Divergent functions of two clades of flavodoxin in diatoms mitigate oxidative stress and iron limitation
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eLife assessment
This study presents valuable findings regarding the functional diversification of flavodoxins from diatoms, a protein initially described as an Fe-sparing substitute for ferredoxin in Fe-poor open ocean environments. The evidence supporting the claims of the authors is solid, although the study could be strengthened by using a wider gradient of oxidative stress in the experiments and using Fe limitation methodology, which allows more certain differentiation between a low Fe and oxidative stress response.
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Abstract
Phytoplankton rely on diverse mechanisms to adapt to the decreased iron bioavailability and oxidative stress-inducing conditions of today’s oxygenated oceans, including replacement of the iron-requiring ferredoxin electron shuttle protein with a less-efficient iron-free flavodoxin under iron-limiting conditions. Yet, diatoms transcribe flavodoxins in high-iron regions in contrast to other phytoplankton. Here, we show that the two clades of flavodoxins present within diatoms exhibit a functional divergence, with only clade II flavodoxins displaying the canonical role in acclimation to iron limitation. We created CRISPR/Cas9 knock-outs of the clade I flavodoxin from the model diatom Thalassiosira pseudonana and found that these cell lines are hypersensitive to oxidative stress, while maintaining a wild-type response to iron limitation. Within natural diatom communities, clade I flavodoxin transcript abundance is regulated over the diel cycle rather than in response to iron availability, whereas clade II transcript abundances increase either in iron-limiting regions or under artificially induced iron limitation. The observed functional specialization of two flavodoxin variants within diatoms reiterates two major stressors associated with contemporary oceans and illustrates diatom strategies to flourish in diverse aquatic ecosystems.
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Author Response
Reviewer #1 (Public Review):
The authors worked towards a better understanding of the functional diversification of flavodoxins among diatoms, and this represents a quantum contribution building on the initial findings of Whitney, Lins, Hughes, Wells, Chappelle, and Jenkins (2011), with the inclusion of metatranscriptomic and other data from field collections and on-deck incubation experiments, relatively new genomic and transcriptomic datasets, and the adoption of reverse genetics tools that are not yet widely used in T. pseudonana. They hypothesize that clade I flavodoxins play a role in mitigating oxidative stress, while additional clade II flavodoxins would respond according to canon, in response to low iron availability.
The authors embarked on several field campaigns across environmental gradients where …
Author Response
Reviewer #1 (Public Review):
The authors worked towards a better understanding of the functional diversification of flavodoxins among diatoms, and this represents a quantum contribution building on the initial findings of Whitney, Lins, Hughes, Wells, Chappelle, and Jenkins (2011), with the inclusion of metatranscriptomic and other data from field collections and on-deck incubation experiments, relatively new genomic and transcriptomic datasets, and the adoption of reverse genetics tools that are not yet widely used in T. pseudonana. They hypothesize that clade I flavodoxins play a role in mitigating oxidative stress, while additional clade II flavodoxins would respond according to canon, in response to low iron availability.
The authors embarked on several field campaigns across environmental gradients where iron-responsive and oxidative stress-responsive flavodoxins were expected to show differential expression. The use of metatranscriptomics allowed taxa-specific assignment of relative transcript expression levels, and the results of both measurements across the environmental gradient and manipulative incubation experiments show the widespread taxonomic distribution of iron-responsive clade II flavodoxin. The fieldwork was well thought out, and biogeochemical trends comported to expectations. It's worth noting that the concomitant inclusion of geochemical data such as dissolved iron further strengthened the work. The authors also found clade I flavodoxins were not iron-responsive (as expected), but rather exhibited diel patterns in transcript abundance that suggest responses to photo-oxidative stress. Taken together, these field data are stunning.
We thank the reviewer for this kind assessment.
Lab experiments with five diatom species grown under varied iron and induced oxidative (H2O2) stress and transcript abundances for flavodoxin genes are reported. One reservation concerns the untoward and unknown effects of inducing outright iron starvation with the strong chelator, DFB (as opposed to achieving steady-state growth rate limitation from low iron by use of weak chelators such as EDTA). With DFB it is also difficult to predict sample timing (when cells have hit that "correct" and reproducible iron-limited space) when independent replicates are collected on different dates. Similarly, the use of DFB also makes it difficult to sample low and high iron cells at the same density or to maintain densities among replicate samples collected on different dates. pH and CO2 availability change with density unless special measures are taken.
