Phenotypic variability in chloroplast redox state predicts cell fate in a marine diatom

Diatoms are photosynthetic microorganisms of great ecological and biogeochemical importance, forming vast blooms in diverse aquatic ecosystems. They are subjected to a wide range of environmental cues, including abiotic stressors and biotic interactions with associated bacteria, viruses and grazers. However, the cellular strategies that underline their ecological success and their rapid acclimation to fluctuating conditions are still underexplored. This study investigates heterogeneity within diatom populations in response to oxidative stress, which mediates a wide range of environmental stress conditions. We combined flow cytometry and microfluidics system for live-imaging microscopy to measure redox dynamics at the single-cell level. Using the redox-sensitive sensor roGFP we measured in vivo organelle-specific oxidation patterns in the model diatom Phaeodactylum tricornutum. Chloroplast targeted roGFP exhibited a light-dependent, bi-stable oxidation pattern in response to oxidative stress, revealing two distinct subpopulations. Remarkably, the “oxidized” subpopulation was sensitive to the stress and subsequently died, while the “reduced” subpopulation was resilient to it and recovered. We further characterized an early phase of “pre-commitment” to cell death following oxidative stress, after which cell death was irreversibly activated in the “oxidized” cells, even upon removal of the stress. Oxidation of the chloroplast glutathione pool preceded commitment to cell death, and was used as a novel predictor of cell fate. We propose that phenotypic variability within diatom populations can provide an ecological strategy to cope with rapid environmental fluctuations in the marine ecosystem. Significance Diatoms are responsible for ~20% of photosynthesis on Earth, and are central to biogeochemical cycles. They are constantly exposed to fluctuating conditions of light, nutrients and chemical cues. Until now, our understanding of acclimation strategies in phytoplankton has been based primarily on analysis at the population level. Here we investigate phenotypic variability in the redox response of diatoms to oxidative stress, which mediates diverse environmental stress conditions. Using single-cell approaches, we revealed two distinct subpopulations differing in their chloroplast redox response and subsequent cell fate. These approaches are used as a novel platform to study intraspecies heterogeneity, and shed light on stress sensing and cell fate regulation. Individuality in stress response may provide a “bet-hedging” strategy beneficial in fluctuating environments. Introduction Diatoms are considered to be amongst the most successful and diverse eukaryotic phytoplankton groups, and are estimated to contribute 20% of global net primary production (1–3). They form massive blooms and are thus central to the biogeochemical cycling of important elements such as carbon, nitrogen, phosphate, iron and silica, in addition to their important role at the base of marine food webs (1, 2, 4–6). As other phytoplankton, diatoms need to constantly acclimate to physicochemical gradients in a fluctuating environment. They are exposed to stress from different biotic and abiotic origins such as grazing, viral and bacterial infections, allelopathic interactions, light availability, and nutrient limitations (7–14). Importantly, induction of programmed cell death (PCD) in response to different stressors has been suggested as a major mechanism contributing to the fast turnover of phytoplankton and the rapid demise of their blooms (7, 8, 15). Recent studies suggested that diatoms possess a signaling mechanism based on compartmentalized redox in response to various environmental cues (16–18). Reactive oxygen species (ROS) are known to play an important role in sensing stress and external signals across kingdoms, from bacteria to plants and animals (19– 23). They are produced as byproducts of oxygen-based metabolism in respiration and photosynthesis, by ROS generating enzymes, and due to various stress conditions that lead to their accumulation (16, 22, 24–27). To maintain redox balance and avoid oxidative damage, cells harbor various ROS scavenging enzymes and small antioxidant molecules that regulate and buffer ROS levels, such as glutathione (GSH), ascorbate and NADPH. ROS can cause fast post-translational modifications of proteins through oxidation, affecting their activity even prior to changes in gene expression (22). The specificity of the ROS signal is derived from the specific chemical species, its concentration, sub-cellular localization, temporal dynamics, and available downstream ROSsensitive targets (16, 22, 24, 28–30). Therefore, ROS production and redox metabolic networks can be used to sense and integrate information of both the metabolic state of the cell and its