Evolutionary divergence of chloroplast FAD synthetase proteins

<p>Abstract</p> <p>Background</p> <p>Flavin adenine dinucleotide synthetases (FADSs) - a group of bifunctional enzymes that carry out the dual functions of riboflavin phosphorylation to produce flavin mononucleotide (FMN) and its subsequent adenylation to generate FAD in most prokaryotes - were studied in plants in terms of sequence, structure and evolutionary history.</p> <p>Results</p> <p>Using a variety of bioinformatics methods we have found that FADS enzymes localized to the chloroplasts, which we term as plant-like FADS proteins, are distributed across a variety of green plant lineages and constitute a divergent protein family clearly of cyanobacterial origin. The <it>C</it>-terminal module of these enzymes does not contain the typical riboflavin kinase active site sequence, while the <it>N</it>-terminal module is broadly conserved. These results agree with a previous work reported by Sandoval <it>et al</it>. in 2008. Furthermore, our observations and preliminary experimental results indicate that the <it>C-</it>terminus of plant-like FADS proteins may contain a catalytic activity, but different to that of their prokaryotic counterparts. In fact, homology models predict that plant-specific conserved residues constitute a distinct active site in the <it>C</it>-terminus.</p> <p>Conclusions</p> <p>A structure-based sequence alignment and an in-depth evolutionary survey of FADS proteins, thought to be crucial in plant metabolism, are reported, which will be essential for the correct annotation of plant genomes and further structural and functional studies. This work is a contribution to our understanding of the evolutionary history of plant-like FADS enzymes, which constitute a new family of FADS proteins whose <it>C</it>-terminal module might be involved in a distinct catalytic activity.</p

Elucidating The Mechanism Of Phenylpropanoid Regulation By The Arabidopsis Mediator Complex

The Mediator complex is a multi-protein co-regulator of eukaryotic transcription which plays a role in the expression of many, if not most, genes of the cell. Two Mediator subunits, REF4 and RFR1, were demonstrated to be important for the normal regulation of phenylpropanoid metabolism in Arabidopsis. Phenylpropanoids are a family of specialized plant metabolites derived from the amino acid phenylalanine and are involved in defense against pathogens, UV protection and structural support. In order to understand how Mediator regulates phenylpropanoid metabolism through REF4 and RFR1, a better basic understanding of this protein complex is required. Here I provide data on the initial steps in characterizing the Arabidopsis Mediator complex. First, I performed partial purification of Mediator using ion-exchange chromatography followed by LC-MS analysis of cation-exchange purified fraction. Then we generated and evaluated antibodies against specific Mediator subunits. Lastly, I used yeast two-hybrid to evaluate previously identified putative interactors of REF4, evaluated interaction between Mediator tail subunits with REF4 and RFR1, and identified new interaction candidates for RFR1

Chlamydomonas reinhardtii, an Algal Model in the Nitrogen Cycle

Nitrogen (N) is an essential constituent of all living organisms and the main limiting macronutrient. Even when dinitrogen gas is the most abundant form of N, it can only be used by fixing bacteria but is inaccessible to most organisms, algae among them. Algae preferentially use ammonium (NH4+) and nitrate (NO3−) for growth, and the reactions for their conversion into amino acids (N assimilation) constitute an important part of the nitrogen cycle by primary producers. Recently, it was claimed that algae are also involved in denitrification, because of the production of nitric oxide (NO), a signal molecule, which is also a substrate of NO reductases to produce nitrous oxide (N2O), a potent greenhouse gas. This review is focused on the microalga Chlamydomonas reinhardtii as an algal model and its participation in different reactions of the N cycle. Emphasis will be paid to new actors, such as putative genes involved in NO and N2O production and their occurrence in other algae genomes. Furthermore, algae/bacteria mutualism will be considered in terms of expanding the N cycle to ammonification and N fixation, which are based on the exchange of carbon and nitrogen between the two organisms

Predicted class-I aminoacyl tRNA synthetase-like proteins in non-ribosomal peptide synthesis

