The induction of pyrenoid synthesis by hyperoxia and its implications for the natural diversity of photosynthetic responses in Chlamydomonas
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Curated by eLife
Evaluation Summary:
Many algae, such as Chlamydomonas, form a pyrenoid under certain conditions to enable high photosynthetic rates during inorganic carbon limitation. The data presented here support that hydrogen peroxide, a common by-product of hyperoxia and CO2 limitation, induces pyrenoid formation in Chlamydomonas, even when CO2 levels are high. Although the underlying genetic mechanisms remain unresolved, these observations offer an exciting starting point to dissect the molecular components that drive pyrenoid formation. Therefore, this paper is of interest to a broad audience of scientists working in the areas of photosynthesis, synthetic biology of agriculture, and algal biotechnology.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Abstract
In algae, it is well established that the pyrenoid, a component of the carbon-concentrating mechanism (CCM), is essential for efficient photosynthesis at low CO 2 . However, the signal that triggers the formation of the pyrenoid has remained elusive. Here, we show that, in Chlamydomonas reinhardtii , the pyrenoid is strongly induced by hyperoxia, even at high CO 2 or bicarbonate levels. These results suggest that the pyrenoid can be induced by a common product of photosynthesis specific to low CO 2 or hyperoxia. Consistent with this view, the photorespiratory by-product, H 2 O 2 , induced the pyrenoid, suggesting that it acts as a signal. Finally, we show evidence for linkages between genetic variations in hyperoxia tolerance, H 2 O 2 signaling, and pyrenoid morphologies.
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Author Response:
Reviewer #1 (Public Review):
[...] An aspect that would greatly strengthen the paper overall is to clarify whether the pyrenoids induced at high CO2 by hyperoxia or hydrogen peroxide participate in the CCM. Do these cells now have high affinities for Ci?
Response: We found that hydrogen peroxide treated cells do have a higher affinity for Ci.
Reviewer #2 (Public Review):
[...] It would be helpful for readers to understand the significance of the work if the authors could put the work into context of ongoing efforts to engineer the pyrenoid into crop plants to increase yield and to highlight the global importance of pyrenoid-mediated algal photosynthesis.
For a better understanding of their proposed model of H2O2 mediated pyrenoid/CCM induction it would be very helpful if the author added a figure of their CCM …
Author Response:
Reviewer #1 (Public Review):
[...] An aspect that would greatly strengthen the paper overall is to clarify whether the pyrenoids induced at high CO2 by hyperoxia or hydrogen peroxide participate in the CCM. Do these cells now have high affinities for Ci?
Response: We found that hydrogen peroxide treated cells do have a higher affinity for Ci.
Reviewer #2 (Public Review):
[...] It would be helpful for readers to understand the significance of the work if the authors could put the work into context of ongoing efforts to engineer the pyrenoid into crop plants to increase yield and to highlight the global importance of pyrenoid-mediated algal photosynthesis.
For a better understanding of their proposed model of H2O2 mediated pyrenoid/CCM induction it would be very helpful if the author added a figure of their CCM induction model to the discussion.
Response: We have updated the introduction with all the references the reviewer suggested, and also added the following text to better close out our introduction:
“Engineering the algal CCM into land plants is seen as a key route to improving crop photosynthesis (Fei et al., 2021; Hennacy and Jonikas, 2020; Mackinder, 2018; Meyer et al., 2016; Rae et al., 2017). If the algal pyrenoid CO2 concentration system were engineered into crops such as rice, wheat, or soya yields could increase by up to 60% (Long et al., 2019); yet photosynthetic improvements is thought to only occur if a complete algal-like CCM is assembled in land plants (Atkinson et al., 2020; Barrett et al., 2021); such ambitions necessitate an understanding of the signals and trade-offs of pyrenoid formation, for which Chlamydomonas is an excellent model system.“
Reviewer #3 (Public Review):
[...] It is not quite clear why the authors included a growth analyses under mixotrophic conditions and solid media and measured of photosystem II efficiency only under these conditions. The results showed faster growth of cells (CC2343) that tend to accumulate fractured pyrenoid starch sheath, however, growth is based on undefined proportions of autotrophy and heterotrophy under these conditions, and changes in metabolism are not well understood or predictable, which - from my point of view - is too confounding for gaining conclusive evidence for hyperoxia tolerance from biomass accumulation. Likewise, the measurements of PSII function which is a product of many factors concertedly impacting photosynthetic electron transport and correlates with growth only conditionally under autotrophy is even less informative under mixotrophy. Differences in respiration rates may have to be considered as well as differential partitioning of metabolites in the two different strains, which is outside the scope of this paper.