microenvironment. H2O2 is a relatively mild and stable ROS that can accumulate in cells due to various stress conditions, and thus often serves as a signaling molecule (19–23, 31). It has a preferential activity towards cysteine residues, and it can remodel the redox-sensitive proteome network (17, 23, 28). In addition, it can diffuse across membranes (depending on membrane properties) and through aquaporins channels (32). Combined properties as lower toxicity, diffusibility and selective reactivity make H2O2 suitable for studying signaling in various biological systems (23, 28). Since many environmental stressors induce ROS generation, application of H2O2 can reproduce the downstream cellular response. H2O2 application in marine diatoms led to oxidation patterns similar to other environmental stressors (16, 17). It also led to the induction of cell death, in a dose-dependent manner, with characteristics of PCD that included externalization of phosphatidylserine, DNA laddering, and compromised cell membrane (16–18). In addition, early oxidation of the mitochondrial GSH redox potential (EGSH), which represents the balance between GSH and its oxidized form (GSSG), preceded subsequent cell death at the population level following exposure to H2O2 and diatom-derived infochemicals in the diatom Phaeodactylum tricornutum (16). In this work, we investigated phenotypic variability within diatom populations in response to oxidative stress, which may expose a differential response within the population to environmental stressors. We established single-cell approaches to measure in vivo oxidation dynamics in the model diatom P. tricornutum using flow cytometry and microfluidics live-imaging. To measure oxidation dynamics of specific organelles, we used P. tricornutum strains expressing redox-sensitive GFP (roGFP) targeted to various sub-cellular compartments. The oxidation of roGFP is reversible, and can be quantified using ratiometric fluorescence measurements (33). The roGFP oxidation degree (OxD) represents EGSH, providing a metabolic input of the redox state of the cell and correlates with the oxidation state of native proteins in the organelle (17, 33). By measuring redox dynamics at single-cell resolution, we uncovered a previously uncharacterized phenotypic heterogeneity in the response of marine diatoms to oxidative stress. We revealed a bi-stable response in the chloroplast EGSH following H2O2 treatment, exposing distinct subpopulations with different cell fates. Our results revealed a specific link between oxidation patterns in the chloroplast and subsequent cell fate regulation.


Introduction
Diatoms are considered to be amongst the most successful and diverse eukaryotic phytoplankton groups, and are estimated to contribute 20% of global net primary production (1)(2)(3). They form massive blooms and are thus central to the biogeochemical cycling of important elements such as carbon, nitrogen, phosphate, iron and silica, in addition to their important role at the base of marine food webs (1,2,(4)(5)(6). As other phytoplankton, diatoms need to constantly acclimate to physicochemical gradients in a fluctuating environment. They are exposed to stress from different biotic and abiotic origins such as grazing, viral and bacterial infections, allelopathic interactions, light availability, and nutrient limitations (7)(8)(9)(10)(11)(12)(13)(14). Importantly, induction of programmed cell death (PCD) in response to different stressors has been suggested as a major mechanism contributing to the fast turnover of phytoplankton and the rapid demise of their blooms (7,8,15).
Recent studies suggested that diatoms possess a signaling mechanism based on compartmentalized redox in response to various environmental cues (16)(17)(18). Reactive oxygen species (ROS) are known to play an important role in sensing stress and external signals across kingdoms, from bacteria to plants and animals (19)(20)(21)(22)(23). They are produced as byproducts of oxygen-based metabolism in respiration and photosynthesis, by ROS generating enzymes, and due to various stress conditions that lead to their accumulation (16,22,(24)(25)(26)(27). To maintain redox balance and avoid oxidative damage, cells harbor various ROS scavenging enzymes and small antioxidant molecules that regulate and buffer ROS levels, such as glutathione (GSH), ascorbate and NADPH.
ROS can cause fast post-translational modifications of proteins through oxidation, affecting their activity even prior to changes in gene expression (22). The specificity of the ROS signal is derived from the specific chemical species, its concentration, sub-cellular localization, temporal dynamics, and available downstream ROSsensitive targets (16,22,24,(28)(29)(30). Therefore, ROS production and redox metabolic networks can be used to sense and integrate information of both the metabolic state of the cell and its microenvironment.
