Avalilação PREreview de Quantitative proteomics uncovers a role for REVEILLE8- like clock genes in osmotic and salt stress response in Arabidopsis

In the manuscript entitled “Quantitative proteomics uncovers a role for REVEILLE8-like clock genes in osmotic and salt stress response in Arabidopsis,” Grubb et al. describe the effects of the transcription factors RVE4, 6, and 8 on the Arabidopsis proteome under salt and osmotic stress. In rve4 6 8 mutants, proteins related to osmotic stress, photosynthesis, and phenylpropanoid metabolism show altered abundance relative to wild-type plants, depending on the time of day these proteomes were obtained. Additionally, plants with mutations in genes that are more abundant following salt stress show a reduced severity in salt stress response. Overall, the findings included in this article are important because of their specific insight into how RVE4, 6, and 8 affect drought and salinity stress responses and general implications for the circadian control of abiotic stress resilience. This can have applications for breeding of climate-resilient crops and underscores the importance of controlling for circadian factors in experiments. The manuscript is logical and easy to read, with evidence that supports the authors’ conclusions, like the in depth GO-enrichment analysis and association network analysis. However, this manuscript does present some limitations, particularly analyzing whole seedlings rather than separating tissue by type, leading to the potential obstruction of tissue-specific changes in protein abundance.

Major points:

1. Below are several comments on the quantitative proteomics experimental design as outlined in Figure 2a and the methods. While it might not be feasible to repeat these experiments, some limitations of the approach related to the following points could be discussed in the manuscript or considered for future experiments.

   1. Whole seedlings were analyzed for the quantitative proteomics experiments, rather than separating plants into root and shoot tissues. It is likely that different proteins would be differentially abundant in these different tissues, especially for osmotic and salinity stress, which are primarily sensed through the roots. Repeating the quantitative proteomics looking at roots and shoots separately could provide valuable information about tissue-specific expression changes.

   2. It is not indicated how many seedlings were analyzed for each condition or how many replicates were included in the analysis. Furthermore, it is unclear whether individual plants were separated and treated as biological replicates, or if multiple seedlings per condition were pooled. The authors could consider clarifying their experimental design and, if they have not already, analyzing at least 4-5 replicates of single seedlings to allow for reduction of noise that might confound pooled tissue analysis and more robust results.

   3. Figure 1F-H shows substantial spread in the root lengths under 50 mM Mannitol and 25, 50, and 100 mM NaCl, especially in the rve4 6 8 strain. As a future area of study, it would be interesting to separately analyze mutant seedlings with root lengths overlapping with wild-type and those with much shorter roots to see if there are differences in DAPs depending on the severity of the root phenotype. This could enhance the understanding of the variability of the stress response in these mutants and elucidate the extent to which root length phenotype correlates with protein expression.

2. The rve4 6 8 strain is used throughout much of this manuscript. However, there is no description of the nature of the alleles or validation that these represent true null alleles. Adding a description of strains to the materials and methods section would be clarifying, as well as qPCR and/or western blotting if appropriate. This could also be valuable to anyone intending to use these strains in the future.

3. In Figure 6, root-length phenotypes are assessed for wild type and strains harboring mutations in three candidate genes identified by the proteomics on media with and without NaCl added. Considering that these genes were found to be upregulated in rve4 6 8 mutants, the authors might consider generating quadruple mutants to assess how lacs3, syp43, and cst affect root length in the rve4 6 8 background. Additionally, assessing other phenotypes responsive to salt stress observed in Figure 5, such as photosynthesis rates or anthocyanin production, could strengthen the validation of these candidates.

Minor points:

1. In Figure 6, the data in panel A is presented differently from the data in panels C and E, despite the experimental designs being the same. Panel A avoids normalization and is a clearer representation of the effects on root length. Maintaining a consistent presentation across all panels and changing panels C and E to be in the form of panel A would make the data easier to interpret.

2. Figure 7 shows a number of pathways downstream of RVE 4,6,8. However, these pathways are not referenced in the manuscript beyond the introduction, and their relationships and post-translational modifications as depicted in the figure are not described. Including an explanation of this figure in the text or a more comprehensive figure legend could clarify the purpose of this figure and improve the comprehension of the manuscript. Alternatively, Figure 7 could be removed from the manuscript entirely to avoid confusion.

3. In lines 225-237, a number of GO-terms that are differentially regulated at ZT12 vs. ZT0 in Col-0 plants are listed in the text. This is presented as a dense list that is difficult to read. The authors could consider displaying this as a table and highlighting the most important terms. This reformatting would allow the GO-terms to be more easily identified by the reader and allow the manuscript text to flow better.

4. In Figure 1 D and E, it appears as though the images of the control conditions are of the same plate for both panels. Combining these images into one panel with the same control images for both perturbations would save space and more accurately represent the experiment.

I reviewed this preprint as part of the Transparent and Accountable Peer Review training program. This report was written with the support of my research mentor

Competing interests

The authors declare that they have no competing interests.

Use of Artificial Intelligence (AI)

The authors declare that they did not use generative AI to come up with new ideas for their review.