the Creative Commons Attribution 4.0 License.
the Creative Commons Attribution 4.0 License.
| 05 May 2025
Reviews and syntheses: Photosynthetic oxygen evolution in plants-A potential inheritance from early abiotic oxygen production on Earth
Abstract. The phenomenon of photosynthetic oxygen evolution by plants, as the basis of life on our planet, has long attracted scientists from various disciplines. This process converts natural energy and inorganic carbon into organic matter and oxygen, which are not only crucial for maintaining terrestrial ecosystems but also reveal the early evolution of the Earth's biosphere. In this review, we present evidence from various disciplines, such as paleontology, biochemistry, stratigraphy, geochemistry, and molecular evolutionary biology, to support the proposition that abiotic processes generated the earliest detected oxygen on Earth. The bicarbonate photolytic oxygen release mechanism in photosynthetic organisms is, in our opinion, an inheritance of the abiotic oxygen release mechanism. In contrast, the water photolytic oxygen release mechanism evolved in response to insufficient availability of inorganic carbon. This review provides insights into the evolution of oxygen production mechanisms and their implications for the design of artificial photosynthetic reactors.
- Preprint
(1624 KB) - Metadata XML
- BibTeX
- EndNote
Status: closed
-
EC1: 'Comment on egusphere-2025-1764', Bertrand Guenet, 13 May 2025
Dear Authors,
we have received the first review and the comments are attached in the document.
The reviewer added some major comments copied below.
Please consider all of them carefully
Best
Bertrand Guenet
1) The idea and the view that bicarbonate is also a “donor” of oxygen is that only of the current authors: Wu et al- not yet accepted by others in the field. It is highly controversial. Editors may investigate it and have an “Editorial Note”- and have someone like Johannes Messinger ( of Sweden) look into this issue - and write an accompanying brief independent “Letter to the Editor"2) The text is quite terse and would gain by having some more diagrams - that are integrated and fully and clearly discussed.3) Regarding carbonic anhydrase- a key recent paper by Alex Shitov must be read by the authors and cited appropriately ( it is available by clicking on the “blue” text ).Shitov AV, Terentyev VV and Govindjee G (2025) High and unique carbonic anhydrase activity of Photosystem II from Pisum sativum: Measurements by a new and very sensitive fluorescence method. Plant Physiology and Biochemistry 221: #109516 (16 pages) DOI 10.1016/j.plaphy.2025.109516.
-
AC3: 'Reply on EC1', Yanyou Wu, 18 Jun 2025
- The idea and the view that bicarbonate is also a “donor” of oxygen is that only of the current authors: Wu et al- not yet accepted by others in the field. It is highly controversial. Editors may investigate it and have an “Editorial Note”- and have someone like Johannes Messinger ( of Sweden) look into this issue - and write an accompanying brief independent “Letter to the Editor"
We appreciate the reviewer’s concern and fully acknowledge that the hypothesis regarding bicarbonate acting as a direct oxygen donor in photosynthetic oxygen evolution is not yet part of the mainstream consensus. Our intent in this review is not to present this model as an established fact, but to synthesize emerging evidence that supports this alternative perspective and to encourage further dialogue and investigation in the scientific community.
To ensure clarity, we have revised the relevant sections of the manuscript to emphasize that the bicarbonate oxygen evolution model is a hypothesis based on recent studies (Wu, 2021; Guo et al., 2024) and not yet widely adopted. We have also included more cautious language such as “we propose,” “this suggests,” and “this hypothesis remains to be tested.” Additionally, we now explicitly state that this model requires further experimental validation and does not yet reflect a field-wide consensus.
We would also welcome the editor’s decision to invite an independent perspective or commentary, as suggested, which we believe would foster constructive scientific debate around the origin and mechanisms of oxygen evolution in photosynthesis.
- The text is quite terse and would gain by having some more diagrams - that are integrated and fully and clearly discussed.
We thank the reviewer for this valuable suggestion. In response, we have added several new integrated and thematically coherent figures (Figures 2, 5, 6, 7, 8, and 9) to clarify and visually support key concepts discussed in the text, especially the proposed evolutionary link between abiotic oxygen evolution and the development of photosynthetic systems.
Each new figure is now directly referenced and explained within the corresponding sections of the manuscript to ensure that they are fully integrated into the narrative. For instance:
Figure 2 illustrates the conceptual transition from abiotic to biotic oxygen evolution.
Figures 5 and 6 provide schematic models of abiotic oxygen generation via mineral photochemistry.
Figures 8 and 9 visualize the dual-substrate (HCO₃⁻ and H₂O) nature of oxygen evolution and its link to global biogeochemical cycles.
We believe these additions enhance the clarity and accessibility of the manuscript, especially for readers from diverse disciplinary backgrounds.
3) Regarding carbonic anhydrase- a key recent paper by Alex Shitov must be read by the authors and cited appropriately ( it is available by clicking on the “blue” text ).
Shitov AV, Terentyev VV and Govindjee G (2025) High and unique carbonic anhydrase activity of Photosystem II from Pisum sativum: Measurements by a new and very sensitive fluorescence method. Plant Physiology and Biochemistry 221: #109516 (16 pages) DOI 10.1016/j.plaphy.2025.109516.
We thank the reviewer for highlighting this important and timely publication. We have carefully reviewed the work by Shitov, Terentyev, and Govindjee (2025), and we have appropriately cited it in our revised manuscript. The findings in this study further support our discussion on the intrinsic carbonic anhydrase (CA) activity within Photosystem II and provide crucial experimental evidence for the physiological role of CA in oxygen evolution.
The reference has been integrated in the discussion of Section 3: Potential ancestor of Photosystem II, and is also included in the updated reference list.
-
AC3: 'Reply on EC1', Yanyou Wu, 18 Jun 2025
-
CC1: 'Comment on egusphere-2025-1764', Arafat Abdel Hamed Abdel Latef, 04 Jun 2025
This review article offers a compelling interdisciplinary synthesis of evidence supporting an abiotic origin for early Earth’s oxygen, which has implications for the evolution of photosynthesis and artificial photosynthetic systems. The hypothesis that bicarbonate photolysis preceded water splitting is intriguing and merits further discussion. While the manuscript is well-structured and timely, some sections need refinement and correction of typographical errors to strengthen the argument and enhance readability.
Citation: https://doi.org/10.5194/egusphere-2025-1764-CC1 -
AC2: 'Reply on CC1', Yanyou Wu, 18 Jun 2025
We sincerely thank the Editor and community for their positive feedback and insightful suggestions. We are pleased that the significance of our hypothesis was acknowledged. We fully agree that certain sections require further refinement and typographical errors need correction. In response, we have carefully revised the manuscript to strengthen our arguments and enhance overall clarity and readability. Specifically, we have made the following improvements:
- Refined Key Sections: We polished the narrative in crucial sections of the paper (particularly the introduction and discussion) to improve logical flow and clarity. This involved reorganizing some paragraphs and tightening the language so that our hypothesis and supporting evidence are presented more coherently.
- Corrected Typos and Grammar: We have corrected all identified typographical and grammatical errors. We also thoroughly proofread the entire manuscript to eliminate any lingering errors, thereby improving the professionalism and readability of the text.
- Improved Figure Integration: We have more clearly integrated the figures into the text. In the revised manuscript, each figure is explicitly referenced and discussed at the appropriate point, and figure captions have been enhanced for better clarity. These changes help to directly tie the visual evidence to our arguments and make it easier for readers to follow the reasoning.
- Enhanced Clarity of Arguments: Beyond correcting errors, we revisited certain explanations (for example, the section describing early abiotic oxygen production) to clarify our reasoning. We added transition sentences to bridge ideas between sections and ensure the hypothesis is consistently and clearly supported by the multidisciplinary evidence presented.
We believe these revisions address the concerns raised. The argument is now stronger and the manuscript more readable. Thank you once again for your valuable feedback and the opportunity to improve our work. We appreciate the collegial and constructive comments, which have undoubtedly helped us to refine the paper.
-
AC2: 'Reply on CC1', Yanyou Wu, 18 Jun 2025
-
CC2: 'Comment on egusphere-2025-1764', Deke Xing, 10 Jun 2025
Traditionally, it was believed that oxygen was produced through the photolysis of water. The proposal of the bicarbonate photosynthetic oxygen evolution theory has deepened our understanding of the role of bicarbonate in the formation and evolution of the Earth. The oxygen released by this process participated in altering the composition of the primitive atmosphere, gradually making it more suitable for the survival of Earth's organisms. Additionally, bicarbonate catalyzes the reversible hydration reaction of carbon dioxide under the action of carbonic anhydrase, providing a rich source of inorganic carbon for plant photosynthesis. This plays a crucial role in enhancing plant photosynthetic efficiency and promoting plant evolution and adaptive survival.
Citation: https://doi.org/10.5194/egusphere-2025-1764-CC2 -
AC1: 'Reply on CC2', Yanyou Wu, 18 Jun 2025
We greatly appreciate this thoughtful and encouraging comment. As highlighted, our central aim in this review is to revisit the conventional understanding of photosynthetic oxygen evolution by proposing that bicarbonate photolysis may have preceded water photolysis in the evolutionary history of photosynthesis.
We fully agree with the observation that oxygen released via bicarbonate photolysis could have contributed to the gradual transformation of the early Earth’s atmosphere, thus facilitating the emergence and evolution of oxygen-dependent life. This idea is consistent with the geochemical evidence we presented (e.g., from deep-sea nodules, mineral coatings, and banded iron formations) and is further supported by the catalytic role of carbonic anhydrase in early enzyme evolution (as detailed in Section 3).
Furthermore, the reviewer is absolutely right in pointing out the dual function of bicarbonate — not only as a potential oxygen donor but also as a key inorganic carbon source regulated by carbonic anhydrase activity. In our manuscript, we have elaborated on how this function improves carbon availability and efficiency of photosynthesis, especially under the changing redox and carbon-limited conditions of the Archean and Proterozoic eons (see Sections 3 and 4, and Figures 6–9).
We thank the reviewer again for recognizing the broader implications of this hypothesis — both for understanding Earth's atmospheric evolution and for enhancing our perspective on plant adaptation and photosynthetic efficiency.
Citation: https://doi.org/10.5194/egusphere-2025-1764-AC1
-
AC1: 'Reply on CC2', Yanyou Wu, 18 Jun 2025
-
AC4: 'Comment on egusphere-2025-1764', Yanyou Wu, 18 Jun 2025
Dear Scientists,
In our recent conference report, we presented evidence regarding the dual-substrate mechanism of photosynthetic oxygen evolution related to the paper titled "Reviews and syntheses: Photosynthetic oxygen evolution in plants—A potential inheritance from early abiotic oxygen production on Earth." Recognizing the complexity and significance of this topic, we sincerely invite you to share your professional insights and evaluations.
Your feedback will play a crucial role in further validating our findings, uncovering potential research gaps, and driving scientific progress in this field. Whether it’s theoretical analysis, experimental design suggestions, or critical reviews of our data interpretation, all perspectives are highly valued.
Please submit your comments. If you have any questions, feel free to contact me. Thank you for your time and contribution to advancing our understanding of photosynthetic oxygen evolution.
Best regards,
Yanyou Wu
Photosynthetic bicarbonate photolysis masked by the rapid oxygen isotopic exchange between water and bicarbonate
https://www.researchgate.net/publication/384113308_Photosynthetic_bicarbonate_photolysis_masked_by_the_rapid_oxygen_isotopic_exchange_between_water_and_bicarbonate
Citation: https://doi.org/10.5194/egusphere-2025-1764-AC4 -
CC3: 'Comment on egusphere-2025-1764', Mahmoud A. Abdelhafiz, 23 Jun 2025
-
CC8: 'Reply on CC3', Mohamed Aboueldahab, 18 Aug 2025
Thank you for your insightful feedback. We hope that our work contributes significantly to advancing the understanding of photosynthetic oxygen evolution in plants, particularly in the context of its potential inheritance from early abiotic oxygen production mechanisms on Earth.
