Cytokinin-deficient Chlamydomonas reinhardtii CRISPR-Cas9 mutants show reduced ability to prime resistance of tobacco against bacterial infection.

Although microalgae have only recently been recognized as part of the plant and soil microbiome, their application as biofertilizers has a tradition in sustainable crop production. Under consideration of their ability to produce the plant growth-stimulating hormone cytokinin (CK), known to also induce pathogen resistance, we have assessed the biocontrol ability of CK-producing microalgae. All pro- and eukaryotic CK-producing microalgae tested were able to enhance the tolerance of tobacco against Pseudomonas syringae pv. tabaci (PsT) infection. Since Chlamydomonas reinhardtii (Cre) proved to be the most efficient, we functionally characterized its biocontrol ability. We employed the CRISPR-Cas9 system to generate the first knockouts of CK biosynthetic genes in microalgae. Specifically, we targeted Cre Lonely Guy (LOG) and isopentenyltransferase (IPT) genes, the key genes of CK biosynthesis. While Cre wild-type exhibits a strong protection, the CK-deficient mutants have a reduced ability to induce plant defence. The degree of protection correlates with the CK levels, with the IPT mutants showing less protection than the LOG mutants. Gene expression analyses showed that Cre strongly stimulates tobacco resistance through defence gene priming. This study functionally verifies that Cre primes defence responses with CK, which contributes to the robustness of the effect. This work contributes to elucidate microalgae-mediated plant defence priming and identifies the role of CKs. In addition, these results underscore the potential of CK-producing microalgae as biologicals in agriculture by combining biofertilizer and biocontrol ability for sustainable and environment-friendly crop management.

APX2 Is an Ascorbate Peroxidase-Related Protein that Regulates the Levels of Plastocyanin in Chlamydomonas.

The function of ascorbate peroxidase-related (APX-R) proteins, present in all green photosynthetic eukaryotes, remains unclear. This study focuses on APX-R from Chlamydomonas reinhardtii, namely, ascorbate peroxidase 2 (APX2). We showed that apx2 mutants exhibited a faster oxidation of the photosystem I primary electron donor, P700, upon sudden light increase and a slower re-reduction rate compared to the wild type, pointing to a limitation of plastocyanin. Spectroscopic, proteomic and immunoblot analyses confirmed that the phenotype was a result of lower levels of plastocyanin in the apx2 mutants. The redox state of P700 did not differ between wild type and apx2 mutants when the loss of function in plastocyanin was nutritionally complemented by growing apx2 mutants under copper deficiency. In this case, cytochrome c6 functionally replaces plastocyanin, confirming that lower levels of plastocyanin were the primary defect caused by the absence of APX2. Overall, the results presented here shed light on an unexpected regulation of plastocyanin level under copper-replete conditions, induced by APX2 in Chlamydomonas.

Improvement of cell growth in green algae Chlamydomonas reinhardtii through co-cultivation with yeast Saccharomyces cerevisiae.

PURPOSE: CO2 fixation methods using green algae have attracted considerable attention because they can be applied for the fixation of dilute CO2 in the atmosphere. However, green algae generally exhibit low CO2 fixation efficiency under atmospheric conditions. Therefore, it is a challenge to improve the CO2 fixation efficiency of green algae under atmospheric conditions. Co-cultivation of certain microalgae with heterotrophic microorganisms can increase the growth potential of microalgae under atmospheric conditions. The objective of this study was to determine the culture conditions under which the growth potential of green algae Chlamydomonas reinhardtii is enhanced by co-culturing with the yeast Saccharomyces cerevisiae, and to identify the cause of the enhanced growth potential. RESULTS: When C. reinhardtii and S. cerevisiae were co-cultured with an initial green algae to yeast inoculum ratio of 1:3, the cell concentration of C. reinhardtii reached 133 × 105 cells/mL on day 18 of culture, which was 1.5 times higher than that of the monoculture. Transcriptome analysis revealed that the expression levels of 363 green algae and 815 yeast genes were altered through co-cultivation. These included genes responsible for ammonium transport and CO2 enrichment mechanism in green algae and the genes responsible for glycolysis and stress responses in yeast. CONCLUSION: We successfully increased C. reinhardtii growth potential by co-culturing it with S. cerevisiae. The main reasons for this are likely to be an increase in inorganic nitrogen available to green algae via yeast metabolism and an increase in energy available for green algae growth instead of CO2 enrichment.