H2O2 is a relatively mild and stable ROS that can accumulate in cells due to various stress conditions, and thus often serves as a signaling molecule (19)(20)(21)(22)(23)31). It has a preferential activity towards cysteine residues, and it can remodel the redox-sensitive proteome network (17,23,28). In addition, it can diffuse across membranes (depending on membrane properties) and through aquaporins channels (32). Combined properties as lower toxicity, diffusibility and selective reactivity make H2O2 suitable for studying signaling in various biological systems (23,28). Since many environmental stressors induce ROS generation, application of H2O2 can reproduce the downstream cellular response. H2O2 application in marine diatoms led to oxidation patterns similar to other environmental stressors (16,17). It also led to the induction of cell death, in a dose-dependent manner, with characteristics of PCD that included externalization of phosphatidylserine, DNA laddering, and compromised cell membrane (16)(17)(18). In addition, early oxidation of the mitochondrial GSH redox potential (EGSH), which represents the balance between GSH and its oxidized form (GSSG), preceded subsequent cell death at the population level following exposure to H2O2 and diatom-derived infochemicals in the diatom Phaeodactylum tricornutum (16).
In this work, we investigated phenotypic variability within diatom populations in response to oxidative stress, which may expose a differential response within the population to environmental stressors. We established single-cell approaches to measure in vivo oxidation dynamics in the model diatom P. tricornutum using flow cytometry and microfluidics live-imaging. To measure oxidation dynamics of specific organelles, we used P. tricornutum strains expressing redox-sensitive GFP (roGFP) targeted to various sub-cellular compartments.
The oxidation of roGFP is reversible, and can be quantified using ratiometric fluorescence measurements (33).
The roGFP oxidation degree (OxD) represents EGSH, providing a metabolic input of the redox state of the cell and correlates with the oxidation state of native proteins in the organelle (17,33). By measuring redox dynamics at single-cell resolution, we uncovered a previously uncharacterized phenotypic heterogeneity in the response of marine diatoms to oxidative stress. We revealed a bi-stable response in the chloroplast EGSH following H2O2 treatment, exposing distinct subpopulations with different cell fates. Our results revealed a specific link between oxidation patterns in the chloroplast and subsequent cell fate regulation.

Results
Bi-stable chloroplast roGFP oxidation in response to oxidative stress reveals distinct subpopulations.
To assess the roGFP oxidation state in an organelle-specific manner, we measured OxD in P. tricornutum strains expressing roGFP targeted to the chloroplast, nucleus and mitochondria using flow cytometry. At steady-state conditions without perturbations, the OxD distribution in the population had a single distinct peak, representing a reduced state at all examined compartments ( Fig. 1 A, E, I and Figs. S1, S2). Application of H2O2 led to organelle-specific and dose-dependent oxidation patterns in these organelles (Fig. 1).
The chl-roGFP exhibited a distinct bi-stable response following treatments of 50-100µM H2O2, revealing two distinct subpopulations of "oxidized" and "reduced" cells ( minutes post treatment compared to later time points, indicating that some cells were able to recover during this time (Fig. 1 M). The proportion between these subpopulations stabilized after 46-51 min post treatment, and was H2O2-dose dependent, as more cells were within the "oxidized" subpopulation at higher H2O2 concentrations ( Fig. 1 A-D, M). The emergence of stable co-existing "oxidized" and "reduced" subpopulations revealed underlying heterogeneity within the diatom population, resulting in a differential response to oxidative stress.
This clear bi-stable pattern was unique to the chloroplast EGSH. The nuclear roGFP displayed a continuous distribution in response to H2O2 treatments, and no distinct subpopulations could be observed ( Fig. 1 F-H).