<p>Abstract</p> <p>Background</p> <p>Recent studies point to a great diversity of non-ribosomal peptide synthesis systems with major roles in amino acid and co-factor biosynthesis, secondary metabolism, and post-translational modifications of proteins by peptide tags. The least studied of these systems are those utilizing tRNAs or aminoacyl-tRNA synthetases (AAtRS) in non-ribosomal peptide ligation.</p> <p>Results</p> <p>Here we describe novel examples of AAtRS related proteins that are likely to be involved in the synthesis of widely distributed peptide-derived metabolites. Using sensitive sequence profile methods we show that the cyclodipeptide synthases (CDPSs) are members of the HUP class of Rossmannoid domains and are likely to be highly derived versions of the class-I AAtRS catalytic domains. We also identify the first eukaryotic CDPSs in fungi and in animals; they might be involved in immune response in the latter organisms. We also identify a paralogous version of the methionyl-tRNA synthetase, which is widespread in bacteria, and present evidence using contextual information that it might function independently of protein synthesis as a peptide ligase in the formation of a peptide- derived secondary metabolite. This metabolite is likely to be heavily modified through multiple reactions catalyzed by a metal-binding cupin domain and a lysine N6 monooxygenase that are strictly associated with this paralogous methionyl-tRNA synthetase (MtRS). We further identify an analogous system wherein the MtRS has been replaced by more typical peptide ligases with the ATP-grasp or modular condensation-domains.</p> <p>Conclusions</p> <p>The prevalence of these predicted biosynthetic pathways in phylogenetically distant, pathogenic or symbiotic bacteria suggests that metabolites synthesized by them might participate in interactions with the host. More generally, these findings point to a complete spectrum of recruitment of AAtRS to various non-ribosomal biosynthetic pathways, ranging from the conventional AAtRS, through closely related paralogous AAtRS dedicated to certain pathways, to highly derived versions of the class-I AAtRS catalytic domain like the CDPSs. Both the conventional AAtRS and their closely related paralogs often provide aminoacylated tRNAs for peptide ligations by MprF/Fem/MurM-type acetyltransferase fold ligases in the synthesis of peptidoglycan, N-end rule modifications of proteins, lipid aminoacylation or biosynthesis of antibiotics, such as valinamycin. Alternatively they might supply aminoacylated tRNAs for other biosynthetic pathways like that for tetrapyrrole or directly function as peptide ligases as in the case of mycothiol and those identified here.</p> <p>Reviewers</p> <p>This article was reviewed by Andrei Osterman and Igor Zhulin.</p

An Enigmatic Stramenopile Sheds Light on Early Evolution in Ochrophyta Plastid Organellogenesis

Ochrophyta is an algal group belonging to the Stramenopiles and comprises diverse lineages of algae which contribute significantly to the oceanic ecosystems as primary producers. However, early evolution of the plastid organelle in Ochrophyta is not fully understood. In this study, we provide a well-supported tree of the Stramenopiles inferred by the large-scale phylogenomic analysis that unveils the eukaryvorous (nonphotosynthetic) protist Actinophrys sol (Actinophryidae) is closely related to Ochrophyta. We used genomic and transcriptomic data generated from A. sol to detect molecular traits of its plastid and we found no evidence of plastid genome and plastid-mediated biosynthesis, consistent with previous ultrastructural studies that did not identify any plastids in Actinophryidae. Moreover, our phylogenetic analyses of particular biosynthetic pathways provide no evidence of a current and past plastid in A. sol. However, we found more than a dozen organellar aminoacyl-tRNA synthases (aaRSs) that are of algal origin. Close relationships between aaRS from A. sol and their ochrophyte homologs document gene transfer of algal genes that happened before the divergence of Actinophryidae and Ochrophyta lineages. We further showed experimentally that organellar aaRSs of A. sol are targeted exclusively to mitochondria, although organellar aaRSs in Ochrophyta are dually targeted to mitochondria and plastids. Together, our findings suggested that the last common ancestor of Actinophryidae and Ochrophyta had not yet completed the establishment of host–plastid partnership as seen in the current Ochrophyta species, but acquired at least certain nuclear-encoded genes for the plastid functions

Molecular interaction and evolution of jasmonate signaling with transport and detoxification of heavy metals and metalloids in plants

