Using Diatom and Apicomplexan Models to Study the Heme Pathway of Chromera velia

Heme biosynthesis is essential for almost all living organisms. Despite its conserved function, the pathway’s enzymes can be located in a remarkable diversity of cellular compartments in different organisms. This location does not always reflect their evolutionary origins, as might be expected from the history of their acquisition through endosymbiosis. Instead, the final subcellular localization of the enzyme reflects multiple factors, including evolutionary origin, demand for the product, availability of the substrate, and mechanism of pathway regulation. The biosynthesis of heme in the apicomonad Chromera velia follows a chimeric pathway combining heme elements from the ancient algal symbiont and the host. Computational analyses using different algorithms predict complex targeting patterns, placing enzymes in the mitochondrion, plastid, endoplasmic reticulum, or the cytoplasm. We employed heterologous reporter gene expression in the apicomplexan parasite Toxoplasma gondii and the diatom Phaeodactylum tricornutum to experimentally test these predictions. 5-aminolevulinate synthase was located in the mitochondria in both transfection systems. In T. gondii, the two 5-aminolevulinate dehydratases were located in the cytosol, uroporphyrinogen synthase in the mitochondrion, and the two ferrochelatases in the plastid. In P. tricornutum, all remaining enzymes, from ALA-dehydratase to ferrochelatase, were placed either in the endoplasmic reticulum or in the periplastidial space.

Transcriptomic Analysis Reveals the Roles of Detoxification Systems in Response to Mercury in Chromera velia

Heavy metal pollution is an increasing global concern. Among heavy metals, mercury (Hg) is especially dangerous because of its massive release into the environment and high toxicity, especially for aquatic organisms. The molecular response mechanisms of algae to Hg exposure are mostly unknown. Here, we combine physiological, biochemical, and transcriptomic analysis to provide, for the first time, a comprehensive view on the pathways activated in Chromera velia in response to toxic levels of Hg. Production of hydrogen peroxide and superoxide anion, two reactive oxygen species (ROS), showed opposite patterns in response to Hg2+ while reactive nitrogen species (RNS) levels did not change. A deep RNA sequencing analysis generated a total of 307,738,790 high-quality reads assembled in 122,874 transcripts, representing 89,853 unigenes successfully annotated in databases. Detailed analysis of the differently expressed genes corroborates the biochemical results observed in ROS production and suggests novel putative molecular mechanisms in the algal response to Hg2+. Moreover, we indicated that important transcription factor (TF) families associated with stress responses differentially expressed in C. velia cultures under Hg stress. Our study presents the first in-depth transcriptomic analysis of C. velia, focusing on the expression of genes involved in different detoxification defense systems in response to heavy metal stress.

Subcellular Compartments Interplay for Carbon and Nitrogen Allocation in Chromera velia and Vitrella brassicaformis

Endosymbioses necessitate functional cooperation of cellular compartments to avoid pathway redundancy and streamline the control of biological processes. To gain insight into the metabolic compartmentation in chromerids, phototrophic relatives to apicomplexan parasites, we prepared a reference set of proteins probably localized to mitochondria, cytosol, and the plastid, taking advantage of available genomic and transcriptomic data. Training of prediction algorithms with the reference set now allows a genome-wide analysis of protein localization in Chromera velia and Vitrella brassicaformis. We confirm that the chromerid plastids house enzymatic pathways needed for their maintenance and photosynthetic activity, but for carbon and nitrogen allocation, metabolite exchange is necessary with the cytosol and mitochondria. This indeed suggests that the regulatory mechanisms operate in the cytosol to control carbon metabolism based on the availability of both light and nutrients. We discuss that this arrangement is largely shared with apicomplexans and dinoflagellates, possibly stemming from a common ancestral metabolic architecture, and supports the mixotrophy of the chromerid algae.

Deep transcriptomic analysis of Chromera velia under Mercury-stress condition

The heavy metal pollution in ecosystems is of increasing global concern. The main sources of the metal toxicity are the industrial waste, mining and the use of pesticides containing heavy metals. Out of all heavy metals, mercury (Hg) is considered to be the one, easily accumulated in terrestrial plants as well as aquatic organism. Hg can induce more severe oxidative stress by triggering the production of reactive oxygen species (ROS) and damage macro-molecules. ROS serve not only as dangerous molecules that damage proteins, lipids and DNA but also as signalling molecules in the regulation of biological processes such as biotic and abiotic stress responses, growth and development. This study shows the change in the Chromera velia transcriptome due to Hg stress. C. velia is the recently discovered closest known relative of apicomplexans. Our study shows the first deep transcriptomic analysis of C. velia, specifically focusing on the expression level of the genes involved in detoxification defence systems under heavy metal stress. The results show that in total 1239 genes are differentially expressed. DEGs (Differentially expressed genes) showed overall up-regulation (1,070 genes) and only 169 up-regulated genes in cultures under Mercury stress (Cvel_mer) when compared to the control level (Cvel_cont). The qPCR expression analysis exhibited a positive correlation with the findings of RNA-seq data.

Deep transcriptomic analysis of Chromera velia under Mercury-stress condition

The heavy metal pollution in ecosystems is of increasing global concern. The main sources of the metal toxicity are the industrial waste, mining and the use of pesticides containing heavy metals. Out of all heavy metals, mercury (Hg) is considered to be the one, easily accumulated in terrestrial plants as well as aquatic organism. Hg can induce more severe oxidative stress by triggering the production of reactive oxygen species (ROS) and damage macro-molecules. ROS serve not only as dangerous molecules that damage proteins, lipids and DNA but also as signalling molecules in the regulation of biological processes such as biotic and abiotic stress responses, growth and development. This study shows the change in the Chromera velia transcriptome due to Hg stress. C. velia is the recently discovered closest known relative of apicomplexans. Our study shows the first deep transcriptomic analysis of C. velia, specifically focusing on the expression level of the genes involved in detoxification defence systems under heavy metal stress. The results show that in total 1239 genes are differentially expressed. DEGs (Differentially expressed genes) showed overall up-regulation (1,070 genes) and only 169 up-regulated genes in cultures under Mercury stress (Cvel_mer) when compared to the control level (Cvel_cont). The qPCR expression analysis exhibited a positive correlation with the findings of RNA-seq data.