PtCP1 Is an Extraplastidial Cysteine Protease Involved in Leaf Protein Degradation of Populus tomentosa Carr

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript investigates the role of a papain-like cysteine protease (PtCP1) in the vegetative growth and photosynthetic efficiency of poplar. The authors demonstrate that PtCP1 is localized to the vacuole and plays a significant role in Rubisco turnover. Through CRISPR/Cas9-mediated knockout and gain-of-function lines, the study provides evidence that PtCP1-mediated proteolysis is a key regulator of carbon metabolism and biomass accumulation in woody plants. While the phenotypic data in this research are compelling, the functional analysis appears to rely heavily on a single representative line. To ensure that the reported phenotypes are consistent and not due to random variation, it is necessary to include data from additional independent lines.

Author Response

Comments 1: While the phenotypic data in this research are compelling, the functional analysis appears to rely heavily on a single representative line. To ensure that the reported phenotypes are consistent and not due to random variation, it is necessary to include data from additional independent lines.

 

Response 1: Thanks for this constructive comment. We agree that using multiple independent lines would further strengthen the conclusions. However, due to the lengthy growth cycle of poplar and the relatively low efficiency of stable transformation, we have so far obtained one homozygous CRISPR Cas9 knockout line (and one early matured line) that passed rigorous molecular validation. To address the possibility of random variation, we have expanded the analysis of our single representative line. This representative line has been characterized across at least three independent growth experiments, each with 15–20 biological replicates, and consistently exhibited the same phenotypic trends. Therefore, we are confident that the observed phenotype is indeed attributable to PtCP1 disruption rather than random variation.

Reviewer 2 Report

Comments and Suggestions for Authors

The article "PtCP1 is an extraplastidial cysteine ​​protease involved in leaf protein degradation of Populus tomentosa Carr" by Yawei Fan, Jingyi Han, Xiatong Liu, Han Liu, Mengyu Zhang, Xincaiyu Cui, Hui Li, and Hai Lu reviews data on the function and localization of cysteine ​​protease in poplar plant tissues.
The manuscript is formatted according to the guidelines and contains the necessary materials.
The main methodological flaw limiting this study is the choice of sample: poplars are dioecious plants and have differences in structure and morphology. This was not taken into account by the authors, despite the fact that sex identification in these plants is available using molecular and other methods.
This significantly limits the validity of the conclusions of this study, given the sample size. I believe this study is still valuable due to its original approach, but limitations should be mentioned in the discussion and conclusion.
Minor comments.
It's inappropriate to describe the article's results at the end of the introduction; they are included in the abstract, results, and conclusions. Formulate a hypothesis, aim, and objectives.
Check the figure captions—they should always indicate the object, its characteristics, and methods, as open-access journals require expanded information under the figures.
On page 275, is the gene expressed in the vacuole?
The diagram is rather questionable, as the authors have no evidence with immunogold, and this is neither a hypothesis nor a conjecture. First, there are many proteins in it besides Rubisco. Second, what does light have to do with it? Similar processes occur in root plastids. This diagram should either be removed or completely reworked and left in the appendix—at this stage, theorizing is premature.
The lack of a conclusion is apparently due to the lack of hypotheses, aims, and objectives? Review the data; it's still crude and, even if well executed, has a weak connection to the "hypothesis" and other studies. The sample should be clearly shown, taking into account randomization.
Expand the discussion of targets and mechanisms.

Author Response

Comments 1: The main methodological flaw limiting this study is the choice of sample: poplars are dioecious plants and have differences in structure and morphology. This was not taken into account by the authors, despite the fact that sex identification in these plants is available using molecular and other methods.

 

Response 1: We thank the reviewer for this comment. The poplar clone used in our study, “741” ([Populus alba × (P. davidiana + P. simonii) × P. tomentosa]), is a highly female‑sterile line（line 85）. Its female reproductive structures are largely non‑functional, and it produces virtually no viable seeds or cottony fluff. Therefore, any potential sexual dimorphism in resource allocation does not affect the non‑reproductive traits examined here (photosynthetic, cell expansion, etc.).

 

Comments 2: This significantly limits the validity of the conclusions of this study, given the sample size. I believe this study is still valuable due to its original approach, but limitations should be mentioned in the discussion and conclusion.

 

Response 2: Agree, a limitation paragraph has been added at the end of the Discussion, acknowledging the sample size constraint (single line per genotype). The added text is marked in gray（line 567-571）.

 

Comments 3: It's inappropriate to describe the article's results at the end of the introduction; they are included in the abstract, results, and conclusions. Formulate a hypothesis, aim, and objectives.

 

Response 3: Agree, we revised the last paragraph of the introduction. The revised text has been highlighted in gray （line 78-82）.