Gene dosage of independent dynein arm motor preassembly factors influences cilia assembly in Chlamydomonas reinhardtii.

Motile cilia assembly utilizes over 800 structural and cytoplasmic proteins. Variants in approximately 58 genes cause primary ciliary dyskinesia (PCD) in humans, including the dynein arm (pre)assembly factor (DNAAF) gene DNAAF4. In humans, outer dynein arms (ODAs) and inner dynein arms (IDAs) fail to assemble motile cilia when DNAAF4 function is disrupted. In Chlamydomonas reinhardtii, a ciliated unicellular alga, the DNAAF4 ortholog is called PF23. The pf23-1 mutant assembles short cilia and lacks IDAs, but partially retains ODAs. The cilia of a new null allele (pf23-4) completely lack ODAs and IDAs and are even shorter than cilia from pf23-1. In addition, PF23 plays a role in the cytoplasmic modification of IC138, a protein of the two-headed IDA (I1/f). As most PCD variants in humans are recessive, we sought to test if heterozygosity at two genes affects ciliary function using a second-site non-complementation (SSNC) screening approach. We asked if phenotypes were observed in diploids with pairwise heterozygous combinations of 21 well-characterized ciliary mutant Chlamydomonas strains. Vegetative cultures of single and double heterozygous diploid cells did not show SSNC for motility phenotypes. When protein synthesis is inhibited, wild-type Chlamydomonas cells utilize the pool of cytoplasmic proteins to assemble half-length cilia. In this sensitized assay, 8 double heterozygous diploids with pf23 and other DNAAF mutations show SSNC; they assemble shorter cilia than wild-type. In contrast, double heterozygosity of the other 203 strains showed no effect on ciliary assembly. Immunoblots of diploids heterozygous for pf23 and wdr92 or oda8 show that PF23 is reduced by half in these strains, and that PF23 dosage affects phenotype severity. Reductions in PF23 and another DNAAF in diploids affect the ability to assemble ODAs and IDAs and impedes ciliary assembly. Thus, dosage of multiple DNAAFs is an important factor in cilia assembly and regeneration.

Potential cellular targets of platinum in the freshwater microalgae Chlamydomonas reinhardtii and Nitzschia palea revealed by transcriptomics.

Platinum group element levels have increased in natural aquatic environments in the last few decades, in particular as a consequence of the use of automobile catalytic converters on a global scale. Concentrations of Pt over tens of µg L-1 have been observed in rivers and effluents. This raises questions regarding its possible impacts on aquatic ecosystems, as Pt natural background concentrations are extremely low to undetectable. Primary producers, such as microalgae, are of great ecological importance, as they are at the base of the food web. The purpose of this work was to better understand the impact of Pt on a cellular level for freshwater unicellular algae. Two species with different characteristics, a green alga C. reinhardtii and a diatom N. palea, were studied. The bioaccumulation of Pt as well as its effect on growth were quantified. Moreover, the induction or repression factors of 16 specific genes were determined and allowed for the determination of possible intracellular effects and pathways of Pt. Both species seemed to be experiencing copper deficiency as suggested by inductions of genes linked to copper transporters. This is an indication that Pt might be internalized through the Cu(I) metabolic pathway. Moreover, Pt could possibly be excreted using an efflux pump. Other highlights include a concentration-dependent negative impact of Pt on mitochondrial metabolism for C. reinhardtii which is not observed for N. palea. These findings allowed for a better understanding of some of the possible impacts of Pt on freshwater primary producers, and also lay the foundations for the investigation of pathways for Pt entry at the base of the aquatic food web.

