Lipid ROS- and Iron-Dependent Ferroptotic Cell Death in Unicellular Algae Chlamydomonas reinhardtii.

The phenomenon of heat stress leading to ferroptosis-like cell death has recently been observed in bacteria as well as plant cells. Despite recent findings, the evidence of ferroptosis, an iron-dependent cell death remains unknown in microalgae. The present study aimed to investigate if heat shock could induce reactive oxygen species (ROS) and iron-dependent ferroptotic cell death in Chlamydomonas reinhardtii in comparison with RSL3-induced ferroptosis. After RSL3 and heat shock (50 °C) treatments with or without inhibitors, Chlamydomonas cells were evaluated for cell viability and the induction of ferroptotic biomarkers. Both the heat shock and RSL3 treatment were found to trigger ferroptotic cell death, with hallmarks of glutathione-ascorbic acid depletion, GPX5 downregulation, mitochondrial dysfunction, an increase in cytosolic calcium, ROS production, lipid peroxidation, and intracellular iron accumulation via heme oxygenase-1 activation (HO-1). Interestingly, the cells preincubated with ferroptosis inhibitors (ferrostatin-1 and ciclopirox) significantly reduced RSL3- and heat-induced cell death by preventing the accumulation of Fe2+ and lipid ROS. These findings reveal that ferroptotic cell death affects the iron homeostasis and lipid peroxidation metabolism of Chlamydomonas, indicating that cell death pathways are evolutionarily conserved among eukaryotes.

Ectopic expression of bacterial 1-aminocyclopropane 1-carboxylate deaminase in Chlamydomonas reinhardtii enhances algal biomass and lipid content under nitrogen deficit condition.

1-Aminocyclopropane-1-carboxylate (ACC) deaminase is a well-known bacterial producing enzyme that helps plants to overcome stress conditions by modulating ethylene biosynthesis. However, the functional role of ACC deaminase and ethylene in microalgae during stress remains to be explored. In this study, to investigate the role of ACC deaminase (acds) from Pseudomonas putida UW4 in enhancing the biomass and lipid content of Chlamydomonas under nitrogen deficit condition. The synthetic codon-optimized acds gene was cloned into vector pChlamy_4 and introduced into Chlamydomonas. Results indicated that Chlamydomonas-expressing acds lines showed significant tolerance to nitrogen-deficit by reducing the ethylene content. The biomass, chlorophyll content and photosynthetic activity of acds-expressing lines were significantly increased during nitrogen deficit condition. Moreover, the intracellular lipid and fatty acid content were much higher in acds-expressing lines than the wild-type. In terms of stress alleviation, the transgenic lines displayed increased antioxidant enzymes, reduced ROS and lipid peroxidation levels.

Regulation of CCM genes in Chlamydomonas reinhardtii during conditions of light-dark cycles in synchronous cultures.

We have investigated transcript level changes of CO(2)-concentrating mechanism (CCM) genes during light-dark (12 h:12 h) cycles in synchronized Chlamydomonas reinhardtii at air-level CO(2). CCM gene transcript levels vary at various times of light-dark cycles, even at same air-level CO(2). Transcripts of inorganic carbon transporter genes (HLA3, LCI1, CCP1, CCP2 and LCIA) and mitochondrial carbonic anhydrase genes (CAH4 and CAH5) are up regulated in light, following which their levels decline in dark. Contrastingly, transcripts of chloroplast carbonic anhydrases namely CAH6, CAH3 and LCIB are up regulated in dark. CAH3 and LCIB transcript levels reached maximum by the end of dark, followed by high expression into early light period. In contrast, CAH6 transcript level stayed high in dark, followed by high level even in light. Moreover, the up regulation of transcripts in dark was undone by high CO(2), suggesting that the dark induced CCM transcripts were regulated by CO(2) even in dark when CCM is absent. Thus while the CAH3 transcript level modulations appear not to positively correlate with that of CCM, the protein regulation matched with CCM status: in spite of high transcript levels in dark, CAH3 protein reached peak level only in light and localized entirely to pyrenoid, a site functionally relevant for CCM. Moreover, in dark, CAH3 protein level not only reduced but also the protein localized as a diffused pattern in chloroplast. We propose that transcription of most CCM genes, followed by protein level changes including their intracellular localization of a subset is subject to light-dark cycles.

UVI31+ is a DNA endonuclease that dynamically localizes to chloroplast pyrenoids in C. reinhardtii.

UVI31+ is an evolutionarily conserved BolA family protein. In this study we examine the presence, localization and possible functions of this protein in the context of a unicellular alga, Chlamydomonas reinhardtii. UVI31+ in C. reinhardtii exhibits DNA endonuclease activity and is induced upon UV stress. Further, UVI31+ that normally localizes to the cell wall and pyrenoid regions gets redistributed into punctate foci within the whole chloroplast, away from the pyrenoid, upon UV stress. The observed induction upon UV-stress as well as the endonuclease activity suggests plausible role of this protein in DNA repair. We have also observed that UV31+ is induced in C. reinhardtii grown in dark conditions, whereby the protein localization is enhanced in the pyrenoid. Biomolecular interaction between the purified pyrenoids and UVI31+ studied by NMR demonstrates the involvement of the disordered loop domain of the protein in its interaction.