Review Reports - Integrated Transcriptomic and Metabolomic Insights into the Molecular Mechanisms of Albino Leaf Formation in Sweetpotato

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This manuscript investigates the molecular mechanisms of albino leaf formation in sweetpotato using integrated transcriptomic and metabolomic profiling. The study confirms strong repression of chlorophyll/carotenoid biosynthesis genes, structural chloroplast defects, and major metabolic shifts. The topic is relevant, experiments are well-chosen, and multi-omics integration is a strength.

However, there are important concerns that need to be addressed:

1/ The mutant is said to differ “solely in leaf coloration”, yet metabolic pathways of defense (camalexin, indole phytoalexins) are discussed. This means stress response is not a direct control variable, and the concept of "sole difference" must be softened or removed.

2/ Please clarify if other tissues were checked for albinism (roots, young shoots, stem cross-sections). Albino phenotypes can sometimes be tissue-dependent.

3/ Statistical test used for Table 1 pigment contents (ANOVA? t-test?) and justify sample size vs variance (mutant SD=0.9 is very high relative to mean 1.3).

4/ Mutant Fv/Fm = 0.59 suggests 30% PSII reduction, but such verbal approximations should be replaced by exact % or statistical interpretation from the test.

5/ The very low Fo value cannot be compared only descriptively. You must provide statistical evidence for the reduction in Fo and the decline in RC density.

6/ Python v2.7.6 and CentOS v6.6 are obsolete. If the analysis was done more recently, update; otherwise, justify.

7/ Fastp cited is version 0.18.0, but the reference shown at the end is Fastp 2018 without explicit version mapping. Make sure versions in text = versions in citations.

8/ UHPLC gradient lines conflict: in “2-85% B, 3 min; 85-100% B, 10 min; 100-2% B, 10.1 min”, this yields a plateau before it starts to fall. A gradient cannot remain at 100% from 10-10.1, then suddenly 2%. Please verify the gradient timing or rewrite it clearly.

9/ The Discussion section could be broadened to include comparative evidence on stress physiology and metabolite reprogramming reported in other crops, in particular: https://doi.org/10.1016/j.jaridenv.2023.105095

10/ Minor grammatical typos were spotted, and the references should be reviewed for consistency in formatting.

Comments on the Quality of English Language

Minor grammatical typos were spotted

Author Response

The mutant is said to differ “solely in leaf coloration”, yet metabolic pathways of defense (camalexin, indole phytoalexins) are discussed. This means stress response is not a direct control variable, and the concept of "sole difference" must be softened or removed. Response: Thank you for pointing this out. We agree that the original statement was overly strong. The sentence has been revised accordingly in the Materials & Methods section (Section 2.1).
Please clarify if other tissues were checked for albinism (roots, young shoots, stem cross-sections). Albino phenotypes can sometimes be tissue-dependent. Response: We appreciate this suggestion. Young shoots, stem cross-sections, and roots were examined, and all exhibited lack of pigmentation. This clarification has been added (Section 2.1).
Statistical test used for Table 1 pigment contents (ANOVA? t-test?) and justify sample size vs variance (mutant SD=0.9 is very high relative to mean 1.3). Response: A two-tailed Student’s t-test (n = 3) was applied. Although the mutant exhibited high SD due to extremely low pigment levels, all differences remained highly significant (p < 0.01). This information has been added (Section 2.2).
Mutant Fv/Fm = 0.59 suggests 30% PSII reduction, but such verbal approximations should be replaced by exact % or statistical interpretation from the test. Response: As suggested, the description now states: “Fv/Fm decreased by 29.8% relative to WT” (Section 3.1).
The very low Fo value cannot be compared only descriptively. You must provide statistical evidence for the reduction in Fo and the decline in RC density. Response: We performed statistical analyses for Fo and related JIP parameters (Table 2). All showed significant reductions (p < 0.01), and the results have been incorporated into the manuscript (Section 3.1).
Python v2.7.6 and CentOS v6.6 are obsolete. If the analysis was done more recently, update; otherwise, justify. Response: Thank you for raising this point. Although the vendor’s pipeline originally used Python 2.7 and CentOS 6.6, the underlying statistical algorithms remain unchanged. All downstream analyses were revalidated using updated environments (Python 3.9/CentOS 7).
Fastp cited is version 0.18.0, but the reference shown at the end is Fastp 2018 without explicit version mapping. Make sure versions in text = versions in citations. Response: Thank you for pointing this out. Fastp v0.18.0 was released on July 5, 2018, and its version number does not correspond to the release year (https://github.com/OpenGene/fastp/releases?page=3).
UHPLC gradient lines conflict: in “2-85% B, 3 min; 85-100% B, 10 min; 100-2% B, 10.1 min”, this yields a plateau before it starts to fall. A gradient cannot remain at 100% from 10-10.1, then suddenly 2%. Please verify the gradient timing or rewrite it clearly. Response: Thank you for identifying this issue. The UHPLC gradient description has been rewritten to accurately reflect the conditions used: 95% B, 0-0.5 min; 95-65% B, 0.5-7 min; 65-40% B,7-8 min; 40% B, 8-9min;40-95% B, 9-9.1min;95%B,9.1-12min (Section 2.5).
The Discussion section could be broadened to include comparative evidence on stress physiology and metabolite reprogramming reported in other crops, in particular: https://doi.org/10.1016/j.jaridenv.2023.105095 Response: We have carefully revised the fourth paragraph of the Discussion section accordingly. The updated paragraph now explicitly compares the metabolic reprogramming observed in our albino sweetpotato mutant with stress-induced responses reported in various crops under drought and salinity. Specifically, we highlight the shared upregulation of stress-protective metabolites (e.g., phenylpropanoid precursors, osmoprotectants, and antioxidants) and enhanced antioxidant enzyme activities, using argan tree under salinity as a representative example [37], alongside the broader patterns summarized in Seleiman et al. (2023).
Minor grammatical typos were spotted, and the references should be reviewed for consistency in formatting. Response: The manuscript has been carefully checked, and the reference list has been standardized according to MDPI guidelines.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Dear Editors and Authors,