Citation: https://doi.org/10.5194/egusphere-2025-1764-CC8
-
CC8: 'Reply on CC3', Mohamed Aboueldahab, 18 Aug 2025
-
RC1: 'Comment on egusphere-2025-1764', Anonymous Referee #1, 18 Jul 2025
This manuscript makes several claims about bicarbonate oxidation resulting in molecular oxygen. The work is not focused and poorly organized.
In a review of recent publications in this area, I am surprised that the authors fail to cite the 2024 paper by Vinyard and Govindjee (Photosynthesis Research, “Bicarbonate is a key regulator but not a substrate for O₂ evolution in Photosystem II”), which directly addresses the core question posed here. That perspective clearly showed that while PSII can oxidize bicarbonate under certain conditions, bicarbonate is not used as a substrate at the donor-side active site. This is not a peripheral detail—it refutes nearly all of the central claims made in this manuscript.
The authors propose speculative ideas without providing experimental data or a clear understanding of current literature. While speculative models can be valuable, they require a foundation in existing evidence. This manuscript reads more like a “what if” exercise than a scientific review. Key findings from well-established studies are either misrepresented or ignored, and the overall framing appears biased against peer-reviewed work that has been replicated and widely cited by others in the field.
If the authors believe the current model is incomplete, they need to provide a strong rationale and support it with data. At minimum, they should acknowledge existing credible literature and explain how their proposal fits within—or diverges from—that framework. Until then, I do not support publication.
Citation: https://doi.org/10.5194/egusphere-2025-1764-RC1 -
AC5: 'Reply on RC1', Yanyou Wu, 26 Jul 2025
This reply provides an excellent opportunity for us to showcase our work. Many people have not read our previous articles and might assume that water is the sole substrate for photosynthetic oxygen release. In this reply, we aim to highlight that oxygen evolution during photosynthesis is not exclusively attributable to water photolysis but also involves bicarbonate photolysis. Both water and bicarbonate photolysis contribute comparably to the overall oxygen-evolving process. Notably, we propose that the photolysis of bicarbonate constitutes the initial step in photosynthetic carbon assimilation. We will divide the reviewer’s critique into individual parts and respond to each one separately.
“This manuscript makes several claims about bicarbonate oxidation resulting in molecular oxygen. The work is not focused and is poorly organized ”.
RPLY:
Our article focuses on the origin of early inorganic oxygen release events on Earth. It synthesizes evidence from paleontology, biochemistry, stratigraphy, geochemistry, and molecular evolutionary biology to demonstrate that abiotic processes were the first mechanism detected on Earth for the generation of oxygen.
This paper follows a logically coherent structure consisting of three key stages: hypothesis formulation → layered argumentation → synthesis and conceptual advancement.
- The overall framework is as follows
- Abstract & Background (including Background and Challenge)
The paper begins by presenting the central hypothesis that the mechanism of oxygen evolution in plant photosynthesis may be evolutionary derived from early abiotic oxygen-producing processes on primordial Earth. Specifically, it posits that bicarbonate photolysis represents a conserved abiotic pathway for oxygen generation, whereas water photolysis evolved later as an adaptive response to the depletion of accessible carbon sources. This section also reviews the history of atmospheric oxygen accumulation, highlighting the two major oxidation events, and critically examines the limitations of conventional interpretations. In doing so, it establishes the scientific rationale and necessity for the present study.
- Early Abiotic Oxygen Production
Geochemical evidence, such as banded iron formations dated prior to 3.77 billion years ago and atmospheric oxygen signatures predating 3.0 billion years ago, alongside modern observations, including oxygen generation from deep-sea polymetallic nodules and mineral photocatalysis, collectively support the existence of abiotic oxygen production on early Earth. Notably, these mechanisms exhibit similarities to the oxygen-evolving processes observed in modern photosynthesis.
- The potential ancestor of Photosystem II (PSII):
Focusing on the evolutionary origin of photosystem II (PSII), this study hypothesizes that manganese-containing γ-class carbonic anhydrase (γ-CA[Mn]) may represent an ancestral precursor to PSII. By examining shared functional traits such as carbonic anhydrase activity and manganese cluster coordination, as well as comparable physicochemical responses to pH and redox potential, the study provides evidence supporting a transitional pathway from abiotic to biologically mediated oxygen evolution.
- Two-Substrate Photosynthetic Oxygen Evolution:
Through a combination of thermodynamic modeling, isotope labeling experiments, and analysis of the Kok–Joliot cycle, this study demonstrates that photosynthetic oxygen evolution arises from the synergistic contributions of bicarbonate, implicated in the S₄→S₀ transition, and water, which is oxidized during the S₂→S₃ transition. The findings elucidate the evolutionary impetus for the development of water-splitting mechanisms, driven by the depletion of readily available carbon sources, and establish a mechanistic link between oxygenic photosynthesis and Earth's broader carbon cycle.
- Conclusions:
The research summarizes the evolutionary continuity between abiotic oxygen production and photosynthetic oxygen release, emphasizing how core hypotheses contribute to understanding life's origin and biochemical process evolution.
- Research Emphasis
This study centers on a core hypothesis: The bicarbonate photolysis mechanism for plant photosynthetic oxygen release originated from early Earth's abiotic oxygen production processes, while water photolysis emerged as an evolutionary adaptation to environmental conditions.
It specifically addresses three key questions
- Evidence and mechanisms of the early Earth's abiotic oxygen generation (e.g., mineral photocatalysis, electrochemical processes in deep-sea polymetallic nodules).
- Evolutionary precursors of the core photosynthetic oxygen-release component Photosystem II (PSII), particularly the role of carbonic anhydrase.
- The synergistic mechanism and evolutionary logic of bicarbonate-water dual substrates in photosynthetic oxygen release, driven by shifts in carbon sources.
- Research logic
This study follows a progressive logic of "geological evidence → biological mechanisms → evolutionary logic" to systematically validate core hypotheses:
- Challenging conventional views by demonstrating through geological records (e.g., oxygen influence on sedimentation before 3.77 Ga and atmospheric oxygen signals before 3.0 Ga) that early Earth's oxygen existed prior to photosynthetic cyanobacteria, suggesting the possibility of abiotic oxygen production.
- Establishing abiotic-biological connections by comparing mechanism similarities between abiotic oxygen generation (e.g., photocatalytic decomposition of bicarbonate via iron-manganese oxides) and photosynthetic oxygen release (bicarbonate photolysis), proposing the "inherited" hypothesis.
- Tracing the origin of biological components through high functional similarity (catalyzing bicarbonate reactions), structural similarity (manganese clusters), and physicochemical properties (light and pH response) between carbonic anhydrase and Photosystem II, demonstrating that Photosystem II may have originated from manganese-containing carbonic anhydrase, completing the transition from abiotic to biological processes.
- Explaining the dual-substrate mechanism by combining thermodynamic efficiency (lower energy consumption in bicarbonate photolysis) and environmental changes (reduced carbon sources), elucidating why photosynthetic oxygen release retains bicarbonate photolysis while evolving water photolysis, ultimately forming a dual-substrate synergistic model.
- Regarding " not focused and poorly organized“
As demonstrated above, our paper is focused and well-organized, as reflected in the following points.
- Thematic Focus: The paper consistently centers on the core hypothesis that "the bicarbonate mechanism of photosynthetic oxygen release is inherited from abiotic oxygen production."All arguments, from geological evidence and the evolution of biological components to detailed biochemical processes, are developed in support of this central premise. For example, the concept of early abiotic oxygen production establishes the foundation for the idea of inheritance. At the same time, the discussion of ancestral Photosystem II illustrates the transition from abiotic to biological mechanisms. The proposed dual-substrate mechanism then explains the cooperative interaction between bicarbonate and water in oxygen evolution, forming a logically consistent and integrated framework.
- Rigorous Structure: The manuscript follows a classic academic structure: posing key questions → building layered arguments → synthesizing insights. The background section identifies the limitations of traditional models, and the subsequent sections develop progressively: from abiotic foundations, to the emergence of biological components, and finally to specific mechanistic pathways. Each section is supported by clear visual aids, such as Fig. 2 (depicting the abiotic-to-biological transition) and Figure 8 (illustrating the dual-substrate cycle), which reinforce continuity and conceptual clarity.
- Progressive Argumentation: Despite covering a wide range of topics, from macro-scale geological evidence (e.g., early atmospheric oxygen) to micro-scale biochemical mechanisms (e.g., the manganese cluster of Photosystem II), and from abiotic photocatalytic processes to modern biological oxygen evolution, the argument remains tightly focused on the central theme of inheritance. Each section is logically linked; for instance, the decomposition of bicarbonate in abiotic oxygen production directly parallels the photolysis of bicarbonate in biological photosynthesis, emphasizing the continuity of the proposed mechanism. This interconnected design demonstrates both conceptual rigor and internal consistency.
Conclusion:
Overall, the manuscript maintains a strong thematic focus, a well-organized structure, and a clear, logically developed line of argumentation throughout.“ In a review of recent publications in this area, I am surprised that the authors fail to cite the 2024 paper by Vinyard and Govindjee (Photosynthesis Research, “Bicarbonate is a key regulator but not a substrate for O₂ evolution in Photosystem II”), which directly addresses the core question posed here. That perspective clearly showed that while PSII can oxidize bicarbonate under certain conditions, bicarbonate is not used as a substrate at the donor-side active site. This is not a peripheral detail; it refutes nearly all of the central claims made in this manuscript ”.
RPLY:
Regarding your comment about the omission of the 2024 paper by Vinyard and Govindjee and their claim that the paper "refutes the core argument of our review," we would like to offer the following clarifications.
Explanation Regarding the Citation of the Paper
The paper entitled “Bicarbonate is a key regulator but not a substrate for O2 evolution in Photosystem II” by Vinyard and Govindjee (2024) was published online on July 22, 2024 (received on June 20, 2024, and accepted on July 11, 2024). The reason it was not cited in our review was not due to intentional omission but rather because our review involves an interdisciplinary approach, and given the vast amount of literature, it was difficult to cover every relevant study. We sincerely apologize for the oversight. We have now added the citation of their paper in the revised manuscript and specifically addressed their core claims.
Rebuttal to "Bicarbonate is not a substrate at the donor-side active site of PSII"
The core conclusion of Vinyard and Govindjee (2024) is that “bicarbonate is a key regulator but not a substrate for oxygen evolution in PSII,” based on the following reasoning:
- Structural and spectroscopic experiments failed to detect bicarbonate at the oxygen-evolving center (OEC) of PSII.
- Thermodynamic analyses support that bicarbonate can be oxidized, but it is argued that it does not participate in the donor-side oxygen-evolving reaction.
However, based on the research presented in this review and supporting evidence, we believe this conclusion has significant limitations and does not refute the central thesis of our review, that bicarbonate and water both serve as substrates for oxygen evolution in PSII, with each contributing 50% to photosynthetic oxygen release. Our reasoning is as follows.
A. Non-physiological experimental conditions limiting the generalizability of Vinyard and Govindjee’s conclusion
The experimental evidence cited by Vinyard and Govindjee (2024) is primarily based on in vitro reconstitution systems (e.g., PSII membrane fragments) or non-physiological conditions (e.g., low pH, removal of carbonic anhydrase), which significantly differ from the physiological environment of photosynthetic organisms (e.g., stroma pH of 8.0-8.5, thylakoid lumen pH of 5.5-6.5).
- pH-dependent binding:Ferreira et al. (2004) showed that bicarbonate binds to the OEC of PSII at pH 7.5, but no binding is observed at pH <7.0 (Refereed:The photosynthetic oxygen evolution does not exclude the important role and contribution of bicarbonate photolysis). Most of the structural experiments referenced in Vinyard and Govindjee’s paper were conducted below pH 6.0, likely missing the bicarbonate binding signal under physiological conditions.
