Reduction of Reactive Oxygen Species reduces the Acetate-Dependent Aging of Chlamydomonas reinhardtii

Reduction of Reactive Oxygen Species reduces the Acetate-Dependent Aging of Chlamydomonas reinhardtii | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL This is a preprint and has not been peer reviewed. Data may be preliminary. 3 October 2025 V1 Latest version Share on Reduction of Reactive Oxygen Species reduces the Acetate-Dependent Aging of Chlamydomonas reinhardtii Authors : Navpreet Kaur Kaur 0009-0002-6672-7299 , Ghaith Zamzam , and Dion Durnford 0000-0001-5232-2333 [email protected] Authors Info & Affiliations https://doi.org/10.22541/au.175952222.29404216/v1 251 views 134 downloads Contents Abstract Chlorophyll Measurement Dissolved Oxygen Measurements Statistical analysis Discussion Acknowledgments References Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract Factors regulating aging and longevity in microalgae remain largely underexplored. The unicellular alga Chlamydomonas reinhardtii serves as an ideal model for aging studies due to its mixotrophic capabilities and ease of culture. While microalgae are immortal under favourable conditions, nutrient limitation induces conditional senescence and ultimately death of the culture. Acetate is a key carbon source for mixotrophic growth in Chlamydomonas and higher acetate concentrations accelerate senescence and reduce longevity in batch culture. We hypothesized that elevated acetate enhances metabolic activity and reactive oxygen species (ROS) production, causing cellular damage and reducing lifespan. We confirmed that high acetate cultures produce more ROS as the culture ages, specifically hydrogen peroxide as measured with DCFH-DA, correlating with reduced longevity. Interestingly, cells in high-acetate conditions exhibited increased resistance to external ROS-inducing agents such as rose bengal and hydrogen peroxide, suggesting the induction of antioxidant defense pathways in these cells, likely related to the higher endogenous production of ROS during growth in high acetate conditions. Treatment with ROS quenchers catalase (hydrogen peroxide), 2,2-dipyridyl (hydroxyl radical), and diphenylamine (singlet oxygen) partially reversed the high-acetate senescence, supporting the role of ROS as a trigger for the acetate-dependent aging. In addition, inhibition of the TOR regulatory kinase with rapamycin also increased longevity in stationary phase cultures growing in high acetate. These findings support a role of ROS and the nutrient-sensing TOR pathway in the regulation of lifespan under high-acetate conditions, providing insights into conserved mechanisms of aging across biological systems. Reduction of Reactive Oxygen Species reduces the Acetate-Dependent Aging of Chlamydomonas reinhardtii Navpreet Kaur 1 , Ghaith Zamzam 1 , 2 , Dion G. Durnford 1 , ∗ 1 Department of Biology, University of New Brunswick, Fredericton, Canada 2 Present Address: Fredericton Research and Development Centre, Agriculture and Agri-Food, Canada ∗ Corresponding author: [email protected] Abstract Factors regulating aging and longevity in microalgae remain largely underexplored. The unicellular alga Chlamydomonas reinhardtii serves as an ideal model for aging studies due to its mixotrophic capabilities and ease of culture. While microalgae are immortal under favourable conditions, nutrient limitation induces conditional senescence and ultimately death of the culture. Acetate is a key carbon source for mixotrophic growth in Chlamydomonas and higher acetate concentrations accelerate senescence and reduce longevity in batch culture. We hypothesized that elevated acetate enhances metabolic activity and reactive oxygen species (ROS) production, causing cellular damage and reducing lifespan. We confirmed that high acetate cultures produce more ROS as the culture ages, specifically hydrogen peroxide as measured with DCFH-DA, correlating with reduced longevity. Interestingly, cells in high-acetate conditions exhibited increased resistance to external ROS-inducing agents such as rose bengal and hydrogen peroxide, suggesting the induction of antioxidant defense pathways in these cells, likely related to the higher endogenous production of ROS during growth in high acetate conditions. Treatment with ROS quenchers catalase (hydrogen peroxide), 2,2-dipyridyl (hydroxyl radical), and diphenylamine (singlet oxygen) partially reversed the high-acetate senescence, supporting the role of ROS as a trigger for the acetate-dependent aging. In addition, inhibition of the TOR regulatory kinase with rapamycin also increased longevity in stationary phase cultures growing in high acetate. These findings support a role of ROS and the nutrient-sensing TOR pathway in the regulation of lifespan under high-acetate conditions, providing insights into conserved mechanisms of aging across biological systems. Keywords Acetate, Longevity, Reactive Oxygen Species, Senescence Introduction Aging is a complex, natural process characterized by a gradual decline in physiological function, an accumulation of deleterious changes in cells and tissues, and an increased susceptibility to diseases and death (Harman, 2006). This physiological decline is driven by several conserved cellular hallmarks, including telomere shortening, genomic instability, epigenetic modifications, mitochondrial dysfunction, loss of proteostasis, and dysregulated nutrient sensing (López-Otín et al., 2023; Maldonado et al., 2023). Free radicals, or reactive oxygen species (ROS), have long been recognized as significant contributors to the aging process due to their harmful effects on cellular components (Harman, 1956). However, ROS also act as essential signaling molecules, leading to their description as a ”double-edged sword” (Martin & Barrett, 2002). In plants, for instance, ROS are crucial for stress perception, signal transduction, development, and differentiation (Foyer et al., 2017; Mittler et al., 2004). ROS encompass a group of highly reactive, oxygen-containing molecules that are an unavoidable consequence of aerobic metabolism. These include highly reactive oxygen-based free radicals, such as superoxide anion (O2•−) and hydroxyl radical (OH−), as well as more stable, diffusible non-radical oxidants like hydrogen peroxide (H 2 O 2 ) and singlet oxygen ( 1 O 2 ). Mitochondria are the primary consumers of oxygen and, thus, the main producers of ROS in aerobic organisms (Perez-Campo et al., 1998), with chloroplasts also contributing significantly in photosynthetic organisms like Chlamydomonas . In aerobic