I read with interest the manuscript entitled "Integrated transcriptomic and metabolomic insights into the molecular mechanisms of albino leaf formation in sweetpotato". In this study, it was identified a naturally occurring sweetpotato leaf albino mutant characterized by a stable white-leaf phenotype throughout development. To uncover the molecular mechanisms underlying this distinct phenotype, integrated transcriptomic and metabolomic analyses were conducted comparing the albino mutant with wildtype leaves. The subject of the article is important and has great relevance for the scientific environment of the study area. Therefore, the manuscript needs some adjustments so that it can then be forwarded to the publication process. The manuscript has the potential for publication in this journal Horticulturae and needs the following adjustments:

ABSTRACT

Line 25–28: I suggest including at least one central quantitative result, such as "percent reduction in total chlorophyll, Fv/Fm value, or number of most relevant DEGs/DAMs."

Line 33–36: I suggest specifying which classes of secondary metabolites were most impacted (flavonoids, phenylpropanoids), avoiding overly generic formulation.

Line 37–38: I suggest explicitly mentioning key regulatory genes (GLK1, PORA/PORB) as prime candidates associated with the albino phenotype.

Replace keywords that are repeated in the title

INTRODUCTION

Row 46–50: I suggest reinforcing the knowledge gap specific to Ipomoea potatoes by differentiating studies on model plants from those on hexaploid crops.

Line 57–69: Add a paragraph of conceptual integration, connecting pigments, chloroplast development, and secondary metabolism.

Line 73–75: Broaden the discussion on the agronomic and ornamental relevance of albino mutants as physiological and genetic tools, even when unfeasible for production.

MATERIAL AND METHODS

Line 89–91: I suggest clarifying whether the albino mutant is phenotypically stable over successive vegetative propagations and whether it presents intra-plant variation.

Line 105–110: Briefly discuss the detection limits of the spectrophotometric method, considering the extremely low levels of pigments in the mutant.

Line 126–127: I suggest including additional JIP-test parameters (e.g., PI_ABS, ABS/RC, DI₀/RC) to further characterize photosynthetic involvement.

Line 151–153: Justify the use of the Human Metabolome Database (HMDB) in plant study or discuss potential limitations in metabolic annotation.

Line 155–157: Indicate whether permutation tests have been performed on the OPLS-DA, considering the high Q² values observed.

Line 178–179: Briefly comment on the analytical challenges associated with the hexaploid genome of sweet potatoes and possible effects on the distinction of paralogous genes.

RESULTS AND DISCUSSION

Line 228–231: Discuss whether the ultrastructural defects seen in chloroplasts represent the primary cause or consequence of pigmentary deficiency.

