Molecular machinery of auxin synthesis,secretion, and perception in the unicellular chlorophyte alga Chlorella sorokiniana UTEX 1230

Indole-3-acetic acid is a ubiquitous small molecule found in all domains of life. It is the predominant and most active auxin in seed plants, where it coordinates a variety of complex growth and development processes. The potential origin of auxin signaling in algae remains a matter of some controversy. In order to clarify the evolutionary context of algal auxin signaling, we undertook a genomic survey to assess whether auxin acts as a signaling molecule in the emerging model chlorophyte Chlorella sorokiniana UTEX 1230. C. sorokiniana produces the auxin indole-3-acetic acid (IAA), which was present in both the cell pellet and in the supernatant at a concentration of ~ 1 nM, and its genome encodes orthologs of genes related to auxin synthesis, transport, and signaling in higher plants. Candidate orthologs for the canonical AUX/IAA signaling pathway were not found; however, auxin-binding protein 1 (ABP1), an alternate auxin receptor, is present and highly conserved at essential auxin binding and zinc coordinating residues. Additionally, candidate orthologs for PIN proteins, responsible for intercellular, vectorial auxin transport in higher plants, were not found, but PILs (PIN-Like) proteins, a recently discovered family that mediates intracellular auxin transport, were identified. The distribution of auxin related gene in this unicellular chlorophyte demonstrates that a core suite of auxin signaling components was present early in the evolution of plants. Understanding the simplified auxin signaling pathways in chlorophytes will aid in understanding phytohormone signaling and crosstalk in seed plants, and in understanding the diversification and integration of developmental signals during the evolution of multicellular plants

Synthesis, secretion, and perception of abscisic acid regulates stress responses in \u3ci\u3eChlorella sorokiniana\u3c/i\u3e

Abscisic acid (ABA) is a phytohormone that has been extensively characterized in higher plants for its roles in seed and bud dormancy, leaf abscission, and stress responses. Genomic studies have identified orthologs for ABA-related genes throughout the Viridiplantae, including in unicellular algae; however, the role of ABA in algal physiology has not been characterized, and the existence of such a role has been a matter of dispute. In this study, we demonstrate that ABA is involved in regulating algal stress responses. Chlorella sorokiniana strain UTEX 1230 contains genes orthologous to those of higher plants which are essential for ABA biosynthesis, sensing, and degradation. RNAseq-based transcriptomic studies reveal that treatment with ABA induces dramatic changes in gene expression profiles, including the induction of a subset of genes involved in DNA replication and repair, a phenomenon which has been demonstrated in higher plants. Pretreatment of C. sorokiniana cultures with ABA exerts a protective effect on cell viability in response to ultraviolet radiation. Additionally, C. sorokiniana produces and secretes biologically relevant amounts of both ABA and the oxylipin 12-oxo-phytodienoic acid (OPDA) into the growth medium in response to abiotic stressors. Taken together, these phenomena suggest that ABA signaling evolved as an intercellular stress response signaling molecule in eukaryotic microalgae prior to the evolution of multicellularity and colonization of land

Comparative genomics, transcriptomics, and physiology distinguish symbiotic from free-living \u3ci\u3eChlorella\u3c/i\u3e strains

Most animal–microbe symbiotic interactions must be advantageous to the host and provide nutritional benefits to the endosymbiont. When the host provides nutrients, it can gain the capacity to control the interaction, promote self-growth, and increase its fitness. Chlorella-like green algae engage in symbiotic relationships with certain protozoans, a partnership that significantly impacts the physiology of both organisms. Consequently, it is often challenging to grow axenic Chlorella cultures after isolation from the host because they are nutrient fastidious and often susceptible to virus infection. We hypothesize that the establishment of a symbiotic relationship resulted in natural selection for nutritional and metabolic traits that differentiate symbiotic algae from their free-living counterparts. Here, we compare metabolic capabilities of 5 symbiotic and 4 free-living Chlorella strains by determining growth levels on combinations of nitrogen and carbon sources. Data analysis by hierarchical clustering revealed clear separation of the symbiotic and free-living Chlorella into two distinct clades. Symbiotic algae did not grow on nitrate but did grow on two symbiont-specific amino acids (Asn and Ser) on which the free-living strains did not grow. The use of these amino acids was exclusively affected by the presence/absence of Ca2+ in the medium, and the differences were magnified if galactose was provided rather than sucrose or glucose. In addition, Chlorella variabilis NC64A genomic and differential expression analysis confirmed the presence of abundant amino acid transporter protein motifs, some of which were expressed constitutively both axenically and within the host. Significantly, all 5 symbiotic strains exhibited similar physiological phenotypes even though they were isolated as symbionts from different host organisms. Such similarities indicate a parallel coevolution of shared metabolic pathways across multiple independent symbiotic events. Collectively, our results suggest that physiological changes drive the Chlorella symbiotic phenotype and contribute to their natural fitness. Includes Supplementary materials

