RAF-like protein kinases mediate a deeply conserved, rapid auxin response.

The plant-signaling molecule auxin triggers fast and slow cellular responses across land plants and algae. The nuclear auxin pathway mediates gene expression and controls growth and development in land plants, but this pathway is absent from algal sister groups. Several components of rapid responses have been identified in Arabidopsis, but it is unknown if these are part of a conserved mechanism. We recently identified a fast, proteome-wide phosphorylation response to auxin. Here, we show that this response occurs across 5 land plant and algal species and converges on a core group of shared targets. We found conserved rapid physiological responses to auxin in the same species and identified rapidly accelerated fibrosarcoma (RAF)-like protein kinases as central mediators of auxin-triggered phosphorylation across species. Genetic analysis connects this kinase to both auxin-triggered protein phosphorylation and rapid cellular response, thus identifying an ancient mechanism for fast auxin responses in the green lineage.

Smart systems in producing algae-based protein to improve functional food ingredients industries.

Production and extraction systems of algal protein and handling process of functional food ingredients need to control several parameters such as temperature, pH, intensity, and turbidity. Many researchers have investigated the Internet of Things (IoT) approach for enhancing the yield of microalgae biomass and machine learning for identifying and classifying microalgae. However, there have been few specific studies on using IoT and artificial intelligence (AI) for production and extraction of algal protein as well as functional food ingredients processing. In order to improve the production of algal protein and functional food ingredients, the implementation of smart system is a must to have real-time monitoring, remote control system, quick response to sudden events, prediction and characterisation. Techniques of IoT and AI are expected to help functional food industries to have a big breakthrough in the future. Manufacturing and implementation of beneficial smart systems are important to provide convenience and to increase the efficiency of work by using the interconnectivity of IoT devices to have good capturing, processing, archiving, analyzing, and automation. This review investigates the possibilities of implementation of IoT and AI in production and extraction of algal protein and processing of functional food ingredients.

Structure of a TOC-TIC supercomplex spanning two chloroplast envelope membranes.

The TOC and TIC complexes are essential translocons that facilitate the import of the nuclear genome-encoded preproteins across the two envelope membranes of chloroplast, but their exact molecular identities and assembly remain unclear. Here, we report a cryoelectron microscopy structure of TOC-TIC supercomplex from Chlamydomonas, containing a total of 14 identified components. The preprotein-conducting pore of TOC is a hybrid ß-barrel co-assembled by Toc120 and Toc75, while the potential translocation path of TIC is formed by transmembrane helices from Tic20 and YlmG, rather than a classic model of Tic110. A rigid intermembrane space (IMS) scaffold bridges two chloroplast membranes, and a large hydrophilic cleft on the IMS scaffold connects TOC and TIC, forming a pathway for preprotein translocation. Our study provides structural insights into the TOC-TIC supercomplex composition, assembly, and preprotein translocation mechanism, and lays a foundation to interpret the evolutionary conservation and diversity of this fundamental translocon machinery.

An Investigation of Pepsin Hydrolysate of Short Antibacterial Peptides Derived from Limnospira Sp.

The antimicrobial peptides derived from microalgae have attracted a huge attention due to the insufficient availability of effective drugs from the natural resources. In this study, the enzymatic hydrolysate of protein derived from Limnospira maxima was prepared using pepsin under optimized conditions. The peptides with range of 10 kDa were isolated and purified using the Ultra membrane filtration, SDS-PAGE, and TLC. Furthermore, the peptide sequence was identified and characterized by MALDI-TOF mass spectrometry in which an algal peptide, KLENCNYAVELGK showed a strong signal at 466.68 m/z among seven peptides derived from the pepsin hydrolysate. The FT-IR spectroscopic study confirmed the presence of a characteristic functional group of amino acids in the sequence. The algal derived peptide showed antibacterial properties against Escherichia coli (27 ± 0.5 mm) and Staphylococcus aureus (14 mm ± 0.5). This study paves a way to explore the antibacterial peptide from a novel species, L. maxima (MZ26519) evident to utilize for the novel drug to overcome the conventional approach.

