PREreview of Enzymes degraded under high light maintain proteostasis by transcriptional regulation in Arabidopsis

The manuscript by Lei Li et al. reveals how plants maintain proteostasis under high light stress via a combined analysis of protein degradation rates, transcripts and proteins abundance in Arabidopsis. The authors performed a partial 13C labeling assay and identified 74 proteins with significant turnover rate changes in high light compared to standard light. Then they compared the transcriptional level and protein abundance of those 74 proteins and found negligible correlation between them, but a strong correlation between the turnover rate of the proteins encoded by nuclear genes and their transcripts. This study significantly advances the field of stress responses in plant biology with the findings of new direct or indirect targets of photodamage and how transcriptional processes counteract protein degradation to maintain proteostasis under high light.

Major comments

• Please provide qPCR data to verify the RNA-seq results on representative genes showing significant changes e.g. RH2A2A, FTSH8, PARG2, BCS1, PUB54 in Fig 2C. For Fig 2A, a Venn diagram or an intersection analysis would be more informative.
• Please describe in more detail how the LPF and especially PTO values were calculated based on the 13CO2 labeling experiment in the method.
• Please explain the lack of change in D1 accumulation in Fig 5B and provide D1 immunoblot for each time point. Also indicate the meaning of NA in the legend.
• In Fig 3, clarify the reasoning behind using the same peptide for THI1 and PIFI to calculate LPF in the two light conditions but different peptides for PSBA. Please provide an explanation in the text for calculating LPF using 2h HL for PSBA, 5h for THI1 and 8h for PIFI. What about the LPF from the same protein, such as PSBA, at different time points? Please provide an explanation of the absence of time points for PSBA, THI and PIFI.
• Line 209, please add a sentence to explain that you are assuming that the translation rates are similar for all the detectable proteins in your manuscript. Indeed if the translation rate is different in HL compared to normal light for a given protein, then this will affect its labeling and thus estimation of the degradation rate.
• Line 128-131, phenylalanine, tryptophan, and tyrosine are not abundant throughout high light treatment, and especially at 8h high light, they are back to the level in standard light. Rewrite these sentences to better reflect the results.
• Line 168, comment on down-regulated proteolytic pathways in cytosol.
• Abstract about plastid-encoded proteins, it should be noted that the distinction is made based on four observed proteins, do you think a generalization can be made for other plastid-encoded proteins?

Minor comments

• Fig 1, A, B, C, D, the Y-axis and ticks on the axes should be added for more readability; A,B, add x-axis legend D, Y-axis should start at 0.
• Line 118, do you mean that heat can induce NPQ by “contribute”? Please provide a reference and the leaf surface temperature measurements.
• In Fig2 C, define pink color for p-values.
• In Fig 3, it is difficult to distinguish the light green and dark green in the histogram. We suggest changing the color for the natural abundance (NA) or the newly synthesized peptides, label the x-axis and to use another acronym for "natural abundance".
• Line 211, "one-third to one-half". Three LPF are presented in standard light conditions, the lowest being 28.5% and the highest 41.2%, that’s not “one-half” or does this refer to other proteins with LPF of 50%? In that case, data is not presented. Clarify or include the data in Table S4.
• Line 216, how is the K_D value calculated?
• Line 235, it is difficult to identify PSBP in Fig 4. Please make it clearer.
• Please show your protein Coomassie Blue staining results from the in-gel digestion for MS as a supplementary figure to see the amount of total proteins compared to explain the variation shown in Fig S2A.
• Throughout text, make sure when you say "high light" to specify which time point (2h, 5h or 8h?).
• Line 301-303, ferredoxin thioredoxin reductase also showed a significant abundance decrease after 8 hours. Please comment this in the text.
• Line 344-347, the lower Fv/Fm level after longer high light exposure is not only due to the uncoupling of D1 degradation from its synthesis rate but also due to sustained NPQ forms such as qI (see Malnoë EEB 2018, doi:10.1016/j.envexpbot.2018.05.005).
• RNA-seq method: which fold-change threshold was selected to consider the candidates?  How many technical replicates were used?
• Line 352, you state that protein degradation is supported by up-regulation of protease gene expression, but what about their degradation rates? In Chlamydomonas, FtsH transcript is upregulated in high light but its rate of degradation is also faster resulting in a modest higher accumulation of the FtsH protease (see Wang et al. Mol Plant 2017, doi:10.1016/j.molp.2016.09.012).
• Line 374, you state that translation failed to keep pace with protein degradation, you could cite work on chloroplastic translation rate being affected by oxidation of translation factors in cyanobacteria (see Jimbo et al. PNAS 2019, doi:10.1073/pnas.1909520116).

Jianli Duan, Jingfang Hao  (Umeå University) - not prompted by a journal; this review was written within a preprint journal club with input from group discussion including Alizée Malnoë, Maria Paola Puggioni, André Graça, Aurélie Crepin, Pierrick Bru.