Response: We feel this the data should be retained to guide future studies, but have moved the mixotrophic growth figure to the Supplemental Materials (SM Figure 7). We have also deleted the line “These observations support that the growth advantage on the TAP plate was related to carboxylation, at least more likely than an aspect of the light reactions.”
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Evaluation Summary:
Many algae, such as Chlamydomonas, form a pyrenoid under certain conditions to enable high photosynthetic rates during inorganic carbon limitation. The data presented here support that hydrogen peroxide, a common by-product of hyperoxia and CO2 limitation, induces pyrenoid formation in Chlamydomonas, even when CO2 levels are high. Although the underlying genetic mechanisms remain unresolved, these observations offer an exciting starting point to dissect the molecular components that drive pyrenoid formation. Therefore, this paper is of interest to a broad audience of scientists working in the areas of photosynthesis, synthetic biology of agriculture, and algal biotechnology.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with …
Evaluation Summary:
Many algae, such as Chlamydomonas, form a pyrenoid under certain conditions to enable high photosynthetic rates during inorganic carbon limitation. The data presented here support that hydrogen peroxide, a common by-product of hyperoxia and CO2 limitation, induces pyrenoid formation in Chlamydomonas, even when CO2 levels are high. Although the underlying genetic mechanisms remain unresolved, these observations offer an exciting starting point to dissect the molecular components that drive pyrenoid formation. Therefore, this paper is of interest to a broad audience of scientists working in the areas of photosynthesis, synthetic biology of agriculture, and algal biotechnology.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Reviewer #1 (Public Review):
The paper by Neofotis and colleagues contains a substantial amount of data that together supports a highly novel and important finding: the pyrenoid of the green alga Chlamydomonas can be induced by hyperoxia, even in the presence of high levels of CO2. More specifically, hyperoxia can be substituted by hydrogen peroxide, indicating that ROS signalling mediates this response. The finding is significant because hyperoxia is a common but understudied event, especially when algae are grown for biotechnological purposes.
The paper leads by exploring the phenotypes of two wild-type strains of Chlamydomonas that show markedly different abilities to grow and tolerate at hyperoxic conditions. Extensive electron microscopy indicates that the resistant strain produces pyrenoids with closed and robust starch sheaths …
Reviewer #1 (Public Review):
The paper by Neofotis and colleagues contains a substantial amount of data that together supports a highly novel and important finding: the pyrenoid of the green alga Chlamydomonas can be induced by hyperoxia, even in the presence of high levels of CO2. More specifically, hyperoxia can be substituted by hydrogen peroxide, indicating that ROS signalling mediates this response. The finding is significant because hyperoxia is a common but understudied event, especially when algae are grown for biotechnological purposes.
The paper leads by exploring the phenotypes of two wild-type strains of Chlamydomonas that show markedly different abilities to grow and tolerate at hyperoxic conditions. Extensive electron microscopy indicates that the resistant strain produces pyrenoids with closed and robust starch sheaths when challenged with oxygen. In contrast the susceptible strain has fragmented starch sheaths surrounding the pyrenoid. Crossing the two strains demonstrated the heritability of this phenotype. Importantly this hyperoxia dependent pyrenoid induction occurs even at the very high levels of 5% CO2, a condition where no pyrenoid would normally be seen.