Structure-activity relationships of oomycete elicitins uncover the role of reactive oxygen and nitrogen species in triggering plant defense responses.

Elicitins are proteinaceous elicitors that induce the hypersensitive response and plant resistance against diverse phytopathogens. Elicitin recognition by membrane receptors or high-affinity sites activates a variety of fast responses including the production of reactive oxygen species (ROS) and nitric oxide (NO), leading to induction of plant defense genes. Beta-cryptogein (CRY) is a basic ß-elicitin secreted by the oomycete Phytophthora cryptogea that shows high necrotic activity in some plant species, whereas infestin 1 (INF1) secreted by the oomycete P. infestans belongs to acidic α-elicitins with a significantly weaker capacity to induce necrosis. We compared several mutated forms of ß-CRY and INF1 with a modulated capacity to trigger ROS and NO production, bind plant sterols and induce cell death responses in cell cultures of Nicotiana tabacum L. cv. Xanthi. We evidenced a key role of the lysine residue in position 13 in basic elicitins for their biological activity and enhancement of necrotic effects of acidic INF1 by the replacement of the valine residue in position 84 by larger phenylalanine. Studied elicitins activated in differing intensity signaling pathways of ROS, NO and phytohormones jasmonic acid, ethylene and salicylic acid, known to be involved in triggering of hypersensitive response and establishment of systemic resistance.

The mammalian-type thioredoxin reductase 1 confers a high-light tolerance to the green alga Chlamydomonas reinhardtii.

Reactive oxygen species (ROS) can both act as a poison causing cell death and important signaling molecules among various organisms. Photosynthetic organisms inevitably produce ROS, making the appropriate elimination of ROS an essential strategy for survival. Interestingly, the unicellular green alga Chlamydomonas reinhardtii expresses a mammalian form of thioredoxin reductase, TR1, which functions as a ROS scavenger in animal cells. To investigate the properties of TR1 in C. reinhardtii, we generated TR1 knockout strains using CRISPR/Cas9-based genome editing. We found a reduced tolerance to high-light and ROS stresses in the TR1 knockout strains compared to the parental strain. In addition, the regulation of phototactic orientation, known to be regulated by ROS, was affected in the knockout strains. These results suggest that TR1 contributes to a ROS-scavenging pathway in C. reinhardtii.

Chlamydomonas ARMC2/PF27 is an obligate cargo adapter for intraflagellar transport of radial spokes.

Intraflagellar transport (IFT) carries proteins into flagella but how IFT trains interact with the large number of diverse proteins required to assemble flagella remains largely unknown. Here, we show that IFT of radial spokes in Chlamydomonas requires ARMC2/PF27, a conserved armadillo repeat protein associated with male infertility and reduced lung function. Chlamydomonas ARMC2 was highly enriched in growing flagella and tagged ARMC2 and the spoke protein RSP3 co-migrated on anterograde trains. In contrast, a cargo and an adapter of inner and outer dynein arms moved independently of ARMC2, indicating that unrelated cargoes distribute stochastically onto the IFT trains. After concomitant unloading at the flagellar tip, RSP3 attached to the axoneme whereas ARMC2 diffused back to the cell body. In armc2/pf27 mutants, IFT of radial spokes was abolished and the presence of radial spokes was limited to the proximal region of flagella. We conclude that ARMC2 is a cargo adapter required for IFT of radial spokes to ensure their assembly along flagella. ARMC2 belongs to a growing class of cargo-specific adapters that enable flagellar transport of preassembled axonemal substructures by IFT.

Size and Fluorescence Properties of Algal Photosynthetic Antenna Proteins Estimated by Microscopy.