organisms, molecular oxygen (O₂) is essential, acting as the terminal electron acceptor in respiration and a byproduct of photosynthesis, ultimately used to generate metabolic energy. The transfer of electrons through the transport chains located in mitochondria and chloroplasts is imperfect and a premature leakage of electrons can lead to the partial reduction of oxygen, resulting in the inevitable formation of ROS (Foyer & Hanke, 2022). In chloroplasts, the primary sites for the generation of reactive oxygen species (ROS) are Photosystem I (PSI) and Photosystem II (PSII), located within the chloroplast thylakoids (Asada, 2006; Foyer & Hanke, 2022; Wakao & Niyogi, 2021). A key process occurring at PSI is the Mehler reaction, which involves the photoreduction of molecular oxygen to the superoxide radical (O₂•⁻) (Mehler, 1951). This highly reactive superoxide is then enzymatically converted to hydrogen peroxide (H₂O₂) by superoxide dismutase (SOD). Because the superoxide anion (O2•−) is a charged molecule, its ability to cross biological membranes is restricted. Therefore, the subcellular compartmentalization of antioxidant defense mechanisms is critical for the efficient neutralization of superoxide at its site of production (Foyer & Noctor, 2005). The subsequent H₂O₂ can be reduced to water through the action of enzymes like ascorbate peroxidase, or it can participate in the Fenton reaction where it interacts with ferrous ions (Fe²⁺) to generate the extremely toxic hydroxyl radical (•OH). Furthermore, another potent ROS, singlet oxygen (¹O₂), is produced when energy from excited chlorophyll molecules in the reaction centers is transferred to ground-state triplet oxygen (³O₂) (Asada, 2006; Foyer & Hanke, 2022; Ledford et al., 2007). These ROS can damage DNA, proteins, and lipids (Gough & Cotter, 2011; Halliwell & Aruoma, 1991). Maintaining a delicate balance between low, beneficial levels of ROS for signaling and adaptive responses, and high, detrimental levels that cause damage, is fundamentally important for normal cellular physiology and overall health (Foyer et al., 2017; Wang et al., 2023). While aging is widely studied in multicellular organisms, its exploration in unicellular eukaryotes has been more limited, and mainly confined to yeast and even bacteria like E. coli as model organisms. Microbial aging generally manifests in two ways: replicative aging and conditional senescence (chronological aging) (Fredriksson & Nyström, 2006). Replicative aging occurs in growth-inducing conditions, where the number of cell divisions before death defines the replicative lifespan (RLS). In asymmetrically dividing unicellular organisms like S. cerevisiae , the mother cell passes old, damaged structures to one daughter cell, while the other—the “rejuvenated cell”—receives a newly synthesized set of cellular components (Yang et al., 2015). Conversely, conditional senescence is characteristic of growth-limiting conditions during the stationary phase, which inhibits cell division. The longevity is measured by the cell viability of the whole population during this phase, termed chronological lifespan (CLS) (Florea, 2017). In microalgae, however, there are only a handful of studies on aging (Damoo & Durnford, 2021; Humby et al., 2013; Machado & Soares, 2022), but aging is both ecologically relevant and an important process to understand, especially as it relates to their handling of light as growth slows as nutrient levels decline (Devkota & Durnford, 2025; Meagher et al., 2021). These approaches generally follow conditional senescence and monitor CLS once one or more nutrients have become exhausted in the media. In batch culture, microalgal populations grow exponentially while nutrients are abundant but transition into declining growth/stationary phases as nutrients become depleted (Damoo & Durnford, 2021). In this state of conditional senescence, cells cease dividing and undergo dramatic reductions in protein content as the chloroplast shrinks in an apparent chloroautophagic process that is not well defined (Humby et al., 2013; Machado & Soares, 2022). If nutrients are not repleted, this can lead to cellular or molecular damage, and ultimately death (Damoo & Durnford, 2021; Humby et al., 2013; Machado & Soares, 2022), the timing of which depends on many external factors (Zamzam et al., 2022). Conditional senescence has been observed in yeast (Longo et al., 2012), bacteria (Fredriksson & Nyström, 2006); and a common microbial response to depleted environmental nutrients. Multiple approaches have been used to unravel the complex phenomenon of aging and longevity, with calorie restriction (CR) standing out as one of the most robust interventions in animals. CR, defined as a reduced energy intake without initiating starvation, has been consistently reported to increase lifespan (López‐Lluch & Navas, 2016). Early observations, such as Peyton Rous’s work in 1914 showing the effect of low calorie on tumor growth in mice, paved the way for later studies, including demonstration of a 50% lifespan extension in mice with 65% calorie restriction (Weindruch et al., 1986). CR has proven effective in extending the lifespan of diverse organisms, including yeast, nematodes, and fruit flies (Kyryakov et al., 2012; Leonov et al., 2017; Li et al., 2024). Recently, a CR-like response was demonstrated in the photosynthetic model organism Chlamydomonas reinhardtii . Cells grown in low-acetate (10 mM) conditions survived significantly longer than those in high-acetate (30 mM) conditions (Zamzam et al., 2022), suggesting that carbon budget was an important feature in determining longevity. This observation in Chlamydomonas raises a crucial mechanistic question: does an elevated carbon nutrient supply shorten lifespan by driving up the metabolic rate, leading to a detrimental increase in ROS production and oxidative stress? While the exact mechanism of CR-related longevity increase is not clear (Fontana et al., 2018), mediating the effects of ROS likely play a role by reducing the production of ROS (López‐Lluch & Navas, 2016; Merry, 2002), or stimulating the antioxidant capacity of the cell to buffer their potential damaging effects (Fontana et al., 2018), which can slow senescence and enhance longevity, features related to the CR response. Increasing autophagy in cells is also a suspected role of CR in animal cells (Bagherniya et al., 2018), suggesting the activation of cellular pathways that directly promote longevity, and implicating the Target of Rapamycin (TOR) pathway (Stallone et al., 2019) as a mediator in these