An increase in environmental pollution resulting from toxic heavy metals and metalloids [e.g., cadmium (Cd), arsenic (As), and lead (Pb)] causes serious health risks to humans and animals. Mitigation strategies need to be developed to reduce the accumulation of the toxic elements in plant-derived foods. Natural and genetically-engineered plants with hyper-tolerant and hyper-accumulating capacity of toxic minerals are valuable for phytoremediation. However, the molecular mechanisms of detoxification and accumulation in plants have only been demonstrated in very few plant species such as Arabidopsis and rice. Here, we review the physiological and molecular aspects of jasmonic acid and the jasmonate derivatives (JAs) in response to toxic heavy metals and metalloids. Jasmonates have been identified in, limiting the accumulation and enhancing the tolerance to the toxic elements, by coordinating the ion transport system, the activity of antioxidant enzymes, and the chelating capacity in plants. We also propose the potential involvement of Ca2+ signaling in the stress-induced production of jasmonates. Comparative transcriptomics analyses using the public datasets reveal the key gene families involved in the JA-responsive routes. Furthermore, we show that JAs may function as a fundamental phytohormone that protects plants from heavy metals and metalloids as demonstrated by the evolutionary conservation and diversity of these gene families in a large number of species of the major green plant lineages. Using ATP-Binding Cassette G (ABCG) transporter subfamily of six representative green plant species, we propose that JA transporters in Subgroup 4 of ABCGs may also have roles in heavy metal detoxification. Our paper may provide guidance toward the selection and development of suitable plant and crop species that are tolerant to toxic heavy metals and metalloids

Uso del nitrógeno en algas: desvelando piezas del rompecabezas de la asimilación del nitrógeno y su regulación en el alga modelo Chlamydomonas reinhardtii