Antenna proteins play a major role in the regulation of light-harvesting in photosynthesis. However, less is known about a possible link between their sizes (oligomerization state) and fluorescence intensity (number of photons emitted). Here, we used a microscopy-based method, Fluorescence Correlation Spectroscopy (FCS), to analyze different antenna proteins at the particle level. The direct comparison indicated that Chromera Light Harvesting (CLH) antenna particles (isolated from Chromera velia) behaved as the monomeric Light Harvesting Complex II (LHCII) (from higher plants), in terms of their radius (based on the diffusion time) and fluorescence yields. FCS data thus indicated a monomeric oligomerization state of algal CLH antenna (at our experimental conditions) that was later confirmed also by biochemical experiments. Additionally, our data provide a proof of concept that the FCS method is well suited to measure proteins sizes (oligomerization state) and fluorescence intensities (photon counts) of antenna proteins per single particle (monomers and oligomers). We proved that antenna monomers (CLH and LHCIIm) are more "quenched" than the corresponding trimers. The FCS measurement thus represents a useful experimental approach that allows studying the role of antenna oligomerization in the mechanism of photoprotection.

Photosynthetic assimilation of CO2 regulates TOR activity.

The target of rapamycin (TOR) kinase is a master regulator that integrates nutrient signals to promote cell growth in all eukaryotes. It is well established that amino acids and glucose are major regulators of TOR signaling in yeast and metazoan, but whether and how TOR responds to carbon availability in photosynthetic organisms is less understood. In this study, we showed that photosynthetic assimilation of CO2 by the Calvin-Benson-Bassham (CBB) cycle regulates TOR activity in the model single-celled microalga Chlamydomonas reinhardtii Stimulation of CO2 fixation boosted TOR activity, whereas inhibition of the CBB cycle and photosynthesis down-regulated TOR. We uncovered a tight link between TOR activity and the endogenous level of a set of amino acids including Ala, Glu, Gln, Leu, and Val through the modulation of CO2 fixation and the use of amino acid synthesis inhibitors. Moreover, the finding that the Chlamydomonas starch-deficient mutant sta6 displayed disproportionate TOR activity and high levels of most amino acids, particularly Gln, further connected carbon assimilation and amino acids to TOR signaling. Thus, our results showed that CO2 fixation regulates TOR signaling, likely through the synthesis of key amino acids.

Nitric oxide synthases from photosynthetic organisms improve growth and confer nitrosative stress tolerance in E. coli. Insights on the pterin cofactor.

Nitric oxide synthase (NOS) catalyzes NO formation from the substrate l-arginine (Arg). Previously, NOS with distinct biochemical properties were characterized from two photosynthetic microorganisms, the unicellular algae Ostreococcus tauri (OtNOS) and the cyanobacteria Synechococcus PCC 7335 (SyNOS). In this work we studied the effect of recombinant OtNOS and SyNOS expressed under IPTG-induced promoter in E. coli, a bacterium that lacks NOS. Results show that OtNOS and SyNOS expression promote E. coli growth in a nutrient replete medium and allow to better metabolize Arg as N source. In LB medium, OtNOS induces the expression of the NO dioxygenase hmp in E. coli, in accordance with high NO levels visualized with the probe DAF-FM DA. In contrast, SyNOS expression does not induce hmp and show a slight increase of NO production compared to OtNOS. NOS expression reduces ROS production and increases viability of E. coli cultures growing in LB. A strong nitrosative stress provoked by the addition of 1 mM of the NO donors sodium nitroprusside (SNP) and nitrosoglutathione (GSNO) inhibits bacterial growth rate. Under these conditions, the expression of OtNOS or SyNOS counteracts NO donor toxicity restoring bacterial growth. Finally, using bioinformatic tools and ligand docking analyses, we postulate that tetrahydromonapterin (MH4), an endogenous pterin found in E. coli, could act as cofactor required for NOS catalytic activity. Our findings could be useful for the development of biotechnological applications using NOS expression to improve growth in NOS-lacking bacteria.