The TEM images are supplemented using light microscopy, where the presence or absence of the pyrenoid starch sheath can be clearly discerned. Following various control conditions (e.g. no pyrenoid at high CO2 alone), it is discovered that H2O2 in the presence of high CO2 alone can bring about pyrenoid formation. Importantly the effect of H2O2 is validated using another Chlamydomonas strain that expresses fluorescently tagged Rubisco. In addition data is presented that indicates that H2O2 in the hyperoxia resistant strain is localized in small clusters that may represent peroxisomes (and would implicate photorespiratory H2O2 production). The resistant strain was also able to operate at higher O2 levels, having a higher O2 compensation point.
The main weakness of the manuscript is its current poor presentation, which needs to be improved to enhance accessibility of the work. The word limits common in selective journals could have been helpful to better communicate the work's impact and importance. This comment relates mostly to the results section. The discussion, albeit also long, is better written and deeply engages with the literature at a high level.
An aspect that would greatly strengthen the paper overall is to clarify whether the pyrenoids induced at high CO2 by hyperoxia or hydrogen peroxide participate in the CCM. Do these cells now have high affinities for Ci?
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Reviewer #2 (Public Review):
A summary of what the authors were trying to achieve:
Under limiting CO2 algae operate CO2-concentrating mechanisms (CCMs) to increase CO2 at the active site of Rubisco to enhance Rubisco's efficiency. To achieve this Rubisco is packaged into a non-membrane bound compartment called the pyrenoid where CO2 is delivered via a series of Ci transporters and carbonic anhydrases. The size and Rubisco content of the pyrenoid has previously been shown to increase when CO2 becomes limiting and the cells are photosynthetically active (i.e. in the light). However, the signal for pyrenoid (and CCM) induction is unclear, with it proposed it could be due to direct sensing of inorganic carbon (i.e. CO2 or HCO3- concentration) or a metabolic signal through reduced photosynthetic capacity (i.e. photorespiratory by-product). …
Reviewer #2 (Public Review):
A summary of what the authors were trying to achieve:
Under limiting CO2 algae operate CO2-concentrating mechanisms (CCMs) to increase CO2 at the active site of Rubisco to enhance Rubisco's efficiency. To achieve this Rubisco is packaged into a non-membrane bound compartment called the pyrenoid where CO2 is delivered via a series of Ci transporters and carbonic anhydrases. The size and Rubisco content of the pyrenoid has previously been shown to increase when CO2 becomes limiting and the cells are photosynthetically active (i.e. in the light). However, the signal for pyrenoid (and CCM) induction is unclear, with it proposed it could be due to direct sensing of inorganic carbon (i.e. CO2 or HCO3- concentration) or a metabolic signal through reduced photosynthetic capacity (i.e. photorespiratory by-product). In this study, Neofotis et al. explore whether inorganic carbon concentration or a photosynthetic by-product induced by hyperoxia leads to pyrenoid induction. They show that hyperoxia most likely due to increased hydrogen peroxide levels is a prime candidate for pyrenoid induction. With pyrenoid induction even in the presence of high CO2 when cells are hyperoxic or exposed to H2O2.
An account of the major strengths and weaknesses of the methods and results:
Strengths:
The authors use two independent strains that show differing morphologocial and growth responses to hyperoxia which correlate to different pyrenoid starch sheath induction levels and morphologies; linking for the first time response to hyperoxia with pyrenoid formation. They further analyze progeny of crosses of these two strains indicating that this is potentially linked to a singular genomic locus, although this would require further investigation. They show that both exogenous and endogenous produced H2O2 induces pyrenoid formation independent of inorganic carbon level and that the addition of H2O2 scavengers prevents pyrenoid induction. As far as we are aware this is the first time in Chlamydomonas that a specific metabolic signal, H2O2, induces pyrenoid formation. The results will strongly support efforts to identify the unknown molecular mechanisms that lead to pyrenoid formation. The authors also analyse a large range of conditions relating to carbon source and light, and record the state of pyrenoid induction in these conditions, providing a useful resource for the pyrenoid community.
