PREreview of Biochemical analysis of deacetylase activity of rice sirtuin OsSRT1, a class IV member in plants

Mitra, N. and Dey, S. have biochemically characterized the deacetylase activity of rice sirtuin, OsSRT1, and provided a glimpse of its role in the context of abiotic stresses in plants. They have asked fundamental questions about the role of different domains of this protein in its enzymatic function and addressed those using mutagenesis and biochemical assays. However, I have a few concerns about some results. I have described these below:

General

1. Towards the end of the Introduction, it is said that “OsSRT1 is capable of influencing the DNA repair pathway as the constituent proteins involved in this pathway can interact with OsSRT1 and eventually get deacetylated”. However, this study does not unambiguously show the effect of OsSRT1 on DNA damage repair pathways. Thus, I suggest conducting additional experiments (such as single cell RNA-sequencing to show OsSRT1 mediated upregulation of DNA damage response genes when subjected to abiotic stress or fluorescence-based experiments to show OsSRT1 mediated increase in recruitment of DNA damage repair enzymes to the damage site) to show the role of OsSRT1 on DNA damage repair pathways. Alternatively, the authors should consider removing this aspect from the Introduction but elaborating more on this in the Conclusions / Discussions section.
2. In Figure 1A, the labels for sample and control are missing. Also, the figure legend lacks key details about the experiment. For example, it does not explain what H134Y is, whether it is a mutation on OsSRT1 or on H3, and what it contributes to the results. In Figure 1B, the deacetylation of several acetylated H3 lysine residues is shown. This figure also provides the histone deacetylation by HsSIRT6 as a control. Here, adding a lane showing the H3 deacetylation by HsSIRT7 will provide a more complete picture. Similarly, adding HsSIRT6 and HsSIRT7 mediated H4 deacetylation as controls in Figure 2A will provide a better picture.
3. The text points out the specificity of OsSRT1 towards NAD+ for its catalytic function. This is a rather interesting finding and I believe, much like myself, many readers would like to look at the results of this experiment. I would strongly recommend authors to add this result as a figure. Also, addressing the structural basis for this specificity might be an aspect that can be explored further.
4. In this study, the effects of various drugs, such as resveratrol on the activity of OsSRT1 have been studied. However, the motivation for testing these drugs and an interpretation of the findings is missing.
5. Figure 4B requires a more detailed legend. Again, the reason for using H134Y is not mentioned. Also, the text does not talk about the activity of these constructs on PARP1. I believe a reader would like to see a comparison between the activities of these constructs, from OsSRT1 and OsSRT2. What additional function does the longer C-terminus of OsSRT1 carry out? Do both OsSRT1 and OsSRT2 exhibit similar deacetylation substrate specificities? Which of the domains confer the deacetylation substrate specificity to these proteins?

Minor comments

1. The abstract lacks a clear distinction between what was previously known and what this study contributes to existing knowledge. I would suggest to add a few sentences to the end of the abstract emphasizing the downstream implications of these findings.
2. In the second line of Introduction, it is said that: “Among all the eukaryotes, human sirtuins are the most studied proteins” which is misleading. I would recommend rephrasing it to something like “Sirtuins from humans are among the most studied isoforms of this protein family”.
3. In the second paragraph of the Introduction, there is a section that says “Most of the plants, monocots and dicots, have these two members (SRT1 and SRT2), except maize having only one sirtuins, SRT1. This class III member is also present in few other plants, including rice”. Here, the second sentence is hanging as this sentence leaves the reader confused if rice and the most other plants mentioned here have only SRT1? Following this, it is said that “OsSRT1 (483aa) has homology with both HsSIRT6 (355aa) and HsSIRT7 (410aa), thus, belonging to class IV sirtuin family” which further leaves a confusion whether SRT1 is a class III or a class IV sirtuin. Also, I would further suggest adding a few sentences to the introduction, summarizing the differences between the different classes of sirtuins.
4. The residue numbers for CC, ΔN1 and ΔN2 does not match between the text and Figure 4A. Figure 4C is cited before Figure 4B.
5. In various places in the manuscript, the phrase “OsSRT1 deacetylation” is used. This phrase is misleading as it seems to point towards the deacetylation of OsSRT1, which is not the case here. I would suggest to change it to something like “OsSRT1 mediated deacetylation”.
6. In the Conclusions section, it is difficult to follow what previous knowledge does this study build on and what are the key insights from this study. However, here, the implications of the study are well highlighted.