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

Reviewers' comments: 

Reviewer #1 (Remarks to the Author): 

This manuscript by Grimmer et al. mainly focused the effect of 26S proteasome mutants in 
photosynthesis, particularly in relation to chloroplast protein import deficiency. The work is 
extensive and impressive, generating numerous Arabidopsis mutants and performing protein 
quantitation with proteome analysis. The main essence of this paper is, if I understand correctly, 
the recovery of pale phenotype in ppi2 when combined with 26S proteasome mutations (rpn8a in 
Figure 1; rpa2b and pad1 in Figure 4). Moreover, the authors showed that rpn8a improves 
photosynthesis performance (Fig. 2). Given the previous observation that similar effects were seen 
in ppi1/sp1 mutants and that SP1 is E3 ligase specifically associated with Toc components, the 
authors propose that general proteasomal degradation of precursor proteins in the cytosol, rather 
than the specific degradation of protein import machineries, controls chloroplast biogenesis. 
While I thought the work is deep and interesting, the manuscript was difficult to follow. It perhaps 
comes from several inconsistencies in the data presented in the figures. Some of those are 
highlighted below. 

1) As mentioned above, suppression phenotype of rpn8a, rpn8b, and pad1 (but not sp1) is split in 
Figure 1 and Figure 4A. I think it should be comparably shown in the same figure with proper 
controls. Also, not only pigment content but also photographs of these mutants need to be shown 
at the several different developmental stages. 

2) In Figure 1, ppi2 is shown as a kind of control against the double mutants, but the actual WT is 
better to be included (perhaps sp1, too). 

3) Figures 1B and 1C: protein abundance is estimated based on total protein amount? If so, the 
change reflects the degree of greening? 

4) Figure 2: I understand the result well. However, readers may like to have other lines included in 
this figure, such as sp1, rpn8b and pad1, to assess how this finding can be generalized. 

5) Figure 3: Why the double mutant was not subjected to MG132 treatment in panel A? In panel B, 
the difference of protease activity between ppi2 and rpn82/ppi2 is significant (statistical analysis is 
lacking here)? 

Although the authors attempted to compare rpn8 mutants to sp1, which I understand well, they 
may want to pay attention to the following publication, in which ubiquitylation of chloroplast 
protease is reported. This suggests a possibility that a defect in cytosolic protein degradation has a 
pleiotropic effect in chloroplastic protein degradation. 

Shen G, Adam Z, Zhang H. (2007) The E3 ligase AtCHIP ubiquitylates FtsH1, a component of the 
chloroplast FtsH protease, and affects protein degradation in chloroplasts. Plant Journal 

Reviewer #2 (Remarks to the Author): 

Comments on the manuscript NCOMMS-19-26321 

In their study” Mild proteasomal stress improves photosynthetic performance in Arabidopsis 
chloroplasts”, Julia Grimmer and her colleagues suggest that a slower degradation of functional 
protein precursors in the cytoplasm, which is caused by mild impairment of the proteasome 
function, would allow fine-tuned systemic regulation of organellar biogenesis and functions. 

The study has some fascinating parts. The rpn8axppi2 mutant, which the authors present in their 
study, is really intriguing. The loss of the rpn8a function in the environment of the ppi2 mutant has 
an amazing effect on restoring a part of the viability of the plant cell. The underlying mechanisms 
would definitely be interesting for both specialists in the field and a wider readership. 

Understanding double mutants is not easy because the mutations may have indirect effects that 
are not obvious. As for the rpn8axppi2 mutant, there would be a relatively easy model that could 
serve as an attempt for an explanation. Loss of the lid-subunit Rpn8a of the 26 S proteasome 
decreases the turnover of cytosolic precursors, which increases the pressure on the import system 
of the chloroplasts. That may allow more precursors to be imported into the chloroplasts of the 
rpn8axppi2 mutant than into those of the ppi2 mutant. The chloroplast can synthesize some 
thylakoids and the viability of the cells in the rpn8axppi2 mutant increases in comparison to the 
ppi2 mutant. That explanation would be easier to understand than a systemic regulatory effect 
through proteasome activity. The authors identify 18 intact cytosolic plastid precursors that are 
1.2 time higher expressed in the rpn8axppi2 mutant than in the ppi2 mutant, which is consistent 
with this explanation. That model would also work as a possible explanation for the observations 
for the pad1xppi2 double mutant. The conclusion that mild impairment of the proteasome function, 
would allow fine-tuned systemic regulation of organellar biogenesis and functions is not entirely 
obvious to me. 