Line 233–240: I suggest integrating OJIP data with strong repression of photosynthetic genes, strengthening the coherence between physiology and transcriptome.

Line 271–274: I suggest discussing the direct physiological relevance of the major DAMs highlighted and their connections to chlorophylls, pigmentary degradation, or stress.

Line 275–279: Separate primary and secondary metabolism more clearly, possibly with an integrative figure.

Line 295–300: Discuss whether the activation of defense pathways reflects a compensatory response to photo-oxidative stress, resulting from the loss of functional chloroplasts.

Line 324–339: To further discuss possible upstream transcriptional regulators as central causes of coordinated repression of pigment pathways.

Line 330–333: Critically comment on the fact that degradation genes are also repressed, suggesting global failure of plastid development.

Line 353–355: Compare the mutant studied with other types of albinos already described in the literature.

CONCLUSIONS

Line 426–431: Reinforce that the identification of the causal mutation (by WGS or genetic mapping) is necessary to consolidate the proposed molecular model.

Line 432–437: Discuss the potential of the mutant as an experimental model for dissociation between photosynthesis and secondary metabolism.

Line 441–444: Emphasize more clearly the originality of the study, highlighting the transcriptomic-metabolomic integration in sweet potatoes.

Author Response

ABSTRACT Line 25–28: I suggest including at least one central quantitative result, such as "percent reduction in total chlorophyll, Fv/Fm value, or number of most relevant DEGs/DAMs." Response: Thank you for this recommendation. We added key quantitative data—98.8% chlorophyll reduction, Fv/Fm = 0.59, 3,520 DEGs, and 270 DAMs—to strengthen the Abstract.

Line 33–36: I suggest specifying which classes of secondary metabolites were most impacted (flavonoids, phenylpropanoids), avoiding overly generic formulation. Response: As suggested, we now explicitly mention flavonoids and phenylpropanoids as the most affected classes (Line 32).

Line 37–38: I suggest explicitly mentioning key regulatory genes (GLK1, PORA/PORB) as prime candidates associated with the albino phenotype. Response: We appreciate this valuable suggestion. In the revised Abstract, we incorporated the names of key regulatory genes (GLK1, PORA, PORB) that were strongly repressed in the mutant.Highlighting these genes increases the mechanistic depth of the summary and clearly links chloroplast development pathways to the observed phenotype.

Replace keywords that are repeated in the title Response: Thank you for pointing this out. Keywords duplicated from the title were replaced with more specific alternatives (e.g., “chloroplast development,” “multi-omics integration”).

INTRODUCTION

Row 46–50: I suggest reinforcing the knowledge gap specific to Ipomoea potatoes by differentiating studies on model plants from those on hexaploid crops. Response: We thank the reviewer for this valuable suggestion. We have revised the first paragraph of the Introduction to clearly distinguish leaf color research in (1) diploid model species, (2) allotetraploid/allohexaploid crops, and (3) autohexaploid sweetpotato, which remains almost unstudied due to its high genomic redundancy. This revision explicitly highlights the unique knowledge gap in sweetpotato that justifies our work.

Line 57–69: Add a paragraph of conceptual integration, connecting pigments, chloroplast development, and secondary metabolism. Response: We thank the reviewer for this helpful suggestion. To provide the requested conceptual integration, we have added a short integrative section at the end of the paragraph. This addition explicitly connects the three processes by illustrating how chloroplast structural defects trigger retrograde signaling that coordinately represses photosynthetic pigments and activates secondary metabolic pathways.

Line 73–75: Broaden the discussion on the agronomic and ornamental relevance of albino mutants as physiological and genetic tools, even when unfeasible for production. Response: Thank you for your suggestion. We have revised and expanded the paragraph to explicitly emphasize the high scientific value of severe leaf color mutants as powerful physiological and genetic tools for dissecting chlorophyll biosynthesis, chloroplast development, retrograde signaling, and homoeologous gene dosage effects in autohexaploid sweetpotato—even though these mutants are themselves unsuitable for field production.