We agree with the reviewer that DFB is a strong iron chelator that may affect diatom physiology in inadvertent ways. We designed the DFB experiments to allow us to screen multiple diatoms for whether they transcribed clade I and II flavodoxins in a short-term response to iron limitation.
We added the logic behind this experimental design (L177-179):
“In order to screen multiple diatoms for whether they transcribed clade I and II flavodoxins in response to iron limitation, we used the strong iron-chelator desferrioxamine B (DFB) and enhanced short-term iron limitation.”
Additionally, we now discuss the possible effect of DFB in our discussion (L395-410):
“Notably, we used the strong iron chelator DFB to enhance iron limitation in a variety of diatoms, as previously described (Andrew et al., 2019; Kranzler et al., 2021; Lampe et al., 2018; Timmermans et al., 2001; Wells, 1999), while recognizing that undesirable effects of DFB, that are not related to iron-limitation per se cannot be ruled out. Here, DFB was used in experiments designed to test whether transcription of the two flavodoxin clades differentially responded to iron limitation. The results from T. oceanica, and T. pseudonana agree with the literature, in which DFB was not added. In T. oceanica only the expression of one clade II flavodoxin was induced (Figure 2B-C, as in Lommer et al., 2012). The minor induction in mRNA of T. pseudonana clade I flavodoxin in response to iron limitation was detected in both long- and short-term adaptation to low iron, without added DFB (Goldman et al., 2019; Thamatrakoln et al., 2012). This flavodoxin seems to have diel regulation, and the observed induction might be specific to the circadian time and the setting of the diel cycle (Goldman et al., 2019).”
Based on the reviewer comments, we realized that our transcriptome sampling protocol was not clear. Because the diatom species have different growth rates, as well as different rates of growth-inhibition by iron limitation, we adjusted the sampling day for each species based on cell counts and photosynthetic efficiency. Importantly, the 9 samples (triplicates of 3 conditions) of each species were sampled together, at the same date and time. We also ensured that the biological replicates of each species and treatment had similar cell density at the time of harvest.
We clarified these concerns in the Results section (L188-206):
“For each diatom 6 replicates were grown in iron-replete conditions and 3 replicates in iron-limiting conditions until the low-iron cultures displayed a decrease in maximum photochemical yield of photosystem II (Fv/Fm), 3-6 days (depending on species, Figure 2 -figure supplement 1A-C, Figure 2A, supplementary file 1c), indicative of iron limitation, at which point transcriptome samples were collected for both the iron-limited and iron-replete conditions. Three of the iron-replete replicates were exposed to oxidative stress, mimicked by a lethal dose of H2O2, and transcriptome samples were collected about 1.5 h after exposure, when the cell phenotype (Fv/Fm or cell abundance) was unaltered from control.”
In the Materials and Methods section (L542-545)
"Cells were harvested by filtration onto 0.22 µm filters. Full details of the number of cells harvested per treatments, per species, and samples that failed library preparation are indicated in supplementary file 1c. The 9 samples of each diatom species were sampled together, at the same date and time. Filters were snap-frozen…”
A second set of lab experiments involved the (non-trivial) establishment and use of "knock out" clones of the clade I flavodoxin gene in the model diatom T. pseudonana to test the oxidative stress hypothesis. This is an exciting idea and the data suggest this flavodoxin may confer resistance to oxidative stress. The conclusion would be greatly strengthened if different phenotypes could be observed between WT and KO clones in response to environmentally relevant oxidative stress (such as supra-optimal irradiance), rather than exogenous H2O2 addition.
Based on the reviewer suggestion, we conducted a preliminary experiment with irradiation of up to 500 µE. As with the light level originally tested, there were no differences in growth rate or Fv/Fm between the WT and KO lines. We agree that future study of these knock-lines a series of much higher irradiation levels, photosynthetic-inhibitors, and other environmental stresses is interesting, but it is out of the scope of the current study.
We now also mention this in the revised manuscript (L417-419):
“Future studies in which the oxidative stress is driven by other environmental conditions as supra-optimal irradiation, UV radiation or biotic interactions are needed to further support the role of clade I flavodoxins in oxidative stress.”