Weaknesses:
A limited number of crossing progeny are analysed, and in some instances the n values for observational experiments are low. Without quantitative analyses of these data, the phenotypes remain observational and purely correlative with other observations the authors make. The authors mention in several occasions that the genetic variation between the two strains used should modulate differing responses, however they do not give any details on the genetic basis for the differing response - particularly the change in H2O2 production between the two strains used for comparison and the differences in starch synthesis - even though genetic information on both strains are available. The claims that pyrenoid formation is induced by endogenously-produced H2O2 by methyl viologen or metronidazole and is repressed by H2O2 scavengers ascorbic acid and dimethylthiourea requires further experimental proof.
An appraisal of whether the authors achieved their aims, and whether the results support their conclusions:
The authors provide strong evidence that H2O2 peroxide provides a signal for pyrenoid induction. The data further supports the importance of the pyrenoid starch sheath in efficient pyrenoid function and the CCM. In addition, the data indicates that pyrenoid formation (and CCM induction) is more complex than just H2O2 signalling but is likely a combination of factors including light, carbon source and availability, plus metabolic signalling, likely via H2O2 production.
A discussion of the likely impact of the work on the field, and the utility of the methods and data to the community:
The work provides a foundation for investigating the regulation and control of pyrenoid assembly at a molecular level in Chlamydomonas. For the first time a metabolite (H2O2) was shown to induce pyrenoid formation. This opens a starting point to investigate potential signaling pathways and to identify other components involved in pyrenoid assembly and CCM induction. The findings of this study are likely applicable to pyrenoids across algal lineages, and this study will provide a starting framework for follow-up studies in Chlamydomonas and other pyrenoid-containing species.
Any additional context you think would help readers interpret or understand the significance of the work:
It would be helpful for readers to understand the significance of the work if the authors could put the work into context of ongoing efforts to engineer the pyrenoid into crop plants to increase yield and to highlight the global importance of pyrenoid-mediated algal photosynthesis.
For a better understanding of their proposed model of H2O2 mediated pyrenoid/CCM induction it would be very helpful if the author added a figure of their CCM induction model to the discussion.
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Reviewer #3 (Public Review):
The paper presents novel insight into a potential signal, H2O2, and environmental conditions that induce the pyrenoid, which is an essential part of the physical CO2/inorganic carbon (Ci) concentrating mechanism (CCM) of many microalgae One central claim of this study is that pyrenoid formation and morphology with regard to its starch sheath is induced by hyperoxia in two Chlamydomonas wild type strains which otherwise differ in their ability to grow under hyperoxic conditions. Yet, the way some conclusions are presented may need clarification to fully appreciate the impact of the findings presented. The authors have carefully devised growth conditions that allow constant monitoring and control of the O2 and inorganic carbon (Ci; dissolved CO2 and bicarbonate) concentrations in liquid cell cultures. Ci was …
Reviewer #3 (Public Review):
The paper presents novel insight into a potential signal, H2O2, and environmental conditions that induce the pyrenoid, which is an essential part of the physical CO2/inorganic carbon (Ci) concentrating mechanism (CCM) of many microalgae One central claim of this study is that pyrenoid formation and morphology with regard to its starch sheath is induced by hyperoxia in two Chlamydomonas wild type strains which otherwise differ in their ability to grow under hyperoxic conditions. Yet, the way some conclusions are presented may need clarification to fully appreciate the impact of the findings presented. The authors have carefully devised growth conditions that allow constant monitoring and control of the O2 and inorganic carbon (Ci; dissolved CO2 and bicarbonate) concentrations in liquid cell cultures. Ci was supplied by addition of NaHCO3, which will provide predominantly bicarbonate as CI source at pH above 8 and by sparging liquid cultures with gas mixes of 5% CO2 with either 21% or 95% O2 for normal, air level or high oxygen conditions respectively. Without hyperoxic stress, cell lines were shown to behave as expected in terms of starch sheath accumulation and formation of pyrenoids in response to light and low Ci. Therefore evidence presented in a series of high quality TEM images for the presence of pyrenoids and their starch sheath morphology specific to hyperoxia is unambiguous. This finding is of great interest with regards to unsolved questions about pyrenoid function and mechanisms underlying responses to Ci and high O2 stress in more general terms.