Bioprospecting of Chlamydomonas reinhardtii for boosting biofuel-related products production based on novel aggregation-induced emission active extracellular polymeric substances nanoprobes.

Biofuel production from microalgae has been greatly restricted by low biomass productivity and long-term photosynthetic efficacy. Here, a novel strategy for selecting high-growing, stress-resistant algal strains with high photosynthetic capacity was proposed based on biocompatible extracellular polymeric substances (EPS) probes with aggregation-induced emission (AIE) properties. Specifically, AIE active EPS probes were synthesized for in-situ long-term monitoring of the EPS productivity at different algal growth stages. By coupling the AIE-based fluorescent techniques, algal cells were classified into four diverse populations based on their chlorophyll and EPS signals. Mechanistic studies on the sorted algal cells revealed their remarkable stress resistance and high expression of cell division, biopolymer production and photosynthesis-related genes. The sorted and subcultured algal cells consistently exhibited relatively higher growth rates and photosynthetic capacities, resulting in an increased (1.2 to 1.8-fold) algal biomass production, chlorophyll, and lipids. This study can potentially open new strategies to boost microalgal-based biofuel production.

Expression, purification, and characterization of transmembrane protein homogentisate solanesyltransferase.

Homogentisate solanesyltransferase (HST) is a crucial enzyme in the plastoquinone biosynthetic pathway and has recently emerged as a promising target for herbicides. In this study, we successfully expressed and purified a stable and highly pure form of seven times transmembrane protein Chlamydomonas reinhardtii HST (CrHST). The final yield of CrHST protein obtained was 12.2 mg per liter of M9 medium. We evaluated the inhibitory effect on CrHST using Des-Morpholinocarbony Cyclopyrimorate (DMC) and found its IC50 value to be 3.63 ± 0.53 µM, indicating significant inhibitory potential. Additionally, we investigated the substrate affinity of CrHST with two substrates, determining the Km values as 22.76 ± 1.70 µM for FPP and 48.54 ± 3.89 µM for HGA. Through sequence alignment analyses and three-dimensional structure predictions, we identified conserved amino acid residues forming the active cavity in the enzyme. The results from molecular docking and binding energy calculations indicate that DMC has a greater binding affinity with HST compared to HGA. These findings represent substantial progress in understanding CrHST's properties and potential for herbicide development. KEY POINTS: • First high-yield transmembrane CrHST protein via E. coli system • Preliminarily identified active cavity composition via activity testing • Determined substrate and inhibitor modes via molecular docking.

The Effects of De Novo Mutation on Gene Expression and the Consequences for Fitness in Chlamydomonas reinhardtii.

Mutation is the ultimate source of genetic variation, the bedrock of evolution. Yet, predicting the consequences of new mutations remains a challenge in biology. Gene expression provides a potential link between a genotype and its phenotype. But the variation in gene expression created by de novo mutation and the fitness consequences of mutational changes to expression remain relatively unexplored. Here, we investigate the effects of >2,600 de novo mutations on gene expression across the transcriptome of 28 mutation accumulation lines derived from 2 independent wild-type genotypes of the green algae Chlamydomonas reinhardtii. We observed that the amount of genetic variance in gene expression created by mutation (Vm) was similar to the variance that mutation generates in typical polygenic phenotypic traits and approximately 15-fold the variance seen in the limited species where Vm in gene expression has been estimated. Despite the clear effect of mutation on expression, we did not observe a simple additive effect of mutation on expression change, with no linear correlation between the total expression change and mutation count of individual MA lines. We therefore inferred the distribution of expression effects of new mutations to connect the number of mutations to the number of differentially expressed genes (DEGs). Our inferred DEE is highly L-shaped with 95% of mutations causing 0-1 DEG while the remaining 5% are spread over a long tail of large effect mutations that cause multiple genes to change expression. The distribution is consistent with many cis-acting mutation targets that affect the expression of only 1 gene and a large target of trans-acting targets that have the potential to affect tens or hundreds of genes. Further evidence for cis-acting mutations can be seen in the overabundance of mutations in or near differentially expressed genes. Supporting evidence for trans-acting mutations comes from a 15:1 ratio of DEGs to mutations and the clusters of DEGs in the co-expression network, indicative of shared regulatory architecture. Lastly, we show that there is a negative correlation with the extent of expression divergence from the ancestor and fitness, providing direct evidence of the deleterious effects of perturbing gene expression.