We clarify that our use of exogenous H2O2 additions was based on previous studies with Phaeodactylum and T. pseudonana that indicate that exogenous addition of micromolar range of H2O2 is representative for other oxidative stress-responses (Graff van Creveld, 2015, Volpert 2018, Mizrachi 2019) (L185-188):
“Oxidative stress was induced by the lowest lethal dose of H2O2 (200-250 µM), as similar treatment was shown to be representative to other environmentally-relevant oxidative stressors in T. pseudonana and Phaeodactylum (Graff van Creveld et al., 2015; Mizrachi et al., 2019; Volpert et al., 2018).”
The relationship between the experimental conditions and results in Figure 3C and Supplemental Figure 3H was not clear.
Figure 3C summarize parts of Figure S3H information, Figure S3D-I present the individual clones, while Figure 3 only shows WT vs Flav-KO.
According to the reviewer comments, we modified Figure S3H (it is now Figure S3I), and specify this relationship in the legend:
“H-I. Percentage of Sytox Green-positive (dead) cells, measured by flow cytometry 24 h after treatment with H2O2 treatment. Orange and gray box plots represent a Flav-KO and WT respectively, single measurements are marked, color-coded by the individual colonies. H. Results of a single dose-response experiment. I. Results from additional experiments, experiments marked with an asterisk are summarized in main Figure 3C.”
In the introduction, the authors suggest that Fe-S-containing proteins are particularly sensitive to damage via oxygen and ROS and that reliance on ferredoxin (Fd) for electron shuttling carries an enhanced sensitivity to the ROS generated during photosynthesis. References would be helpful here. Fe-S cluster-containing proteins are not monolithic regarding their behavior or susceptibility towards ROS. My limited understanding is that (i) several 4Fe-4S cluster proteins (such as aconitase, isopropylmalate isomerase) are particularly sensitive but that (ii) this is less so for canonical 2Fe-2S cluster ferredoxins; (iii) in some phototrophs Fd catalyzes the reduction of molecular oxygen to superoxide, as part of a mechanism that keeps the electron transport chain less reduced under extremely high light. Thus, ferredoxins may not necessarily be susceptible to in vivo ROS-mediated damage.
Thank you for these comments.
We modified our original sentence (L37-39):
“Moreover, iron-sulfur-containing proteins are particularly sensitive to damage via oxygen and reactive oxygen species (ROS).”
Corrected sentence:
“Moreover, iron containing proteins are sensitive to damage via oxygen and reactive oxygen species (ROS), and Fd is down-regulated in response to oxidative stress (Singh et al., 2010, 2004).”
Reviewer #2 (Public Review):
In their manuscript, Van Creveld et al. set out to demonstrate divergent functions for two clades of flavodoxin in diatoms. To achieve their goals, the authors combined metatranscriptomic results originating from three separate research cruises in the North Pacific Ocean with laboratory experiments with a clade I flavodoxin knock-out mutant in the diatom Thalassiosira pseudonana. Overall, their field study confirmed that Clade II flavodoxin is mostly up-regulated under iron limitation in most diatoms that were represented in their metatranscriptomic data (Figure 5 A-F). Their field study also demonstrated that clade I flavodoxin is expressed at levels that are several orders of magnitude lower than clade II flavodoxin (figure 5H). The lower expression of clade I flavodoxin was also observed in laboratory culture experiments (Figure 2). The laboratory experiments also demonstrated that the clade I flavodoxins were responsive to iron limitation in some of the species studied (Their Figure 2C), such that the assignment of function based solely on the clade I and clade II flavodoxin classification may not always be straight forward, and that exceptions will likely be found as more diatom species are studied.
In their quest to determine whether Clade I flavodoxin plays a role in adaptation to oxidative stress, the authors created several knock-out mutants where the clade I flavodoxin is not functional. These mutant strains responded to iron limitation in the same way as the WT strains. However, the mutant strains defective in the clade I flavodoxin were more slightly more sensitive to oxidative stress (created by exposure to lethal doses of hydrogen peroxide) than the wild-type strains. The results of the oxidative stress challenges would have been stronger if a broader concentration range of hydrogen peroxide had been used in the experiments leading to a dose-response curve for both the mutant and wild-type strains.