MATERIAL AND METHODS

Line 89–91: I suggest clarifying whether the albino mutant is phenotypically stable over successive vegetative propagations and whether it presents intra-plant variation. Response: Thank you for your suggestion. To address this point, we have added the following clarification in the Materials and Methods section: “The albino phenotype was visually stable across multiple vegetative generations, presenting in leaves, stems, young shoots, and roots, showing no sectorial or tissue-specific variation”. (Section 2.1)

Line 105–110: Briefly discuss the detection limits of the spectrophotometric method, considering the extremely low levels of pigments in the mutant. Response: We thank for pointing this out. Although trace pigment readings were recorded in the albino mutant (1.3 ± 0.9 mg/100 g FW chlorophyll a+b and 0.9 ± 0.05 mg/100 g FW carotenoids), these values fall within or very close to the detection/quantification limit of the 80% acetone spectrophotometric method under our exact conditions (≈0.8–1.5 mg/100 g FW for total chlorophyll and ≈0.5–1.0 mg/100 g FW for carotenoids). (Section 2.2)

Line 126–127: I suggest including additional JIP-test parameters (e.g., PI_ABS, ABS/RC, DI₀/RC) to further characterize photosynthetic involvement. Response: We have revised the “Fast Chlorophyll Fluorescence (OJIP) Measurement” section to explicitly list additional key JIP-test parameters, including PI_ABS, ABS/RC, DI₀/RC, which were already calculated by the Handy-PEA software.

Line 151–153: Justify the use of the Human Metabolome Database (HMDB) in plant study or discuss potential limitations in metabolic annotation. Response: We added a statement explaining that HMDB was used primarily for annotating common primary metabolites and lipid molecules that are shared between plants and other organisms. We also acknowledged that HMDB has certain limitations for exclusively plant-specific metabolites, which is why it was used in combination with plant-oriented databases. (Section 2.5)

Line 155–157: Indicate whether permutation tests have been performed on the OPLS-DA, considering the high Q² values observed. Response: We agree that high Q² values in OPLS-DA require validation to rule out potential overfitting. In the revised manuscript, we now clarify that a 200-time permutation test was performed in SIMCA to assess the reliability of the OPLS-DA model. The permuted R² and Q² intercepts were markedly lower than those of the original model, indicating that the model is statistically robust and not overfitted.

Line 178–179: Briefly comment on the analytical challenges associated with the hexaploid genome of sweet potatoes and possible effects on the distinction of paralogous genes. Response: Thank you for your suggestion. To address this point, we have added a new paragraph in the Discussion section (in the revised manuscript with tracked changes) that explicitly discusses the analytical challenges posed by the hexaploid nature of sweet potato and its implications for distinguishing paralogous genes.

RESULTS AND DISCUSSION

Line 228–231: Discuss whether the ultrastructural defects seen in chloroplasts represent the primary cause or consequence of pigmentary deficiency. Response: Thank you for the insightful suggestion. We have revised the paragraph to clearly address whether the observed ultrastructural defects represent a primary cause or a downstream consequence of pigment deficiency. We added a discussion indicating that the severe and early-stage defects in chloroplast organization—such as vesiculated thylakoids and reduced stroma density—are more consistent with impaired chloroplast biogenesis rather than degeneration caused by pigment loss. This clarification strengthens the interpretation that defective plastid development is the primary driver of the albino phenotype.

Line 233–240: I suggest integrating OJIP data with strong repression of photosynthetic genes, strengthening the coherence between physiology and transcriptome. Response: Thank you for your suggestion. We fully agree that linking OJIP fluorescence data to the biological processes underlying photosynthesis improves the coherence of the Results. As suggested, we have revised the paragraph to highlight that the impaired PSII efficiency and altered electron transport dynamics revealed by OJIP analysis are characteristic of a physiological state in which photosystem assembly and light-harvesting processes are strongly compromised. To preserve the logical flow of the manuscript, the detailed molecular evidence supporting this interpretation is presented in Discusstion sections. This adjustment strengthens the connection between physiology and downstream mechanisms while maintaining the organizational structure of the Results.

Line 271–274: I suggest discussing the direct physiological relevance of the major DAMs highlighted and their connections to chlorophylls, pigmentary degradation, or stress. Response: Thank you for this valuable suggestion. We agree that interpreting the biological relevance of the major DAMs improves the clarity and depth of the metabolomic analysis. In the revised manuscript, we expanded the description of the key metabolites enriched in the mutant and linked them to known physiological processes. Specifically, we now discuss how the accumulation of phenylalanine and phenylacetaldehyde reflects enhanced flux toward the phenylpropanoid/stress-response pathway, likely compensating for impaired photosynthetic activity. We also note that Dl-ethionine is associated with metabolic stress and may indicate disrupted amino acid homeostasis. Additionally, we clarified that the reduced abundance of pheophorbide a—a chlorophyll-degradation intermediate—supports the interpretation that chlorophyll deficiency in the mutant is caused by impaired biosynthesis rather than increased degradation. These additions provide clearer physiological context for the DAMs and strengthen the connection between metabolite changes, pigment phenotypes, and stress responses.

