Perturbation of protein homeostasis brings plastids at the crossroad between repair and dismantling

The chloroplast proteome is a dynamic mosaic of plastid- and nuclear-encoded proteins. Plastid protein homeostasis is maintained through the balance between de novo synthesis and proteolysis. Intracellular communication pathways, including the plastid-to-nucleus signalling and the protein homeostasis machinery, made of stromal chaperones and proteases, shape chloroplast proteome based on developmental and physiological needs. However, the maintenance of fully functional chloroplasts is costly and under specific stress conditions the degradation of damaged chloroplasts is essential to the maintenance of a healthy population of photosynthesising organelles while promoting nutrient redistribution to sink tissues. In this work, we have addressed this complex regulatory chloroplast-quality-control pathway by modulating the expression of two nuclear genes encoding plastid ribosomal proteins PRPS1 and PRPL4. By transcriptomics, proteomics and transmission electron microscopy analyses, we show that the increased expression of PRPS1 gene leads to chloroplast degradation and early flowering, as an escape strategy from stress. On the contrary, the overaccumulation of PRPL4 protein is kept under control by increasing the amount of plastid chaperones and components of the unfolded protein response (cpUPR) regulatory mechanism. This study advances our understanding of molecular mechanisms underlying chloroplast retrograde communication and provides new insights into cellular responses to impaired plastid protein homeostasis.

plastid-encoded genes are represented in the library and their differential expression cannot be inferred from our transcriptome data.
3. PRPS1 as a member of plastid ribosome, the amount of PRPS1 protein was significantly reduced when the indPRPS1 plants infiltrated in presence of DEX for 6 hours. I think the chloroplast may not be under the normal condition, the transcription level and translation level still remained unchanged? And the author also investigated the possible negative effect of PRPS1 inducible expression on plastid protein translation. In this paper, I think the transcription level and protein level of chloroplast-encode genes should be shown because the authors aimed to describe the effect of perturbance chloroplast protein homeostasis.  (Fig. 4D). Furthermore, an alteration of chloroplast-related proteins emerges from the proteomic investigation. As reported (see Tables S7, S8 and the corresponding text in the Results section), among over-represented GO terms in down-accumulated proteins we found "chloroplast stroma" (GO:0009570) and "chloroplast organization" (GO:0009658). In the latter category, we observed reduced amounts of proteins, such as the chloroplast-localized nifu-like protein 2 (AT5G49940), the plastid transcriptionally active 15 (mTERF8; AT5G54180) and a close homolog of the Cauliflower OR (AT5G61670), which suggest a possible impairment of chloroplast functions.
4. In the Fig4D, the contents of RbcL and D1/D2 showed a big difference in vivo. The blots may be over exposed in order to quantitative analysis. The author can check the two proteins in different blot. 5. The author think PRPS1 accumulation is negatively regulated by chloroplast Clp protease complex. The accumulation of PRPS1 protein increased about two-fold in all the double mutants tested with respect to prps1-1. In the Fig6C, the CBB stain is not precise in my eyes. Thought I believe the results, the authors should check the results of protein quantification by examining gene Actin.
Reply: To address this point we used the nuclear-located histone H3 as loading control, since it appears to be a better control than Actin in mutants with a marked leaf phenotype, where defects in cell cytoskeleton cannot be excluded (see new Fig. 6).
6. As for the overexpression PRPS1 material, I am also curious the protein level of PRPS1 was dramatically decreased. This point should be clarified. One point is that if all PRPPS1 proteins can enter the chloroplast, or part of them stay in the cytoplasm and then be degraded. Or is it likely the transgenetic plants carry a second mutation?

Reply: To address this point and to exclude the side-effects of T-DNA insertion itself, data from independent over-expressor and inducible PRPS1 lines have been provided (see new Fig. S1).
Moreover, in oePRPS1 lines, PRPS1 protein accumulates in the chloroplast fraction only (Fig. S8), while there is no trace of it in extra-plastid fractions, suggesting that PRPS1 is imported in the plastid before degradation occurs. To strengthen both these points, we crossed oePRPS1 with clpc1-1 and clpd-1 mutants, lacking two subunits of CLP plastid-located protease complex, showing that the accumulation of PRPS1 protein is partially recovered, pointing to a major role CLP in plastid degradation of PRPS1 protein (Fig. S7). Similarly, the increased accumulation of PRPS1 protein could be observed upon short-term induction of PRPS1 in clpc1-1 and clpd-1 mutant backgrounds, when compared to indPRPS1 +DEX samples (Fig. 6 D and Fig. S7 A). Finally, no peptides from PRPS1 cTP could be detected in our proteomics studies. Overall, this set of evidences point to a complete import of PRPS1 protein into the chloroplast stroma, where its abundance is modulated by the CLP protease complex, as part of the the plastid proteostasis machinery.