Las algas, formando parte de la base de la cadena trófica de ecosistemas marinos y de agua dulce, son clave para la vida acuática. Estos organismos fotosintéticos, sometidos a fluctuaciones constantes de disponibilidad de nutrientes, muestran un alto nivel de adaptabilidad a estos ambientes dinámicos. Aunque el nitrógeno (N), nutriente esencial para la vida, es comúnmente usado por las algas en su forma inorgánica, algunas especies de algas pueden usar compuestos de N orgánico, los cuales pueden ser especialmente abundantes debido a la escorrentía y filtrado de áreas fertilizadas de forma intensiva. El alga modelo Chlamydomonas reinhardtii (Chlamydomonas) puede consumir fuentes de nitrógeno inorgánico (amonio, nitrato y nitrito), así como L-arginina y urea. Además, este alga presenta una Laminoácido oxidasa extracelular (LAO1) que desamina un amplio rango de aminoácidos. En este trabajo hemos estudiado el control de la señalización que da lugar a la preferencia de nitrato sobre N orgánico en Chlamydomonas, el papel clave de LAO1 en el uso de aminoácidos y péptidos, así como el establecimiento de nuevas interacciones mutualistas con bacterias que promueven el crecimiento en N orgánico. Capítulo 1 El factor de transcripción NIT2 es el regulador clave de los genes de la asimilación de nitrato en Chlamydomonas. En primer lugar, comparamos el transcriptoma de una estirpe silvestre y otra mutante nit2 de Chlamydomonas en respuesta a nitrato. Observamos que nitrato y NIT2 reprimen los genes involucrados en el uso de fuentes de N orgánicas, incluyendo LAO1. Mediante el uso de mutantes de Chlamydomonas demostramos que tanto el nitrato como el nitrito afectan negativamente el uso de aminoácidos por este alga. Capítulo 2 Las enzimas L-aminoácido oxidasa (LAAO, L-Amino Acid Oxidase) están ampliamente distribuidas en la naturaleza y se propone que su papel principal en hongos y algas es la captación de nitrógeno. Mediante búsquedas genómicas comparativas, no pudimos encontrar ningún ortólogo de LAO1 en ningún alga verde ni en plantas, pero identificamos ortólogos en 10 de otras 27 especies de algas, incluyendo Rhodophyta, Alveolata, Heterokonta, Haptophyta y Dinophyta. La construcción de un árbol filogenético de enzimas LAAO reveló que las secuencias identificadas como ortólogas de LAO1 -denominadas aquí como ALAAOs (Algal LAAOs)-, se agrupaban en la misma rama evolutiva. Observamos que en Chlamydomonas el gen LAO1 está situado adyacente a un gen que codifica una putativa proteína RidA, que resultó estar evolutivamente cercana a la de cianobacterias. Nuestro análisis filogenético apoya la idea de que las proteínas ALAAOs pueden tener un origen en el ancestro común de las algas, el cual se originó por la endosimbiosis de una cianobacteria por un protista. Mediante el uso de un mutante lao1 hemos mostrado que LAO1 era crucial para el crecimiento de Chlamydomonas en 16 de 20 aminoácidos proteinogénicos, así como para algunos di-/tri-péptidos. Además de amonio, las enzimas LAAO producen el correspondiente cetoácido y peróxido de hidrógeno. Hemos demostrado que la reacción espontánea de los productos derivados de la desaminación por LAO1 de Lalanina -ácido pirúvico y peróxido de hidrógeno- genera ácido acético. Capítulo 3 Aunque Chlamydomonas puede crecer en la mayoría de los L-aminoácidos y en algunos di-/tri-péptidos como únicas fuentes de N, este crecimiento es mucho menos eficiente que en fuentes de N inorgánicas, y además, algunos aminoácidos y péptidos no pueden ser usados por este alga. De forma fortuita descubrimos una contaminación Methylobacterium sp. que permitió el crecimiento de Chlamydomonas en un di-péptido que no puede asimilar. Las especies de Methylobacterium están incluidas en el grupo de bacterias promotoras del crecimiento de plantas (PGPB, del inglés Plant Growth- Promoting Bacteria), las cuales mejoran el crecimiento de las plantas. Tras el muestreo en campo, aislamiento e identificación de bacterias, encontramos que algunas especies salvajes, incluidas en los géneros Methylobacterium, Sphingomonas, Deinococcus y Chitinophagaceae, mejoran el crecimiento de Chlamydomonas en L-serina. Además, algunas especies de Methylobacterium permitieron el crecimiento de Chlamydomonas en algunos aminoácidos y péptidos que este alga no puede usar. Hemos demostrado un nuevo mutualismo basado en un intercambio metabólico de carbono y nitrógeno entre Chlamydomonas y M. aquaticum. Por otro lado, algunas especies de Methylobacterium mejoraron el crecimiento de Chlamydomonas en aminoácidos asimilables. Para esta mejora, la enzima LAO1 fue esencial para el crecimiento del consorcio con algunas estirpes de Methylobacterium, incluyendo M. extorquens, M. hispanicum y M. organophilum. La comunicación química en la interacción entre organismos diferentes media las relaciones simbióticas. Entre estas moléculas de señalización, el ácido indolacético es una de las más estudiadas. Descubrimos que la producción de índoles dependiente de L-triptófano por Chlamydomonas, observada aquí por primera vez, disminuyó significativamente en el mutante lao1. Además, observamos que altas concentraciones de ácido indolacético (> 30 μM) inhibe el crecimiento de Chlamydomonas y que esta inhibición se puede reducir por la presencia de especies de Methylobacterium.Algae, lying on the basis of food webs in marine and freshwater ecosystems, are key for aquatic life. These photosynthetic organisms live under continuously fluctuating nutrients availability, showing a high level of adaptability to these dynamic environments. Although the essential nutrient Nitrogen (N) is usually used by algae in its inorganic form, some algal species can use organic N compounds, which may become especially abundant due to terrestrial leaking and runoff of highly fertilized areas. The model alga Chlamydomonas reinhardtii (Chlamydomonas) uptakes inorganic N sources (i.e. ammonium, nitrate and nitrite), as well as L-arginine and urea. Moreover, this alga presents an extracellular L-amino acid oxidase (LAO1) with a broad substrate specificity that scavenges N from L-amino acids. In this work we studied the signaling control that leads the preference for nitrate over organic N in Chlamydomonas, the key role of LAO1 in the use of amino acids and peptides, as well as the establishment of new mutualistic interactions with bacteria to facilitate growth on organic N. Chapter 1 The transcription factor NIT2 is the key regulator of nitrate assimilation genes in Chlamydomonas. First, we compared the transcriptome of Chlamydomonas wild type (WT) and a nit2 mutant in response to nitrate. We observed that nitrate and NIT2 down-regulated genes involved in organic N scavenging, including LAO1. By the use of Chlamydomonas mutant strains we demonstrated that both nitrate and nitrite negatively impact the use of amino acids by this alga. Chapter 2 L-amino acid oxidase (LAAO) enzymes are widely present in nature and a major role as N scavenger has been proposed in fungal and algal LAAOs. By comparative genomic searches, we could not find any LAO1 ortholog in any green plant or plant, but we identified orthologs in 10 out of 27 other algal species, including Rhodophyta, Alveolata, Heterokonta, Haptophyta and Dinophyta algae. The construction of a LAAO phylogenetic tree revealed that algal protein sequences identified as LAO1 orthologs -named here as ALAAOs (Algal LAAOs)-, clustered on the same evolutionary branch. We observed that Chlamydomonas LAO1 gene is clustered to a putative RidA gene (LAO2), which resulted to be closely related to cyanobacterial members. Our phylogenetic analysis favoured the idea that ALAAOs may have a common origin in the archaeplastidan ancestor, originated by a protist engulfing cyanobacteria. By the use of a lao1 mutant, we showed that LAO1 was crucial for Chlamydomonas growth on 16 out of 20 proteinogenic amino acids, as well as for some di- and tripeptides. Besides ammonium, LAAO produces keto acids and hydrogen peroxide. We have demonstrated that the spontaneous reaction of the LAO1-derived products generated by L-alanine deamination, pyruvic acid and hydrogen peroxide, generates acetic acid. Chapter 3 Although Chlamydomonas can grow on most amino acids and some di-/tripeptides as the sole N sources, this growth is far less efficient than that on inorganic N, and yet, there are some amino acids and peptides that cannot be used by this alga. We serendipitously found a contaminating Methylobacterium sp. that allowed Chlamydomonas growth on a dipeptide that is not readily assimilated by this alga. Methylobacterium spp. are included in the PGPB group of bacteria (Plant Growth-Promoting Bacteria), which improve plant growth and fitness. After field sampling, isolation and identification of some bacteria, we found that some wild species, included in Methylobacterium, Sphingomonas, Deinococcus, and Chitinophagaceae genera, promoted Chlamydomonas growth on L-serine. Moreover, some Methylobacterium spp. allowed Chlamydomonas growth on amino acids and peptides that are not used by this alga. We have demonstrated a new mutualism based on carbon-nitrogen metabolic exchange between Chlamydomonas and M. aquaticum. Otherwise, some Methylobacterium spp. improved Chlamydomonas growth on assimilable amino acids. For this growth promotion, LAO1 was crucial for consortia growth with some Methylobacterium spp., including M. extorquens, M. hispanicum and M. organophilum. The chemical cross-talk between interacting organisms mediates the beneficial and pathogenic symbiotic relationships. Within these inter-kingdom signal molecules IAA (Indole-3-Acetic Acid) is one of the best studied. We found that L-tryptophan-dependent indoles production in Chlamydomonas, observed here for the first time, was significantly reduced in the lao1 mutant. Moreover, we observed that high levels of exogenously added IAA (> 30 μM) inhibits Chlamydomonas growth and that this inhibition may be relieved by the presence of Methylobacterium spp