- Inevitability of carbonic anhydrase activity:The works cited by Vinyard and Govindjee (2024) hypothesize that “there is no carbonic anhydrase activity in the experimental system,” but PSII itself possesses carbonic anhydrase activity (Refereed:Combined effect of bicarbonate and water in photosynthetic oxygen evolution and carbon neutrality; Rapid oxygen isotopic exchange between bicarbonate and water during photosynthesis; The photosynthetic oxygen evolution does not exclude the important role and contribution of bicarbonate photolysis; Photosynthetic bicarbonate photolysis masked by the rapid oxygen isotopic exchange between water and bicarbonate (Presentation of GPMB 2024)), and the carbonic anhydrase on the thylakoid membrane (tCA) cannot be completely removed (Refereed:Combined effect of bicarbonate and water in photosynthetic oxygen evolution and carbon neutrality), leading to rapid oxygen isotope exchange between bicarbonate and water (Refereed:Rapid oxygen isotopic exchange between bicarbonate and water during photosynthesis; Photosynthetic bicarbonate photolysis masked by the rapid oxygen isotopic exchange between water and bicarbonate (Presentation of GPMB 2024)), which may obscure direct evidence for bicarbonate as a substrate.
B. Dual-Isotope Experiment Directly Demonstrates Bicarbonate’s Role in Oxygen Evolution
One of the core pieces of evidence in this review comes from dual-element isotope tracing experiments (Refereed:Rapid oxygen isotopic exchange between bicarbonate and water during photosynthesis; Photosynthetic bicarbonate photolysis masked by the rapid oxygen isotopic exchange between water and bicarbonate (Presentation of GPMB 2024)):
- In microalgae (Microcystis aeruginosaand Chlamydomonas reinhardtii), when exogenous bicarbonate is labeled, carbon isotope signals significantly accumulate in biomass (16%-34% contribution), but oxygen isotope signals remain indistinguishable from unlabeled controls. This suggests that the carbon from bicarbonate is directly used in photosynthesis, while its oxygen exchanges rapidly with water (catalyzed by PSII and carbonic anhydrase) and is not retained. Thus, the oxygen released, although reflecting the water isotope signature, also contains a contribution from bicarbonate (Refereed:Rapid oxygen isotopic exchange between bicarbonate and water during photosynthesis).
- In calcium and magnesium-deficient media, the carbon/oxygen utilization ratio in microalgae approaches 1:1 (Refereed:Photosynthetic bicarbonate photolysis masked by the rapid oxygen isotopic exchange between water and bicarbonate (Presentation of GPMB 2024)), demonstrating that under physiological conditions with no isotope exchange, the oxygen from bicarbonate directly participates in oxygen evolution, with contributions from both bicarbonate and water being equal.
C. Thermodynamic and Geochemical Evidence Supports Bicarbonate’s Substrate Role
- Thermodynamic Advantage:The standard free energy of bicarbonate photolysis (24.8 kcal/mol) is significantly lower than that of water photolysis (37.3 kcal/mol) (Refereed:Combined effect of bicarbonate and water in photosynthetic oxygen evolution and carbon neutrality; The photosynthetic oxygen evolution does not exclude the important role and contribution of bicarbonate photolysis; Photosynthetic bicarbonate photolysis masked by the rapid oxygen isotopic exchange between water and bicarbonate (Presentation of GPMB 2024)), making it more readily oxidizable by the OEC of PSII, which aligns with evolutionary logic favoring low-energy pathways.
- Dole Effect Explanation:The oxygen isotope enrichment of atmospheric oxygen (24‰ higher than seawater) cannot be explained solely by water photolysis but fits perfectly with a model in which bicarbonate and water photolysis each contribute 50%. The ¹⁸O enrichment of bicarbonate (47.62‰) and the 0‰ enrichment of water match the observed values (Refereed:Combined effect of bicarbonate and water in photosynthetic oxygen evolution and carbon neutrality; The photosynthetic oxygen evolution does not exclude the important role and contribution of bicarbonate photolysis; Photosynthetic bicarbonate photolysis masked by the rapid oxygen isotopic exchange between water and bicarbonate (Presentation of GPMB 2024)).
D. Modified Kok Cycle Supports Dynamic Involvement of Bicarbonate
Vinyard and Govindjee (2024) argue that the S₃→S₄→S₀ stages of the OEC exclusively bind water. Still, we propose that bicarbonate may participate in the S₄→S₀ transition due to its rapid reaction rate (thermodynamic advantage), which current instrumentation is unable to capture in the S₄ state (Refereed:Rapid oxygen isotopic exchange between bicarbonate and water during photosynthesis; The photosynthetic oxygen evolution does not exclude the important role and contribution of bicarbonate photolysis; Photosynthetic bicarbonate photolysis masked by the rapid oxygen isotopic exchange between water and bicarbonate (Presentation of GPMB 2024)). This explains why structural experiments failed to detect their binding, not because it does not participate, but because it binds dynamically with a very short lifetime.
The revised Kok cycle model (1 molecule of bicarbonate + 1 molecule of water) can simultaneously explain the “bicarbonate effect” (multiple-fold enhancement in oxygen release rates), isotope exchange phenomena, and the Dole effect, whereas the sole water photolysis model cannot accommodate these observations (Refereed:Rapid oxygen isotopic exchange between bicarbonate and water during photosynthesis; Photosynthetic bicarbonate photolysis masked by the rapid oxygen isotopic exchange between water and bicarbonate (Presentation of GPMB 2024)).
- Conclusion
While the experiments cited by Vinyard and Govindjee (2024) provide important evidence for the regulatory role of bicarbonate, their non-physiological experimental conditions prevent them from identifying bicarbonate's core role as a substrate. The central thesis of this review, that bicarbonate and water both serve as substrates for oxygen evolution in PSII, has been supported by isotope tracing, thermodynamic analyses, geochemical phenomena (the Dole effect), and the modified Kok cycle model under physiological conditions. This conclusion is not contradictory to the findings of Vinyard and Govindjee (2024) but rather provides a more complete explanation of the photosynthetic oxygen evolution mechanism.
We have now added a citation for Vinyard and Govindjee (2024) in the revised manuscript and clearly discussed the impact of experimental condition differences on their conclusions to present a more comprehensive view of the current debates and consensus in the field.
“The authors propose speculative ideas without providing experimental data or a clear understanding of current literature. While speculative models can be valuable, they require a foundation in existing evidence. This manuscript reads more like a “what if” exercise than a scientific review. Key findings from well-established studies are either misrepresented or ignored, and the overall framing appears biased against peer-reviewed work that has been replicated and widely cited by others in the field ”.
RPLY:
In response to your concerns regarding “speculative hypotheses lacking experimental data, misinterpretation or distortion of existing literature, and potential bias,” We provide the following detailed reply, supported by the cited documentation:
1. This review is not based on speculation, but on robust experimental evidence and systematic analysis
The central conclusion of our review is that bicarbonate and water both serve as substrates for oxygen evolution in PSII, each contributing approximately 50%. This is not a "what-if" speculation. It is derived from multidimensional experimental evidence and logical validation as a fellow.
A. Direct Isotopic Evidence for Bicarbonate as a Substrate
Paper entitled “Rapid oxygen isotopic exchange between bicarbonate and water during photosynthesis”, presents a dual-element (C–O) isotope labeling study in Microcystis aeruginosa and Chlamydomonas reinhardtii. The carbon from labeled bicarbonate was directly incorporated into biomass, with 16–34% incorporation in experimental groups, confirming the use of bicarbonate carbon in photosynthesis. However, oxygen isotope signals in biomass remained indistinguishable from the control, indicating rapid oxygen exchange between bicarbonate and water, catalyzed by PSII’s intrinsic carbonic anhydrase activity. This demonstrates that while the released oxygen displays water's isotopic signature, it also reflects a biochemical contribution from bicarbonate. These results directly support the hypothesis that bicarbonate functions as an oxygen-evolving substrate, not merely a regulator.
B. Physiological Isotope Exchange and Quantitative Validation of the Dole Effect
Paper entitled “Combined effect of bicarbonate and water in photosynthetic oxygen evolution and carbon neutrality”, and paper entitled “The photosynthetic oxygen evolution does not exclude the important role and contribution of bicarbonate photolysis”, reanalyzed classic ¹⁸O-labeling experiments (e.g., Ruben et al. 1941; Stemler et al. 1975), revealing that their conclusions depend on the assumption of "slow oxygen isotope exchange." However, PSII possesses carbonic anhydrase activity(Refereed:Combined effect of bicarbonate and water in photosynthetic oxygen evolution and carbon neutrality; The photosynthetic oxygen evolution does not exclude the important role and contribution of bicarbonate photolysis), which accelerates this exchange, potentially obscuring the true oxygen source in prior studies. Furthermore, by modeling a 50:50 contribution of bicarbonate and water photolysis, the calculated atmospheric oxygen ¹⁸O enrichment (23.06‰) aligns closely with empirical observations (23.56‰), thus providing geochemical validation for our mechanism ·(Refereed:Combined effect of bicarbonate and water in photosynthetic oxygen evolution and carbon neutrality; Rapid oxygen isotopic exchange between bicarbonate and water during photosynthesis).
C. Thermodynamic Justification and Kok Cycle Revision
Using thermodynamic data (standard free energy of bicarbonate photolysis = 24.8 kcal/mol vs. water photolysis = 37.3 kcal/mol) (Refereed:Combined effect of bicarbonate and water in photosynthetic oxygen evolution and carbon neutrality; The photosynthetic oxygen evolution does not exclude the important role and contribution of bicarbonate photolysis; Photosynthetic bicarbonate photolysis masked by the rapid oxygen isotopic exchange between water and bicarbonate (Presentation of GPMB 2024)), our presentation(Refereed:Photosynthetic bicarbonate photolysis masked by the rapid oxygen isotopic exchange between water and bicarbonate (Presentation of GPMB 2024)), proposes that bicarbonate participates in the S₄→S₀ transition of the Kok cycle. The reaction occurs too rapidly to be detected with current instrumentation.
The revised Kok cycle model (incorporating 1 bicarbonate + 1 water molecule) coherently explains:
- The "bicarbonate effect" (a multi-fold increase in O₂ release rate),
- The lack of detectable S₄-state intermediates,
- And mechanistic compatibility with thermodynamic favorability.
2. Our review accurately reflects and engages with the existing literature; it does not distort or ignore prior work
The experimental conditions referenced in Vinyard and Govindjee (2024), such as PSII crystals prepared at pH < 7.0 or removal of carbonic anhydrase, do not reflect physiological settings.
- Ferreira et al. (2004) showed that bicarbonate binds to the OEC of PSII at pH 7.5, but not below pH 7.0(Refereed:Combined effect of bicarbonate and water in photosynthetic oxygen evolution and carbon neutrality; The photosynthetic oxygen evolution does not exclude the important role and contribution of bicarbonate photolysis), explaining why their experiments failed to detect bicarbonate binding.
Moreover, Vinyard and Govindjee (2024) acknowledge that “bicarbonate can be oxidized by PSII”, which is not in contradiction with our view. The divergence lies in the interpretation of whether bicarbonate participates as a substrate under physiological conditions, not in the fundamental redox potential.
- Reinterpretation, Not Rejection, of Classic Studies
We do not reject the findings of Ruben, Stemler, or other classical studies. Rather, we point out that their conclusions may not account for PSII’s carbonic anhydrase activity.
- For instance, paper entitled “Combined effect of bicarbonate and water in photosynthetic oxygen evolution and carbon neutrality”,and paper entitled “The photosynthetic oxygen evolution does not exclude the important role and contribution of bicarbonate photolysis”, cite these foundational works while presenting supplementary experiments (e.g.,dual-isotope tracing under Ca/Mg-deficient media in our presentation (Refereed:Photosynthetic bicarbonate photolysis masked by the rapid oxygen isotopic exchange between water and bicarbonate (Presentation of GPMB 2024)) to demonstrate that, under non-exchange conditions, oxygen from bicarbonate can directly contribute to evolved O₂.