Thank you for this suggestion. We now tested a broader range of H2O2 concentrations on the WT and KO strains and added a new Figure S3H, which includes responses to 0, 25, 50, 75, 100, 150, 200, 250 µM H2O2.
The supplemental information provided in the main manuscript holds a lot of important information. Take for example Figure S4 showing the placement of reads for Clade I and Clade II in a Maximum-likelihood tree for flavodoxin in the North Pacific Ocean. The results show that clade II flavodoxin is much more commonly found in the transcripts than clade I flavodoxin.
Perhaps different results would have been obtained by conducting a similar sampling of metatranscriptome in the Atlantic Ocean that is less subject to iron limitation.
We agree completely and would love to analyze metatranscriptomes from the Atlantic Ocean in the future.
Overall, the authors have provided results that support a role for Clade I flavodoxin in alleviating oxidative stress in Thalassiosira pseudonana, however, whether or not this role is universal for clade I flavodoxin in other diatom species will require further studies.
We agree with this assessment that additional experiments with additional diatoms is a fruitful research area into the future.
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eLife assessment
This study presents valuable findings regarding the functional diversification of flavodoxins from diatoms, a protein initially described as an Fe-sparing substitute for ferredoxin in Fe-poor open ocean environments. The evidence supporting the claims of the authors is solid, although the study could be strengthened by using a wider gradient of oxidative stress in the experiments and using Fe limitation methodology, which allows more certain differentiation between a low Fe and oxidative stress response.
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Reviewer #1 (Public Review):
The authors worked towards a better understanding of the functional diversification of flavodoxins among diatoms, and this represents a quantum contribution building on the initial findings of Whitney, Lins, Hughes, Wells, Chappelle, and Jenkins (2011), with the inclusion of metatranscriptomic and other data from field collections and on-deck incubation experiments, relatively new genomic and transcriptomic datasets, and the adoption of reverse genetics tools that are not yet widely used in T. pseudonana. They hypothesize that clade I flavodoxins play a role in mitigating oxidative stress, while additional clade II flavodoxins would respond according to canon, in response to low iron availability.
The authors embarked on several field campaigns across environmental gradients where iron-responsive and …
Reviewer #1 (Public Review):
The authors worked towards a better understanding of the functional diversification of flavodoxins among diatoms, and this represents a quantum contribution building on the initial findings of Whitney, Lins, Hughes, Wells, Chappelle, and Jenkins (2011), with the inclusion of metatranscriptomic and other data from field collections and on-deck incubation experiments, relatively new genomic and transcriptomic datasets, and the adoption of reverse genetics tools that are not yet widely used in T. pseudonana. They hypothesize that clade I flavodoxins play a role in mitigating oxidative stress, while additional clade II flavodoxins would respond according to canon, in response to low iron availability.
The authors embarked on several field campaigns across environmental gradients where iron-responsive and oxidative stress-responsive flavodoxins were expected to show differential expression. The use of metatranscriptomics allowed taxa-specific assignment of relative transcript expression levels, and the results of both measurements across the environmental gradient and manipulative incubation experiments show the widespread taxonomic distribution of iron-responsive clade II flavodoxin. The fieldwork was well thought out, and biogeochemical trends comported to expectations. It's worth noting that the concomitant inclusion of geochemical data such as dissolved iron further strengthened the work. The authors also found clade I flavodoxins were not iron-responsive (as expected), but rather exhibited diel patterns in transcript abundance that suggest responses to photo-oxidative stress. Taken together, these field data are stunning.
Lab experiments with five diatom species grown under varied iron and induced oxidative (H2O2) stress and transcript abundances for flavodoxin genes are reported. One reservation concerns the untoward and unknown effects of inducing outright iron starvation with the strong chelator, DFB (as opposed to achieving steady-state growth rate limitation from low iron by use of weak chelators such as EDTA). With DFB it is also difficult to predict sample timing (when cells have hit that "correct" and reproducible iron-limited space) when independent replicates are collected on different dates. Similarly, the use of DFB also makes it difficult to sample low and high iron cells at the same density or to maintain densities among replicate samples collected on different dates. pH and CO2 availability change with density unless special measures are taken.