Reviewer #3
Major points: 1. The lack of genetic material makes the genetic data of this manuscript unconvincing. The transgenic plants (oePRSP1, indPRPS1, oePRPL4 and indPRPL4) used in this paper have only one line respectively. Please provide other independently transformed transgenic lines and their corresponding data, including the phenotypes and the expression levels of relevant RNA and protein.
Reply: To address this point, data from three independent over-expressor and inducible lines for each genotype have been added. Visible phenotypes, photosynthetic efficiency, expression of PRPS1 and PRPL4 at transcript and protein level are now shown in the new Fig. S1. Moreover, several oePRPS1 lines have been already described in literature (Yu et al. 2012 andTadini et al. 2016). The text referring to the new Fig S1 has been added in the paragraph "PRPS1 over-expression impairs chloroplast activity and biogenesis" of the Result section and in the legend of Fig. 2. Additionally, a new Fig. S1 and the corresponding legend has been added to the supplementary figure section.
2. The authors concluded that "the accumulation of PRPS1 is negatively regulated by chloroplast CLP protease complex" by comparing the accumulation of PRPS1 in the prps1-1 mutant and prps1-1 clp double mutants. prps1-1 is a knockdown mutant of PRPS1, and the increased amount of PRPS1 protein in prps1-1 clp double mutants is not sufficient to draw this conclusion. Why is the accumulation of PRPS1 protein not higher in clp mutants than in wild-type plants? This problem can be better explained if indPRPS1 is crossed with the clp mutants.
Reply: To address this concern, two different indPRPS1 transgenic lines have been crossed with clpc1-1 and clpd-1 mutant backgrounds and the accumulation of PRPS1 protein was tested upon DEX induction (0-24 h) (see Fig. 6 and Fig. S7). Compared to indPRPS1 lines, in which PRPS1 accumulation decreases over time, PRPS1 accumulation level remained rather stable upon induction in plastids with defects in the CLP-mediated protein degradation. Similarly, oePRPS1 clpc1-1 and oePRPS1 clpd-1 lines showed higher PRPS1 accumulation and partial rescue of the photosynthetic phenotype when compared with oePRPS1 plants (Fig. S7), corroborating the notion that the CLP protease is responsible for PRPS1 degradation.
3. To investigate the molecular responses to the increased expression of PRPS1 gene, transcriptome and proteome analysis was performed on leaf discs harvested from indPRPS1 that were vacuum infiltrated for 6 hours in either the absence or presence of DEX. As shown in Figure 4, after 6 hours DEX induction, the RNA expression level of PRPS1 increased 50-fold, while the protein level of PRPS1 decreased to 40%. Therefore, it is difficult to tell whether the transcriptomic and proteomic results are caused by increased PRPS1 gene expression or decreased PRPS1 protein content. Figure  2 showed that compared to WT, the expression level of PRPS1 gene is 20% and the protein content is 45% in prps1-1 mutant. If the prps1-1 mutant is added as a reference in transcriptome and proteome analysis, it will be helpful to draw the correct conclusion.
Reply: While we agree with R#3 that this point needs a clarification, we do not think that the strategy he/she suggested provide the desired information. Indeed, the comparative analysis of indPRPS1, where the transient over-expression PRPS1 is triggered, with prps1-stable lines, in which PRPS1 expression is reduced throughout the entire plant life cycle, represent two completely different experimental setups difficult to be compared. However, to clarify the reviewer's concerns, we have analyzed the accumulation of TULP5 and SWEET13 (see new Fig. S14) transcripts (highly upregulated in indPRPS1 in response to DEX) by comparing their expression indPRPS1 + DEX, in which the up-regulation of PRPS1 transcript is followed by PRPS1 degradation, to indPRPS1 clpd-1 + DEX, in which PRPS1 degradation is inhibited, despite similar PRPS1 transcript upregulation (Fig 6 and S7). Interestingly, TULP5 and SWEET13 transcripts did not over-accumulate in the absence of a functional CLP protease, indicating that the re-orchestration of nuclear gene expression requires the accumulation/degradation of PRPS1 protein (Fig. S14). Similarly, transgenic lines in which the prps1-2 null allele is either under the control of CaMV35S promoter or the inducible promoter (see Materials and Methods) in Col-0 wild-type background did not show any virescent phenotype, supporting the notion that, in order to trigger its own degradation, the transient overaccumulation of PRPS1 protein is required (Fig. S14).
Minor points: 4. In Figure 2, the alteration of PRPS1 expression has different effects on true leaves and cotyledons. Please explain it.
Reply: Chloroplasts in cotyledons and true leaves were often found to behave differently in mutants lacking factors involved in plastid biogenesis, protein homeostasis and degradation (Chen et al. 2000;Albrecht et al. 2006;Jeran et al. 2020;Tadini et al. 2020). Thus, it is possible that alterations in PRPS1 homeostasis has different impacts on the two organs. This aspect has been highlighted in the discussion section.

Reply: Information related to the Arabidopsis prps1-1 mutant was already included in the Materials
and Methods section and in the Introduction section together with the corresponding reference (Romani et al. 2012). A further piece of information related to the position of T-DNA insertion responsible for the prps1-1 knock-down allele has been added in the Introduction section.
6. There are three pathways of chloroplast degradation: senescence-associated vacuoles (SAVs), chloroplast vesiculation, and autophagy. In order to accurately analyze chloroplast degradation in Figure 3, marker lines of different degradation pathways should be added as controls, or marker genes of different degradation pathways should be comprehensively examined.
Reply: The expression level of several genes involved in different vacuole-mediated chloroplast degradation pathways was already reported in the former Fig. S1 that in the current version of the manuscript is indicated as Fig. S2. We believed to have provided a comprehensive picture of the chloroplast degradation mechanisms by including genes involved in ATG-dependent andindependent pathways. However, the SAV pathway (Izuma and Nakamura, 2018) that was not considered in the first version of the manuscript, was now investigated by monitoring the expression of SAG12 gene. However, the SAG12 transcripts were below the limit of detection, probably because the SAV pathway is mainly activated during senescence rather than upon stress conditions. 7. Please explain the calculation method of Figure 4F.