- Therefore, the “negative” results in earlier studies likely stem from experimental limitations rather than definitive evidence against bicarbonate involvement.
4. This review is unbiased and aims to synthesize, not refute, existing work
The debate over whether bicarbonate serves as a substrate for oxygen evolution in PSII has persisted for decades. Our review aims to reconcile this debate using new experimental insights and a synthesis of interdisciplinary data:
- We acknowledge bicarbonate's regulatory role, as discussed by Vinyard and Govindjee (2024), including its involvement in electron transport modulation and proton transfer.
- At the same time, we present evidence supporting its role as a substrate, based on dual-isotope tracing, thermodynamic favorability, and geochemical validation.
- For studies favoring exclusive water oxidation (e.g., Hillier et al., 2006), we show that the absence of labeled oxygen signals can be explained by rapid isotopic exchange (Refereed:Rapid oxygen isotopic exchange between bicarbonate and water during photosynthesis; Photosynthetic bicarbonate photolysis masked by the rapid oxygen isotopic exchange between water and bicarbonate (Presentation of GPMB 2024))., not by bicarbonate exclusion, an interpretation grounded in data, not bias.
“If the authors believe the current model is incomplete, they need to provide a strong rationale and support it with data. At a minimum, they should acknowledge existing credible literature and explain how their proposal fits within, or diverges from, that framework. Until then, I did not support publication ”.
RPLY:
In response to your request for strong justification, supporting data, acknowledgment of existing literature, and clarification of our conceptual framework, we provide the following evidence-based clarification.
- Strong justification and experimental evidence supporting the incompleteness of the current model
Our proposed “dual-substrate model,” in which both bicarbonate and water function as substrates for oxygen evolution in PSII, each contributing approximately 50%, is not intended to reject existing models, but rather to extend them based on new evidence.
The following key findings
A. Dual-element isotope tracing demonstrates bicarbonate's direct involvement in o₂ evolution
Paper entitled “Rapid oxygen isotopic exchange between bicarbonate and water during photosynthesis”, employed C–O dual-element bidirectional isotope labeling in Microcystis aeruginosa and Chlamydomonas reinhardtii and found that: The carbon from exogenously added bicarbonate was incorporated into algal biomass at a rate of 16%–34% (vs. control), providing direct evidence that bicarbonate-derived carbon is used in photosynthesis.
- However, oxygen isotope ratios in biomass showed no significant difference between labeled and unlabeled groups. This is attributable to rapid oxygen exchange between bicarbonate and water, catalyzed by PSII-associated carbonic anhydrase (Refereed:Rapid oxygen isotopic exchange between bicarbonate and water during photosynthesis; 4). As a result, the evolved oxygen carries the isotopic signature of water. However, it includes a contribution from bicarbonate.
- This experiment offers molecular-level evidence that bicarbonate is not merely a regulatory cofactor but a true substrate in oxygen evolution, thereby supporting the dual-substrate model.
B. Quantitative Validation of the Dole Effect and Thermodynamic Advantage
- Paper entitled “Combined effect of bicarbonate and water in photosynthetic oxygen evolution and carbon neutrality”,and paper entitled “The photosynthetic oxygen evolution does not exclude the important role and contribution of bicarbonate photolysis”, quantitatively demonstrate that:
- The observed ¹⁸O enrichment of atmospheric oxygen (~24‰ greater than seawater) cannot be explained by water photolysis alone. However, a 50:50 model of bicarbonate and water photolysis perfectly fits this enrichment pattern. Specifically, the combined contribution of bicarbonate (¹⁸O = 47.62‰) and water (0‰) yields a predicted enrichment of 23.81‰, which closely matches the observed value of 23.56‰ (Paper entitled “The photosynthetic oxygen evolution does not exclude the important role and contribution of bicarbonate photolysis, Table 1).
- The standard Gibbs free energy for bicarbonate photolysis (24.8 kcal/mol) is substantially lower than that for water photolysis (37.3 kcal/mol), making bicarbonate more thermodynamically favorable for oxidation by the PSII oxygen-evolving complex (OEC). This supports an evolutionary preference for low-energy oxidation pathways.
C. Revised Kok Cycle Model and Compatibility with Physiological Conditions
In our presentation (Refereed: Photosynthetic bicarbonate photolysis masked by the rapid oxygen isotopic exchange between water and bicarbonate (Presentation of GPMB 2024)), we address:
- The “energy barrier” associated with the S₃→S₄→S₀ transition in existing Kok models, and the failure to detect S₄ intermediates. We propose that bicarbonate is involved in the S₄→S₀ step, proceeding so rapidly (due to its thermodynamic favorability) that S₄ has an extremely short lifetime ( Refereed: Photosynthetic bicarbonate photolysis masked by the rapid oxygen isotopic exchange between water and bicarbonate (Presentation of GPMB 2024), Section 4.2).
- The revised model (1 molecule of bicarbonate + 1 molecule of water) explains both the "bicarbonate effect" (a several-fold increase in oxygen evolution rate) and the isotopic discrepancies seen in classical labeling studies. Importantly, it remains fully compatible with physiological parameters, such as the alkaline microenvironment of the thylakoid lumen.
- We acknowledge and systematically address the existing literature, clarifying the relationship between our framework and prior studies.
We have engaged with key publications in this field, including Vinyard and Govindjee (2024), and clarified the root of conceptual differences:
- Vinyard and Govindjee (2024) (paper entitled “Bicarbonate is a key regulator but not a substrate for O2 evolution in Photosystem II”) argues that bicarbonate is not an O₂-evolving substrate in PSII, primarily based on the absence of bicarbonate in structural studies at the OEC. However,paper entitled “The photosynthetic oxygen evolution does not exclude the important role and contribution of bicarbonate photolysis, highlights that.
- These PSII crystals were prepared under non-physiological conditions (pH < 7.0), whereas Ferreira et al. (2004) showed bicarbonate binding to the OEC at pH 7.5(Refereed:The photosynthetic oxygen evolution does not exclude the important role and contribution of bicarbonate photolysis).
- Thus, the failure to detect bicarbonate binding may be attributed to experimental conditions (e.g., acidic pH) that do not reflect physiological environments, rather than to the actual absence of bicarbonate involvement(Refereed:The photosynthetic oxygen evolution does not exclude the important role and contribution of bicarbonate photolysis).
- We fully acknowledge the regulatory roles of bicarbonate (e.g., in electron transport and proton transfer) but argue that its substrate function is masked under non-physiological conditions.
- Reinterpretation of Classical ¹⁸O-Labeling Experiments
- Paper entitled “Combined effect of bicarbonate and water in photosynthetic oxygen evolution and carbon neutrality”,and paper entitled “The photosynthetic oxygen evolution does not exclude the important role and contribution of bicarbonate photolysis”, directly cite and reanalyze the classic ¹⁸O experiments by Ruben et al. (1941) and Stemler et al. (1975).
- These early studies assumed “slow oxygen isotope exchange,” but paper entitled “Combined effect of bicarbonate and water in photosynthetic oxygen evolution and carbon neutrality”, shows that PSII's carbonic anhydrase activity accelerates isotope exchange, obscuring distinctions between oxygen sources.
- We do not reject the validity of their data but rather reinterpret their results using complementary experiments. For example, in calcium- and magnesium-deficient media, where isotope exchange is minimized, oxygen from bicarbonate directly contributes to the evolved O₂ (Refereed: Photosynthetic bicarbonate photolysis masked by the rapid oxygen isotopic exchange between water and bicarbonate (Presentation of GPMB 2024), Section 2.4). This suggests that prior “negative” results stemmed from experimental limitations, not from bicarbonate's lack of involvement.
4. Conclusion
The dual-substrate model proposed in this review is not speculative, nor is it incompatible with existing frameworks. It is built upon:
- Direct experimental evidence (dual-element isotope tracing),
- Quantitative geochemical validation (Dole effect),
- Mechanistic plausibility (thermodynamics and a revised Kok cycle),
- And thorough engagement with existing literature.
We clearly define the points of compatibility with the classical model (e.g., acknowledging bicarbonate’s regulatory role), and the areas of extension (e.g., incorporating bicarbonate as a true substrate). In the revised manuscript, we have strengthened the discussion of prior literature, explicitly cited all relevant studies, and clarified the experimental basis for our differing interpretations. We respectfully request that you reconsider your assessment in light of this additional evidence and analysis.
-
AC5: 'Reply on RC1', Yanyou Wu, 26 Jul 2025
-
CC4: 'Comment on egusphere-2025-1764', Fatemeh Rezaei Ashtiani, 07 Aug 2025
This article was so beneficial for future life on Earth. It can be one step for the removal of pollution on Earth with oxygen structure and Photocytogenic plants and Microbes.
Thank you for sharing this knowledge
Citation: https://doi.org/10.5194/egusphere-2025-1764-CC4 -
CC7: 'Reply on CC4', Mohamed Aboueldahab, 17 Aug 2025
Thank you very much for your valuable comments. The authors greatly appreciate your support
Citation: https://doi.org/10.5194/egusphere-2025-1764-CC7
-
CC7: 'Reply on CC4', Mohamed Aboueldahab, 17 Aug 2025
-
CC5: 'Comment on egusphere-2025-1764', Eslam Rashad, 08 Aug 2025
This interdisciplinary review explores the hypothesis that modern photosynthetic oxygen evolution may have inherited key elements from abiotic oxygen-producing mechanisms on early Earth. The authors use evidence from geochemistry, biochemistry, mineral photochemistry, and evolutionary biology to argue that bicarbonate photolysis may play a fundamental role alongside water photolysis in the photosynthetic oxygen-evolving complex (PSII). However, the paper needs enhancements, such as clearly defining "bicarbonate photolysis" for non-specialist readers, balancing the argument, addressing methodological critiques, improving visual clarity, and streamlined proofreading.
Citation: https://doi.org/10.5194/egusphere-2025-1764-CC5 -
CC6: 'Reply on CC5', Mohamed Aboueldahab, 17 Aug 2025
We appreciate the reviewer’s constructive feedback and suggestions. Below is our detailed response to each point raised:
-
Clarification of "Bicarbonate Photolysis":
We understand the need for clarity regarding the term "bicarbonate photolysis." To address this, we have added a concise definition in the revised manuscript. Bicarbonate photolysis refers to the light-driven process where bicarbonate (HCO₃⁻) undergoes decomposition, releasing oxygen (O₂) and carbon dioxide (CO₂). This reaction occurs under specific photochemical conditions and is hypothesized to play a role similar to water photolysis in the context of photosynthetic oxygen evolution. A more detailed explanation is now provided in the introduction and methodology sections to ensure accessibility for non-specialist readers. -
Balancing the Argument:
In response to the concern about balancing the argument, we have made revisions to provide a more nuanced discussion. While we continue to emphasize the role of bicarbonate photolysis in parallel with water photolysis, we have included additional references and evidence that present alternative perspectives and limitations of our hypothesis. This will ensure that both sides of the argument are well-represented and discussed in the context of existing literature. -
Addressing Methodological Critiques:
Regarding the methodological critique, we acknowledge the need for further elaboration on the experimental setup and data interpretation. We have expanded the methodology section to provide clearer descriptions of the techniques used in evaluating bicarbonate photolysis, particularly regarding isotope labeling experiments and thermodynamic efficiency analysis. We have also added a discussion on the potential limitations and uncertainties of the methods used. -
Improving Visual Clarity:
We have revisited all figures and diagrams to enhance their clarity and visual appeal. The legends have been revised for better readability, and we have ensured that all visuals are properly labeled with high-quality images. Specifically, Figure 1 and Figure 5 have been modified to better illustrate the dual-substrate nature of photosynthetic oxygen evolution, and we believe these revisions improve both the aesthetic and informative quality of the paper. -
Streamlined Proofreading:
The manuscript has undergone a thorough proofreading process. We have corrected minor typographical errors, improved sentence flow, and ensured consistency in terminology across sections. We believe this enhances the overall readability of the paper.