pathways. In this study, we investigate the role of ROS in the longevity of Chlamydomonas reinhardtii under low and high acetate concentrations, that causes a CR-like effect. We hypothesized that the acetate-dependent rapid senescence was related to a higher metabolic activity and faster growth that disrupted cellular ROS homeostasis, triggering accelerated senescence and ultimately cell death. By using the fast-senescence system developed by Zamzam et al. (2022) and using treatments that either stimulate or quench ROS’s under high acetate, we demonstrate that ROS clearly have a role in longevity of Chlamydomonas . We also show that by inhibiting the TOR pathway, we can modulate longevity under conditions that promote accelerated senescence, indicating a conserved pathway in taxonomically broad organisms for responding to CR-like conditions. Overall, this work aims to uncover the molecular underpinnings that govern microalgal survival and shed light on potential parallels in other eukaryotic systems. 1 Materials and Methods 1.1 Cell Strains and Culture Conditions The wildtype strain CC125 was obtained from the Chlamydomonas Resource Center (chlamycollection.org) at the University of Minnesota. CC125 was maintained on Tris-Acetate Phosphate (TAP; Harris, 2009) media plates grown at room temperature and under 200 µmol quanta m-2 sec-1 under continuous light. Prior to the start of an experiment, cells were inoculated into 70 mL of TAP liquid media in a 250 mL Erlenmeyer flask and grown for 3 days under continuous light (83 µmol quanta m-2 sec-1, Sylvania 17W cool white, fluorescent lights) at 22 °C and mixed at 110 rpm on an orbital shaker. After 3 days, the cultures reached a cell abundance of approximately 2 × 106 cells ml-1. These cultures were then diluted to 3×105 cells mL-1 in Tris-Phosphate media supplemented with 10 mM or 30 mM acetate (Harris, 2009). Cell abundance was confirmed by counting on a hemocytometer. Experiments were done in a rapid senescence system (Zamzam et al., 2022) in which a 9 mL aliquot of the diluted cells was transferred into 24 mL capped glass vials (6.5-dram glass vials) and cultured under continuous light (83 µmol quanta m-2 sec-1) at 22 °C and 110 rpm on a shaker. Experiments were usually completed within 4-6 days. Normal caps were replaced with silicone-septa caps when different treatment chemicals need to be added on different days. 1.2 ROS Assay To detect reactive oxygen species in the form of H2O2 we used 2′,7′-Dichlorodihydrofluorescein diacetate (DCFH-DA) (Cayman, Ann Arbor, Michigan). A 10mM solution of DCFH-DA was prepared in 100% DMSO and then added to 10 mM and 30 mM acetate-grown Chlamydomonas cells at a final concentration of 100 µM at the appropriate day by injecting through the silicon-septa caps (Millipore, Sigma) with a syringe, without opening the vials. The vials were incubated on a shaker for 40 minutes prior to sampling. Experiments were conducted in four biological replicates. Cell media was centrifuged at 13000g for 10 minutes and after removing the supernatant, the cell pellet was resuspended in water. This allowed an estimation of ROS levels in cells only because DCFH-DA can pass through the membrane and into cells during incubation (Hempel et al., 1999). The fluorescence of DCFH-DA was measured (excitation 485 nm, auto cutoff 515nm, Emission 525 nm) using a SpectraMax® M3 multi-Mode microplate reader (Molecular Devices, U.S.A). Cell abundance was estimated by measuring the absorption at 750nm and fluorescence readings were normalized to the relative cell abundance. 1.3 ROS Inducer Treatments We used a variety of chemicals to induce a different types of ROS stress in cultures to test their resilience under high and low acetate. Rose bengal (ICN Biomedicals, Aurora, Ohio) is an exogenous photosensitizing dye that generates a singlet oxygen (1O2* ) stress in the presence of light (Stiel et al., 1996). Rose bengal was dissolved in water and used at a range of concentrations. To generate a superoxide (O2•-) stress, we used methyl viologen (Sigma-Aldrich, St. Louis, MO), that accepts electrons from the electron transport chain and can pass electron to oxygen, which rapidly generates superoxide (O₂•⁻) (Hassan, 1984). MV was dissolved in water to make 1mM stock and used at different concentrations from 0 to 20μM. Hydrogen peroxide (H2O2) is a non-radical ROS, which can damage cellular components like DNA and lipids (Gough & Cotter, 2011). Hydrogen peroxide (Avantor, Radnor, PA) was diluted in water and added to cultures at concentrations ranging from 0 to 12mM. The ROS-stress experiments were conducted on cells grown for 3 days in either 10 mM or 30 mM acetate containing media. Cells were diluted to 106 cells ml-1 using spent media (supernatant-media collected after centrifugation of the cells from same experiment) and cell concentration confirmed by counting on a hemocytometer. One milliliter of diluted culture was transferred to each treatment vial, and the required concentrations of ROS inducers were added. Cultures were incubated for 1.5 hours under the light (83 µmol quanta m-2 sec-1) at 22 °C on an orbital shaker (110 rpm). For rose bengal treatments, a dark control was also used. Here the vials were wrapped in black polybags and put on the shaker. Following incubation, cells were pelleted in a microfuge for 10 minutes, the supernatant was removed, and the cells were resuspended in the same volume of fresh TAP medium to keep the cell density same. Five microliter droplets of collected cells were placed on TAP plates and incubated under continuous light (200 µE) for 5 days. 1.4 ROS Scavenger Treatments We used a variety of chemical scavengers/antioxidants to reduce ROS stresses in high-acetate cultures to test the effect on viability, cell abundance, and chlorophyll content after 6 days. For these experiments, 24 mL glass vials with silicon-septa caps (Millipore-Sigma, Burlington, Massachusetts) were used. ROS scavengers were injected directly through the lid to prevent air exchange, which could affect the timing of senescence in the culturing system. 2,2-dipyridyl (Sigma-Aldrich, St. Louis, MO) was used to reduce production of hydroxyl radicals by reacting with Fe2+ to prevent the Fenton reaction (Nedelcu & Michod, 2003). 