Peer review of: Mild proteasomal stress improves photosynthetic performance in Arabidopsis chloroplasts (2020)

 

REVIEWERS' COMMENTS: 

Reviewer #1 (Remarks to the Author): 

Inclusion of the images from single and double mutants in Figure 1 makes the manuscript easier to 
follow, and at least we assess how the double mutants look. However, my honest impression is 
that the suppression of growth in rpn8a/ppi2 is, when compared to ppi2, is VERY weak. Then the 
authors decided to show the ratio of pigments/fresh weight instead of pigment content - this is fine 
to demonstrate the significant difference between the single and double mutants, but I am not 
fully satisfied because the substantial difference between WT and the mutants is neglected. At 
least, the authors should mention explicitly somewhere in the text that Chl content of ppi2 
rpn8a/ppi2 is 10-fold lower than WT, thus the suppression of ppi2 phenotype is only minor when 
compared to WT. 

In this context, I did not agree with the statement in Discussion (316-317) ‘Mutations in the lid 
subunit Rpn8a and the core subunit PAD1 partially complement the ppi2 mutant phenotype’. 
Perhaps the phenotype observed in the double mutants is not to the extent that we can state it as 
partial complementation. 

Figure 6. I was puzzled when I read the legend pointing GFP antibodies here, because the fact that 
the precursor proteins were GFP fusions was not mentioned in the text or the figure at all. This 
panel therefore needs some revision, like putting caption like what are seen in Figure S6. 

Reviewer #2 (Remarks to the Author): 

Comments on the manuscript 

The revised version of this manuscript has a much improved text and presentation of the results. 
The study is fascinating and of interest for both specialist in the field and a broad readership. 
Nevertheless, there are some point, which I would like the authors to consider for a revision. 

Until figure 5, the text is excellent and a reader can follow it without difficulties. I suggest to move 
the the section about the elevated precursor level in the cytosol of the double mutants in the end 
of the manuscript. It would perhaps better fit into the manuscript at a place between figure 4 and 
5? There is also a minor question about figure 1 C. Has the y-axis the same scale for ppi2 and the 
double mutants as for the wildtype and the other single mutants? It appears that the y-axis for 
ppi2 and the double mutants is missing. 

Figure 5 is not easy to understand. I wonder, if the authors possibly could consider more the 
different mechanisms of function that are known for the 26S and the 20S proteasome, when they 
present these results? 

The rpn8a mutant has no lid and it shows no phenotype under the controlled growth conditions of 
this study. This observation fits together with the observation by Paci et al. (2016) that a region 
close to the C-terminus of yeast Rpn8 is needed for assembling the intact 26S proteasome. As the 
rpn8a mutant cannot perform de-ubiquitination, it relies on 20S activity. As reviewed by Ben-
Nissan (2014), 20S proteasome can recognize and degrade proteins that contain unstructured 
regions because of oxidation, mutation or aging. Apparently, the proteolytic capacity of 20S in the 
rpn8a mutant is sufficient to ensure that this mutant can live with 20S without turning into a 
phenotype. The ability of the proteasome to function without the lid can in part also explain the 
data for the ppi2xrpn8a double mutant, although this mutant has a lid and would be expected to 
also have the ubiquitination-controlled pathway of 26S proteasome function. Taking this into 

account, the data for the rpn8a mutants in figure 5 would not simply suggest a compensatory 
upregulation of the 20S proteasome but also a shift to different pathways for proteasome function. 

The authors use the MG132 proteasome inhibitor to mimic the effect of rpn8a mutation on 
photosynthetic protein abundance. Is this convincing? The data that the authors present show that 
the MG132-treatment is accompanied by a significant increase of the amount of 20S, which in turn 
may affect the expression of the lid and base subunits. Mechanistically, this would be different 
from mimicking loss of Rpn8a. The authors suggest that loss of Rpn8a and inhibition of 20S by 
MG132 have different effects on the import of chloroplast proteins in wildtype plants and in plants 
with ppi2 background. That is reasonable, but the line of reasoning is not entirely clear and the 
authors might consider clarifying it and try to achieve a better separation between presenting and 
discussing their results in this part of the manuscript (line 251 and following). 

To add a brief technical comment, I understand that space for presenting figures is limited, but 
perhaps there is solution to present the data for the lid and the base with the same scale for the y-
axis? 

References 
Ben-Nissan G, Sharon M. Regulating the 20S proteasome ubiquitin-independent degradation 
pathway. Biomolecules. 4:862-884, 2014. doi: 10.3390/biom4030862. 

Paci A, Liu PX, Zhang L, Zhao R. The proteasome subunit Rpn8 interacts with the small nucleolar 
RNA protein (snoRNP) assembly protein Pih1 and mediates its ubiquitin-independent degradation 
in Saccharomyces cerevisiae. J Biol Chem. 291:11761-11775, 2016. doi: 
10.1074/jbc.M115.702043. 

Comments on supplementary material and the data submitted to EBI-Pride 

The document 219310_1_supp_4359005_q494t2.pdf is a valuable supplement and may well be 
added to the manuscript, but it would help the reader if the figures would have a description that 
integrates them better into the context of the manuscript. 

Please consider some additions and corrections to the data that you submitted to EBI-Pride. You 
can only make changes to the data that you submitted before they are published. When your data 
have received a DOI, they are locked and the Pride team will not be able to add corrections or 
revisions. 

Private Project PXD014560 
Please give the full reference of Helm et al. 2014. Please avoid empty key words as far as possible 
(“unknown”) and add software and experiment type. Some characters were converted to 
unreadable symbols. Please replace them by simple text. 

Private Project PXD014531 
Please add a csv file with descriptions of the raw data file like in your project PXD014560. Please 
give the full reference of the publication of Helm et al. (2014). The description of the data in a 
repository should be independent from an article, for which these data were used and the 
metadata should be rich. Like in project PXD014560, please avoid empty key words and add 
acquisition software and experiment type. 

Private Project PXD017126 
Please correct undefined characters such as Â and remove them or replace them by simple text. 
Please give the full reference of the publication of Shema et al. Like in the previous projects please 
avoid empty key words and add the data acquisition software. 

As for the Excel file ChaFRADI_JG.xlsx, you may keep it in this dataset, but please provide the 

sheets of this file also as single csv files and add a readme file that describes their content. The 
reason for that is that CSV is a recommended data format for archiving, which XLSX is not. Please, 
have also a look at the sheet of table 3 (Tabelle 3) in the file ChaFRADI_JG.xlsx. Was it supposed 
to be empty?