PREreview of The ROS-producing enzyme NADPH oxidase/RBOH is a Ca2+ channel regulated by protons in the eukaryotic kingdom

The ROS-producing enzyme NADPH oxidase/RBOH is a Ca2+ channel regulated by protons in the eukaryotic kingdom. Liangyu Liu et al., 2024

General Impressions

This paper by Liangyu Liu et al., explores the dual role of NADPH oxidase (NOX) enzymes as both ROS-producing enzymes and calcium/cation channels in eukaryotes, with a focus on their role in integrating ROS-Ca²⁺ signaling. By identifying NOX/RBOHs as novel calcium channels regulated by protons, the study introduces a paradigm shift in understanding ROS-Ca²⁺ interactions in diverse biological processes. The findings reveal that NOX enzymes, like plant RBOHD, can mediate calcium influx in a proton-dependent manner, with characteristics analogous to human TRP channels. These results provide insights into the interplay between ROS, Ca²⁺, and membrane potential, highlighting the potential of NOX/RBOHs as key hubs in systemic signal transmission and as candidates for further exploration in calcium-redox biology.

The manuscript is concise yet comprehensive, however sometimes we lack a bit of introducing some of the previous work. However, it could benefit from more context regarding some of the prior work in the field to enhance the reader's understanding. We believe they may improve clarity/readability and enhance some of the analyses performed. 

Nonetheless, the manuscript is very interesting and disruptive.

Introduction: No comments 

Figure 1:

• It would be necessary to clarify what CNGC2 is, as well as its function and regulation. When Figure 1b is presented, it is observed that cngc2 plants become smaller with increased calcium. If RBOHD also acts as a calcium transporter, why are contrasting phenotypes observed with RBOHD?

• Clarify the N number of replicates for Figure 1a and b.

• It is unclear what the origin of the RBOHD mutant is. Was it generated in a transgenic plant by your group, or is it part of a collection from another institution, such as the Salk Institute? If it was generated by your group, please specify the method.

• Why are AtGNC14 (in Xenopus) and AtCGNC2 (in Arabidopsis) used for different assays? Please clarify why you are using different genes.

• Including an inset showing a single cell would greatly enhance the clarity and detail of the data. This would help highlight the specific localization or dynamics of calcium signals within individual cells, making the figure more informative (figure 1f)

• For those who do not work with yeast, it would be helpful to include a brief explanation of how the growth inhibition experiment with alpha factor works and what is shown in the figure.

Figure 2:

• We wonder if the dose-dependency observed with calcium is also observable with other cations, such as Mg²⁺ and K⁺.

• Panel f: There are some doubts regarding the Xenopus experiment using the chloride transporter inhibitor DIDS. Please explain why an increase in calcium signaling is expected. It would also be valuable to observe this in an experiment with CNGC (for example). Is it possible that DIDS also inhibits RBOHD?

Figure 4:

• It would be interesting to include AtCNGC14 in the root experiment under different pH conditions, as this channel does not appear to be sensitive to calcium, according to the experiment in Xenopus (Figure 4a).

Discussion:

• Changes in apoplastic pH have been reported during pathogen attack (pathogen-induced alkalinization) and cell growth (acidification). It would be beneficial to discuss this in the corresponding section. The authors propose that RBOH is activated by protons, but given that the apoplastic pH is generally around 5.7, how do the authors reconcile this with the activation of Ca2+ channels? Could we speculate that Ca2+ channels are normally active at the resting state of the cell and become inhibited during a pathogen response or induced during cell elongation? Alternatively, could it be that these channels are inhibited at high pH rather than being activated by protons? Clarifying this point could provide valuable insights into the mechanism.

• LaCl₃ is known to act as a Ca²⁺ channel inhibitor and here the authors report to inhibit RBOH channel activity. Could the authors clarify whether LaCl₃ also inhibits RBOHD-mediated H₂O₂ production, specifically as an NADPH oxidase? Have there been any studies or experiments conducted to assess this inhibition both in vivo and in vitro? Including such data or discussing its implications could add valuable insight into the role of Ca²⁺ signaling in RBOHD function.

Supplemental Figure 8

• It would be appropriate to include controls without the addition of external calcium in the infection experiments with Pseudomonas and stomatal closure assays.

• Calcium treatment induces stomatal closure (Schroeder et al., 1989). How could the lack of stomatal closure observed in your experiment (Supplemental Figure 8C) be reconciled with the previously reported findings?

Competing interests

The authors declare that they have no competing interests.