Optimized transgene expression in the red alga Porphyridium purpureum and efficient recombinant protein secretion into the culture medium.

Microalgae represent a promising but yet underexplored production platform for biotechnology. The vast majority of studies on recombinant protein expression in algae have been conducted in a single species, the green alga Chlamydomonas reinhardtii. However, due to epigenetic silencing, transgene expression in Chlamydomonas is often inefficient. Here we have investigated parameters that govern efficient transgene expression in the red microalga Porphyridium purpureum. Porphyridium is unique in that the introduced transformation vectors are episomally maintained as autonomously replicating plasmids in the nucleus. We show that full codon optimization to the preferred codon usage in the Porphyridium genome confers superior transgene expression, not only at the level of protein accumulation, but also at the level of mRNA accumulation, indicating that high translation rates increase mRNA stability. Our optimized expression constructs resulted in YFP accumulation to unprecedented levels of up to 5% of the total soluble protein. We also designed expression cassettes that target foreign proteins to the secretory pathway and lead to efficient protein secretion into the culture medium, thus simplifying recombinant protein harvest and purification. Our study paves the way to the exploration of red microalgae as expression hosts in molecular farming for recombinant proteins and metabolites.

GATA family transcription factors in alga Chlamydomonas reinhardtii.

GATA family transcription factors (GATA-TFs) are metalloproteins that regulate many metabolic pathways. These conserved proteins recognize the consensus sequence (A/T)GATA(A/G) in the promoter regions of many genes and regulate their transcription in response to environmental signals. Currently, the study of GATA-TFs is of increasing interest. GATA genes and their proteins are most actively studied in vascular plants and fungi. Based on the results of numerous studies, it has been shown that GATA factors regulate the metabolic pathways of nitrogen and carbon, and also play a major role in the processes induced by light and circadian rhythms. In algae, GATA-TFs remain poorly studied, and information about them is scattered. In this work, all known data on GATA-TFs in the unicellular green alga Chlamydomonas reinhardtii has been collected and systematized. The genome of this alga contains 12 GATA coding genes. Using the phylogenetic analysis, we identified three classes of GATA factors in C. reinhardtii according to the structure of the zinc finger domain and showed their difference from the classification of GATA factors developed on vascular plants.

Extraction and selection of high-molecular-weight DNA for long-read sequencing from Chlamydomonas reinhardtii.

Recent advances in long-read sequencing technologies have enabled the complete assembly of eukaryotic genomes from telomere to telomere by allowing repeated regions to be fully sequenced and assembled, thus filling the gaps left by previous short-read sequencing methods. Furthermore, long-read sequencing can also help characterizing structural variants, with applications in the fields of genome evolution or cancer genomics. For many organisms, the main bottleneck to sequence long reads remains the lack of robust methods to obtain high-molecular-weight (HMW) DNA. For this purpose, we developed an optimized protocol to extract DNA suitable for long-read sequencing from the unicellular green alga Chlamydomonas reinhardtii, based on CTAB/phenol extraction followed by a size selection step for long DNA molecules. We provide validation results for the extraction protocol, as well as statistics obtained with Oxford Nanopore Technologies sequencing.

Protocol to isolate nuclei from Chlamydomonas reinhardtii for ATAC sequencing.