Study on the Interaction of Algal Peptides on Virulence Factors of Helicobacter pylori: In Silico Approach.

In the Asian region, Helicobacter pylori infects about 80% populations, which is most leading cause of peptic ulcers, and it is an asymptomatic infection. Studies reported that the particular bacteria carry specific virulence factors that leads to severe complications. These virulence factors can be used as a drug targets to inhibit their growth and pathogenicity. Chronic infection with H. pylori virulence factors are CagA, VacA and HtrA positive strains the risk factor of gastric cancer. In this study, we aimed to study the antagonistic interaction pattern between the potential eight algal peptides against the virulence factors of H. pylori through in silico analysis intended to treat peptic ulcer and prevent the further complications such as cancer. The proteins of virulent factors are docked using C-Docker algorithm and calculated the bind energy of the complexes. The results showed that the peptide derived from a green alga, Tetradesmus sp. are active against the three virulent factors such as cag-A, vac-A, and Htr-A with multiple hydrogen, vdW, electrostatic interactions, and mild π-hydrophobic bindings with the libdock energy score for CagA, VacA and HtrA are 175.625, 158.603 and 89.397 kcal/mol. These primes and the peptide lead to develop a better and potential inhibitors against H. pylori infection.

Consensus mutagenesis and computational simulation provide insight into the desaturation catalytic mechanism for delta 6 fatty acid desaturase.

Fatty acid desaturase (FADS) generates double bond at a certain position of the corresponding polyunsaturated fatty acids (PUFAs) with high selectivity, the enzyme activity and PUFAs products of which are essential to biological systems and are associated with a variety of physiological diseases. Little is known about the structure of FADSs and their amino acid residues related to catalytic activities. Identifying key residues of Micromonas pusilla delta 6 desaturase (MpFADS6) provides a point of departure for a better understanding of desaturation. In this study, conserved amino acids were anchored through gene consensus analysis, thereby generating corresponding variants by site-directed mutagenesis. To achieve stable and high-efficiency expression of MpFADS6 and its variants in Saccharomyces cerevisiae, the key points of induced expression were optimized. The contribution of conserved residues to the function of enzyme was determined by analyzing enzyme activity of the variants. Molecular modeling indicated that these residues are essential to catalytic activities, or substrate binding. Mutants MpFADS6[Q409R] and MpFADS6[M242P] abolished desaturation, while MpFADS6[F419V] and MpFADS6[A374Q] significantly reduced catalytic activities. Given that certain residues have been identified to have a significant impact on MpFADS6 activities, it is put forward that histidine-conserved region III of FADS6 is related to electronic transfer during desaturation, while histidine-conserved regions I and II are related to desaturation. These findings provide new insights and methods to determine the structure, mechanism and directed transformation of membrane-bound desaturases.

Microalgal protein AstaP is a potent carotenoid solubilizer and delivery module with a broad carotenoid binding repertoire.

Carotenoids are lipophilic substances with many biological functions, from coloration to photoprotection. Being potent antioxidants, carotenoids have multiple biomedical applications, including the treatment of neurodegenerative disorders and retina degeneration. Nevertheless, the delivery of carotenoids is substantially limited by their poor solubility in the aqueous phase. Natural water-soluble carotenoproteins can facilitate this task, necessitating studies on their ability to uptake and deliver carotenoids. One such promising carotenoprotein, AstaP (astaxanthin-binding protein), was recently identified in eukaryotic microalgae, but its structure and functional properties remained largely uncharacterized. By using a correctly folded recombinant protein, here we show that AstaP is an efficient carotenoid solubilizer that can stably bind not only astaxanthin but also zeaxanthin, canthaxanthin, and, to a lesser extent, ß-carotene, that is, carotenoids especially valuable to human health. AstaP accepts carotenoids provided as acetone solutions or embedded in membranes, forming carotenoid-protein complexes with an apparent stoichiometry of 1:1. We successfully produced AstaP holoproteins in specific carotenoid-producing strains of Escherichia coli, proving it is amenable to cost-efficient biotechnology processes. Regardless of the carotenoid type, AstaP remains monomeric in both apo- and holoform, while its rather minimalistic mass (~ 20 kDa) makes it an especially attractive antioxidant delivery module. In vitro, AstaP transfers different carotenoids to liposomes and to unrelated proteins from cyanobacteria, which can modulate their photoactivity and/or oligomerization. These findings expand the toolkit of the characterized carotenoid binding proteins and outline the perspective of the use of AstaP as a unique monomeric antioxidant nanocarrier with an extensive carotenoid binding repertoire.

