, despite their high complexity, experimental investigation of IDP content is lacking. We identified by mass spectrometry 682 heat-resistant proteins from the green alga, Chlamydomonas reinhardtii. Using a phosphoproteome database, we found that 331 of these proteins are targets of phosphorylation. We analyzed the flexibility propensity of the heat-resistant proteins and their specific features as well as those of predicted IDPs from the same organism. Their mean percentage of disorder was about 20%. Most of the IDPs (~70%) were addressed to other compartments than mitochondrion and chloroplast. Their amino acid composition was biased compared to other classic IDPs. Their molecular functions were diverse; the predominant ones were nucleic acid binding and unfolded protein binding and the less abundant one was catalytic activity. The most represented proteins were ribosomal proteins, proteins associated to flagella, chaperones and histones. We also found CP12, the only experimental IDP from C. reinhardtii that is referenced in disordered protein database. This is the first experimental investigation of IDPs in C. reinhardtii that also combines in silico analysis.

Photosynthetic organisms are frequently exposed to light intensities that surpass the photosynthetic electron transport capacity. Under these conditions, the excess absorbed energy can be transferred from excited chlorophyll in the triplet state (3Chl*) to molecular O 2 , which leads to the production of harmful reactive oxygen species. To avoid this photooxidative stress, photosynthetic organisms must respond to excess light. In the green alga Chlamydomonas reinhardtii , the fastest response to high light is nonphotochemical quenching, a process that allows safe dissipation of the excess energy as heat. The two proteins, UV-inducible LHCSR1 and blue light-inducible LHCSR3, appear to be responsible for this function. While the LHCSR3 protein has been intensively studied, the role of LHCSR1 has been only partially elucidated. To investigate the molecular functions of LHCSR1 in C. reinhardtii , we performed biochemical and spectroscopic experiments and found that the protein mediates excitation energy transfer from light-harvesting complexes for Photosystem II (LHCII) to Photosystem I (PSI), rather than Photosystem II, at a low pH. This altered excitation transfer allows remarkable fluorescence quenching under high light. Our findings suggest that there is a PSI-dependent photoprotection mechanism that is facilitated by LHCSR1., Competing Interests: The authors declare no conflict of interest.

Despite the impressive progress made in recent years in understanding the early steps in charge separation within the photosynthetic reaction centers, our knowledge of how ferredoxin (Fd) interacts with the acceptor side of photosystem I (PSI) is not as well developed. Fd accepts electrons after transiently docking to a binding site on the acceptor side of PSI. However, the exact location, as well as the stoichiometry, of this binding have been a matter of debate for more than two decades. Here, using Isothermal Titration Calorimetry (ITC) and purified components from wild type and mutant strains of the green algae Chlamydomonas reinhardtii we show that PSI has a single binding site for Fd, and that the association consists of two distinct binding events, each with a specific association constant., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Key Message: Several amino acid motifs required for Rop-dependent activity were found to form a common surface on RLCKVI&#95;A kinases. This indicates a unique mechanism for Rho-type GTPase-mediated kinase activation in plants. Rho-of-plants (Rop) G-proteins are implicated in the regulation of various cellular processes, including cell growth, cell polarity, hormonal and pathogen responses. Our knowledge about the signalling pathways downstream of Rops is continuously increasing. However, there are still substantial gaps in this knowledge. One reason for this is that these pathways are considerably different from those described for yeast and/or animal Rho-type GTPases. Among others, plants lack all Rho/Rac/Cdc42-activated kinase families. Only a small group of plant-specific receptor-like cytoplasmic kinases (RLCK VI&#95;A) has been shown to exhibit Rop-binding-dependent in vitro activity. These kinases do not carry any known GTPase-binding motifs. Based on the sequence comparison of the Rop-activated RLCK VI&#95;A and the closely related but constitutively active RLCK VI&#95;B kinases, several distinguishing amino acid residues/motifs were identified. All but one of these were found to be required for the Rop-mediated regulation of the in vitro activity of two RLCK VI&#95;A kinases. Structural modelling indicated that these motifs might form a common Rop-binding surface. Based on in silico data mining, kinases that have the identified Rop-binding motifs are present in Embryophyta but not in unicellular green algae. It can, therefore, be supposed that Rops recruited these plant-specific kinases for signalling at an early stage of land plant evolution.