50 µM final concentration of 2,2-Dipyridyl, was added to cultures on day 3 by injecting the solution through the septa of the lids. An equivalent volume of water was added to the controls. Sampling was performed on day 6 to measure cell viability, cell density, and chlorophyll content. To reduce superoxide production, copper (II) 3,5-diisopropyl salicylate hydrate (CuDIPSH) (Aldrich, Milwaukee, WI) was added to cultures at a final concentration of 20 µM in DMSO to cells on day 3. CuDIPSH reportedly has superoxide dismutase-like activity (Leuthauser et al., 1981; Nedelcu & Michod, 2003). An equivalent amount of DMSO was added to the controls and sampling was performed on day To reduce hydrogen peroxide concentration in the cultures, we added catalase (bovine liver) (Sigma, St. Louis, MO), an enzyme that converts the ROS to water (Baker et al., 2023; Nedelcu et al., 2004). Catalase was prepared in Tris-HCl (50mM/pH 7) and added to the cultures at a final concentration of 50 µg mL-1 to the treatment cultures on days 3 and 4 by injecting directly through the septa of the lids. 50 µg mL-1 BSA (Invitrogen, Waltham, Massachusetts) was added to control cultures to control for the addition of protein that could be a nitrogen source. Sampling was performed on day 6. We also used diphenylamine (DPA) (Sigma-Aldrich, St. Louis, MO), a quencher of singlet oxygen, to reduce singlet oxygen stress (Kruk et al., 2005; Trebst et al., 2002). DPA was dissolved in DMSO and added to cultures at a final concentration of 20 µM on day 3, using a syringe as before. The equivalent volume of DMSO was added to the controls and sampling was again performed on day 6. 1.5 Cell Survival and Abundance Cell Abundance was determined by fixing the cells with Lugol’s iodine (Throndsen, 1978) and counted under a light microscope using a Neubauer hemocytometer. 1 Materials and Methods To assess cell survival, we used SYTOX Green that is excluded from living cells. To assay the cells, 0.5 µL of a 5 mM SYTOX Green nucleic acid stain (S7020, Invitrogen, USA) was added to 1 mL of 10mM growing culture and 30mM were diluted two-fold to normalize it to the similar cell abundance as 10mM for staining with SYTOX. Cells were incubated for 10 minutes in the dark, followed by fixation with 4% paraformaldehyde (v/v). Brightfield and GFP filter images were captured using a fluorescence microscope with a 504 nm excitation wavelength. 300-400 cells were counted for each replicate and analyzed using Fiji (ImageJ) software. To qualitatively assess viability, we used a plate growth assay. Cells were diluted to 10 6 cells mL -1 in sterile water, and 5 µL spotted on TAP plates. The plates were incubated under continuous light (200 µE) for 5 days and then photographed. Chlorophyll Measurement For chlorophyll content, 200 µL of the cell culture was centrifuged at 13,000 g at 4°C for 10 minutes. The supernatant was discarded, and the cell pellet was resuspended in 100% methanol and vortexed. Samples were incubated at 4°C for 15-20 minutes with intermittent vortexing. The mixture was centrifuged at 13,000 g for 10 minutes, and the supernatant was used to measure the chlorophyll content at 664, 647, 750, and 470 nm using a Beckman-Coulter DU720 spectrophotometer (Beckman Coulter Inc., Fullerton, CA, USA). Chlorophyll content was calculated using methanol-based equations (Porra et al., 1989) and normalized to cell abundance. Dissolved Oxygen Measurements Oxygen evolution and consumption, which represent photosynthesis and respiration respectively, were measured to assess the metabolic activity of the 10 mM and 30 mM cultures on different days. Oxygen evolution rates were determined using a Clark-type electrode (Hansatech Instruments, Norfolk, UK). Photosynthetic rates were measured as the change in oxygen evolution under a light intensity of 100 µmol quanta m⁻² s⁻¹ followed by high light of 1000 µmol quanta m⁻² s⁻¹ and then, immediately after, samples were placed in the dark to determine oxygen consumption rates as a proxy for respiration. The dissolved oxygen readings concentration for each vial was then normalized to cell number. ‘ Statistical analysis The data was transferred to Microsoft Excel for bar graphing and analysed it with student t-test (one-tailed, paired variance). Box and whisker plots were prepared using RStudio to show better data variation across all the replicates (RStudio Team, 2023). Two-way ANOVAs and Tukey’s HSD tests were also performed using RStudio (RStudio Team, 2023). 1 Results Cell abundance plateaus in both low and high acetate growing cells within 3-4 days signifying the start of stationary phase (Fig. 1). However, the biomass accumulation is different in high-acetate growing cells compared to low-acetate growing cells. Initially, both cultures have a comparable accumulation of biomass, but after 2 days in culture, the low acetate culture exits exponential growth while the high acetate continues, doubling cell number over the next day, which is about twice that in the low acetate (Fig. 1). Due to this difference, our results are normalized to cell abundance. 1.1 ROS Levels Differ in the High and Low-Acetate Cultures We investigated the acetate-dependent production of reactive oxygen species (ROS) in Chlamydomonas using 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA). DCFH-DA detects ROS, particularly H2O2 and peroxide-derived oxidants. Upon reaction with ROS, DCFH-DA is converted into the fluorescent compound 2,7-dichlorofluorescein (Gomes et al., 2005; Hempel et al., 1999). On days 2 and 3, ROS levels were low and no significant difference between high and low-acetate cultures (Fig. 2). By day 4, however, ROS levels increased in both cultures, but were two-fold higher in the high-acetate conditions (Fig. 2). 1.2 Metabolic Activity of Chlamydomonas reinhardtii Cultures Based on oxygen consumption measurements, Chlamydomonas reinhardtii cultures grown in 10 mM and 30 mM acetate media have significant metabolic shifts over time (Fig. 3). For the first three days, both the low and high-acetate culture had similar respiration rates (Fig. 3), which showed a gradual decline over that time. Interestingly, on Day 4, there were notable spikes in dark oxygen consumption in both the low and high acetate, though the spike is significantly larger under high acetate. After day 4, oxygen consumption rates in the dark were negligible, indicating a substantially reduce metabolic activity under any acetate condition. 