 

Comments 4: Check the figure captions—they should always indicate the object, its characteristics, and methods, as open-access journals require expanded information under the figures.

 

Response 4: Thanks. We have revised the caption of Figures accordingly, marking in gray shading. Line 252-253, 256, 314, 317-318, 322, 382.

 

Comments 5: On page 275, is the gene expressed in the vacuole?
The diagram is rather questionable, as the authors have no evidence with immunogold, and this is neither a hypothesis nor a conjecture. First, there are many proteins in it besides Rubisco. Second, what does light have to do with it? Similar processes occur in root plastids. This diagram should either be removed or completely reworked and left in the appendix—at this stage, theorizing is premature.

 

Response 5: We appreciate your comment. The localization of PtCP1 to the central vacuole is supported by immunogold labeling (Fig. 1G). We agree that the diagram was problematic and lacked sufficient evidence. Accordingly, we have removed it from the Discussion. Thanks for your suggestion.

 

Comments 6: The lack of a conclusion is apparently due to the lack of hypotheses, aims, and objectives? Review the data; it's still crude and, even if well executed, has a weak connection to the "hypothesis" and other studies. The sample should be clearly shown, taking into account randomization.
Expand the discussion of targets and mechanisms.

 

Response 6: Thanks, we have revised the manuscript according to your comments. Specifically, we have rewritten the last paragraph of the Introduction (line 78-82) to clearly state our hypothesis, aims, and objectives. We have also restructured the final paragraph of the Discussion (line 551-571) to provide a concise conclusion that directly links our findings back to the hypothesis, and we have expanded the discussion of targets (Rubisco) and mechanisms (vacuolar targeting, pH-dependent auto-activation, extraplastidial chloroplast protein degradation in non-senescing leaves). Regarding sample randomization, we have clarified in the Materials and Methods that all experiments used a completely randomized design with multiple biological replicates.

 

Reviewer 3 Report

Comments and Suggestions for Authors

Protein degradation, as well as their biosynthesis, are crucial for plant life. Papain-like cysteine ​​proteases (PLCPs) play a special role in this process. The authors identified PLCP from Populus tomentosa. It is synthesized as a biologically inactive enzyme but is activated at acidic pH. A mutant with an inactivated PLCP gene was obtained, which exhibited suppressed growth and reduced photosynthesis. Transgenic plants overexpressing this gene exhibited accelerated growth and improved photosynthetic parameters. In d7 plants, excessive accumulation of the large Rubisco subunit occurred, leading to impaired regulation of carbon metabolism, while in plants with enhanced PLCP accumulation, no impairments were observed. It was shown that PtCP1 proteolysis regulates photosynthetic carbon assimilation by altering Rubisco turnover, which positively impacts the vegetative growth of woody plants.

 This paper explores a very important question. It is beautifully executed, employing modern research methods and yielding fundamentally important results. There are no serious criticisms, but some corrections are necessary.

1. The authors generated and analyzed transcriptomes, but the method for obtaining them and assessing their quality is not provided. The methods section makes no mention of transcriptome generation. Before analyzing transcriptome data, the accuracy of the obtained results must be demonstrated. This stage of the work is called validation, and the authors are aware of this. Several genes must be selected and transcript levels determined using qRT-PCR in the main experimental setup and compared with the transcriptome data. If the results match, the transcriptome results can be analyzed. The validation results can be included either in the main text of the article or in the Suppl. Materials.
2. For all genes whose expression was determined by qRT-PCR, the primers used must be provided. Which genes served as reference genes?
3. For all genes studied, please provide the Locus number and a link to the gene database used.
4. Line 161, Fig. 1g, and others in the manuscript refer to organs, not tissues. Correct throughout the text.
5. Line 164, etc. 4°C, 10,000 rpm, 10. Specify the centrifuge type and rotor type.
6. Fig. 1d: Why did such a small portion of the protein convert to the mature form?
7. In Fig. 2C, the first leaf has a very poor actin control. What amount of protein was applied to each variant? Fig. 3J: Why is there such a large difference between the variants in the controls.
8. Line 131: Solution S is usually indicated by pH.
9. It would be a good idea to digitize the Western blot to quantify the results (Figs. 1d and 1m).

Author Response

Comments 1: The authors generated and analyzed transcriptomes, but the method for obtaining them and assessing their quality is not provided. The methods section makes no mention of transcriptome generation. Before analyzing transcriptome data, the accuracy of the obtained results must be demonstrated. This stage of the work is called validation, and the authors are aware of this. Several genes must be selected and transcript levels determined using qRT-PCR in the main experimental setup and compared with the transcriptome data. If the results match, the transcriptome results can be analyzed. The validation results can be included either in the main text of the article or in the Suppl. Materials.