CBM20CP, a novel functional protein of starch metabolism in green algae.

Ostreococcus tauri is a picoalga that contains a small and compact genome, which resembles that of higher plants in the multiplicity of enzymes involved in starch synthesis (ADP-glucose pyrophosphorylase, ADPGlc PPase; granule bound starch synthase, GBSS; starch synthases, SSI, SSII, SSIII; and starch branching enzyme, SBE, between others), except starch synthase IV (SSIV). Although its genome is fully sequenced, there are still many genes and proteins to which no function was assigned. Here, we identify the OT_ostta06g01880 gene that encodes CBM20CP, a plastidial protein which contains a central carbohydrate binding domain of the CBM20 family, and a coiled coil domain at the C-terminus that lacks catalytic activity. We demonstrate that CBM20CP has the ability to bind starch, amylose and amylopectin with different affinities. Furthermore, this protein interacts with OsttaSSIII-B, increasing its binding to starch granules, its catalytic efficiency and promoting granule growth. The results allow us to postulate a functional role for CBM20CP in starch metabolism in green algae. KEY MESSAGE: CBM20CP, a plastidial protein that has a modular structure but lacks catalytic activity, regulates the synthesis of starch in Ostreococcus tauri.

Genome sequencing of the multicellular alga Astrephomene provides insights into convergent evolution of germ-soma differentiation.

Germ-soma differentiation evolved independently in many eukaryotic lineages and contributed to complex multicellular organizations. However, the molecular genetic bases of such convergent evolution remain unresolved. Two multicellular volvocine green algae, Volvox and Astrephomene, exhibit convergent evolution of germ-soma differentiation. The complete genome sequence is now available for Volvox, while genome information is scarce for Astrephomene. Here, we generated the de novo whole genome sequence of Astrephomene gubernaculifera and conducted RNA-seq analysis of isolated somatic and reproductive cells. In Volvox, tandem duplication and neofunctionalization of the ancestral transcription factor gene (RLS1/rlsD) might have led to the evolution of regA, the master regulator for Volvox germ-soma differentiation. However, our genome data demonstrated that Astrephomene has not undergone tandem duplication of the RLS1/rlsD homolog or acquisition of a regA-like gene. Our RNA-seq analysis revealed the downregulation of photosynthetic and anabolic gene expression in Astrephomene somatic cells, as in Volvox. Among genes with high expression in somatic cells of Astrephomene, we identified three genes encoding putative transcription factors, which may regulate somatic cell differentiation. Thus, the convergent evolution of germ-soma differentiation in the volvocine algae may have occurred by the acquisition of different regulatory circuits that generate a similar division of labor.

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.

Fatty Acid Photodecarboxylase Is an Interfacial Enzyme That Binds to Lipid-Water Interfaces to Access Its Insoluble Substrate.