Nuclear roGFP exhibited fast oxidation within a few minutes post treatment, even in response to a low H2O2 concentration of 50µM, which had only a mild effect on the chloroplast ( Fig. 1 B, F). At that concentration, nuc-roGFP oxidation was followed by a gradual and much slower recovery, which lasted >5 hours post the "oxidized" subpopulation over time upon exposure to 0-100µM H2O2. Data is shown as mean ± SEM, n=3. SEM lower than 0.018 is not shown. treatment ( Fig. 1 F). At higher concentrations, the entire population was oxidized within 3 minutes post treatment, and most cells remained stably oxidized >5 hours post treatment ( Fig. 1 G-H). The mitochondrial roGFP exhibited a heterogeneous redox response within the diatom population, as seen in the 80µM and 100µM H2O2 treatments starting at ~24 minutes post treatment ( Fig. 1 K-L). However, distinct subpopulations were not clearly separated until later stages, and were not detected consistently between experiments ( Fig. 1 K-L and Fig. S4). Therefore, we chose to focus on the chl-roGFP strain, which revealed two discrete subpopulations.

The bi-stable chloroplast redox response is light dependent.
After observing the distinct chloroplast redox response, we hypothesized that light regime will affect the bi-stable pattern of chl-roGFP following oxidative stress. Photosynthesis is the major source for reductive power as well as ROS in algal cells, and exposure to dark was shown to increase sensitivity to oxidative stress in another marine diatom (18). Therefore, we investigated the effects of short exposure to darkness on the chloroplast redox response. Cells were treated with 0-100µM H2O2 and were immediately moved to the dark for 90 minutes, and were then returned to the light ("dark" treated, Fig . Therefore, we conclude that the differential redox response of the chloroplast to oxidative stress in the "oxidized" and "reduced" subpopulations was light dependent. Next, we investigated whether the transition to the dark also affected the diatoms' cell fate. We quantified cell death 24 hours post treatment using flow cytometry measurements of Sytox green staining, which selectively stains nuclei of dead cells. The transition to the dark increased H2O2 sensitivity in the entire population, as seen both by the stronger chl-roGFP oxidation and by the larger fraction of dead cells in "dark" compared to "light" treated cells ( Fig. 2 and Fig. S5). Taken together, these results demonstrate an important role for chloroplast redox homeostasis and light-dependent reactions in cell fate regulation in diatoms. The "oxidized" and "reduced" subpopulations differ in their cell fate.
We further examined the possible link between early chloroplast EGSH oxidation and subsequent cell fate determination. We compared the fraction of the chl-roGFP "oxidized" subpopulation 1-2 hours post H2O2 treatments to the fraction of dead cells 24 hours post treatment as measured by Sytox staining (Fig. 3 A). These parameters exhibited a strong correlation (R 2 =0.835, slope of the linear fit: 0.945, Fig. 3 A), supporting our hypothesis that detection of early chloroplast oxidation in distinct subpopulations predicts cell fate at much later stages. To investigate directly the link between early chl-roGFP oxidation and subsequent cell death, we sorted single cells based on chl-roGFP oxidation at different time-points following addition of 80µM H2O2 using fluorescence-activated cell sorting (FACS) into fresh media, and measured their survival by their ability to grow and form a colony (Fig. 3 B-D). When sorted 30 minutes post treatment, the "oxidized" subpopulation exhibited a high survival rate of 92.3 ± 1.4%, similar to the "reduced" subpopulation (94.1 ± 1.1%, P=0.32), though slightly but significantly lower than sorted untreated control (96 ± 0.9%, P=0.029, Fig. 3 D). However, at later time-points the survival of the "oxidized" subpopulation gradually diminished. When sorted 60 min  post treatment almost half of the "oxidized" cells were still able to recover (45.1 ± 2%), but only 12.7 ± 2.1% survived following sorting at 100 min (Fig. 3 D). These results suggest that after a distinct exposure time, cell death is induced in an irreversible manner in the "oxidized" subpopulation. In contrast, the "reduced" subpopulation from the same culture and treatment exhibited a high survival rate similar to the control at all time-points examined, demonstrating its resilience to the stress (Fig. 3 D). In agreement with these findings, cell death measurements using Sytox staining of sorted enriched subpopulations also showed higher mortality in the "oxidized" compared to "reduced" and control cells, which remained viable (Fig. S7). Taken together, these results demonstrate that the "oxidized" subpopulation was sensitive to the oxidative stress applied, which led to induction of cell death in those cells, while the "reduced" subpopulation was able to survive. In addition, we revealed a distinct phase of "pre-commitment" to cell death, ranging approximately 30-100 min in most cells, during which the fate of the "oxidized" subpopulation is still reversible upon removal of the stress.