1.3 High acetate cultures Cells Better Tolerate ROS Inducer Treatment Given that high-acetate-grown cells were already dealing with higher peroxide stress than low-acetate cells, we wanted to determine if they had consequently induced detoxification enzymes. This was investigated by screening for sensitivity to different concentrations of ROS inducers. We hypothesized that the high-acetate cells would have upregulated ROS detoxification enzymes in response to the high endogenous ROS production that we detected by DCFH-DA. As a result, we predicted they would be better protected during a short-term challenge with external ROS-inducing agents before being plated onto fresh media. To test this, we used three different ROS inducers: rose bengal, hydrogen peroxide, and methyl viologen. Rose bengal is an exogenous photosensitizing dye that generates singlet oxygen (¹O₂) in the presence of light (Stiel et al., 1996). Singlet oxygen is highly reactive and can modify lipids (Girotti & Kriska, 2004), nucleic acids (Martinez et al., 2003), and proteins (Davies, 2004). Cells growing in high- and low-acetate conditions were treated with rose bengal under both light and dark conditions. Since rose bengal requires light to produce ¹O₂, no effect on cell growth was observed in the dark at any concentration tested (Fig. 4A). However, under light conditions, cells grown in the high-acetate medium demonstrated better tolerance to higher concentrations of rose bengal. In the light, the low-acetate-grown cells were more sensitive, showing signs of growth inhibition at concentrations as low as 14 µM. In contrast, cells from the high-acetate cultures were unaffected at 25 µM (Fig. 4A). We also tested the effects of hydrogen peroxide (H₂O₂), a non-radical ROS that can react with metal ions (e.g., Fe²⁺) via the Fenton reaction to produce hydroxyl radicals (•OH), which are highly reactive (Winterbourn, 1995). These radicals can damage cellular components like DNA (Halliwell & Aruoma, 1991), lipids (Gough & Cotter, 2011), and can oxidize amino acid side chains of proteins, particularly cysteine and methionine (Stadtman & Levine, 2003). Cells grown in high-acetate conditions demonstrated a better tolerance to H₂O₂, showing growth in up to 12 mM whereas, low-acetate cultures only showed growth at up to 6 mM (Fig. 4B). Methyl Viologen is a herbicide that can rapidly generate superoxide (O₂•⁻) (Hassan, 1984). Following treatments of MV up to 20 µM, both the low and high-acetate cultures show comparable sensitivity to the treatment (Fig. 4C). 1.4 ROS Scavenger Treatments Mitigate High-Acetate Induced Mortality Reactive oxygen species (ROS) levels were elevated in cells growing in high-acetate conditions (Fig. 2), suggesting a possible disruption in ROS homeostasis. To further explore the high acetate-driven mortality and the relationship to ROS, we tested the effects of a variety of ROS quenchers on the longevity of high-acetate cultures once they reach stationary phase. Diphenylamine (DPA), a chemical quencher of singlet oxygen (¹O₂) (Trebst et al., 2002), significantly improved survival and slowed the progression of senescence in high-acetate-grown cells. The SYTOX assay showed that an average (median) of 32.3% of DPA-treated cells were alive, a significant increase compared to only 13.6% in the controls (p ≤ 0.001; Fig. 5B). This improved viability was also evident in the plate assay (Fig. 5A). Furthermore, the chlorophyll a and b content per cell was significantly higher in treated cells (2.6 pg cell-1) compared to controls (1.5 pg cell-1) (p ≤ 0.01; Fig 5C-5D). This suggests that DPA-treated cells were physiologically younger and maintained their photosynthetic machinery more effectively. The Fenton reaction plays a major role in producing highly reactive hydroxyl radicals from hydrogen peroxide, a process catalyzed by ferrous iron (Fe²⁺) (Winterbourn, 1995). To quench the contribution of this reaction to the production of highly reactive hydroxyl radicals, we added 2,2-dipyridyl to the cultures at day 3, when the culture approaches stationary (Fig. 1) and before the peak ROS production (Fig. 2). 2,2-dipyridyl is an Fe²⁺ chelator that forms a stable complex with the ion and inhibits the Fenton reaction (Aljuwayd et al., 2024; Nedelcu & Michod, 2003). When 2,2-dipyridyl was added to high-acetate cultures the treated samples showed improved survival and slowed senescence compared to the untreated control (Fig. 5B). Survival assessed using the SYTOX assay showed that the treated cells were partially rescued from the high-acetate-induced senescence; on day 6, 77.7% of the treated cells were alive compared to only 49.3% in the control cultures (p ≤ 0.05; Fig. 5B). This was supported by the qualitative plate assay where the treated culture had an obviously more robust growth after 5 days (Fig. 5A). Furthermore, the 2,2-dipyridyl treated cells also had a slowing of senescence as indicated by the higher chlorophyll a and b levels per cell. Treated cells exhibited a combined chlorophyll a and b content of approximately 4.1 pg cell-1, significantly higher than the 3.4 pg cell-1 observed in controls (p ≤ 0.05) (Figure 5C-5D). Also, the chl a/b ratio was considerably lower in the untreated culture approximately 2. pg cell-1and significantly higher around 2.8 pg cell-1in treated (p ≤ 0.01) (Figure 5C-5D), which is a characteristic of a senescing culture. When catalase, an enzyme that catalyzes the decomposition of H₂O₂ into water and oxygen, was injected into the vials it substantially improved cell survival and slowed senescence in high acetate cultures. On average (median) around 77.5% of catalase-treated cells remained alive, compared to 54.7% for the controls (p ≤ 0.0001 Fig. 5A). These findings were corroborated by the qualitative plate assay, where treated cells demonstrated better growth on the TAP plate after 5 days (Fig. 5B). The improved health of the culture was also reflected in the average chlorophyll a and b, which was 3.8 pg cell-1 in treated cells versus 3.4 pg cell-1 in controls (p ≤ 0.05; Fig. 5C-5D), indicating that the progression toward senescence where chlorophyll levels decline was slowed. The greater progression of senescence was also apparent by the lower Chl a/b ratio in the in the treated culture with 3.2 pg cell-1 and 2.3 pg cell-1 in controls (p ≤ 0.0001; Fig. 5D). In contrast to the other ROS quenchers, treatment with CuDIPSH (Copper (II) 3,5-diisopropyl salicylate hydrate), a cell-permeable superoxide dismutase mimic that converts superoxide anions (O₂•⁻) into hydrogen peroxide (H₂O₂), showed no significant effect. There was no difference in survival between treated and control samples in either the SYTOX assay or the viability plate assay (Figure 5A-B). Similarly, regarding chlorophyll content, no significant difference was observed (Fig. 5C-D). 