We hope that these revisions satisfactorily address the reviewer’s concerns. We have attached the updated manuscript for further consideration.
Thank you again for the valuable feedback.
Citation: https://doi.org/10.5194/egusphere-2025-1764-CC6 -
-
CC6: 'Reply on CC5', Mohamed Aboueldahab, 17 Aug 2025
Status: closed
-
EC1: 'Comment on egusphere-2025-1764', Bertrand Guenet, 13 May 2025
Dear Authors,
we have received the first review and the comments are attached in the document.
The reviewer added some major comments copied below.
Please consider all of them carefully
Best
Bertrand Guenet
1) The idea and the view that bicarbonate is also a “donor” of oxygen is that only of the current authors: Wu et al- not yet accepted by others in the field. It is highly controversial. Editors may investigate it and have an “Editorial Note”- and have someone like Johannes Messinger ( of Sweden) look into this issue - and write an accompanying brief independent “Letter to the Editor"2) The text is quite terse and would gain by having some more diagrams - that are integrated and fully and clearly discussed.3) Regarding carbonic anhydrase- a key recent paper by Alex Shitov must be read by the authors and cited appropriately ( it is available by clicking on the “blue” text ).Shitov AV, Terentyev VV and Govindjee G (2025) High and unique carbonic anhydrase activity of Photosystem II from Pisum sativum: Measurements by a new and very sensitive fluorescence method. Plant Physiology and Biochemistry 221: #109516 (16 pages) DOI 10.1016/j.plaphy.2025.109516.
-
AC3: 'Reply on EC1', Yanyou Wu, 18 Jun 2025
- The idea and the view that bicarbonate is also a “donor” of oxygen is that only of the current authors: Wu et al- not yet accepted by others in the field. It is highly controversial. Editors may investigate it and have an “Editorial Note”- and have someone like Johannes Messinger ( of Sweden) look into this issue - and write an accompanying brief independent “Letter to the Editor"
We appreciate the reviewer’s concern and fully acknowledge that the hypothesis regarding bicarbonate acting as a direct oxygen donor in photosynthetic oxygen evolution is not yet part of the mainstream consensus. Our intent in this review is not to present this model as an established fact, but to synthesize emerging evidence that supports this alternative perspective and to encourage further dialogue and investigation in the scientific community.
To ensure clarity, we have revised the relevant sections of the manuscript to emphasize that the bicarbonate oxygen evolution model is a hypothesis based on recent studies (Wu, 2021; Guo et al., 2024) and not yet widely adopted. We have also included more cautious language such as “we propose,” “this suggests,” and “this hypothesis remains to be tested.” Additionally, we now explicitly state that this model requires further experimental validation and does not yet reflect a field-wide consensus.
We would also welcome the editor’s decision to invite an independent perspective or commentary, as suggested, which we believe would foster constructive scientific debate around the origin and mechanisms of oxygen evolution in photosynthesis.
- The text is quite terse and would gain by having some more diagrams - that are integrated and fully and clearly discussed.
We thank the reviewer for this valuable suggestion. In response, we have added several new integrated and thematically coherent figures (Figures 2, 5, 6, 7, 8, and 9) to clarify and visually support key concepts discussed in the text, especially the proposed evolutionary link between abiotic oxygen evolution and the development of photosynthetic systems.
Each new figure is now directly referenced and explained within the corresponding sections of the manuscript to ensure that they are fully integrated into the narrative. For instance:
Figure 2 illustrates the conceptual transition from abiotic to biotic oxygen evolution.
Figures 5 and 6 provide schematic models of abiotic oxygen generation via mineral photochemistry.
Figures 8 and 9 visualize the dual-substrate (HCO₃⁻ and H₂O) nature of oxygen evolution and its link to global biogeochemical cycles.
We believe these additions enhance the clarity and accessibility of the manuscript, especially for readers from diverse disciplinary backgrounds.
3) Regarding carbonic anhydrase- a key recent paper by Alex Shitov must be read by the authors and cited appropriately ( it is available by clicking on the “blue” text ).
Shitov AV, Terentyev VV and Govindjee G (2025) High and unique carbonic anhydrase activity of Photosystem II from Pisum sativum: Measurements by a new and very sensitive fluorescence method. Plant Physiology and Biochemistry 221: #109516 (16 pages) DOI 10.1016/j.plaphy.2025.109516.
We thank the reviewer for highlighting this important and timely publication. We have carefully reviewed the work by Shitov, Terentyev, and Govindjee (2025), and we have appropriately cited it in our revised manuscript. The findings in this study further support our discussion on the intrinsic carbonic anhydrase (CA) activity within Photosystem II and provide crucial experimental evidence for the physiological role of CA in oxygen evolution.
The reference has been integrated in the discussion of Section 3: Potential ancestor of Photosystem II, and is also included in the updated reference list.
-
AC3: 'Reply on EC1', Yanyou Wu, 18 Jun 2025
-
CC1: 'Comment on egusphere-2025-1764', Arafat Abdel Hamed Abdel Latef, 04 Jun 2025
This review article offers a compelling interdisciplinary synthesis of evidence supporting an abiotic origin for early Earth’s oxygen, which has implications for the evolution of photosynthesis and artificial photosynthetic systems. The hypothesis that bicarbonate photolysis preceded water splitting is intriguing and merits further discussion. While the manuscript is well-structured and timely, some sections need refinement and correction of typographical errors to strengthen the argument and enhance readability.
Citation: https://doi.org/10.5194/egusphere-2025-1764-CC1 -
AC2: 'Reply on CC1', Yanyou Wu, 18 Jun 2025
We sincerely thank the Editor and community for their positive feedback and insightful suggestions. We are pleased that the significance of our hypothesis was acknowledged. We fully agree that certain sections require further refinement and typographical errors need correction. In response, we have carefully revised the manuscript to strengthen our arguments and enhance overall clarity and readability. Specifically, we have made the following improvements:
- Refined Key Sections: We polished the narrative in crucial sections of the paper (particularly the introduction and discussion) to improve logical flow and clarity. This involved reorganizing some paragraphs and tightening the language so that our hypothesis and supporting evidence are presented more coherently.
- Corrected Typos and Grammar: We have corrected all identified typographical and grammatical errors. We also thoroughly proofread the entire manuscript to eliminate any lingering errors, thereby improving the professionalism and readability of the text.
- Improved Figure Integration: We have more clearly integrated the figures into the text. In the revised manuscript, each figure is explicitly referenced and discussed at the appropriate point, and figure captions have been enhanced for better clarity. These changes help to directly tie the visual evidence to our arguments and make it easier for readers to follow the reasoning.
- Enhanced Clarity of Arguments: Beyond correcting errors, we revisited certain explanations (for example, the section describing early abiotic oxygen production) to clarify our reasoning. We added transition sentences to bridge ideas between sections and ensure the hypothesis is consistently and clearly supported by the multidisciplinary evidence presented.
We believe these revisions address the concerns raised. The argument is now stronger and the manuscript more readable. Thank you once again for your valuable feedback and the opportunity to improve our work. We appreciate the collegial and constructive comments, which have undoubtedly helped us to refine the paper.
-
AC2: 'Reply on CC1', Yanyou Wu, 18 Jun 2025
-
CC2: 'Comment on egusphere-2025-1764', Deke Xing, 10 Jun 2025
Traditionally, it was believed that oxygen was produced through the photolysis of water. The proposal of the bicarbonate photosynthetic oxygen evolution theory has deepened our understanding of the role of bicarbonate in the formation and evolution of the Earth. The oxygen released by this process participated in altering the composition of the primitive atmosphere, gradually making it more suitable for the survival of Earth's organisms. Additionally, bicarbonate catalyzes the reversible hydration reaction of carbon dioxide under the action of carbonic anhydrase, providing a rich source of inorganic carbon for plant photosynthesis. This plays a crucial role in enhancing plant photosynthetic efficiency and promoting plant evolution and adaptive survival.
Citation: https://doi.org/10.5194/egusphere-2025-1764-CC2 -
AC1: 'Reply on CC2', Yanyou Wu, 18 Jun 2025
We greatly appreciate this thoughtful and encouraging comment. As highlighted, our central aim in this review is to revisit the conventional understanding of photosynthetic oxygen evolution by proposing that bicarbonate photolysis may have preceded water photolysis in the evolutionary history of photosynthesis.
We fully agree with the observation that oxygen released via bicarbonate photolysis could have contributed to the gradual transformation of the early Earth’s atmosphere, thus facilitating the emergence and evolution of oxygen-dependent life. This idea is consistent with the geochemical evidence we presented (e.g., from deep-sea nodules, mineral coatings, and banded iron formations) and is further supported by the catalytic role of carbonic anhydrase in early enzyme evolution (as detailed in Section 3).
Furthermore, the reviewer is absolutely right in pointing out the dual function of bicarbonate — not only as a potential oxygen donor but also as a key inorganic carbon source regulated by carbonic anhydrase activity. In our manuscript, we have elaborated on how this function improves carbon availability and efficiency of photosynthesis, especially under the changing redox and carbon-limited conditions of the Archean and Proterozoic eons (see Sections 3 and 4, and Figures 6–9).
We thank the reviewer again for recognizing the broader implications of this hypothesis — both for understanding Earth's atmospheric evolution and for enhancing our perspective on plant adaptation and photosynthetic efficiency.
Citation: https://doi.org/10.5194/egusphere-2025-1764-AC1
-
AC1: 'Reply on CC2', Yanyou Wu, 18 Jun 2025
-
AC4: 'Comment on egusphere-2025-1764', Yanyou Wu, 18 Jun 2025
Dear Scientists,
In our recent conference report, we presented evidence regarding the dual-substrate mechanism of photosynthetic oxygen evolution related to the paper titled "Reviews and syntheses: Photosynthetic oxygen evolution in plants—A potential inheritance from early abiotic oxygen production on Earth." Recognizing the complexity and significance of this topic, we sincerely invite you to share your professional insights and evaluations.
Your feedback will play a crucial role in further validating our findings, uncovering potential research gaps, and driving scientific progress in this field. Whether it’s theoretical analysis, experimental design suggestions, or critical reviews of our data interpretation, all perspectives are highly valued.
Please submit your comments. If you have any questions, feel free to contact me. Thank you for your time and contribution to advancing our understanding of photosynthetic oxygen evolution.
Best regards,
Yanyou Wu
Photosynthetic bicarbonate photolysis masked by the rapid oxygen isotopic exchange between water and bicarbonate
https://www.researchgate.net/publication/384113308_Photosynthetic_bicarbonate_photolysis_masked_by_the_rapid_oxygen_isotopic_exchange_between_water_and_bicarbonate
Citation: https://doi.org/10.5194/egusphere-2025-1764-AC4 -
CC3: 'Comment on egusphere-2025-1764', Mahmoud A. Abdelhafiz, 23 Jun 2025
-
CC8: 'Reply on CC3', Mohamed Aboueldahab, 18 Aug 2025
Thank you for your insightful feedback. We hope that our work contributes significantly to advancing the understanding of photosynthetic oxygen evolution in plants, particularly in the context of its potential inheritance from early abiotic oxygen production mechanisms on Earth.
Citation: https://doi.org/10.5194/egusphere-2025-1764-CC8
-
CC8: 'Reply on CC3', Mohamed Aboueldahab, 18 Aug 2025
-
RC1: 'Comment on egusphere-2025-1764', Anonymous Referee #1, 18 Jul 2025
This manuscript makes several claims about bicarbonate oxidation resulting in molecular oxygen. The work is not focused and poorly organized.
In a review of recent publications in this area, I am surprised that the authors fail to cite the 2024 paper by Vinyard and Govindjee (Photosynthesis Research, “Bicarbonate is a key regulator but not a substrate for O₂ evolution in Photosystem II”), which directly addresses the core question posed here. That perspective clearly showed that while PSII can oxidize bicarbonate under certain conditions, bicarbonate is not used as a substrate at the donor-side active site. This is not a peripheral detail—it refutes nearly all of the central claims made in this manuscript.