A second set of lab experiments involved the (non-trivial) establishment and use of "knock out" clones of the clade I flavodoxin gene in the model diatom T. pseudonana to test the oxidative stress hypothesis. This is an exciting idea and the data suggest this flavodoxin may confer resistance to oxidative stress. The conclusion would be greatly strengthened if different phenotypes could be observed between WT and KO clones in response to environmentally relevant oxidative stress (such as supra-optimal irradiance), rather than exogenous H2O2 addition. The relationship between the experimental conditions and results in Figure 3C and Supplemental Figure 3H was not clear.
In the introduction, the authors suggest that Fe-S-containing proteins are particularly sensitive to damage via oxygen and ROS and that reliance on ferredoxin (Fd) for electron shuttling carries an enhanced sensitivity to the ROS generated during photosynthesis. References would be helpful here. Fe-S cluster-containing proteins are not monolithic regarding their behavior or susceptibility towards ROS. My limited understanding is that (i) several 4Fe-4S cluster proteins (such as aconitase, isopropylmalate isomerase) are particularly sensitive but that (ii) this is less so for canonical 2Fe-2S cluster ferredoxins; (iii) in some phototrophs Fd catalyzes the reduction of molecular oxygen to superoxide, as part of a mechanism that keeps the electron transport chain less reduced under extremely high light. Thus, ferredoxins may not necessarily be susceptible to in vivo ROS-mediated damage.
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Reviewer #2 (Public Review):
In their manuscript, Van Creveld et al. set out to demonstrate divergent functions for two clades of flavodoxin in diatoms. To achieve their goals, the authors combined metatranscriptomic results originating from three separate research cruises in the North Pacific Ocean with laboratory experiments with a clade I flavodoxin knock-out mutant in the diatom Thalassiosira pseudonana. Overall, their field study confirmed that Clade II flavodoxin is mostly up-regulated under iron limitation in most diatoms that were represented in their metatranscriptomic data (Figure 5 A-F). Their field study also demonstrated that clade I flavodoxin is expressed at levels that are several orders of magnitude lower than clade II flavodoxin (figure 5H). The lower expression of clade I flavodoxin was also observed in laboratory …
Reviewer #2 (Public Review):
In their manuscript, Van Creveld et al. set out to demonstrate divergent functions for two clades of flavodoxin in diatoms. To achieve their goals, the authors combined metatranscriptomic results originating from three separate research cruises in the North Pacific Ocean with laboratory experiments with a clade I flavodoxin knock-out mutant in the diatom Thalassiosira pseudonana. Overall, their field study confirmed that Clade II flavodoxin is mostly up-regulated under iron limitation in most diatoms that were represented in their metatranscriptomic data (Figure 5 A-F). Their field study also demonstrated that clade I flavodoxin is expressed at levels that are several orders of magnitude lower than clade II flavodoxin (figure 5H). The lower expression of clade I flavodoxin was also observed in laboratory culture experiments (Figure 2). The laboratory experiments also demonstrated that the clade I flavodoxins were responsive to iron limitation in some of the species studied (Their Figure 2C), such that the assignment of function based solely on the clade I and clade II flavodoxin classification may not always be straight forward, and that exceptions will likely be found as more diatom species are studied.
In their quest to determine whether Clade I flavodoxin plays a role in adaptation to oxidative stress, the authors created several knock-out mutants where the clade I flavodoxin is not functional. These mutant strains responded to iron limitation in the same way as the WT strains. However, the mutant strains defective in the clade I flavodoxin were more slightly more sensitive to oxidative stress (created by exposure to lethal doses of hydrogen peroxide) than the wild-type strains. The results of the oxidative stress challenges would have been stronger if a broader concentration range of hydrogen peroxide had been used in the experiments leading to a dose-response curve for both the mutant and wild-type strains.
The supplemental information provided in the main manuscript holds a lot of important information. Take for example Figure S4 showing the placement of reads for Clade I and Clade II in a Maximum-likelihood tree for flavodoxin in the North Pacific Ocean. The results show that clade II flavodoxin is much more commonly found in the transcripts than clade I flavodoxin. Perhaps different results would have been obtained by conducting a similar sampling of metatranscriptome in the Atlantic Ocean that is less subject to iron limitation.
Overall, the authors have provided results that support a role for Clade I flavodoxin in alleviating oxidative stress in Thalassiosira pseudonana, however, whether or not this role is universal for clade I flavodoxin in other diatom species will require further studies.
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