Comparative genomics, transcriptomics, and physiology distinguish symbiotic from free-living \u3ci\u3eChlorella\u3c/i\u3e strains

Most animal–microbe symbiotic interactions must be advantageous to the host and provide nutritional benefits to the endosymbiont. When the host provides nutrients, it can gain the capacity to control the interaction, promote self-growth, and increase its fitness. Chlorella-like green algae engage in symbiotic relationships with certain protozoans, a partnership that significantly impacts the physiology of both organisms. Consequently, it is often challenging to grow axenic Chlorella cultures after isolation from the host because they are nutrient fastidious and often susceptible to virus infection. We hypothesize that the establishment of a symbiotic relationship resulted in natural selection for nutritional and metabolic traits that differentiate symbiotic algae from their free-living counterparts. Here, we compare metabolic capabilities of 5 symbiotic and 4 free-living Chlorella strains by determining growth levels on combinations of nitrogen and carbon sources. Data analysis by hierarchical clustering revealed clear separation of the symbiotic and free-living Chlorella into two distinct clades. Symbiotic algae did not grow on nitrate but did grow on two symbiont-specific amino acids (Asn and Ser) on which the free-living strains did not grow. The use of these amino acids was exclusively affected by the presence/absence of Ca2+ in the medium, and the differences were magnified if galactose was provided rather than sucrose or glucose. In addition, Chlorella variabilis NC64A genomic and differential expression analysis confirmed the presence of abundant amino acid transporter protein motifs, some of which were expressed constitutively both axenically and within the host. Significantly, all 5 symbiotic strains exhibited similar physiological phenotypes even though they were isolated as symbionts from different host organisms. Such similarities indicate a parallel coevolution of shared metabolic pathways across multiple independent symbiotic events. Collectively, our results suggest that physiological changes drive the Chlorella symbiotic phenotype and contribute to their natural fitness. Includes Supplementary materials

Phytohormone signaling in Chlorella sorokiniana: perspectives on the evolution of plant cell-to-cell signaling

Cell-to-cell communication is a key aspect of microbial physiology and population dynamics, and a cornerstone in understanding the evolution of multicellularity. Quorum sensing in bacteria is a canonical example of microbial cell-to-cell signaling, in which bacteria use small molecule signals in order to monitor their population size and modulate their physiology accordingly. We propose that the evolution of plant hormone signaling arose in unicellular green algae, analogously to quorum sensing in bacteria, and that the complexity of these pathways required the recruitment of increasingly specific enzymes to increasingly sophisticated gene networks throughout the course of phytohormone signaling evolution. Using Chlorella sorokiniana UTEX 1230 as a model system, we address the evolution of cell-to-cell signaling from the perspective of phytohormone signaling evolution, particularly the evolution of indole-3-acetic acid (IAA) signaling and abscisic acid (ABA) signaling. We demonstrate that key components of these phytohormone signaling pathways are present in C. sorokiniana, and that these hormones are present and active in the physiology of these organisms. Indeed, the distribution of early auxin signaling related orthologs in the chlorophytes suggests that some auxin signaling machinery was available early in the evolution of plants. Abscisic acid (ABA) is a phytohormone that has been extensively characterized in higher plants for its role in stress response. This dissertation demonstrates that ABA is involved in regulating algal stress responses in Chlorella; additionally, the genome contains orthologs to essential genes in higher plants that control ABA biosynthesis, sensing, and degradation. Transcriptomic studies reveal that treatment with ABA induces dramatic changes in gene expression profiles, including transcripts associated with ABA signaling in higher plants. The physiological effects of phytohormones, together with the presence of phytohormone signaling orthologs, suggest that phytohormone signaling evolved as an intercellular stress response signaling molecule in eukaryotic microalgae prior to the evolution of multicellularity and colonization of land. Advisers: Kenneth W. Nickerson, Wayne R. Riekho

Molecular machinery of auxin synthesis,secretion, and perception in the unicellular chlorophyte alga Chlorella sorokiniana UTEX 1230