The assembly of membranous extensions such as microvilli and cilia in polarized cells is a tightly regulated, yet poorly understood, process. Peptidylglycine α-amidating monooxygenase (PAM), a membrane enzyme essential for the synthesis of amidated bioactive peptides, was recently identified in motile and non-motile (primary) cilia and has an essential role in ciliogenesis in Chlamydomonas, Schmidtea and mouse. In mammalian cells, changes in PAM levels alter secretion and organization of the actin cytoskeleton. Here we show that lack of Pam in zebrafish recapitulates the lethal edematous phenotype observed in Pam -/- mice and reveals additional defects. The pam -/- zebrafish embryos display an initial striking loss of microvilli and subsequently impaired ciliogenesis in the pronephros. In multiciliated mouse tracheal epithelial cells, vesicular PAM staining colocalizes with apical actin, below the microvilli. In PAM-deficient Chlamydomonas, the actin cytoskeleton is dramatically reorganized, and expression of an actin paralogue is upregulated. Biochemical assays reveal that the cytosolic PAM C-terminal domain interacts directly with filamentous actin but does not alter the rate of actin polymerization or disassembly. Our results point to a critical role for PAM in organizing the actin cytoskeleton during development, which could in turn impact both microvillus formation and ciliogenesis.

Intraflagellar transport moves proteins in and out of flagella/cilia and it is essential for the assembly of these organelles. Using whole-genome sequencing, we identified splice site mutations in two IFT genes, IFT81 ( fla9 ) and IFT121 ( ift121-2 ), which lead to flagellar assembly defects in the unicellular green alga Chlamydomonas reinhardtii The splicing defects in these ift mutants are partially corrected by mutations in two conserved spliceosome proteins, DGR14 and FRA10. We identified a dgr14 deletion mutant, which suppresses the 3' splice site mutation in IFT81 , and a frameshift mutant of FRA10 , which suppresses the 5' splice site mutation in IFT121 Surprisingly, we found dgr14-1 and fra10 mutations suppress both splice site mutations. We suggest these two proteins are involved in facilitating splice site recognition/interaction; in their absence some splice site mutations are tolerated. Nonsense mutations in SMG1 , which is involved in nonsense-mediated decay, lead to accumulation of aberrant transcripts and partial restoration of flagellar assembly in the ift mutants. The high density of introns and the conservation of noncore splicing factors, together with the ease of scoring the ift mutant phenotype, make Chlamydomonas an attractive organism to identify new proteins involved in splicing through suppressor screening., (© 2018 The Authors.)

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.

Germ-soma differentiation is a hallmark of complex multicellular organisms, yet its origins are not well understood. Volvox carteri is a simple multicellular green alga that has recently evolved a simple germ-soma dichotomy with only two cell-types: large germ cells called gonidia and small terminally differentiated somatic cells. Here, we provide a comprehensive characterization of the gonidial and somatic transcriptomes of V. carteri to uncover fundamental differences between the molecular and metabolic programming of these cell-types. We found extensive transcriptome differentiation between cell-types, with somatic cells expressing a more specialized program overrepresented in younger, lineage-specific genes, and gonidial cells expressing a more generalist program overrepresented in more ancient genes that shared striking overlap with stem cell-specific genes from animals and land plants. Directed analyses of different pathways revealed a strong dichotomy between cell-types with gonidial cells expressing growth-related genes and somatic cells expressing an altruistic metabolic program geared toward the assembly of flagella, which support organismal motility, and the conversion of storage carbon to sugars, which act as donors for production of extracellular matrix (ECM) glycoproteins whose secretion enables massive organismal expansion. V. carteri orthologs of diurnally controlled genes from C. reinhardtii, a single-celled relative, were analyzed for cell-type distribution and found to be strongly partitioned, with expression of dark-phase genes overrepresented in somatic cells and light-phase genes overrepresented in gonidial cells- a result that is consistent with cell-type programs in V. carteri arising by cooption of temporal regulons in a unicellular ancestor. Together, our findings reveal fundamental molecular, metabolic, and evolutionary mechanisms that underlie the origins of germ-soma differentiation in V. carteri and provide a template for understanding the acquisition of germ-soma differentiation in other multicellular lineages., (Copyright © 2018 Matt and Umen.)

