In Search of Monocot Phosphodiesterases: Identification of a Calmodulin Stimulated Phosphodiesterase from Brachypodium distachyon

Round 1

Reviewer 1 Report

In the paper entitled “In search of Monocot Phosphodiesterases: Identification of a

Calmodulin Stimulated Phosphodiesterase from Brachypodium distachyon”, the authors identified and characterised phosphodiesterases in Brachypodium distachyon, which will be useful and advance the scientific knowledge of phosphodiesterases in monocot plants. However, I recommend this paper for publication after addressing the below-mentioned issues.

Gene and protein names were wrongly used in several places- those should be corrected to the standard nomenclature (GENE and PROTEIN) throughout the manuscript.

The scientific names of all the species were not italicised and differentially abbreviated in several instances (e.g., A. thaliana, Brachypodium distachyon, A.t. and B.d. etc.) - it should be corrected following the standard nomenclature throughout the manuscript, including the figure legends.

Supplementary figures are not cited in sequence- they should be changed to the sequence.

Author Response

Reviewer 1: In the paper entitled “In search of Monocot Phosphodiesterases: Identification of a Calmodulin Stimulated Phosphodiesterase from Brachypodium distachyon”, the authors identified and characterised phosphodiesterases in Brachypodium distachyon, which will be useful and advance the scientific knowledge of phosphodiesterases in monocot plants. However, I recommend this paper for publication after addressing the below-mentioned issues.

In response:

Firstly, we wish to thank the reviewer for his careful and considerate assessment of our manuscript and calling our work useful.

Gene and protein names were wrongly used in several places- those should be corrected to the standard nomenclature (GENE and PROTEIN) throughout the manuscript.

In response:

The gene and protein names are corrected. Specifically, the gene names were italized while protein names were not, and both are capitalized.

The scientific names of all the species were not italicised and differentially abbreviated in several instances (e.g., A. thaliana, Brachypodium distachyon, A.t. and B.d. etc.) - it should be corrected following the standard nomenclature throughout the manuscript, including the figure legends.

In response:

The scientific names of species are italicized. The first appearance of the species name is complete, the next is abbreviated.

Supplementary figures are not cited in sequence- they should be changed to the sequence.

In response:

Supplementary figures are corrected and cited in order.

Author Response File: Author Response.docx

Reviewer 2 Report

In this manuscript, authors present PDE from the monocot and analyze its activity and interactions. The work shows that BdPDE1 can hydrolyze cNMPs and also that CaM interaction further increase the PDE activity. The work is highly interesting, but there is few open questions still needing attention.

1. The overall quality of the text is good, but at some places there are some highly complicated sentences and part. As an example, chapter starting end of page 2 and continuing to page 3. Authors should think how the data is presented and if table is needed or not. Authors show some data in Table 1, which isn't really needed.
2. Some important data from M&M is missing and some of the important concentrations should also be mentioned in the text. For example, IBMX and in similar experiments it is important that all concentrations are known. 2x drop in PDE activity is quite small so it is difficult to estimate why. Similarly the Ca2+ concentrations are not known and thus EGTA chelating is impossible to estimate. In Ca2+ titration the concentration range was in low µM range, as normally e.g. with CaM it is in low mM level ( in vitro assays). Authors must include all the needed concentrations and details. For example the results in figure 2 is impossible to understand without concentration data.
3. Figure legends must be informative enough and the statistical analysis or used programs are not the info should be there but M&M. If there is enzymatic reaction, concentrations, assay times and so on are the infos needed.
4. Letters marked in figures are not explained. Those tell something about the statistical analysis, but are those really needed and what extra that brings?
5. Manuscript needs conclusions section.

Author Response

Reviewer 2: In this manuscript, authors present PDE from the monocot and analyze its activity and interactions. The work shows that BdPDE1 can hydrolyze cNMPs and also that CaM interaction further increase the PDE activity. The work is highly interesting, but there is few open questions still needing attention.

In response:

Firstly, we wish to thank the reviewer for his careful and considerate assessment of our manuscript and finding our work highly interesting.

The overall quality of the text is good, but at some places there are some highly complicated sentences and part. As an example, chapter starting end of page 2 and continuing to page 3. Authors should think how the data is presented and if table is needed or not. Authors show some data in Table 1, which isn't really needed.

In response:

The enzyme kinetic data has been presented in less complicated sentences. The table has been deleted and the values have been inserted into the text.

Some important data from M&M is missing and some of the important concentrations should also be mentioned in the text. For example, IBMX and in similar experiments it is important that all concentrations are known. 2x drop in PDE activity is quite small so it is difficult to estimate why. Similarly the Ca2+ concentrations are not known and thus EGTA chelating is impossible to estimate. In Ca2+ titration the concentration range was in low µM range, as normally e.g. with CaM it is in low mM level ( in vitro assays). Authors must include all the needed concentrations and details. For example the results in figure 2 is impossible to understand without concentration data.

In response:

The information about concentrations has been added in the text, figure legends and M&M. Ca²⁺ concentration used to activate CaM complex was in low µM level to simulate intracellular Ca²⁺ plant concentration. Moreover, our previous experiments with CaM complex showed that to activate various CaM isoforms using 10 µM concentration is sufficient. The addition of high EGTA concentration, compared to Ca²⁺, chelates all Ca²⁺ ions, but this does not disturb the enzymatic reaction, what confirms formation of CaM/Ca²⁺ active complex.

Figure legends must be informative enough and the statistical analysis or used programs are not the info should be there but M&M. If there is enzymatic reaction, concentrations, assay times and so on are the infos needed.

In response:

Statistical analyses have been moved to the M&M section and all details regarding enzymatic reaction are in the figure legends. To avoid repetitions, we used term standard reaction mixture, which contains constants chemical reagents and its composition is explained in detail in M&M and in the Figure 1 legend. Every addition to the standard reaction mixture is listed in the figure legend.

Letters marked in figures are not explained. Those tell something about the statistical analysis, but are those really needed and what extra that brings?

In response:

Letters above bars in figures are now better explained. We think it is worth keeping the letters as in a few cases like in Figure 7B, there is a slight difference in activity between the WT and the L124E mutation where after several repetitions, the statistical analysis eventually indicated that the mutation of this amino acid has no significant effect on the enzymatic activity of BdPDE1.

Manuscript needs conclusions section.

In response:

The conclusions section has been added.

Author Response File: Author Response.docx