The authors propose speculative ideas without providing experimental data or a clear understanding of current literature. While speculative models can be valuable, they require a foundation in existing evidence. This manuscript reads more like a “what if” exercise than a scientific review. Key findings from well-established studies are either misrepresented or ignored, and the overall framing appears biased against peer-reviewed work that has been replicated and widely cited by others in the field.
If the authors believe the current model is incomplete, they need to provide a strong rationale and support it with data. At minimum, they should acknowledge existing credible literature and explain how their proposal fits within—or diverges from—that framework. Until then, I do not support publication.
Citation: https://doi.org/10.5194/egusphere-2025-1764-RC1 -
AC5: 'Reply on RC1', Yanyou Wu, 26 Jul 2025
This reply provides an excellent opportunity for us to showcase our work. Many people have not read our previous articles and might assume that water is the sole substrate for photosynthetic oxygen release. In this reply, we aim to highlight that oxygen evolution during photosynthesis is not exclusively attributable to water photolysis but also involves bicarbonate photolysis. Both water and bicarbonate photolysis contribute comparably to the overall oxygen-evolving process. Notably, we propose that the photolysis of bicarbonate constitutes the initial step in photosynthetic carbon assimilation. We will divide the reviewer’s critique into individual parts and respond to each one separately.
“This manuscript makes several claims about bicarbonate oxidation resulting in molecular oxygen. The work is not focused and is poorly organized ”.
RPLY:
Our article focuses on the origin of early inorganic oxygen release events on Earth. It synthesizes evidence from paleontology, biochemistry, stratigraphy, geochemistry, and molecular evolutionary biology to demonstrate that abiotic processes were the first mechanism detected on Earth for the generation of oxygen.
This paper follows a logically coherent structure consisting of three key stages: hypothesis formulation → layered argumentation → synthesis and conceptual advancement.
- The overall framework is as follows
- Abstract & Background (including Background and Challenge)
The paper begins by presenting the central hypothesis that the mechanism of oxygen evolution in plant photosynthesis may be evolutionary derived from early abiotic oxygen-producing processes on primordial Earth. Specifically, it posits that bicarbonate photolysis represents a conserved abiotic pathway for oxygen generation, whereas water photolysis evolved later as an adaptive response to the depletion of accessible carbon sources. This section also reviews the history of atmospheric oxygen accumulation, highlighting the two major oxidation events, and critically examines the limitations of conventional interpretations. In doing so, it establishes the scientific rationale and necessity for the present study.
- Early Abiotic Oxygen Production
Geochemical evidence, such as banded iron formations dated prior to 3.77 billion years ago and atmospheric oxygen signatures predating 3.0 billion years ago, alongside modern observations, including oxygen generation from deep-sea polymetallic nodules and mineral photocatalysis, collectively support the existence of abiotic oxygen production on early Earth. Notably, these mechanisms exhibit similarities to the oxygen-evolving processes observed in modern photosynthesis.
- The potential ancestor of Photosystem II (PSII):
Focusing on the evolutionary origin of photosystem II (PSII), this study hypothesizes that manganese-containing γ-class carbonic anhydrase (γ-CA[Mn]) may represent an ancestral precursor to PSII. By examining shared functional traits such as carbonic anhydrase activity and manganese cluster coordination, as well as comparable physicochemical responses to pH and redox potential, the study provides evidence supporting a transitional pathway from abiotic to biologically mediated oxygen evolution.
- Two-Substrate Photosynthetic Oxygen Evolution:
Through a combination of thermodynamic modeling, isotope labeling experiments, and analysis of the Kok–Joliot cycle, this study demonstrates that photosynthetic oxygen evolution arises from the synergistic contributions of bicarbonate, implicated in the S₄→S₀ transition, and water, which is oxidized during the S₂→S₃ transition. The findings elucidate the evolutionary impetus for the development of water-splitting mechanisms, driven by the depletion of readily available carbon sources, and establish a mechanistic link between oxygenic photosynthesis and Earth's broader carbon cycle.
- Conclusions:
The research summarizes the evolutionary continuity between abiotic oxygen production and photosynthetic oxygen release, emphasizing how core hypotheses contribute to understanding life's origin and biochemical process evolution.
- Research Emphasis
This study centers on a core hypothesis: The bicarbonate photolysis mechanism for plant photosynthetic oxygen release originated from early Earth's abiotic oxygen production processes, while water photolysis emerged as an evolutionary adaptation to environmental conditions.
It specifically addresses three key questions
- Evidence and mechanisms of the early Earth's abiotic oxygen generation (e.g., mineral photocatalysis, electrochemical processes in deep-sea polymetallic nodules).
- Evolutionary precursors of the core photosynthetic oxygen-release component Photosystem II (PSII), particularly the role of carbonic anhydrase.
- The synergistic mechanism and evolutionary logic of bicarbonate-water dual substrates in photosynthetic oxygen release, driven by shifts in carbon sources.
- Research logic
This study follows a progressive logic of "geological evidence → biological mechanisms → evolutionary logic" to systematically validate core hypotheses:
- Challenging conventional views by demonstrating through geological records (e.g., oxygen influence on sedimentation before 3.77 Ga and atmospheric oxygen signals before 3.0 Ga) that early Earth's oxygen existed prior to photosynthetic cyanobacteria, suggesting the possibility of abiotic oxygen production.
- Establishing abiotic-biological connections by comparing mechanism similarities between abiotic oxygen generation (e.g., photocatalytic decomposition of bicarbonate via iron-manganese oxides) and photosynthetic oxygen release (bicarbonate photolysis), proposing the "inherited" hypothesis.
- Tracing the origin of biological components through high functional similarity (catalyzing bicarbonate reactions), structural similarity (manganese clusters), and physicochemical properties (light and pH response) between carbonic anhydrase and Photosystem II, demonstrating that Photosystem II may have originated from manganese-containing carbonic anhydrase, completing the transition from abiotic to biological processes.
- Explaining the dual-substrate mechanism by combining thermodynamic efficiency (lower energy consumption in bicarbonate photolysis) and environmental changes (reduced carbon sources), elucidating why photosynthetic oxygen release retains bicarbonate photolysis while evolving water photolysis, ultimately forming a dual-substrate synergistic model.
- Regarding " not focused and poorly organized“
As demonstrated above, our paper is focused and well-organized, as reflected in the following points.
- Thematic Focus: The paper consistently centers on the core hypothesis that "the bicarbonate mechanism of photosynthetic oxygen release is inherited from abiotic oxygen production."All arguments, from geological evidence and the evolution of biological components to detailed biochemical processes, are developed in support of this central premise. For example, the concept of early abiotic oxygen production establishes the foundation for the idea of inheritance. At the same time, the discussion of ancestral Photosystem II illustrates the transition from abiotic to biological mechanisms. The proposed dual-substrate mechanism then explains the cooperative interaction between bicarbonate and water in oxygen evolution, forming a logically consistent and integrated framework.
- Rigorous Structure: The manuscript follows a classic academic structure: posing key questions → building layered arguments → synthesizing insights. The background section identifies the limitations of traditional models, and the subsequent sections develop progressively: from abiotic foundations, to the emergence of biological components, and finally to specific mechanistic pathways. Each section is supported by clear visual aids, such as Fig. 2 (depicting the abiotic-to-biological transition) and Figure 8 (illustrating the dual-substrate cycle), which reinforce continuity and conceptual clarity.
- Progressive Argumentation: Despite covering a wide range of topics, from macro-scale geological evidence (e.g., early atmospheric oxygen) to micro-scale biochemical mechanisms (e.g., the manganese cluster of Photosystem II), and from abiotic photocatalytic processes to modern biological oxygen evolution, the argument remains tightly focused on the central theme of inheritance. Each section is logically linked; for instance, the decomposition of bicarbonate in abiotic oxygen production directly parallels the photolysis of bicarbonate in biological photosynthesis, emphasizing the continuity of the proposed mechanism. This interconnected design demonstrates both conceptual rigor and internal consistency.
Conclusion:
Overall, the manuscript maintains a strong thematic focus, a well-organized structure, and a clear, logically developed line of argumentation throughout.“ In a review of recent publications in this area, I am surprised that the authors fail to cite the 2024 paper by Vinyard and Govindjee (Photosynthesis Research, “Bicarbonate is a key regulator but not a substrate for O₂ evolution in Photosystem II”), which directly addresses the core question posed here. That perspective clearly showed that while PSII can oxidize bicarbonate under certain conditions, bicarbonate is not used as a substrate at the donor-side active site. This is not a peripheral detail; it refutes nearly all of the central claims made in this manuscript ”.
RPLY:
Regarding your comment about the omission of the 2024 paper by Vinyard and Govindjee and their claim that the paper "refutes the core argument of our review," we would like to offer the following clarifications.
Explanation Regarding the Citation of the Paper
The paper entitled “Bicarbonate is a key regulator but not a substrate for O2 evolution in Photosystem II” by Vinyard and Govindjee (2024) was published online on July 22, 2024 (received on June 20, 2024, and accepted on July 11, 2024). The reason it was not cited in our review was not due to intentional omission but rather because our review involves an interdisciplinary approach, and given the vast amount of literature, it was difficult to cover every relevant study. We sincerely apologize for the oversight. We have now added the citation of their paper in the revised manuscript and specifically addressed their core claims.
Rebuttal to "Bicarbonate is not a substrate at the donor-side active site of PSII"
The core conclusion of Vinyard and Govindjee (2024) is that “bicarbonate is a key regulator but not a substrate for oxygen evolution in PSII,” based on the following reasoning:
- Structural and spectroscopic experiments failed to detect bicarbonate at the oxygen-evolving center (OEC) of PSII.
- Thermodynamic analyses support that bicarbonate can be oxidized, but it is argued that it does not participate in the donor-side oxygen-evolving reaction.
However, based on the research presented in this review and supporting evidence, we believe this conclusion has significant limitations and does not refute the central thesis of our review, that bicarbonate and water both serve as substrates for oxygen evolution in PSII, with each contributing 50% to photosynthetic oxygen release. Our reasoning is as follows.
A. Non-physiological experimental conditions limiting the generalizability of Vinyard and Govindjee’s conclusion
The experimental evidence cited by Vinyard and Govindjee (2024) is primarily based on in vitro reconstitution systems (e.g., PSII membrane fragments) or non-physiological conditions (e.g., low pH, removal of carbonic anhydrase), which significantly differ from the physiological environment of photosynthetic organisms (e.g., stroma pH of 8.0-8.5, thylakoid lumen pH of 5.5-6.5).
- pH-dependent binding:Ferreira et al. (2004) showed that bicarbonate binds to the OEC of PSII at pH 7.5, but no binding is observed at pH <7.0 (Refereed:The photosynthetic oxygen evolution does not exclude the important role and contribution of bicarbonate photolysis). Most of the structural experiments referenced in Vinyard and Govindjee’s paper were conducted below pH 6.0, likely missing the bicarbonate binding signal under physiological conditions.
- Inevitability of carbonic anhydrase activity:The works cited by Vinyard and Govindjee (2024) hypothesize that “there is no carbonic anhydrase activity in the experimental system,” but PSII itself possesses carbonic anhydrase activity (Refereed:Combined effect of bicarbonate and water in photosynthetic oxygen evolution and carbon neutrality; Rapid oxygen isotopic exchange between bicarbonate and water during photosynthesis; The photosynthetic oxygen evolution does not exclude the important role and contribution of bicarbonate photolysis; Photosynthetic bicarbonate photolysis masked by the rapid oxygen isotopic exchange between water and bicarbonate (Presentation of GPMB 2024)), and the carbonic anhydrase on the thylakoid membrane (tCA) cannot be completely removed (Refereed:Combined effect of bicarbonate and water in photosynthetic oxygen evolution and carbon neutrality), leading to rapid oxygen isotope exchange between bicarbonate and water (Refereed:Rapid oxygen isotopic exchange between bicarbonate and water during photosynthesis; Photosynthetic bicarbonate photolysis masked by the rapid oxygen isotopic exchange between water and bicarbonate (Presentation of GPMB 2024)), which may obscure direct evidence for bicarbonate as a substrate.