Line 275–279: Separate primary and secondary metabolism more clearly, possibly with an integrative figure. Response: Thank you for your valuable suggestion. Figure 3E already clearly highlights the key primary metabolic pathways that directly account for the reduced chlorophyll content and yellow-leaf phenotype in the mutant. Adding an integrative figure to further separate primary and secondary metabolism would not alter our core conclusions.

Line 295–300: Discuss whether the activation of defense pathways reflects a compensatory response to photo-oxidative stress, resulting from the loss of functional chloroplasts. Response: Thank you very much for this insightful suggestion. Following your comment, we have expanded the Discussion section to directly address whether the activation of defense pathways represents a compensatory response to photo-oxidative stress. As newly added in the Discussion, we now explain that the loss of functional chloroplasts in the albino mutant likely leads to increased ROS accumulation, which in turn triggers the upregulation of defense- and detoxification-related metabolic pathways. This interpretation integrates the transcriptomic evidence with the physiological context of impaired chloroplast function, providing a more coherent mechanistic explanation for the observed gene expression patterns.

Line 324–339: To further discuss possible upstream transcriptional regulators as central causes of coordinated repression of pigment pathways. Response: Thank you very much for this valuable suggestion. We have revised the manuscript to include a concise discussion of potential upstream transcriptional regulators. Specifically, we now highlight the reduced expression of IbGLK1 and other chloroplast-associated regulators, and explain how this regulatory impairment may underlie the coordinated repression of pigment biosynthetic pathways. (Paragraph 9 of Discussion section)

Line 330–333: Critically comment on the fact that degradation genes are also repressed, suggesting global failure of plastid development. Response: Thank you for this helpful suggestion. We agree that the simultaneous repression of chlorophyll biosynthesis and degradation genes warrants critical discussion. We have revised the manuscript accordingly to emphasize that this pattern suggests a global failure of plastid development rather than a pathway-specific effect. The newly added explanation has been incorporated into both the Results and Discussion sections.

Line 353–355: Compare the mutant studied with other types of albinos already described in the literature. Response: Thank you for this valuable suggestion. We have revised the Results section to include a comparative statement highlighting that the coordinated repression of photosystem-related genes observed in our sweetpotato mutant is consistent with transcriptomic patterns reported in albino mutants of Hydrangea macrophylla and Cymbidium longibracteatum. This addition clarifies how the mutant aligns with well-characterized albino phenotypes in other species.

CONCLUSIONS

Line 426–431: Reinforce that the identification of the causal mutation (by WGS or genetic mapping) is necessary to consolidate the proposed molecular model. Response: Thank you for this suggestion. We have revised the paragraph to more clearly emphasize that identifying the causal mutation is essential for validating the proposed molecular framework. Specifically, we added statements highlighting the need for whole-genome resequencing or genetic mapping to pinpoint the responsible allele and reinforce the necessity of functional verification through CRISPR/Cas9.

Line 432–437: Discuss the potential of the mutant as an experimental model for dissociation between photosynthesis and secondary metabolism. Response: We appreciate this suggestion. To address it, we expanded the paragraph to explicitly discuss how the mutant can function as a model for dissecting the dissociation between photosynthesis and secondary metabolism. We highlighted that the severe loss of chloroplast function, combined with persistent or enhanced secondary metabolic activity, creates a unique biological context for exploring how metabolic fluxes are reorganized in plants lacking photosynthetic capacity. These additions clarify the broader scientific value of the mutant beyond phenotypic description.

Line 441–444: Emphasize more clearly the originality of the study, highlighting the transcriptomic-metabolomic integration in sweet potatoes. Response: Thank you for this suggestion. We revised this paragraph to more explicitly emphasize the novelty of the study, stating that this is the first work to integrate transcriptomic and metabolomic profiling to investigate albinism in sweetpotato. We further highlighted the significance of this multi-omics approach in providing a systems-level understanding of chloroplast dysfunction in a hexaploid species.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors have carefully reviewed and rigorously addressed all the comments. Each point was carefully considered, and the manuscript was substantially revised. The revisions demonstrate a strong commitment. Therefore, the manuscript can now be considered suitable for publication.