The distinct subpopulations derive from phenotypic variability and not due to variable genetic background.
Next, we investigated whether the differential chloroplast oxidation of the observed subpopulations is due to genetic variability or whether it represents phenotypic plasticity within the population. To this aim, we sorted chl-roGFP individual cells of the "oxidized" and "reduced" subpopulations 30 and 100 minutes post 80µM H2O2 treatment as well as untreated control cells, and regrew them to generate clonal populations derived from cells exhibiting specific phenotypes. The clonal progeny cultures were subsequently exposed to 80µM H2O2 and their chl-roGFP oxidation was measured. The two distinct subpopulations were detected in all the clones measured, and the fraction of the "oxidized" subpopulation was correlated with cell death (Fig. 4 and Fig. S8).
Therefore, the different subpopulations observed did not originate from genetic differences, but rather represent phenotypic variability within clonal populations.

Early chloroplast oxidation predicts cell fate at the single-cell level.
In order to track oxidation dynamics and subsequent cell fate of individual cells, we established a microfluidics platform for in-vivo long-term epifluorescence imaging adapted for diatom cells, under controlled flow, light and temperature conditions (Fig. 5, Fig. S9 and movies S1, S2). We introduced cells expressing chl-roGFP into a custom-made microfluidics device, and after settlement of the cells we introduced treatments of either 80µM H2O2 or fresh media (control) continuously for 2.5-3 hours (see methods). The use of microfluidics enabled also to image the basal state of single cells prior to treatment, as well as the introduction of Sytox green at the end of the experiment to visualize cell viability. We detected the distinct "oxidized" and "reduced" subpopulations following 80µM H2O2 treatment, similar to the flow cytometry experiments (Fig. 5 C, F, and movie S1). However, no clear differences were observed in their basal OxD prior to treatment ( Fig. S10 and movie S1). The separation between the subpopulations emerged within 20 minutes of exposure to 80µM H2O2, and remained stable over the course of the experiment with the "oxidized" subpopulation maintaining a high OxD above 80% (Fig. 5 F, Fig. S10 B and movie S1). The "reduced" subpopulation exhibited an immediate response to the treatment, from 25-45% OxD at basal state to 30-65% OxD during the first 20 minutes post H2O2 treatment (Fig. 5 F, Fig. S10 B, and movie S1), comparable with flow cytometry measurements. Following this initial oxidation, the "reduced" cells recovered gradually over the next hours, reducing to 5-25% oxidation 8 hours post treatment, below the initial basal state (Fig. 5 F, movie S1). A gradual slow reduction was also observed in the control cells over the course of the experiment (Fig. 5 E, movie S2), and may represent acclimation to the experimental setup or a diurnal redox alteration. Control cells did not oxidize in response to addition of fresh media (Fig. 5 E and Fig. S10 A), excluding the possibility that the oxidation observed in 80µM H2O2 treated cells was due to shear stress during treatment.
We detected a clear correlation between initial oxidation in the chloroplast in response to oxidative stress and subsequent cell fate (Fig. 5 A-G). Cells that exhibited within 42 minutes high chl-roGFP oxidation also underwent cell death at a much later stage, while cells that maintained lower OxD were able to recover and remained viable (Fig. 5 G). The Sytox positive cells showed nuclear staining as expected and lower chlorophyll auto-fluorescence intensity (Fig. 5 D). To discriminate between the "oxidized" and "reduced" subpopulations  time post treatment. Color is based on subsequent cell fate as measured by Sytox staining ~23 h post treatment: bluealive; red -dead. Horizontal dashed line represents the threshold used for discrimination between the "oxidized" and "reduced" subpopulations and for cell fate predictions. N≥250 tracks from ≥3 different fields. The same phenomenon was observed in at least two independent experiments, data of one experiment is shown. (G) Box-plot of chl-roGFP OxD 42 min post treatment (arrow in E, F) of cells that were grouped based on their cell fate. Grey -control alive; blue -80µM H2O2 alive; red -80µM H2O2 dead. Circles -single cells; cross -mean; colored box -1 st to 3 rd quartiles; horizontal line within the box -median. N≥250 cells per treatment from ≥3 different fields. (H) Side view of the microfluidics chip design.