Sulfate assimilation in eukaryotes: fusions, relocations and lateral transfers

<p>Abstract</p> <p>Background</p> <p>The sulfate assimilation pathway is present in photosynthetic organisms, fungi, and many bacteria, providing reduced sulfur for the synthesis of cysteine and methionine and a range of other metabolites. In photosynthetic eukaryotes sulfate is reduced in the plastids whereas in aplastidic eukaryotes the pathway is cytosolic. The only known exception is <it>Euglena gracilis</it>, where the pathway is localized in mitochondria. To obtain an insight into the evolution of the sulfate assimilation pathway in eukaryotes and relationships of the differently compartmentalized isoforms we determined the locations of the pathway in lineages for which this was unknown and performed detailed phylogenetic analyses of three enzymes involved in sulfate reduction: ATP sulfurylase (ATPS), adenosine 5'-phosphosulfate reductase (APR) and sulfite reductase (SiR).</p> <p>Results</p> <p>The inheritance of ATPS, APR and the related 3'-phosphoadenosine 5'-phosphosulfate reductase (PAPR) are remarkable, with multiple origins in the lineages that comprise the opisthokonts, different isoforms in chlorophytes and streptophytes, gene fusions with other enzymes of the pathway, evidence a eukaryote to prokaryote lateral gene transfer, changes in substrate specificity and two reversals of cellular location of host- and endosymbiont-originating enzymes. We also found that the ATPS and APR active in the mitochondria of <it>Euglena </it>were inherited from its secondary, green algal plastid.</p> <p>Conclusion</p> <p>Our results reveal a complex history for the enzymes of the sulfate assimilation pathway. Whilst they shed light on the origin of some characterised novelties, such as a recently described novel isoform of APR from Bryophytes and the origin of the pathway active in the mitochondria of Euglenids, the many distinct and novel isoforms identified here represent an excellent resource for detailed biochemical studies of the enzyme structure/function relationships.</p