Indole-3-acetic acid is a ubiquitous small molecule found in all domains of life. It is the predominant and most active auxin in seed plants, where it coordinates a variety of complex growth and development processes. The potential origin of auxin signaling in algae remains a matter of some controversy. In order to clarify the evolutionary context of algal auxin signaling, we undertook a genomic survey to assess whether auxin acts as a signaling molecule in the emerging model chlorophyte Chlorella sorokiniana UTEX 1230. C. sorokiniana produces the auxin indole-3-acetic acid (IAA), which was present in both the cell pellet and in the supernatant at a concentration of ~ 1 nM, and its genome encodes orthologs of genes related to auxin synthesis, transport, and signaling in higher plants. Candidate orthologs for the canonical AUX/IAA signaling pathway were not found; however, auxin-binding protein 1 (ABP1), an alternate auxin receptor, is present and highly conserved at essential auxin binding and zinc coordinating residues. Additionally, candidate orthologs for PIN proteins, responsible for intercellular, vectorial auxin transport in higher plants, were not found, but PILs (PIN-Like) proteins, a recently discovered family that mediates intracellular auxin transport, were identified. The distribution of auxin related gene in this unicellular chlorophyte demonstrates that a core suite of auxin signaling components was present early in the evolution of plants. Understanding the simplified auxin signaling pathways in chlorophytes will aid in understanding phytohormone signaling and crosstalk in seed plants, and in understanding the diversification and integration of developmental signals during the evolution of multicellular plants

Sterol Biosynthesis in Four Green Algae: A Bioinformatic Analysis of the Ergosterol Versus Phytosterol Decision Point

Animals and fungi produce cholesterol and ergosterol, respectively, while plants produce the phytosterols stigmasterol, campesterol, and bsitosterol in various combinations. The recent sequencing of many algal genomes allows the detailed reconstruction of the sterol metabolic pathways. Here, we characterized sterol synthesis in two sequenced Chlorella spp., the free-living C. sorokiniana, and symbiotic C. variabilis NC64A. Chlamydomonas reinhardtii was included as an internal control and Coccomyxa subellipsoidea as a plant-like outlier. We found that ergosterol was the major sterol produced by Chlorella spp. and C. reinhardtii, while C. subellipsoidea produced the three phytosterols found in plants. In silico analysis of the C. variabilis NC64A, C. sorokiniana, and C. subellipsoidea genomes identified 22 homologs of sterol biosynthetic genes from Arabidopsis thaliana, Saccharomyces cerevisiae, and C. reinhardtii. The presence of CAS1, CPI1, and HYD1 in the four algal genomes suggests the higher plant cycloartenol branch for sterol biosynthesis, confirming that algae and fungi use different pathways for ergosterol synthesis. Phylogenetic analysis for 40 oxidosqualene cyclases (OSCs) showed that the nine algal OSCs clustered with the cycloartenol cyclases, rather than the lanosterol cyclases, with the OSC for C. subellipsoidea positioned in between the higher plants and the eight other algae. With regard to why C. subellipsoidea produced phytosterols instead of ergosterol, we identified 22 differentially conserved positions where C. subellipsoidea CAS and A. thaliana CAS1 have one amino acid while the three ergosterol producing algae have another. Together, these results emphasize the position of the unicellular algae as an evolutionary transition point for sterols

Sterol Biosynthesis in Four Green Algae: A Bioinformatic Analysis of the Ergosterol Versus Phytosterol Decision Point

Animals and fungi produce cholesterol and ergosterol, respectively, while plants produce the phytosterols stigmasterol, campesterol, and β‐sitosterol in various combinations. The recent sequencing of many algal genomes allows the detailed reconstruction of the sterol metabolic pathways. Here, we characterized sterol synthesis in two sequenced Chlorella spp., the free‐living C. sorokiniana, and symbiotic C. variabilis NC64A. Chlamydomonas reinhardtii was included as an internal control and Coccomyxa subellipsoidea as a plant‐like outlier. We found that ergosterol was the major sterol produced by Chlorella spp. and C. reinhardtii, while C. subellipsoidea produced the three phytosterols found in plants. In silico analysis of the C. variabilis NC64A, C. sorokiniana, and C. subellipsoidea genomes identified 22 homologs of sterol biosynthetic genes from Arabidopsis thaliana, Saccharomyces cerevisiae, and C. reinhardtii. The presence of CAS1, CPI1, and HYD1 in the four algal genomes suggests the higher plant cycloartenol branch for sterol biosynthesis, confirming that algae and fungi use different pathways for ergosterol synthesis. Phylogenetic analysis for 40 oxidosqualene cyclases (OSCs) showed that the nine algal OSCs clustered with the cycloartenol cyclases, rather than the lanosterol cyclases, with the OSC for C. subellipsoidea positioned in between the higher plants and the eight other algae. With regard to why C. subellipsoidea produced phytosterols instead of ergosterol, we identified 22 differentially conserved positions where C. subellipsoidea CAS and A. thaliana CAS1 have one amino acid while the three ergosterol producing algae have another. Together, these results emphasize the position of the unicellular algae as an evolutionary transition point for sterols