1.5 TOR pathway inhibition improves viability of Chlamydomonas in higher acetate. Reactive Oxygen Species (ROS) are vital for cell signaling, so we decided to investigate their link to the nutrient-sensing TOR pathway. To do this, we used rapamycin (a TOR inhibitor) (Crespo et al., 2005), on cells growing in a high-acetate medium. We added the rapamycin in different replicates right before the cells’ growth plateaued and took samples after they had reached senescence. Data from different sets of experiments were combined even though they were done on different days (4 or 6). There can be variation in timing of senescence, however, chlorophyll levels of control cells between these experiments were similar at these time points indicating that the different timing reflected variation in the progression of senescence, and so combining these data were reasonable as they were the same physiological age. Nevertheless, cells treated with rapamycin showed significantly better survival, with an average (median) viability of 42.3% compared to only 27.7% in the control group (p ≤ 0.001; Fig. 6A). Interestingly, this survival boost didn’t seem to affect chlorophyll levels, as there was no significant difference between the treated and untreated cells (p > 0.05; Fig. 6B). 1 Results Discussion Our findings establish a direct role for reactive oxygen species (ROS) in the acetate-induced senescence of Chlamydomonas reinhardtii and the extension of longevity in stationary phase. We confirmed that high-acetate (high-calorie) conditions accelerate mortality, as previously reported by Zamzam et al., (2022), and provide evidence that this phenomenon is mechanistically linked to a disruption in ROS homeostasis. The key evidence supporting this conclusion is a significant increase in endogenous peroxide levels during the conditional senescence phase in high-acetate cultures and the successful rescue of the accelerated aging phenotype by treating cells with specific antioxidants. A central effect of high-acetate growth is an accelerated production of ROS as cells transition into the stationary phase. This was apparent through our direct measurement of cellular peroxides like hydrogen peroxide (H₂O₂) using the DCFH-DA over 4 days in our culture system (Fig. 2). The accumulation of ROS is a conserved hallmark of senescence and metabolic stress across eukaryotes. This link is clearly demonstrated during the stationary or senescent phase of life, where a notable increase in ROS and autophagy markers is a well-established characteristic in Chlamydomonas (Esperanza et al., 2017; Tran et al., 2019) and in yeast (Núñez et al., 2015) . This principle is not limited to microorganisms; ROS are also recognized as major players in the process of senescence in plants (Lee et al., 2012). Furthermore, the impact of high acetate was clearly reflected in the culture’s metabolic activity. At the early stages (Days 2-3), both cultures exhibited comparable respiratory rates, providing a standardized baseline to accurately assess their differential responses to oxidative stress. An abrupt increase in metabolic rates around Day 4 in both cultures, however, appeared to mark the transition into the stationary phase (Fig. 3) and the exhaustion of the acetate in the media. The observed spike was larger in high-acetate cultures compared to low-acetate (Fig. 3). This metabolic trend is different with previous reports (Zamzam et al., 2022), where no spike was observed in low-acetate cultures. This difference is due to the different methodology. In the Zamzam et al (2022) paper, the readings were done directly in the glass vials, though in this study we used a classic Clark-type electrode and the vials were opened to sample. We predict that this introduced CO 2 that stimulated photosynthesis and respiration in the low-acetate culture. In the high-acetate culture, the metabolism of acetate would liberate CO 2 , thus that culture would not be CO 2 limited as it approached stationary. Nevertheless, in high acetate cultures, the transition to stationary is marked by a respiratory-like spike in oxygen consumption, which may be due to the transition from a heterotrophic, as acetate is depleted, to phototrophic metabolism driven by higher CO 2 levels from acetate metabolism in the closed cultures (Heifetz et al., 2000). This spike in respiration was likely the source for the ROS production upon entry into stationary phase. The burst of ROS as cells approached stationary contributes to the apparent paradox of ROS resilience in the high-acetate cultures. Despite suffering from higher endogenous ROS and reduced longevity, high-acetate cells demonstrated enhanced tolerance to external challenges from the ROS inducers Rose Bengal (¹O₂) and H₂O₂ (Fig. 4A-B). This suggests a state of stress acclimation, where chronic internal oxidative stress triggers the upregulation of robust cellular defense mechanisms. Such pre-conditioning has been observed in yeast and Chlamydomonas, where sub-lethal ROS exposure induces resistance to subsequent lethal oxidant conditions (Ledford et al., 2007; Pereira et al., 2001). However, this qualitative assay, while reflecting the existing detoxification systems when transferred to fresh media, it seems likely that the high acetate cultures under senescent conditions were ultimately overwhelmed by the sustained production of ROS causing unmitigated damage or triggering a programmed cell death response (Dat et al., 2003; Tiwari et al., 2002; Yoshinaga et al., 2005) Direct evidence for the involvement of ROS stress in aging and senescence in microalgae came from the antioxidant experiments where ROS scavengers partially rescued the acetate-dependent decline in survival, an approach that was successful in identifying the role of ROS in triggering sexual reproduction in Volvox (Nedelcu et al., 2004; Nedelcu & Michod, 2003). Acetate-dependent mortality was partially reversed by catalase (reduces hydrogen peroxide), 2,2-dipyridyl (reduces •OH formation), and diphenylamine (quenches singlet oxygen) treatments (Fig. 5), pointing towards a collection of different ROSs involved in inducing senescence and cell death. The success of the catalase and 2,2-dipyridyl suggests that controlling hydrogen peroxide concentrations, and hence the production of hydroxyl radicals via the Fenton reaction, is a significant factor in taming the high acetate-induced senescence. This finding makes sense, as H 2 O 2 itself is known to induce senescence and programmed cell death in multiple organisms, including Chlamydomonas (Chen & Ames, 1994; Esperanza et al., 2017; Prochazkova et al., 2001; Vavilala et al., 2015; Zhou et al., 2018). In the case of catalase, it is the removal of extracellular hydrogen peroxide that remained