Another point that puzzles me is the experiment with the proteasome inhibitor MG132. The 
inhibitor MG132 targets the 20S core of the 26S proteasome (reviewed by Momose and Watanabe, 
2017), while loss of the Rpn8a subunit affects the lid. It is not obvious to me that the cell stress 
that is caused by MG132 in the wildtype and the ppi2 mutant resembles the situation in the cells of 
the rpn8a and rpn8axppi2 mutants. The authors observe that both the rpn8a and rpn8axppi2 
mutants and the MG132 wildtype and ppi2 mutant have higher amounts of the 20S core, but they 
might have it for different reasons and assemble their proteasomes in different ways. The protein 
quantification by proteomics cannot give any detailed information about it, and therefore it is hard 
to tell what is really going on in the rpn8a and rpn8axppi2 mutants. 

Changing subjects, the text in the manuscript includes some jumps, and it might not be easy to 
read for readers who have not followed the field in detail for some time. For instance, it is not 
obvious what inspired the authors to study the loss of rpn8a function in the wildtype and the ppi2 
mutant of Arabidopsis. The rpn8axppi2 mutant and the rpn8a mutant appears in the manuscript a 
bit like a rabbit that jumps out of the hat of a magician. Studies on the function of the Rpn11-Rpn8 
heterodimer such as, for instance, Pathare et al. (2014) and Worden et al. (2014) might be 
worthwhile to mention? In general, if the authors would like to reach out to a wider readership, I 
would suggest to consider to make the manuscript more reader friendly and to give the 
presentation of the ideas behind the design of the experiments a clearer structure. On a side note, 
reference 15 is cited in a way that can be misleading. 

Switching topics and moving to some details of the experimental data, the Western blots shown in 
figure 1B do not look entirely excellent. The bands of Lhcb4.1 in the 50 and 100 microgram 
samples of the rpn8axppi2 mutant indicate transfer issues and some other bands show streaking. 
Are these Western blots really suitable for quantitation? As for figure 1 A and 2A, please explain 
the meaning of the stars. That applies also to all following figures. In addition, how is the rate of 
green plants in figure 2B defined and what do the light gray bars mean? 

As for figure 2C, I suggest a better presentation of the PAM-measurements. It is much squeezed 
together and it is difficult to see the zero values for the PSII quantum yields. Please, also explain 
what the red crosses mean. I also suggest to add the diagram for the electron transfer rate, which 
would not require any new experiments, as it can be extracted for the Pam-measurements that 
the authors already have performed. 

The datasets PXD014531 and PXD014560 would benefit from a better description of the datasets. 
One important point is that it is not always obvious from the names of the raw data files which 
samples were analyzed. The _Header files do not include this information either. Please add a 
sample key to the raw data files, which is not difficult to do. In addition, I suggest to include all 
relevant experimental details, which are needed to understand the proteomics experiments, in the 
description of the datasets PXD014531 and PXD014560 that it is possible to understand and use 
these datasets independently of the manuscript (FAIR data). In addition, I would like to ask why 
the authors did not submit the dataset of the ChaFRADIC experiment to ProteomeXchange? It 
seems that it would be worthwhile to do it. 

References 
Momose I, Watanabe T. Tyropeptins, proteasome inhibitors produced by Kitasatospora sp. MK993-
dF2. J Antibiot (Tokyo). 2017 May;70(5):542-550. doi:10.1038/ja.2017.9. 

Pathare GR, Nagy I, Śledź P, Anderson DJ, Zhou HJ, Pardon E, Steyaert J, Förster F, Bracher A, 
Baumeister W. Crystal structure of the proteasomal deubiquitylation module Rpn8-Rpn11. Proc 
Natl Acad Sci U S A. 2014 Feb 25;111(8):2984-9. doi: 10.1073/pnas.1400546111. 

Worden EJ, Padovani C, Martin A. Structure of the Rpn11-Rpn8 dimer reveals mechanisms of 
substrate deubiquitination during proteasomal degradation. Nat Struct Mol Biol. 2014 
Mar;21(3):220-7. doi: 10.1038/nsmb.2771.