The isolation of sufficient amounts of intact nuclei is essential to obtain high-resolution maps of chromatin accessibility via assay for transposase-accessible chromatin using sequencing (ATAC-seq). Here, we present a protocol for tag-free isolation of nuclei from both cell walled and cell wall-deficient strains of the green model alga Chlamydomonas reinhardtii at a suitable quality for ATAC-seq. We describe steps for nuclei isolation, quantification, and downstream ATAC-seq. This protocol is optimized to shorten the time of isolation and quantification of nuclei.

SAGA1 and SAGA2 promote starch formation around proto-pyrenoids in Arabidopsis chloroplasts.

The pyrenoid is a chloroplastic microcompartment in which most algae and some terrestrial plants condense the primary carboxylase, Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase) as part of a CO2-concentrating mechanism that improves the efficiency of CO2 capture. Engineering a pyrenoid-based CO2-concentrating mechanism (pCCM) into C3 crop plants is a promising strategy to enhance yield capacities and resilience to the changing climate. Many pyrenoids are characterized by a sheath of starch plates that is proposed to act as a barrier to limit CO2 diffusion. Recently, we have reconstituted a phase-separated "proto-pyrenoid" Rubisco matrix in the model C3 plant Arabidopsis thaliana using proteins from the alga with the most well-studied pyrenoid, Chlamydomonas reinhardtii [N. Atkinson, Y. Mao, K. X. Chan, A. J. McCormick, Nat. Commun. 11, 6303 (2020)]. Here, we describe the impact of introducing the Chlamydomonas proteins StArch Granules Abnormal 1 (SAGA1) and SAGA2, which are associated with the regulation of pyrenoid starch biogenesis and morphology. We show that SAGA1 localizes to the proto-pyrenoid in engineered Arabidopsis plants, which results in the formation of atypical spherical starch granules enclosed within the proto-pyrenoid condensate and adjacent plate-like granules that partially cover the condensate, but without modifying the total amount of chloroplastic starch accrued. Additional expression of SAGA2 further increases the proportion of starch synthesized as adjacent plate-like granules that fully encircle the proto-pyrenoid. Our findings pave the way to assembling a diffusion barrier as part of a functional pCCM in vascular plants, while also advancing our understanding of the roles of SAGA1 and SAGA2 in starch sheath formation and broadening the avenues for engineering starch morphology.

Protocol for precision editing of endogenous Chlamydomonas reinhardtii genes with CRISPR-Cas.

CRISPR-Cas genome engineering in the unicellular green algal model Chlamydomonas reinhardtii has until recently suffered from low integration efficiencies despite traditional genetics being well established. Here, we present a protocol for efficient homology-directed knockin mutagenesis in all commonly used strains of Chlamydomonas. We describe steps for scarless integration of fusion tags and sequence modifications of almost all proteins without the need for a preceding mutant line. We further empower this genetic-editing approach by efficient crossing and highly robust screening protocols. For complete details on the use and execution of this protocol, please refer to Nievergelt et al. (2023).1.

Successful reversal of transgene silencing in Chlamydomonas reinhardtii.

Chlamydomonas reinhardtii has been successfully engineered to produce compounds of interest following transgene integration and heterologous protein expression. The advantages of this model include the availability of validated tools for bioengineering, its photosynthetic ability, and its potential use as biofuel. Despite this, breakthroughs have been hindered by its ability to silence transgene expression through epigenetic changes. Histone deacetylases (HDAC) are main players in gene expression. We hypothesized that transgene silencing can be reverted with chemical treatments using HDAC inhibitors. To analyze this, we transformed C. reinhardtii, integrating into its genome the mVenus reporter gene under the HSP70-rbcs2 promoter. From 384 transformed clones, 88 (22.9%) displayed mVenus positive (mVenus+ ) cells upon flow-cytometry analysis. Five clones with different fluorescence intensities were selected. The number of integrated copies was measured by qPCR. Transgene expression levels were followed over the growth cycle and upon SAHA treatment, using a microplate reader, flow cytometry, RT-qPCR, and western blot analysis. First, we observed that expression varies with the cell cycle, reaching a maximum level just before the stationary phase in all clones. Second, we uncovered that supplementation with HDAC inhibitors of the hydroxamate family, such as vorinostat (suberoylanilide-hydroxamic-acid, SAHA) at the initiation of culture increases the frequency (% of mVenus+ cells) and the level of transgene expression per cell over the whole growth cycle, through histone deacetylase inhibition. Thus, we propose a new tool to successfully trigger the expression of heterologous proteins in the green algae C. reinhardtii, overcoming its main obstacle as an expression platform.