effective as it is unlikely that this enzyme would cross the plasma membrane and gain access to the cell interior. This suggests H 2 O 2 escapes across the plasma membrane and builds-up up in the culture media, which could be a problem in algal biomass production. Interestingly, superoxide radicals (O₂•⁻) do not appear to be the primary drivers of cell death in this context (Fig. 5). There was no acetate-dependent protection of cells from methyl viologen, a superoxide generator (Hassan, 1984) nor did a superoxide antioxidant (CuDIPSH) improve survival. It is difficult to prove that such antioxidants are reaching their intended target for a sufficient time to mediate a response, and thus the superoxide quenching data could be considered inconclusive. Since superoxide is a localized molecule which usually remains confined to its generation site due to its charged nature (Apostolova & Victor, 2015), failure of the cell-permeable CuDIPSH to affect a response (Leuthauser et al., 1981) may be due to its inability to accumulate to a sufficient concentration within the mitochondrial matrix (Murphy & Smith, 2007) or the plastid. It could suggest superoxide is less important ROS generated in algal cells as they age and that there may be an alternate source to produce H 2 O 2 other than from superoxide, but that remains to be tested. A key characteristic of microalgal cultures is that as cells approach stationary phase, they begin to senesce as nutrients are depleted and there is a turnover of macromolecules in the cell (Damoo & Durnford, 2021; Humby et al., 2013; Machado & Soares, 2022). This is readily observed by a change in chlorophyll content, which is a proxy for the progression of senescence, with the dismantling of the thylakoid membranes within the chloroplast and the catabolism of the photosynthetic machinery, which is a type of chlorophagy (Nakamura & Izumi, 2019). Similarly in plants, the progressive loss of chlorophyll is a definitive and highly regulated hallmark of senescence in photosynthetic organisms (Hörtensteiner, 2006; Thomas & Ougham, 2014) . A reduction in the senescence-related decline in chlorophyll content following treatment with the antioxidants provides a direct link between oxidative stress and the functional decline observed in senescence. In plants, the onset of senescence is often associated with an increase in ROS production (Jajic et al., 2015; Lee et al., 2012; Prochazkova et al., 2001). This increase can be stress associated (Choudhury et al., 2013; Silva et al., 2010) or part of their development process, where ROS act as an important signal in the highly regulated senescence of leaves and flowers (Rogers & Munné-Bosch, 2016; Zimmermann & Zentgraf, 2005). This highlights the delicate balance of ROS homeostasis and the well-established dual nature of these molecules: they function as critical signaling molecules that trigger protective pathways at lower levels but can become cytotoxic at high concentrations (Foyer et al., 2017; Martin & Barrett, 2002). The ROS-mediated functional decline we observed is consistent with the established role of reactive oxygen species as key signaling molecules and executioners in programmed cell death (PCD) across diverse biological systems (Foyer & Noctor, 2005; Martin & Barrett, 2002; Volpe et al., 2018). This is particularly evident under conditions of high metabolic activity, which exacerbate ROS production. For instance, in mammalian systems, high-glucose or high-calorie metabolic environments are known to substantially increase ROS, triggering a cascade of detrimental effects that include oxidative damage, apoptosis, and widespread metabolic dysfunction (Volpe et al., 2018). This phenomenon is not limited to animals; in Chlamydomonas , rapid metabolism stimulated by other inputs like high light and excess carbon dioxide also leads directly to elevated levels of H 2 O 2 and other ROS (Roach et al., 2015). Upadhyaya et al (2020) also observed enhanced ROS production as acetate levels increased in cultures of Chlamydomonas , agreeing with what we found in our senescence studies under high acetate. This was also accompanied by enhanced autophagy as indicated by the elevation of the autophagy marker, ATG8 (Upadhyaya et al., 2020). This ROS mediated activation of PCD is backed by many other studies (Y. Chen et al., 2019; Vavilala et al., 2015; Zhou et al., 2018). In yeast also, ROS are a trigger of apoptosis in aging cells (Laun et al., 2001; Perrone et al., 2008), and the often-rapid death of cells in high acetate conditions points toward a PCD-like mechanism (Volpe et al., 2018). In our experiments, there was a transient spike in respiratory activity at day 4 (Fig. 3) corresponded with high ROS production (Fig. 2) and the onset of accelerated senescence under high acetate conditions (Fig. 5). Antioxidants partially reversed this trend toward senescence, suggesting an important role of ROS as a central regulator of cellular lifespan (Harman, 1956; Karagianni & Bazopoulou, 2024), mediated through the control of PCD signalling in both plants (Van Breusegem & Dat, 2006) and microalgae (Pérez-Martín et al., 2014; Pérez-Pérez et al., 2012). Our acetate-dependent decline in longevity of Chlamydomonas aligns well with the broader principles of the free radical theory of aging (Harman, 1956) and calorie restriction (CR) (López‐Lluch & Navas, 2016) where elevated respiration and ROS levels lead to shortened lifespan. A central hypothesis of CR is that reduced energy intake lowers metabolic rate, thereby decreasing by-product ROS production and mitigating oxidative damage by elevated antioxidant activity (Fontana et al., 2018; López‐Lluch & Navas, 2016; Sohal & Weindruch, 1996). This relationship between CR, ROS, and lifespan extends across diverse species where longevity is consistently correlated with a reduction in mitochondrial free radical generation, enhanced antioxidant activity, and lower overall oxidative damage to macromolecules (Barja, 2004; Gredilla & Barja, 2005; Sohal et al., 1994). ROS are a key factor in acetate-induced aging but the anti-aging effects of CR-like effect are orchestrated by a complex and highly conserved nutrient-sensing pathway regulated by the kinase Target of Rapamycin (TOR) (Johnson et al., 2013). We found that chemical inhibition of TOR with rapamycin significantly increased cell survival in high-acetate cultures, although it did not affect chlorophyll content (Fig. 6). This finding aligns with extensive evidence that TOR inhibition extends lifespan in diverse model organisms, including yeast (Powers