 

Response 1: We appreciate the reviewer’s suggestion. We have now added a complete description of the transcriptome method in the revised Materials and Methods (subsection 2.13, highlighted in gray, line 205-222). In addition, qRT‑PCR validation of the RNA‑seq data has been performed and the results are provided in the Supplementary Materials Figure 2. The primer sequences and corresponding gene IDs are presented in Supplementary Table 1. Also, the corresponding description in the main text was added, highlighted in gray (line 457-459). We thank the reviewer for pointing out this oversight.

 

Comments 2: For all genes whose expression was determined by qRT-PCR, the primers used must be provided. Which genes served as reference genes?

 

Response 2: Thanks. Primer sequences for all qRT‑PCR genes have been added in Supplementary Table 1, and the use of Actin as the reference gene was stated in Materials and Methods (section 2.4, line 111). The revised text in 2.4 is highlighted in gray.

 

Comments 3: For all genes studied, please provide the Locus number and a link to the gene database used.

 

Response 3: We thank you for this request. For the primary gene of interest, PtCP1, the accession number (Gene ID: XM_006381559.1) has been provided in the revised manuscript at line 226. For the genes involved in sugar metabolism pathways mentioned in the transcriptome analysis (Fig. 4C), their protein accession numbers have been listed in the second column of Fig. 4C (from left to right). For those validated by qRT‑PCR in Supplementary Table 1, their locus numbers were added in Supplementary Table 1. We hope this fulfills the reviewer's requirement.

 

Comments 4: Line 161, Fig. 1g, and others in the manuscript refer to organs, not tissues. Correct throughout the text.

 

Response 4: Agree, the term “tissues” has been replaced with “organs” throughout line 258. All revisions are marked in gray.

 

Comments 5: Line 164, etc. 4°C, 10,000 rpm, 10. Specify the centrifuge type and rotor type

 

Response 5: Thank you. The centrifuge type (Beckman Allegra X‑30R) and rotor (F0850) have been added in the Methods section (line 162), and the text is now highlighted in gray.

 

Comments 6: Fig. 1d: Why did such a small portion of the protein convert to the mature form?

 

Response 6: We thank you for this perceptive question. In our in vitro experiment, the recombinant PtCP1 protein was expressed in E. coli and purified. The proenzyme (zymogen) was then incubated at pH 3.0 to induce autocatalytic processing into the mature form under acidic conditions. As shown in the figure, only a small proportion of the proprotein converted to the mature enzyme. We proposed that this was primarily due to the inherent inefficiency of autocatalytic activation in a simplified in vitro system. The conversion efficiency of papain‑like cysteine proteases is highly dependent on proper protein folding, correct disulfide bond formation, and optimal conformational transitions, which may not be fully achieved in a prokaryotic expression system lacking plant‑specific chaperones or post‑translational modifications. Additionally, the acidic activation is a pH‑dependent self‑cleavage event; factors such as precise pH value, incubation time, protein concentration, and ionic strength can all affect the yield of the mature form. Despite the low conversion rate, the clear appearance of the mature band confirms that PtCP1 possessed the intrinsic ability for pH‑dependent autocatalytic activation, which was consistent with the canonical behavior of papain‑like cysteine proteases. The results reported here were therefore qualitative evidence of activation competence rather than quantitative measurements of processing efficiency.

 

Comments 7: In Fig. 2C, the first leaf has a very poor actin control. What amount of protein was applied to each variant? Fig. 3J: Why is there such a large difference between the variants in the controls.

 

Response 7: We thank the reviewer for pointing this out. We confirm that for all samples shown in Fig. 2C and Fig. 3J, equal amounts of total protein were loaded. Protein extraction was performed from identical dry weight of leaf tissue, and total protein concentration was quantified using a Bradford assay before loading. The same amount was loaded per lane.We are aware that the actin signals do not appear perfectly uniform. Although actin is used as a loading internal reference, its expression level is not absolutely constant across different transgenic events or physiological states and may show modest biological fluctuation. Importantly, the key observations—the accumulation of RbcL fragments and the maturation pattern of PtCP1—were highly reproducible across multiple independent biological replicates and different exposure times. We have tried for times, the data shown here are the best results we obtained, and we have tried our best.

 

Comments 8: Line 131: Solution S is usually indicated by pH.

 

Response 7: Thanks, we have added “pH 8” in the revised manuscript (line 130). The corrected text is highlighted in gray.

 

Comments 9: It would be a good idea to digitize the Western blot to quantify the results (Figs. 1d and 1m).

 

Response 9: We appreciate your suggestion. Fig. 1d was a qualitative experiment to confirm the acidic activation of PtCP1, so quantification was not performed. For Fig. 1m, we have now quantified the bands and added the results as a bar graph in Supplementary Figure S1.

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

The comments have been taken into account and the errors have been corrected. The article may be accepted for publication.