Properties and Functional Analysis of Two Chorismate Mutases from Maritime Pine

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This is a well-written study, and I have only few technical points to suggest:

Line 96: Please put a space between „from“ and "Saccharomyces" and italicise Saccharomyces cerevisiae

Line 97: Please put space before “and”

Please make a consistent spelling of °C and consult Instruction for Authors: write it together with the number or number-space-°C

Author Response

Thanks for the comments, we have fixed these mistakes.

All changes are highlighted on the new version of the manuscript. Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Summary

 

This study explored the regulation and evolution of chorismate mutase (CM) enzymes in maritime pine (Pinus pinaster). Two genes, PpCM1 and PpCM2, were identified as encoding plastid-localized CM isoenzymes that differ in function. PpCM2 plays a significant role in phenylalanine and lignin biosynthesis, exhibiting higher expression levels and resistance to phenylalanine inhibition compared to PpCM1. Both enzymes are very sensitive to tryptophan activation. The study also seems to suggest that PpCM1 and PpCM2 can form heterodimers, suggesting potential cooperative regulation of plastid phenylalanine biosynthesis. 

 

 

Comments to the authors

 

The paper is well written, and I have only minor comments. 

 

Line 21; Unclear whether ‘activities’ refers to enzyme activity or expression levels. 

Line 72; “CM activity” Line 96; “fromSaccharomyces” Line 97; “muscle15and” Line 114; “described by19” Line 115; same as line 114

Line 118; The °C symbol is incorrect 

Line 119; Please rephrase, the ratios do not indicate concentration. 

Line 121; “through both agarose gel electrophoresis” Line 131; This is a very high cycle number (50), please comment on its necessity. Is the template expected to be found at extremely low concentrations?

Line 136; same as line 114

Line 182; Please explain the reason for this gentle shaking protocol.

Line 192; Please italicise organism name

Line 199; please describe centrifugation conditions

Line 208; Please provide more detail on the antibodies used. 

Line 236; “conifers in overall” Line 291; “autofluorescence chlorophyll” Line 311; “performed” Line 313; same as line 114

Line 339; “candidates PpCMs” Line 349; “at A274nm” Line 448; Please confirm that this generalization is valid. 

Line 542; same as line 448

 

 

Author Response

The paper is well written, and I have only minor comments.

Thanks for the comments

Line 21; Unclear whether ‘activities’ refers to enzyme activity or expression levels. We have changed the sentence to make clear this point.

Line 72; “CM activity” Line 96; “fromSaccharomyces” Line 97; “muscle15and” Line 114; “described by19” Line 115; same as line 114. Fixed

Line 118; The °C symbol is incorrect Fixed

Line 119; Please rephrase, the ratios do not indicate concentration. We have now rewritten the sentence

Line 121; “through both agarose gel electrophoresis” fixed Line 131; This is a very high cycle number (50), please comment on its necessity. Is the template expected to be found at extremely low concentrations? Yes, the expression level of these genes are very low, also most of the genes from the shikimate pathway.

Line 136; same as line 114 Fixed

Line 182; Please explain the reason for this gentle shaking protocol. We already optimized recombinat protein protocols having the best results using this shaking step.

Line 192; Please italicise organism name Fixed

Line 199; please describe centrifugation conditions Now included

Line 208; Please provide more detail on the antibodies used. This information is now included 

Line 236; “conifers in overall” Fixed, we have remove "overall" and replaced with globally

Line 291; “autofluorescence chlorophyll” Fixed

Line 311; “performed” Fixed using generated instead of preformed, thanks.

Line 313; same as line 114 Fixed

Line 339; “candidates PpCMs” Fixed, thanks for the indication

Line 349; “at A274nm” Fixed

Line 448; Please confirm that this generalization is valid. We have rewritten the sentence to recognize that only in the case of the CMs of Pinus pinaster we have experimentally determined the subcellular localization. However, analyzes of all identified conifer CM sequences strongly suggest the existence of plastidial localization signals and thus plastidial localization.

Line 542; same as line 448 Through this sentence we suggest that the absence of cytosolic CM in the species Pinus pinaster may be a common characteristic of conifers. This suggestion is reinforced by the fact, described in the work, that all conifer CM proteins analyzed have amino terminal signals that, according to predictions, correspond to plastidial localization peptides.

 

All changes are highlighted on the new version of the manuscript. Please see the attachment.

Author Response File: Author Response.docx