et al., 2006), C. elegans (Robida-Stubbs et al., 2012), Drosophila (Bjedov et al., 2010), and mice (Anisimov et al., 2011; Harrison et al., 2009). It is widely accepted that reduced TOR signaling is a primary mechanism through which CR confers its benefits to longevity (Kenyon, 2010), with the effects of TOR inhibition often overlapping those of CR in yeast (Kaeberlein et al., 2005), C.elegans (Hansen et al., 2007), and Drosophila (Kapahi et al., 2004). Mechanistically, the pro-longevity effects of TOR inhibition are typically attributed to the suppression of protein synthesis or the induction of autophagy (Díaz-Troya et al., 2008; Hands et al., 2009; Maldonado et al., 2023). There is a link between ROS and TOR, as increased ROS production can activate autophagy (Pérez-Martín et al., 2014; Pérez-Pérez et al., 2012), via inhibiting TOR activity (Pérez-Pérez et al., 2010; Upadhyaya et al., 2020). However, the interactions are difficult to decipher. TOR inhibition with rapamycin in our system improved longevity without dramatic effects on chlorophagy, unlike with the ROS quenchers. So while ROS could function by modulating the TOR pathway, it is possible that under high acetate conditions, TOR has a higher activation state at this stage of senescence due to an increase in amino acid pools from chlorophagy or a higher carbon fixation (Mallén-Ponce et al., 2022), the latter because of the liberation of CO 2 during acetate metabolism. Thus, the inhibition of TOR in high acetate cultures as they enter stationary would shift the activation state so that TOR is inhibited and leading to a greater activation of autophagy (Pérez-Pérez et al., 2010; Upadhyaya et al., 2020). Therefore, we propose that the increased lifespan in our high-acetate cultures following rapamycin treatment is primarily due to the induction of autophagy, which enhances cell survival by clearing damaged proteins and organelles (Alvers et al., 2009; Mizushima et al., 2008). TOR inhibition, however, had less of an affect the progression of chlorophagy as did the ROS quenchers. This agrees with the argument that photooxidative damage is not directly related to autophagy induction (Pérez-Pérez et al., 2012). This suggests that these processes are separately regulated and that ROS signals or oxidative damage are a key trigger for chlorophagy that occurs during aging. We demonstrate that high-acetate concentrations shorten the lifespan of Chlamydomonas by inducing chronic oxidative stress driven predominantly by hydrogen peroxide (H 2 O 2 ) and singlet oxygen ( 1 O 2 ). Significantly, ROS quenchers and TOR inhibition by rapamycin extend longevity strongly indicate that a disruption of the redox balance and/or oxidative damage are critical regulators of this aging process that intersect with the nutrient-sensing pathways regulated by TOR. The redox imbalance inherent in high-acetate cultures as they approach stationary phase ultimately drives cells toward programmed cell death (PCD) as suggested by the rapid decline in viability in this culture system, a possibility that can be explored by studying PCD-specific markers. Ultimately, this work establishes acetate-fed Chlamydomonas as a powerful model to investigate CR-related aging and untangle the interplay between metabolism, ROS signaling, and cellular longevity. Acknowledgments This work was supported by a Natural Sciences and Engineering Research Council (NSERC) Discovery Grant (2019-04428) awarded to DGD. Figure 1: Cell Abundance of Chlamydomonas reinhardtii Cultures Under Varying Acetate Concentrations. Changes in cell abundance for Chlamydomonas reinhardtii cultures grown in either 10 mM or 30 mM acetate media over a six-day period. Markers with different notation letters indicate statistically significant differences (P < 0.01), as determined by Two-way ANOVAs followed by Tukey’s HSD post-hoc tests (n = 4 biological replicates). Figure 2: Reactive Oxygen Species (ROS) Levels in Chlamydomonas reinhardtii Cultures Under Varying Acetate Concentrations. This box-whisker plot illustrating the normalized fluorescence as a measure of intracellular ROS, estimated using 2’,7’-dichlorodihydrofluorescein diacetate (H₂DCFDA), at Days 2, 3, and 4 post-inoculation (n = 12 biological replicates). Cultures were grown in either 10 mM or 30 mM acetate media. Statistical significance was determined using Two-way ANOVAs followed by Tukey’s HSD post-hoc tests. Different lowercase letters (a, b, c) above the box plots indicate statistically significant differences (p < 0.05) between groups. Figure 3: Respiration in Chlamydomonas reinhardtii cultures under low and high acetate conditions. Oxygen consumption rates in the dark, a proxy for respiration, of Chlamydomonas reinhardtii cultures grown in 10 mM (grey squares) or 30 mM (black circles) acetate over a 6-day period. Respiration rates, measured as oxygen consumption in the dark, are normalized to cell abundance. Error bars consistently represent ±2 standard errors (n = 3). Figure 4: Resilience of Chlamydomonas reinhardtii Cultures to External Reactive Oxygen Species (ROS)-Inducing Agents. This qualitative growth assay of Chlamydomonas reinhardtii cells from high-acetate and low-acetate cultures after exposure to various external ROS-inducing agents. Cells were treated at Day 3 post-inoculation with different concentrations of: (A) Rose Bengal, (B) Hydrogen Peroxide (H2O2), and (C) Methyl Viologen. Figure 5: Effect of the ROS Scavengers on Chlamydomonas reinhardtii Cultures Under High-Acetate Conditions. 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The correlation between oxidative stress and leaf senescence during plant development. Cellular & Molecular Biology Letters , 10 (3), 515–534. Crossref Google Scholar Information & Authors Information Version history V1 Version 1 03 October 2025 Copyright This work is licensed under a Non Exclusive No Reuse License. Keywords acetate longevity photosynthesis: electron transport reactive oxygen species respiration senescence Authors Affiliations Navpreet Kaur Kaur 0009-0002-6672-7299 University of New Brunswick Department of Biology View all articles by this author Ghaith Zamzam Fredericton Research and Development Centre View all articles by this author Dion Durnford 0000-0001-5232-2333 [email protected] University of New Brunswick Department of Biology View all articles by this author Metrics & Citations Metrics Article Usage 251 views 134 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Navpreet Kaur Kaur, Ghaith Zamzam, Dion Durnford. 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