Furthermore, the authors provide strong evidence in support of their hypothesis that induction and/or formation of the pyrenoid is related to the ability of cells to grow efficiently under hyperoxia conditions (p.3). The study establishes that two wild type isolates show differences in starch sheath morphology. Cell lines with relatively continuous (sealed) starch sheath around pyrenoids (CC1009) accumulated significantly more biomass than cell lines with lesser developed (fragmented) starch sheath (CC2343). The same correlation was found in the meiotic progeny of crosses between CC1009 and CC2343, showing a 2:2 segregation with a more severe growth phenotype in some of the progeny. However, the analysis was not taken any further to investigate the genetic basis and concluded that genetic factors aside from pyrenoid morphology may play a role in tolerance to hyperoxia.
It is not quite clear why the authors included a growth analyses under mixotrophic conditions and solid media and measured of photosystem II efficiency only under these conditions. The results showed faster growth of cells (CC2343) that tend to accumulate fractured pyrenoid starch sheath, however, growth is based on undefined proportions of autotrophy and heterotrophy under these conditions, and changes in metabolism are not well understood or predictable, which - from my point of view - is too confounding for gaining conclusive evidence for hyperoxia tolerance from biomass accumulation. Likewise, the measurements of PSII function which is a product of many factors concertedly impacting photosynthetic electron transport and correlates with growth only conditionally under autotrophy is even less informative under mixotrophy. Differences in respiration rates may have to be considered as well as differential partitioning of metabolites in the two different strains, which is outside the scope of this paper.
Physiological analyses (Rubisco function, photosynthetic O2 evolution and ROS and H2O2 levels), which might have also provided clues as to which processes diverge among progenies, were focussed on the parental strains. A potential correlation between pyrenoid morphology and photosynthetic performance was based to rubisco function (Rubisco content and activation state, rubisco activase levels) and O2 compensation points. It is to note that the O2 compensation point measurements, in contrast to the author's interpretation, are not a very specific measurement of CO2/O2 discrimination by rubisco.
The authors develop the hypothesis that a photosynthesis-derived signal common to low Ci and hyperoxia induces pyrenoid and starch sheath formation, since both conditions are associated with low CO2:O2 ratios in the chloroplast, potentially enhancing H2O2 by photorespiration and O2 acting as alternative electron acceptor in the light reaction. The study firmly establishes that the formation of starch sheath-coated pyrenoids was mediated through elevated H2O2 when applied exogenously or induced endogenously by addition of H2O2 generating chemicals (methylviologen etc.), which is consistent with this hypothesis. However, evidence for elevated H2O2 levels under hypoxia are based on indirect measurement of total ROS, using a general ROS indicator stain, rather than a more specific indicator for H2O2. It was not tested whether other ROS species could induce pyrenoids/starch.
For the reader it is not straight-forward to reconcile, and an explanation is not offered, an increase in H2O2 as the pyrenoid inducing signal in wild type CC2343 which showed no differences in ROS levels before and during hypoxia treatment (Figure12), but forms pyrenoids and starch sheaths similar to CC1009 as opposed to hyperoxia when CC2343 had more fractured starch sheaths.As to the discussion, the implications for H2O2 signalling and the yet to be investigated interaction with other signals are discussed highlighting the differences and commonalities of CCM induction by low CI compared to high Ci and hyperoxia. The eco-physiological implications are presented less comprehensively and focus on functional implications and less on the correlation of species distribution, with presence of pyrenoids and adaptation to a habitat.
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