Fatty acid photodecarboxylase (FAP), one of the few natural photoenzymes characterized so far, is a promising biocatalyst for lipid-to-hydrocarbon conversion using light. However, the optimum supramolecular organization under which the fatty acid (FA) substrate should be presented to FAP has not been addressed. Using palmitic acid embedded in phospholipid liposomes, phospholipid-stabilized microemulsions, and mixed micelles, we show that FAP displays a preference for FAs present in liposomes and at the surface of microemulsions. The kinetics of adsorption onto phospholipid and galactolipid monomolecular films further suggests the ability of FAP to bind to and penetrate into membranes, with a higher affinity in the presence of FAs. The FAP structure reveals a potential interfacial recognition site with clusters of hydrophobic and basic residues surrounding the active site entrance. The resulting dipolar moment suggests the orientation of FAP at negatively charged interfaces. These findings provide important clues about the mode of action of FAP and the development of FAP-based bioconversion processes.

Deep-coverage spatiotemporal proteome of the picoeukaryote Ostreococcus tauri reveals differential effects of environmental and endogenous 24-hour rhythms.

The cellular landscape changes dramatically over the course of a 24 h day. The proteome responds directly to daily environmental cycles and is additionally regulated by the circadian clock. To quantify the relative contribution of diurnal versus circadian regulation, we mapped proteome dynamics under light:dark cycles compared with constant light. Using Ostreococcus tauri, a prototypical eukaryotic cell, we achieved 85% coverage, which allowed an unprecedented insight into the identity of proteins that facilitate rhythmic cellular functions. The overlap between diurnally- and circadian-regulated proteins was modest and these proteins exhibited different phases of oscillation between the two conditions. Transcript oscillations were generally poorly predictive of protein oscillations, in which a far lower relative amplitude was observed. We observed coordination between the rhythmic regulation of organelle-encoded proteins with the nuclear-encoded proteins that are targeted to organelles. Rhythmic transmembrane proteins showed a different phase distribution compared with rhythmic soluble proteins, indicating the existence of a circadian regulatory process specific to the biogenesis and/or degradation of membrane proteins. Our observations argue that the cellular spatiotemporal proteome is shaped by a complex interaction between intrinsic and extrinsic regulatory factors through rhythmic regulation at the transcriptional as well as post-transcriptional, translational, and post-translational levels.

Co-targeting strategy for precise, scarless gene editing with CRISPR/Cas9 and donor ssODNs in Chlamydomonas.

Programmable site-specific nucleases, such as the clustered regularly interspaced short palindromic repeat (CRISPR)/ CRISPR-associated protein 9 (Cas9) ribonucleoproteins (RNPs), have allowed creation of valuable knockout mutations and targeted gene modifications in Chlamydomonas (Chlamydomonas reinhardtii). However, in walled strains, present methods for editing genes lacking a selectable phenotype involve co-transfection of RNPs and exogenous double-stranded DNA (dsDNA) encoding a selectable marker gene. Repair of the dsDNA breaks induced by the RNPs is usually accompanied by genomic insertion of exogenous dsDNA fragments, hindering the recovery of precise, scarless mutations in target genes of interest. Here, we tested whether co-targeting two genes by electroporation of pairs of CRISPR/Cas9 RNPs and single-stranded oligodeoxynucleotides (ssODNs) would facilitate the recovery of precise edits in a gene of interest (lacking a selectable phenotype) by selection for precise editing of another gene (creating a selectable marker)-in a process completely lacking exogenous dsDNA. We used PPX1 (encoding protoporphyrinogen IX oxidase) as the generated selectable marker, conferring resistance to oxyfluorfen, and identified precise edits in the homolog of bacterial ftsY or the WD and TetratriCopeptide repeats protein 1 genes in ∼1% of the oxyfluorfen resistant colonies. Analysis of the target site sequences in edited mutants suggested that ssODNs were used as templates for DNA synthesis during homology directed repair, a process prone to replicative errors. The Chlamydomonas acetolactate synthase gene could also be efficiently edited to serve as an alternative selectable marker. This transgene-free strategy may allow creation of individual strains containing precise mutations in multiple target genes, to study complex cellular processes, pathways, or structures.