Evolution of Plant Nucleotide-Sugar Interconversion Enzymes

Nucleotide-diphospho-sugars (NDP-sugars) are the building blocks of diverse polysaccharides and glycoconjugates in all organisms. In plants, 11 families of NDP-sugar interconversion enzymes (NSEs) have been identified, each of which interconverts one NDP-sugar to another. While the functions of these enzyme families have been characterized in various plants, very little is known about their evolution and origin. Our phylogenetic analyses indicate that all the 11 plant NSE families are distantly related and most of them originated from different progenitor genes, which have already diverged in ancient prokaryotes. For instance, all NSE families are found in the lower land plant mosses and most of them are also found in aquatic algae, implicating that they have already evolved to be capable of synthesizing all the 11 different NDP-sugars. Particularly interesting is that the evolution of RHM (UDP-L-rhamnose synthase) manifests the fusion of genes of three enzymatic activities in early eukaryotes in a rather intriguing manner. The plant NRS/ER (nucleotide-rhamnose synthase/epimerase-reductase), on the other hand, evolved much later from the ancient plant RHMs through losing the N-terminal domain. Based on these findings, an evolutionary model is proposed to explain the origin and evolution of different NSE families. For instance, the UGlcAE (UDP-D-glucuronic acid 4-epimerase) family is suggested to have evolved from some chlamydial bacteria. Our data also show considerably higher sequence diversity among NSE-like genes in modern prokaryotes, consistent with the higher sugar diversity found in prokaryotes. All the NSE families are widely found in plants and algae containing carbohydrate-rich cell walls, while sporadically found in animals, fungi and other eukaryotes, which do not have or have cell walls with distinct compositions. Results of this study were shown to be highly useful for identifying unknown genes for further experimental characterization to determine their functions in the synthesis of diverse glycosylated molecules

Molecular Mechanisms Regulating Chronological Aging and Cell Death in the Toxic Dinoflagellate, Karenia brevis

The toxic dinoflagellate, Karenia brevis, forms nearly annual blooms in the Gulf of Mexico that persist for many months in coastal waters, causing extensive marine animal mortalities and human health impacts. The molecular mechanisms that contribute to cell survival in high density, low growth blooms, and the mechanisms leading to often rapid bloom demise are not well understood. The studies presented in this dissertation investigate the existence and involvement of a programmed cell death-like (PCD-like) pathway in the demise of K. brevis cultures following oxidative stress and chronological aging. Firstly, to gain an understanding of the molecular processes that underlie chronological aging in this dinoflagellate, a microarray study was carried out and identified extensive transcriptomic remodeling during the transition into stationary phase indicative of a shift in the metabolic and signaling requirements for survival in a quiescent non-dividing phase. To better understand the connection between the transcriptomic context identified in the microarray study and the presence of a PCO-like pathway in K. brevis, hallmark morphological and biochemical changes (DNA fragmentation, caspase-like activity, and caspase 3-like protein expression) were used to define PCD-like morphological changes following chronological aging and oxidative stress. Targeted in silico bioinformatic mining was used to identify enzymes potentially responsible for the activities observed, as well as the substrates. Finally, K. brevis S-adenosylmethionine synthetase (KbAdoMetS), a putative caspase substrate predicted from the bioinformatics screen, was examined using MALDI-TOF MS to confirm the validity of the bioinformatics approach. Taken together, this work identified that K. brevis contains morphological changes indicative of a caspase-dependent PCD-like pathway and that KbAdoMetS is a caspase 3-like substrate. Finally, we sought to characterize the presence of metacaspases in Karenia brevis, and specifically evaluated the role of metacaspase 1 (KbMC1) during chronological aging and death in culture. Immunocytochemistry, subcellular fractionation, and western blotting results using a custom KbMC1 peptide antibody indicate that KbMC1 may be involved in PCD-like execution through its chloroplastic localization with proposed interactions with the photosynthetic machinery. This study provides the first comprehensive investigation of the molecular processes regulating chronological aging and execution of PCD-like death in a toxic dinoflagellate