The role of the pigment-protein complex LHCBM1 in nonphotochemical quenching in Chlamydomonas reinhardtii.

Nonphotochemical quenching (NPQ) is the process that protects photosynthetic organisms from photodamage by dissipating the energy absorbed in excess as heat. In the model green alga Chlamydomonas reinhardtii, NPQ is abolished in the knock-out mutants of the pigment-protein complexes LHCSR3 and LHCBM1. However, while LHCSR3 is a pH sensor and switches to a quenched conformation at low pH, the role of LHCBM1 in NPQ has not been elucidated yet. In this work, we combined biochemical and physiological measurements to study short-term high-light acclimation of npq5, the mutant lacking LHCBM1. In low light in the absence of this complex, the antenna size of PSII was smaller than in its presence; this effect was marginal in high light (HL), implying that a reduction of the antenna was not responsible for the low NPQ. The mutant expressed LHCSR3 at the wild-type level in HL, indicating that the absence of this complex is also not the reason. Finally, NPQ remained low in the mutant even when the pH was artificially lowered to values that can switch LHCSR3 to the quenched conformation. We concluded that both LHCSR3 and LHCBM1 are required for the induction of NPQ and that LHCBM1 is the interacting partner of LHCSR3. This interaction can either enhance the quenching capacity of LHCSR3 or connect this complex with the PSII supercomplex.

The UV-A Receptor CRY-DASH1 Up- and Downregulates Proteins Involved in Different Plastidial Pathways.

Algae encode up to five different types of cryptochrome photoreceptors. So far, relatively little is known about the biological functions of the DASH (Drosophila, Arabidopsis, Synechocystis and Homo)-type cryptochromes. The green alga Chlamydomonas reinhardtii encodes two of them. CRY-DASH1 also called DCRY1 has its maximal absorption peak in the UV-A range. It is localized in the chloroplast and plays an important role in balancing the photosynthetic machinery. Here, we performed a comparative analysis of chloroplast proteins from wild type and a knockout mutant of CRY-DASH1 named cry-dash1mut, using label-free quantitative proteomics as well as immunoblotting. Our results show upregulation of enzymes involved in specific pathways in the mutant including key enzymes of chlorophyll and carotenoid biosynthesis consistent with increased levels of photosynthetic pigments in cry-dash1mut. There is also an increase in certain redox as well as photosystem I and II proteins, including D1. Strikingly, CRY-DASH1 is coregulated in a D1 deletion mutant, where its amount is increased. In contrast, key proteins of the central carbon metabolism, including glycolysis/gluconeogenesis, dark fermentation and the oxidative pentose phosphate pathway are downregulated in cry-dash1mut. Similarly, enzymes of histidine biosynthesis are downregulated in cry-dash1mut leading to a reduction in the amount of free histidine. Yet, transcripts encoding for several of these proteins are at a similar level in the wild type and cry-dash1mut or even opposite. We show that CRY-DASH1 can bind to RNA, taking the psbA RNA encoding D1 as target. These data suggest that CRY-DASH1 regulates plastidial metabolic pathways at the posttranscriptional level.

Immunophilin FKB20-2 participates in oligomerization of Photosystem I in Chlamydomonas.

