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Peer Review of A copper-dependent, redox-based hydrogen peroxide perception in plants

Summary

This manuscript presents the structural and functional characterization of the Arabidopsis receptor CARD1/HPCA1, revealing that it coordinates Cu⁺ via a conserved histidine triad and uses this cofactor to perceive hydrogen peroxide. The work challenges previous models that emphasized cysteine-based disulfide switching and instead proposes a novel copper-dependent redox sensing mechanism. This is an important and timely contribution, offering the first example of a copper-based receptor mechanism in plants. The experiments are generally well designed, and the results are compelling. However, there are several areas where the text, figures, and framing could be improved to enhance clarity, rigor, and accessibility.

Major Comments

1. Title The current title is too broad. We recommend making it more specific and result-oriented by explicitly mentioning CARD1 and the copper-dependent mechanism. This would immediately convey the novelty and main finding.

2. Abstract

   • Line 22: Please clarify that the cysteines are not essential for direct signal perception, rather than implying they are functionally irrelevant.

   • Final sentence: The conclusion is vague. It should explicitly state that this study identifies a new receptor mechanism for hydrogen peroxide perception in plants.

3. Introduction

   • Expand the introduction with more information on the biological role of CARD1/HPCA1, particularly its role in long-range ROS signaling.

   • Mention the Ca²⁺ channels that CARD1 activates, to better connect perception to downstream signaling.

4. PEGylation analysis The PEG-maleimide experiments are confusing as written. Please clearly explain which cysteines are free thiols(e.g. Cys124) versus those in disulfide bonds (Cys421–434, Cys424–436), and state explicitly that these disulfides are constitutive rather than stimulus-induced. This will help readers understand why cysteine oxidation is not central to ROS perception.

5. Cysteine mutants (lines 158–159, Supplemental Fig. 4b) The text states “no effect was observed,” but Supplemental Fig. 4b shows that C395S and C405S mutants did affect signaling, while other single Cys mutants and quadruple C421/424/434/436 mutant did not. Please revise to make this distinction clear.

6. Enzyme Kinetics comparison (lines 66–67) The comparison of Km values for H₂O₂ (this study) and DMBQ (from Laohavisit et al. 2020) should be clarified. As written, it appears both were determined here. Please make clear that the DMBQ value comes from previous work, with explicit reference.

7. Figure 4e The legend states “three independent T₃ lines,” but it appears these may actually be three individuals from the same homozygous line. Please clarify whether these represent truly independent transformation events, or replicates from a single line. This distinction is important for genetic rigor.

8. Mechanistic models (Discussion) The terms “on-Cu” and “off-Cu” are not intuitive. We suggest renaming them for clarity — e.g. “Direct model”(Cu–oxyl intermediate on the receptor) and “Release model” (hydroxyl radical release) — to better convey the distinction. A schematic diagram would also help readers follow these models.

9. Supplemental Fig. 10 This figure shows functional data, but the text extends into speculative discussion of downstream pathways. We recommend keeping the focus here on the functional findings and reserving speculation for the main Discussion.

Minor Comments

1. Line 59 – Missing a word after “redox-based” (likely “mechanism”).

2. Figure 1 – Please label the stimulus injection point and annotate Ca²⁺ peak numbers for clarity.

3. Figure 1 legend – Indicate how many species (57) and sequences (405) were used for the phylogeny.

4. Supplemental Fig. 1b – Expand/replot the H₂O₂ dose–response curve to give clearer resolution around the 100–200 μM range.

5. Figure 2 legend – Clarify what the yellow sites in panel A represent.

6. Supplemental Fig. 2b – Lane numbering is reversed; lane 1 should be on the left, not the right.

7. Receptor acronyms – Spell out receptor names at first mention. FLS2 = FLAGELLIN-SENSITIVE 2; HAESA is an Arabidopsis abscission receptor kinase (not an acronym). Similar clarification for other abbreviations (OSCA, PEPR, etc.) would improve accessibility.

8. Mutagenesis rationale – Please explain why cysteines were mutated to serine (structurally similar but non-redox-active) rather than glycine (which could alter backbone flexibility).

Overall Assessment

This study presents a novel and important discovery: a copper-based mechanism for hydrogen peroxide perception in plants. The structural and functional data are strong, and the work is of broad significance to plant biology and redox signaling. Addressing the points above — especially clarifying the cysteine mutagenesis results, improving figure legends, refining the title/abstract, and tightening presentation in the supplement — will significantly strengthen the manuscript and make it more accessible to a wide readership.

Competing interests

The authors declare that they have no competing interests.