Discussion
Our current understanding of the mechanisms that mediate acclimation to environmental stressors in marine microorganisms, including diatoms, is derived primarily from observations at the population level, neglecting any heterogeneity at the single-cell level. Averaging the phenotypes of a whole population could mask the coexistence of distinct subpopulations that induce diverse cellular strategies to improve the survival of this globally important phytoplankton group. In this study, we established a novel system for studying phenotypic variability in the marine diatom P. tricornutum using flow cytometry and microfluidics live-imaging. Using organelle-specific measurements of EGSH dynamics we assessed the in vivo metabolic state of individual diatom cells, and were able to detect a differential response to oxidative stress within the population. We identified two distinct subpopulations that emerged early following exposure to oxidative stress, differing in their chloroplast EGSH and eventual cell fate, demonstrating the importance of phenotypic variability in cell fate regulation in diatoms. We propose that the cell redox state determines the probability of a cell to survive a stress event, and could be affected by various factors such as local ROS levels, antioxidant capacity, metabolic activity, growth phase, cell cycle position, microenvironment etc. If a cell crosses a suggested "death threshold", it is likely to commit to cell death. At steady-state conditions, there is a continuous distribution of metabolic states in the population and most cells are below this threshold. Following a stress event, ROS may accumulate at sub-cellular compartments in the cell, including the chloroplast, and the population distribution will be shifted towards a more oxidized state. The magnitude of the shift depends on the stress applied and the prior metabolic state of the cells, affecting the fraction of cells that will cross the threshold. Depending on these factors, due to the shift some cells will cross the threshold and will commit to cell death, as was observed in the "oxidized" subpopulation. Chloroplast EGSH oxidation is an early stage in this stress response and precedes the commitment to cell fate. Cells that do not cross the threshold, as the "reduced" subpopulation, are able to recover from the stress and survive.
We propose that chloroplast EGSH in diatoms is involved in sensing specific stress cues that induce oxidative stress, and in cell fate regulation (Fig. 6). Depending on the specific stress, ROS can accumulate at various subcellular compartments (16,17), resulting in a shift of the population towards a more oxidized state. Cells that  5 and 6). Cells that do not cross this threshold are able to recover and acclimate, as detected in the "reduced" subpopulation (Figs. 5 and 6). Using a microfluidics setup that allowed cell tracking during exposure to H2O2, we revealed such a "life-death threshold" based on early chl-roGFP oxidation (Fig. 5 G). This early response provided accurate cell fate predictions at the single-cell level. We propose that the balance between the basal metabolic state, the antioxidant capacity, and the magnitude of the applied stress leads to a differential response within the population. This could also explain the increase in the proportion of the "oxidized" subpopulation in a H2O2 dose-dependent manner (Fig. 1 M) and may account for the increased sensitivity of the entire population in the absence of light (Fig. 2). Moreover, the later further supports an involvement of the chloroplast in cell fate determination, and suggests that fluctuating light conditions experienced by diatoms in natural environments may greatly affect their susceptibility to additional stressors.