PSI is a sophisticated photosynthesis protein complex that fuels the light reaction of photosynthesis in algae and vascular plants. While the structure and function of PSI have been studied extensively, the dynamic regulation on PSI oligomerization and high light response is less understood. In this work, we characterized a high light-responsive immunophilin gene FKB20-2 (FK506-binding protein 20-2) required for PSI oligomerization and high light tolerance in Chlamydomonas (Chlamydomonas reinhardtii). Biochemical assays and 77-K fluorescence measurement showed that loss of FKB20-2 led to the reduced accumulation of PSI core subunits and abnormal oligomerization of PSI complexes and, particularly, reduced PSI intermediate complexes in fkb20-2. It is noteworthy that the abnormal PSI oligomerization was observed in fkb20-2 even under dark and dim light growth conditions. Coimmunoprecipitation, MS, and yeast 2-hybrid assay revealed that FKB20-2 directly interacted with the low molecular weight PSI subunit PsaG, which might be involved in the dynamic regulation of PSI-light-harvesting complex I supercomplexes. Moreover, abnormal PSI oligomerization caused accelerated photodamage to PSII in fkb20-2 under high light stress. Together, we demonstrated that immunophilin FKB20-2 affects PSI oligomerization probably by interacting with PsaG and plays pivotal roles during Chlamydomonas tolerance to high light.

Conserved cobalamin acquisition protein 1 is essential for vitamin B12 uptake in both Chlamydomonas and Phaeodactylum.

Microalgae play an essential role in global net primary productivity and global biogeochemical cycling. Despite their phototrophic lifestyle, over half of algal species depend for growth on acquiring an external supply of the corrinoid vitamin B12 (cobalamin), a micronutrient produced only by a subset of prokaryotic organisms. Previous studies have identified protein components involved in vitamin B12 uptake in bacterial species and humans. However, little is known about its uptake in algae. Here, we demonstrate the essential role of a protein, cobalamin acquisition protein 1 (CBA1), in B12 uptake in Phaeodactylum tricornutum using CRISPR-Cas9 to generate targeted knockouts and in Chlamydomonas reinhardtii by insertional mutagenesis. In both cases, CBA1 knockout lines could not take up exogenous vitamin B12. Complementation of the C. reinhardtii mutants with the wild-type CBA1 gene restored B12 uptake, and regulation of CBA1 expression via a riboswitch element enabled control of the phenotype. When visualized by confocal microscopy, a YFP-fusion with C. reinhardtii CBA1 showed association with membranes. Bioinformatics analysis found that CBA1-like sequences are present in all major eukaryotic phyla. In algal taxa, the majority that encoded CBA1 also had genes for B12-dependent enzymes, suggesting CBA1 plays a conserved role. Our results thus provide insight into the molecular basis of algal B12 acquisition, a process that likely underpins many interactions in aquatic microbial communities.

Type II metacaspase mediates light-dependent programmed cell death in Chlamydomonas reinhardtii.

Among the crucial processes that preside over the destiny of cells from any type of organism are those involving their self-destruction. This process is well characterized and conceptually logical to understand in multicellular organisms; however, the levels of knowledge and comprehension of its existence are still quite enigmatic in unicellular organisms. We use Chlamydomonas (Chlamydomonas reinhardtii) to lay the foundation for understanding the mechanisms of programmed cell death (PCD) in a unicellular photosynthetic organism. In this paper, we show that while PCD induces the death of a proportion of cells, it allows the survival of the remaining population. A quantitative proteomic analysis aiming at unveiling the proteome of PCD in Chlamydomonas allowed us to identify key proteins that led to the discovery of essential mechanisms. We show that in Chlamydomonas, PCD relies on the light dependence of a photosynthetic organism to generate reactive oxygen species and induce cell death. Finally, we obtained and characterized mutants for the 2 metacaspase genes in Chlamydomonas and showed that a type II metacaspase is essential for PCD execution.