B. Dual-Isotope Experiment Directly Demonstrates Bicarbonate’s Role in Oxygen Evolution
One of the core pieces of evidence in this review comes from dual-element isotope tracing experiments (Refereed:Rapid oxygen isotopic exchange between bicarbonate and water during photosynthesis; Photosynthetic bicarbonate photolysis masked by the rapid oxygen isotopic exchange between water and bicarbonate (Presentation of GPMB 2024)):
- In microalgae (Microcystis aeruginosaand Chlamydomonas reinhardtii), when exogenous bicarbonate is labeled, carbon isotope signals significantly accumulate in biomass (16%-34% contribution), but oxygen isotope signals remain indistinguishable from unlabeled controls. This suggests that the carbon from bicarbonate is directly used in photosynthesis, while its oxygen exchanges rapidly with water (catalyzed by PSII and carbonic anhydrase) and is not retained. Thus, the oxygen released, although reflecting the water isotope signature, also contains a contribution from bicarbonate (Refereed:Rapid oxygen isotopic exchange between bicarbonate and water during photosynthesis).
- In calcium and magnesium-deficient media, the carbon/oxygen utilization ratio in microalgae approaches 1:1 (Refereed:Photosynthetic bicarbonate photolysis masked by the rapid oxygen isotopic exchange between water and bicarbonate (Presentation of GPMB 2024)), demonstrating that under physiological conditions with no isotope exchange, the oxygen from bicarbonate directly participates in oxygen evolution, with contributions from both bicarbonate and water being equal.
C. Thermodynamic and Geochemical Evidence Supports Bicarbonate’s Substrate Role
- Thermodynamic Advantage:The standard free energy of bicarbonate photolysis (24.8 kcal/mol) is significantly lower than that of water photolysis (37.3 kcal/mol) (Refereed:Combined effect of bicarbonate and water in photosynthetic oxygen evolution and carbon neutrality; The photosynthetic oxygen evolution does not exclude the important role and contribution of bicarbonate photolysis; Photosynthetic bicarbonate photolysis masked by the rapid oxygen isotopic exchange between water and bicarbonate (Presentation of GPMB 2024)), making it more readily oxidizable by the OEC of PSII, which aligns with evolutionary logic favoring low-energy pathways.
- Dole Effect Explanation:The oxygen isotope enrichment of atmospheric oxygen (24‰ higher than seawater) cannot be explained solely by water photolysis but fits perfectly with a model in which bicarbonate and water photolysis each contribute 50%. The ¹⁸O enrichment of bicarbonate (47.62‰) and the 0‰ enrichment of water match the observed values (Refereed:Combined effect of bicarbonate and water in photosynthetic oxygen evolution and carbon neutrality; The photosynthetic oxygen evolution does not exclude the important role and contribution of bicarbonate photolysis; Photosynthetic bicarbonate photolysis masked by the rapid oxygen isotopic exchange between water and bicarbonate (Presentation of GPMB 2024)).
D. Modified Kok Cycle Supports Dynamic Involvement of Bicarbonate
Vinyard and Govindjee (2024) argue that the S₃→S₄→S₀ stages of the OEC exclusively bind water. Still, we propose that bicarbonate may participate in the S₄→S₀ transition due to its rapid reaction rate (thermodynamic advantage), which current instrumentation is unable to capture in the S₄ state (Refereed:Rapid oxygen isotopic exchange between bicarbonate and water during photosynthesis; The photosynthetic oxygen evolution does not exclude the important role and contribution of bicarbonate photolysis; Photosynthetic bicarbonate photolysis masked by the rapid oxygen isotopic exchange between water and bicarbonate (Presentation of GPMB 2024)). This explains why structural experiments failed to detect their binding, not because it does not participate, but because it binds dynamically with a very short lifetime.
The revised Kok cycle model (1 molecule of bicarbonate + 1 molecule of water) can simultaneously explain the “bicarbonate effect” (multiple-fold enhancement in oxygen release rates), isotope exchange phenomena, and the Dole effect, whereas the sole water photolysis model cannot accommodate these observations (Refereed:Rapid oxygen isotopic exchange between bicarbonate and water during photosynthesis; Photosynthetic bicarbonate photolysis masked by the rapid oxygen isotopic exchange between water and bicarbonate (Presentation of GPMB 2024)).
- Conclusion
While the experiments cited by Vinyard and Govindjee (2024) provide important evidence for the regulatory role of bicarbonate, their non-physiological experimental conditions prevent them from identifying bicarbonate's core role as a substrate. The central thesis of this review, that bicarbonate and water both serve as substrates for oxygen evolution in PSII, has been supported by isotope tracing, thermodynamic analyses, geochemical phenomena (the Dole effect), and the modified Kok cycle model under physiological conditions. This conclusion is not contradictory to the findings of Vinyard and Govindjee (2024) but rather provides a more complete explanation of the photosynthetic oxygen evolution mechanism.
We have now added a citation for Vinyard and Govindjee (2024) in the revised manuscript and clearly discussed the impact of experimental condition differences on their conclusions to present a more comprehensive view of the current debates and consensus in the field.
“The authors propose speculative ideas without providing experimental data or a clear understanding of current literature. While speculative models can be valuable, they require a foundation in existing evidence. This manuscript reads more like a “what if” exercise than a scientific review. Key findings from well-established studies are either misrepresented or ignored, and the overall framing appears biased against peer-reviewed work that has been replicated and widely cited by others in the field ”.
RPLY:
In response to your concerns regarding “speculative hypotheses lacking experimental data, misinterpretation or distortion of existing literature, and potential bias,” We provide the following detailed reply, supported by the cited documentation:
1. This review is not based on speculation, but on robust experimental evidence and systematic analysis
The central conclusion of our review is that bicarbonate and water both serve as substrates for oxygen evolution in PSII, each contributing approximately 50%. This is not a "what-if" speculation. It is derived from multidimensional experimental evidence and logical validation as a fellow.
A. Direct Isotopic Evidence for Bicarbonate as a Substrate
Paper entitled “Rapid oxygen isotopic exchange between bicarbonate and water during photosynthesis”, presents a dual-element (C–O) isotope labeling study in Microcystis aeruginosa and Chlamydomonas reinhardtii. The carbon from labeled bicarbonate was directly incorporated into biomass, with 16–34% incorporation in experimental groups, confirming the use of bicarbonate carbon in photosynthesis. However, oxygen isotope signals in biomass remained indistinguishable from the control, indicating rapid oxygen exchange between bicarbonate and water, catalyzed by PSII’s intrinsic carbonic anhydrase activity. This demonstrates that while the released oxygen displays water's isotopic signature, it also reflects a biochemical contribution from bicarbonate. These results directly support the hypothesis that bicarbonate functions as an oxygen-evolving substrate, not merely a regulator.
B. Physiological Isotope Exchange and Quantitative Validation of the Dole Effect
Paper entitled “Combined effect of bicarbonate and water in photosynthetic oxygen evolution and carbon neutrality”, and paper entitled “The photosynthetic oxygen evolution does not exclude the important role and contribution of bicarbonate photolysis”, reanalyzed classic ¹⁸O-labeling experiments (e.g., Ruben et al. 1941; Stemler et al. 1975), revealing that their conclusions depend on the assumption of "slow oxygen isotope exchange." However, PSII possesses carbonic anhydrase activity(Refereed:Combined effect of bicarbonate and water in photosynthetic oxygen evolution and carbon neutrality; The photosynthetic oxygen evolution does not exclude the important role and contribution of bicarbonate photolysis), which accelerates this exchange, potentially obscuring the true oxygen source in prior studies. Furthermore, by modeling a 50:50 contribution of bicarbonate and water photolysis, the calculated atmospheric oxygen ¹⁸O enrichment (23.06‰) aligns closely with empirical observations (23.56‰), thus providing geochemical validation for our mechanism ·(Refereed:Combined effect of bicarbonate and water in photosynthetic oxygen evolution and carbon neutrality; Rapid oxygen isotopic exchange between bicarbonate and water during photosynthesis).
C. Thermodynamic Justification and Kok Cycle Revision
Using thermodynamic data (standard free energy of bicarbonate photolysis = 24.8 kcal/mol vs. water photolysis = 37.3 kcal/mol) (Refereed:Combined effect of bicarbonate and water in photosynthetic oxygen evolution and carbon neutrality; The photosynthetic oxygen evolution does not exclude the important role and contribution of bicarbonate photolysis; Photosynthetic bicarbonate photolysis masked by the rapid oxygen isotopic exchange between water and bicarbonate (Presentation of GPMB 2024)), our presentation(Refereed:Photosynthetic bicarbonate photolysis masked by the rapid oxygen isotopic exchange between water and bicarbonate (Presentation of GPMB 2024)), proposes that bicarbonate participates in the S₄→S₀ transition of the Kok cycle. The reaction occurs too rapidly to be detected with current instrumentation.
The revised Kok cycle model (incorporating 1 bicarbonate + 1 water molecule) coherently explains:
- The "bicarbonate effect" (a multi-fold increase in O₂ release rate),
- The lack of detectable S₄-state intermediates,
- And mechanistic compatibility with thermodynamic favorability.
2. Our review accurately reflects and engages with the existing literature; it does not distort or ignore prior work
The experimental conditions referenced in Vinyard and Govindjee (2024), such as PSII crystals prepared at pH < 7.0 or removal of carbonic anhydrase, do not reflect physiological settings.
- Ferreira et al. (2004) showed that bicarbonate binds to the OEC of PSII at pH 7.5, but not below pH 7.0(Refereed:Combined effect of bicarbonate and water in photosynthetic oxygen evolution and carbon neutrality; The photosynthetic oxygen evolution does not exclude the important role and contribution of bicarbonate photolysis), explaining why their experiments failed to detect bicarbonate binding.
Moreover, Vinyard and Govindjee (2024) acknowledge that “bicarbonate can be oxidized by PSII”, which is not in contradiction with our view. The divergence lies in the interpretation of whether bicarbonate participates as a substrate under physiological conditions, not in the fundamental redox potential.
- Reinterpretation, Not Rejection, of Classic Studies
We do not reject the findings of Ruben, Stemler, or other classical studies. Rather, we point out that their conclusions may not account for PSII’s carbonic anhydrase activity.
- For instance, paper entitled “Combined effect of bicarbonate and water in photosynthetic oxygen evolution and carbon neutrality”,and paper entitled “The photosynthetic oxygen evolution does not exclude the important role and contribution of bicarbonate photolysis”, cite these foundational works while presenting supplementary experiments (e.g.,dual-isotope tracing under Ca/Mg-deficient media in our presentation (Refereed:Photosynthetic bicarbonate photolysis masked by the rapid oxygen isotopic exchange between water and bicarbonate (Presentation of GPMB 2024)) to demonstrate that, under non-exchange conditions, oxygen from bicarbonate can directly contribute to evolved O₂.
- Therefore, the “negative” results in earlier studies likely stem from experimental limitations rather than definitive evidence against bicarbonate involvement.
4. This review is unbiased and aims to synthesize, not refute, existing work
The debate over whether bicarbonate serves as a substrate for oxygen evolution in PSII has persisted for decades. Our review aims to reconcile this debate using new experimental insights and a synthesis of interdisciplinary data:
- We acknowledge bicarbonate's regulatory role, as discussed by Vinyard and Govindjee (2024), including its involvement in electron transport modulation and proton transfer.
- At the same time, we present evidence supporting its role as a substrate, based on dual-isotope tracing, thermodynamic favorability, and geochemical validation.