Redox fluctuations in the chloroplast can serve as a rapid mechanism to perceive specific environmental cues, by regulating key metabolic pathways on the post-translational level prior to gene expression. It is notable that chloroplast EGSH oxidation preceded the "point of no return", after which cell death is irreversibly activated (Fig. 3 D). This "pre-commitment" phase provides an opportunity for cells to recover if conditions change during a narrow time frame, which ranges in most of the population between ~30-100 min following the stress event, before the cell has accumulated damage beyond repair (Fig. 3 D). A similar "pre-commitment" phase was shown when diatom cells were rescued from infochemicals or H2O2 treatments by exogenous application of the antioxidant GSH only within a similar time frame (16,18). Furthermore, analysis of the redox-sensitive proteome in P. tricornutum revealed over-representation of chloroplast-targeted proteins, that were also oxidized to a greater degree under H2O2 treatment as compared to other subcellular compartments (17).
These findings underscore the prevalent role of redox regulation in chloroplast metabolic reactions, and may be involved in cell fate determination.
The role of the chloroplast in mediating PCD remains elusive, although mitochondria-generated ROS are known to play a key role in PCD in plants and animals (34,35). This gap of knowledge is even greater in unicellular marine algae for which the molecular basis for the PCD machinery is largely unknown (8). The chloroplast is a major source for generation of both ROS and reductive power in the form of reduced ferredoxin or NADPH (23). Chloroplast generated ROS were demonstrated to be involved in plants in retrograde signaling from the chloroplast and in hypersensitive response cell death (23,31,34,36). A recent model suggested possible mitochondria-chloroplast cooperative interactions in the execution of ROS-mediated PCD (34). In diatoms, mitochondrial ROS were linked to cell death in response to diatomderived infochemicals (16), and chloroplast EGSH was shown to mediate changes in oxidative stress sensitivity upon light-dark transitions (18). Taken together with the results presented here, these findings suggest that redox dynamics of both the mitochondria and the chloroplast are involved in cell fate regulation in diatoms.
As universal signaling molecules, ROS have been suggested to play a conserved role in stress sensing and cell fate regulation (22,34,37). Interestingly, a similar phenomenon of phenotypic heterogeneity in redox state that was linked to subsequent cell fate was also documented in diverse biological systems. In a mouse model for Alzheimer's disease, EGSH oxidation in a small subpopulation of neurons preceded apoptotic PCD in those cells (38). Mammalian cell lines treated with H2O2 revealed two distinct subpopulations that activated genes mediating either pro-survival (through ERK) or pro-apoptotic (through p53) pathways, and the proportions between these subpopulations were H2O2-dose dependent (39). In yeast, subpopulations with different growth arrest phenotypes were detected in response to H2O2, and their proportions were also H2O2 dosedependent (40). These findings suggest a functionally conserved role for redox heterogeneity in cell fate determination across kingdoms, and could represent a more general phenomenon.
The mechanism driving the variability observed in our system is yet to be explored, but the results presented here provide insights into factors that can affect it. Since clonal populations originated from single-cell isolation maintained the bi-stable chloroplast response, the source of variability was not genetic differences between the subpopulations (Fig. 4). Inter-individual phenotypic variability is known to exist also in genetically homogenous clonal populations, and can derive from various sources such as cell life history, cell cycle phase, cell age, unequal partitioning of molecules during cell division, metabolic activity, heterogeneous microenvironment and biological noise (41)(42)(43)(44)(45)(46)(47)(48)(49)(50)(51). The combination of these factors results in a distribution of cells with different metabolic states within the population (49,50). The metabolic state of the cell can affect its antioxidant capacity, therefore resulting in variability in sensitivity to oxidative stress as observed in our system. The antioxidant capacity of a cell to detoxify ROS depends on photosynthesis-generated NADPH, which is also used for GSH recycling. Importantly, the chl-roGFP bi-stable pattern was abolished when the cells were under darkness, as the entire population became more sensitive to oxidative stress (Fig. 2). The transition to the dark may have compromised the biosynthesis and recycling of GSH therefore enhancing sensitivity to oxidative stress, as was recently demonstrated in another marine diatom (18). Taken together, heterogeneity in sensitivity to oxidative stress within the population can result from variability in photosynthesis-derived redox equivalents that regulate the antioxidant capacity of the chloroplast.