The cyclin-dependent kinase CDK1 is essential for mitosis in fungi and animals. Plant genomes contain the CDK1 ortholog CDKA and a plant kingdom-specific relative, CDKB. The green alga Chlamydomonas reinhardtii has a long G1 growth period followed by rapid cycles of DNA replication and cell division. We show that null alleles of CDKA extend the growth period prior to the first division cycle and modestly extend the subsequent division cycles, but do not prevent cell division, indicating at most a minor role for the CDK1 ortholog in mitosis in Chlamydomonas. A null allele of cyclin A has a similar though less extreme phenotype. In contrast, both CDKB and cyclin B are essential for mitosis. CDK kinase activity measurements imply that the predominant in vivo complexes are probably cyclin A-CDKA and cyclin B-CDKB. We propose a negative feedback loop: CDKA activates cyclin B-CDKB. Cyclin B-CDKB in turn promotes mitotic entry and inactivates cyclin A-CDKA. Cyclin A-CDKA and cyclin B-CDKB may redundantly promote DNA replication. We show that the anaphase-promoting complex is required for inactivation of both CDKA and CDKB and is essential for anaphase. These results are consistent with findings in Arabidopsis thaliana and may delineate the core of plant kingdom cell cycle control that, compared with the well-studied yeast and animal systems, exhibits deep conservation in some respects and striking divergence in others., (© 2018 American Society of Plant Biologists. All rights reserved.)

Ability of actin to polymerise and depolymerise makes it essential to key functions of eukaryotic cell. The functioning of actin is controlled by a host of regulatory proteins, the repertoire of which in diatoms is known to remarkably differ from other organisms. We have performed a phylogenetic analysis of 521 actin and actin-related proteins' aminoacid sequences, as well as 190 sequences of gelsolin family proteins from various genomic and transcriptomic datasets. Based on the results of this analysis, as well as on the presence of clade-specific indels in some of the actin-related proteins, we describe a novel ARP subfamily, dubbed ARP12, which is specific to heterokonts and related organisms. We also describe two novel diatom-specific subfamilies, dGRC1 and dGRC2, among short gelsolin repeat-containing proteins., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Photosynthesis occurs in the thylakoid membrane, where the predominant lipid is monogalactosyldiacylglycerol (MGDG). As environmental conditions change, photosynthetic membranes have to adjust. In this study, we used a loss-of-function Chlamydomonas reinhardtii mutant deficient in the MGDG-specific lipase PGD1 (PLASTID GALACTOGLYCEROLIPID DEGRADATION1) to investigate the link between MGDG turnover, chloroplast ultrastructure, and the production of reactive oxygen species (ROS) in response to different adverse environmental conditions. The pgd1 mutant showed altered MGDG abundance and acyl composition and altered abundance of photosynthesis complexes, with an increased PSII/PSI ratio. Transmission electron microscopy showed hyperstacking of the thylakoid grana in the pgd1 mutant. The mutant also exhibited increased ROS production during N deprivation and high light exposure. Supplementation with bicarbonate or treatment with the photosynthetic electron transport blocker DCMU protected the cells against oxidative stress in the light and reverted chlorosis of pgd1 cells during N deprivation. Furthermore, exposure to stress conditions such as cold and high osmolarity induced the expression of PGD1 , and loss of PGD1 in the mutant led to increased ROS production and inhibited cell growth. These findings suggest that PGD1 plays essential roles in maintaining appropriate thylakoid membrane composition and structure, thereby affecting growth and stress tolerance when cells are challenged under adverse conditions., (© 2018 American Society of Plant Biologists. All rights reserved.)

Objective: The eukaryotic plasma membrane localized light-gated proton-pumping rhodopsins possesses great optogenetic applications for repolarization (silencing) of the neuronal activity simply by light illumination. Very few plasma membrane localized proton-pumping rhodopsins of a eukaryotic origin are known that have optogenetic potential. Our objective was to identify and characterize microbial rhodopsin of an eukaryotic origin that expresses on plasma membrane. The plasma membrane localized light-gated proton pump of an eukaryotic origin hold great promise to be used as an optogenetic tools for the neurobiology., Results: Here, we had characterized the cellular expression and membrane localization of a new rhodopsin in Antarctican algae Coccomyxa subellipsoidea. It is the first algal ion pumping rhodopsin that localizes to the plasma membrane of the eukaryotic cells. Coccomyxa subellipsoidea rhodopsin exists in the monomeric and dimeric state both the in vivo and in vitro. The dimeric form of the Coccomyxa subellipsoidea rhodopsin is resistant to heat and detergent denaturants.