- For studies favoring exclusive water oxidation (e.g., Hillier et al., 2006), we show that the absence of labeled oxygen signals can be explained by rapid isotopic exchange (Refereed:Rapid oxygen isotopic exchange between bicarbonate and water during photosynthesis; Photosynthetic bicarbonate photolysis masked by the rapid oxygen isotopic exchange between water and bicarbonate (Presentation of GPMB 2024))., not by bicarbonate exclusion, an interpretation grounded in data, not bias.
“If the authors believe the current model is incomplete, they need to provide a strong rationale and support it with data. At a minimum, they should acknowledge existing credible literature and explain how their proposal fits within, or diverges from, that framework. Until then, I did not support publication ”.
RPLY:
In response to your request for strong justification, supporting data, acknowledgment of existing literature, and clarification of our conceptual framework, we provide the following evidence-based clarification.
- Strong justification and experimental evidence supporting the incompleteness of the current model
Our proposed “dual-substrate model,” in which both bicarbonate and water function as substrates for oxygen evolution in PSII, each contributing approximately 50%, is not intended to reject existing models, but rather to extend them based on new evidence.
The following key findings
A. Dual-element isotope tracing demonstrates bicarbonate's direct involvement in o₂ evolution
Paper entitled “Rapid oxygen isotopic exchange between bicarbonate and water during photosynthesis”, employed C–O dual-element bidirectional isotope labeling in Microcystis aeruginosa and Chlamydomonas reinhardtii and found that: The carbon from exogenously added bicarbonate was incorporated into algal biomass at a rate of 16%–34% (vs. control), providing direct evidence that bicarbonate-derived carbon is used in photosynthesis.
- However, oxygen isotope ratios in biomass showed no significant difference between labeled and unlabeled groups. This is attributable to rapid oxygen exchange between bicarbonate and water, catalyzed by PSII-associated carbonic anhydrase (Refereed:Rapid oxygen isotopic exchange between bicarbonate and water during photosynthesis; 4). As a result, the evolved oxygen carries the isotopic signature of water. However, it includes a contribution from bicarbonate.
- This experiment offers molecular-level evidence that bicarbonate is not merely a regulatory cofactor but a true substrate in oxygen evolution, thereby supporting the dual-substrate model.
B. Quantitative Validation of the Dole Effect and Thermodynamic Advantage
- Paper entitled “Combined effect of bicarbonate and water in photosynthetic oxygen evolution and carbon neutrality”,and paper entitled “The photosynthetic oxygen evolution does not exclude the important role and contribution of bicarbonate photolysis”, quantitatively demonstrate that:
- The observed ¹⁸O enrichment of atmospheric oxygen (~24‰ greater than seawater) cannot be explained by water photolysis alone. However, a 50:50 model of bicarbonate and water photolysis perfectly fits this enrichment pattern. Specifically, the combined contribution of bicarbonate (¹⁸O = 47.62‰) and water (0‰) yields a predicted enrichment of 23.81‰, which closely matches the observed value of 23.56‰ (Paper entitled “The photosynthetic oxygen evolution does not exclude the important role and contribution of bicarbonate photolysis, Table 1).
- The standard Gibbs free energy for bicarbonate photolysis (24.8 kcal/mol) is substantially lower than that for water photolysis (37.3 kcal/mol), making bicarbonate more thermodynamically favorable for oxidation by the PSII oxygen-evolving complex (OEC). This supports an evolutionary preference for low-energy oxidation pathways.
C. Revised Kok Cycle Model and Compatibility with Physiological Conditions
In our presentation (Refereed: Photosynthetic bicarbonate photolysis masked by the rapid oxygen isotopic exchange between water and bicarbonate (Presentation of GPMB 2024)), we address:
- The “energy barrier” associated with the S₃→S₄→S₀ transition in existing Kok models, and the failure to detect S₄ intermediates. We propose that bicarbonate is involved in the S₄→S₀ step, proceeding so rapidly (due to its thermodynamic favorability) that S₄ has an extremely short lifetime ( Refereed: Photosynthetic bicarbonate photolysis masked by the rapid oxygen isotopic exchange between water and bicarbonate (Presentation of GPMB 2024), Section 4.2).
- The revised model (1 molecule of bicarbonate + 1 molecule of water) explains both the "bicarbonate effect" (a several-fold increase in oxygen evolution rate) and the isotopic discrepancies seen in classical labeling studies. Importantly, it remains fully compatible with physiological parameters, such as the alkaline microenvironment of the thylakoid lumen.
- We acknowledge and systematically address the existing literature, clarifying the relationship between our framework and prior studies.
We have engaged with key publications in this field, including Vinyard and Govindjee (2024), and clarified the root of conceptual differences:
- Vinyard and Govindjee (2024) (paper entitled “Bicarbonate is a key regulator but not a substrate for O2 evolution in Photosystem II”) argues that bicarbonate is not an O₂-evolving substrate in PSII, primarily based on the absence of bicarbonate in structural studies at the OEC. However,paper entitled “The photosynthetic oxygen evolution does not exclude the important role and contribution of bicarbonate photolysis, highlights that.
- These PSII crystals were prepared under non-physiological conditions (pH < 7.0), whereas Ferreira et al. (2004) showed bicarbonate binding to the OEC at pH 7.5(Refereed:The photosynthetic oxygen evolution does not exclude the important role and contribution of bicarbonate photolysis).
- Thus, the failure to detect bicarbonate binding may be attributed to experimental conditions (e.g., acidic pH) that do not reflect physiological environments, rather than to the actual absence of bicarbonate involvement(Refereed:The photosynthetic oxygen evolution does not exclude the important role and contribution of bicarbonate photolysis).
- We fully acknowledge the regulatory roles of bicarbonate (e.g., in electron transport and proton transfer) but argue that its substrate function is masked under non-physiological conditions.
- Reinterpretation of Classical ¹⁸O-Labeling Experiments
- Paper entitled “Combined effect of bicarbonate and water in photosynthetic oxygen evolution and carbon neutrality”,and paper entitled “The photosynthetic oxygen evolution does not exclude the important role and contribution of bicarbonate photolysis”, directly cite and reanalyze the classic ¹⁸O experiments by Ruben et al. (1941) and Stemler et al. (1975).
- These early studies assumed “slow oxygen isotope exchange,” but paper entitled “Combined effect of bicarbonate and water in photosynthetic oxygen evolution and carbon neutrality”, shows that PSII's carbonic anhydrase activity accelerates isotope exchange, obscuring distinctions between oxygen sources.
- We do not reject the validity of their data but rather reinterpret their results using complementary experiments. For example, in calcium- and magnesium-deficient media, where isotope exchange is minimized, oxygen from bicarbonate directly contributes to the evolved O₂ (Refereed: Photosynthetic bicarbonate photolysis masked by the rapid oxygen isotopic exchange between water and bicarbonate (Presentation of GPMB 2024), Section 2.4). This suggests that prior “negative” results stemmed from experimental limitations, not from bicarbonate's lack of involvement.
4. Conclusion
The dual-substrate model proposed in this review is not speculative, nor is it incompatible with existing frameworks. It is built upon:
- Direct experimental evidence (dual-element isotope tracing),
- Quantitative geochemical validation (Dole effect),
- Mechanistic plausibility (thermodynamics and a revised Kok cycle),
- And thorough engagement with existing literature.
We clearly define the points of compatibility with the classical model (e.g., acknowledging bicarbonate’s regulatory role), and the areas of extension (e.g., incorporating bicarbonate as a true substrate). In the revised manuscript, we have strengthened the discussion of prior literature, explicitly cited all relevant studies, and clarified the experimental basis for our differing interpretations. We respectfully request that you reconsider your assessment in light of this additional evidence and analysis.
-
AC5: 'Reply on RC1', Yanyou Wu, 26 Jul 2025
-
CC4: 'Comment on egusphere-2025-1764', Fatemeh Rezaei Ashtiani, 07 Aug 2025
This article was so beneficial for future life on Earth. It can be one step for the removal of pollution on Earth with oxygen structure and Photocytogenic plants and Microbes.
Thank you for sharing this knowledge
Citation: https://doi.org/10.5194/egusphere-2025-1764-CC4 -
CC7: 'Reply on CC4', Mohamed Aboueldahab, 17 Aug 2025
Thank you very much for your valuable comments. The authors greatly appreciate your support
Citation: https://doi.org/10.5194/egusphere-2025-1764-CC7
-
CC7: 'Reply on CC4', Mohamed Aboueldahab, 17 Aug 2025
-
CC5: 'Comment on egusphere-2025-1764', Eslam Rashad, 08 Aug 2025
This interdisciplinary review explores the hypothesis that modern photosynthetic oxygen evolution may have inherited key elements from abiotic oxygen-producing mechanisms on early Earth. The authors use evidence from geochemistry, biochemistry, mineral photochemistry, and evolutionary biology to argue that bicarbonate photolysis may play a fundamental role alongside water photolysis in the photosynthetic oxygen-evolving complex (PSII). However, the paper needs enhancements, such as clearly defining "bicarbonate photolysis" for non-specialist readers, balancing the argument, addressing methodological critiques, improving visual clarity, and streamlined proofreading.
Citation: https://doi.org/10.5194/egusphere-2025-1764-CC5 -
CC6: 'Reply on CC5', Mohamed Aboueldahab, 17 Aug 2025
We appreciate the reviewer’s constructive feedback and suggestions. Below is our detailed response to each point raised:
-
Clarification of "Bicarbonate Photolysis":
We understand the need for clarity regarding the term "bicarbonate photolysis." To address this, we have added a concise definition in the revised manuscript. Bicarbonate photolysis refers to the light-driven process where bicarbonate (HCO₃⁻) undergoes decomposition, releasing oxygen (O₂) and carbon dioxide (CO₂). This reaction occurs under specific photochemical conditions and is hypothesized to play a role similar to water photolysis in the context of photosynthetic oxygen evolution. A more detailed explanation is now provided in the introduction and methodology sections to ensure accessibility for non-specialist readers. -
Balancing the Argument:
In response to the concern about balancing the argument, we have made revisions to provide a more nuanced discussion. While we continue to emphasize the role of bicarbonate photolysis in parallel with water photolysis, we have included additional references and evidence that present alternative perspectives and limitations of our hypothesis. This will ensure that both sides of the argument are well-represented and discussed in the context of existing literature. -
Addressing Methodological Critiques:
Regarding the methodological critique, we acknowledge the need for further elaboration on the experimental setup and data interpretation. We have expanded the methodology section to provide clearer descriptions of the techniques used in evaluating bicarbonate photolysis, particularly regarding isotope labeling experiments and thermodynamic efficiency analysis. We have also added a discussion on the potential limitations and uncertainties of the methods used. -
Improving Visual Clarity:
We have revisited all figures and diagrams to enhance their clarity and visual appeal. The legends have been revised for better readability, and we have ensured that all visuals are properly labeled with high-quality images. Specifically, Figure 1 and Figure 5 have been modified to better illustrate the dual-substrate nature of photosynthetic oxygen evolution, and we believe these revisions improve both the aesthetic and informative quality of the paper. -
Streamlined Proofreading:
The manuscript has undergone a thorough proofreading process. We have corrected minor typographical errors, improved sentence flow, and ensured consistency in terminology across sections. We believe this enhances the overall readability of the paper.
We hope that these revisions satisfactorily address the reviewer’s concerns. We have attached the updated manuscript for further consideration.
Thank you again for the valuable feedback.
Citation: https://doi.org/10.5194/egusphere-2025-1764-CC6 -
-
CC6: 'Reply on CC5', Mohamed Aboueldahab, 17 Aug 2025
Viewed
| HTML | XML | Total | BibTeX | EndNote | |
|---|---|---|---|---|---|
| 441 | 103 | 36 | 580 | 13 | 34 |
- HTML: 441
- PDF: 103
- XML: 36
- Total: 580
- BibTeX: 13
- EndNote: 34
Viewed (geographical distribution)
| Country | # | Views | % |
|---|
| Total: | 0 |
| HTML: | 0 |
| PDF: | 0 |
| XML: | 0 |
- 1