Phenotypic variability can provide an important strategy to cope with fluctuating environments in microbial populations (44). Co-existence of subpopulations with different susceptibilities to certain stressors can be viewed as a "bet-hedging" strategy of the population, enabling at least a portion of the population to survive unpredicted stress events and subsequently leads to a growth benefit at the population level (44,46,52,53).
Furthermore, such variability enables individual cells within isogenic populations to exhibit various cell fates, including PCD, despite possible deleterious effects at the single-cell level. In diatoms, phenotypic variability in cell size, shape and susceptibility to stress conditions were suggested (1,16,18,54). However, until now the experimental setups were not designed to study individuality in stress response. Redox-based phenotypic variability can provide a rapid and adjustable strategy to cope with unpredicted stress conditions as compared to relying only on genetic diversity. There may be a tradeoff between resilience to oxidative stress and the ability to sense with high precision environmental cues and metabolic perturbations (37). Since mild redox perturbations can be used for sensing and acclimation, high ROS buffering capacity may mask some of these signals. In that case, such heterogeneity in oxidative stress sensitivity may provide a benefit under fluctuating environments.
The novel approaches developed here can provide new insights into individuality in marine microbes, and will enable studying dynamic processes at the single-cell level in diatoms and other ecologically relevant microorganisms. Future studies are required to investigate the possible tradeoff between maintaining high antioxidant capacity and the ability to sense environmental cues at high precision. The ecological importance of variability in redox state in the chloroplast and the mechanisms that underlie differential sensitivity to oxidative stress are yet to be explored. Methods roGFP measurements: roGFP oxidation was measured using the ratio between two fluorescence channels, i405 and i488, by fluorescence microscopy (see SI methods) and by flow cytometry using BD LSRII analyzer, BD FACS AriaII and BD FACS AriaIII. The roGFP ratio (i405/i488) increases upon oxidation of the probe (55). The oxidation degree of roGFP (OxD) was calculated according to Schwarzländer  Where R is the roGFP ratio of i405/i488, Rred is the ratio of fully reduced form (15-50 minutes post treatment with 1-2mM DTT), Rox is the ratio of the fully oxidized form (7-30 minutes post treatment with 200μM H2O2), and i488ox and i488red are the i488 of the maximum oxidized and maximum reduced forms respectively. In flow cytometry measurements, i405 was measured using ex 407nm, em 530/30nm or 525/25nm, and i488 was measured using ex 488nm, em 530/30nm. Relative expression level of roGFP was measured by multiplication of i405 and i488. The dynamic range of roGFP was calculated by ratio of Rox/Rred (table S1). Flow cytometry measurements were done under ambient light and temperature conditions, except for dark treatment during which samples were covered with aluminum foil. For sorting purposes, roGFP ratio was used, as exact OxD cannot be calculated prior to sorting. Both parameters give similar partition between the subpopulations (data not shown).
Microfluidics experiments: Cells were introduced into a pre-washed home-made microfluidics chip. Following settlement on the glass bottom and at least 1 hour after cells were introduced to the system, cells were imaged for roGFP measurements, chlorophyll auto-fluorescence and bright field (BF). Cells were imaged every 20 min to avoid phototoxicity, and were illuminated between imaging using BF light. After imaging the basal state of the cells, treatments of either 80µM H2O2 in fresh media or fresh media control were introduced to the system continuously for ~2.5-3 hours, after which they were gradually washed away by fresh media. To quantify cell death, Sytox green was introduced into the system at 21.5-23 hours post treatment (see SI appendix). Only a small fraction of cells within the control treatment were Sytox positive (0.0054%), indicating that cells remained viable in this experimental setup. Furthermore, cells of the "reduced" subpopulation and of control treatment were able to proliferate, further demonstrating their viability under these conditions (movies S1, S2). Flow rate was kept at 1 µl/min for the duration of the experiment, except during treatment introduction (10 µl/min for the initial 10 minutes for rapid replacement of media), cell introduction (100 µl/min) and cell settlement (up to 20µl/min with occasional stops). Experimental setup is further described in SI appendix and Fig. S9. Data was analyzed using a tailored MATLAB script (Fig. S11, SI appendix).
Additional methods are described in SI appendix.