Transcriptome Analysis and Functional Validation of JAZ Subfamily Genes of Sesuvium portulacastrum Under Salt and Cadmium Stresses

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

To author:

The research holds potential application value for saline-alkali land remediation, plant stress-resistant breeding, and environmental restoration, offering a novel perspective. I agree that these are exciting research topics. However, the research work in the manuscript was not systematic enough. There are significant issues in aspects such as design, experimental methods, and result completeness. Here, I express the main reasons for rejection and suggest how the authors can improve it.

1. The experimental data are relatively insufficient. The article includes extensive omics analysis on salt stress but lacks omics analysis on heavy metal Cd or combined stress data involving both factors.
2. The rationale for selecting the JAZ pathway for investigation is not clearly articulated. Moreover, while the text emphasizes that all members of the JAZ gene family can respond to both salt and Cd stress, it does not explain the specific advantages of the cloned JAZ genes within the entire gene family or justify why these particular genes were chosen, resulting in inadequate discussion.
3. In the overexpression line experiments, the conclusion that the JA pathway plays a critical role in stress alleviation is based solely on the improved performance of plants under salt and Cd stress after gene overexpression. This evidence is clearly insufficient and requires additional molecular verification, such as JA content measurement, verification of related protein interactions, and analysis of downstream gene expression changes.
4. Only heterologous overexpression verification experiments were carried out, so it is impossible to fully reflect the true function of the gene in the natural host。
5. The potential for environmental remediation, mentioned in the research background, was not revisited in subsequent experiments. Specifically, there were no experiments examining the synergistic effects of salt and Cd, and the conclusions did not address the plant’s potential role in environmental remediation.
6. Were statistical significance tests (e.g., p-values) performed? Error bars and significance markers are not indicated in the figures.
7. The image is relatively blurry and the text labels are not clear, especially in Figure 3.

Author Response

Comments1: The experimental data are relatively insufficient. The article includes extensive omics analysis on salt stress but lacks omics analysis on heavy metal Cd or combined stress data involving both factors.

Response1: The transcriptomic and physiological analyses associated with Cd tolerance in S. portulacastrum forms the content of our another manuscripts. This work has beening accepted by a journal but has not yet been published online. Therefore, we are unable to provide its DOI number. We can confirm that the differentially expressed genes under Cd stress are significantly enriched in pathways associated with plant hormone signal transduction, and the JA pathway is markedly activated. This aligns closely with our observation under salt stress, where JAZ subfamily genes are rapidly induced, further supporting the central regulatory role of the JA signaling pathway in response of S. portulacastrum to diverse abiotic stresses. Although comprehensive Cd-related omics analysis could not be fully presented here due to data attribution constraints, our functional validation in transgenic Arabidopsis has demonstrated that SpJAZ7 significantly enhances plant growth performance, maintains photosynthetic pigment stability, and improves water regulation under Cd stress. These physiological results substantiate the practical role of this gene in Cd tolerance. Regarding the research direction of cross-stress, it is indeed a prominent research hotspot in the field. We have also conducted combined salt and Cd cross-stress treatments. Currently, the multi-omics samples are still undergoing sequencing. After we obtained the transcriptomic data, we will perform in-depth analyses to investigate the affects and underlying mechanisms about cross-stress on S. portulacastrum.

Comments1: The rationale for selecting the JAZ pathway for investigation is not clearly articulated. Moreover, while the text emphasizes that all members of the JAZ gene family can respond to both salt and Cd stress, it does not explain the specific advantages of the cloned JAZ genes within the entire gene family or justify why these particular genes were chosen, resulting in inadequate discussion.

Response1: Among the pathways enriched by differentially expressed genes in S. portulacastrum under salt stress, we focused on the plant hormone signal transduction pathway. The results showed that the related genes of IAA, JA, cytokinin, ABA, brassinosteroid, and ethylene signaling pathways were all significantly upregulated, suggesting that multiple plant hormone signals are involved in the resistance of S. portulacastrum to salt stress. Among them, the JAZ and MYC2 genes on the JA pathway also experienced significant upregulation, we focused on the JA signaling pathway. Consequently, our attention was drawn to the JAZ genes, which are key components of the JA pathway. Transcriptomic data revealed differential expression of JAZ genes under salt stress. Subsequent qPCR validation identified SpJAZ1, SpJAZ5, and SpJAZ7 as members of the JAZ subfamily with relatively high expression levels.

Furthermore, the amino acid sequences of these SpJAZ1, SpJAZ5, and SpJAZ7 proteins showed the closest similarity to Arabidopsis AtTIFY10a, a protein previously demonstrated to enhance salt tolerance in Arabidopsis. Evolutionary analysis of the TIFY gene family indicated conservation of JAZ genes between S. portulacastrum and Arabidopsis. Based on these findings, we hypothesized that SpJAZ1/5/7 in S. portulacastrum may play a functional role in salt stress resistance. To investigate this hypothesis, we proceeded to generate overexpression lines for further functional characterization.

Comments1: In the overexpression line experiments, the conclusion that the JA pathway plays a critical role in stress alleviation is based solely on the improved performance of plants under salt and Cd stress after gene overexpression. This evidence is clearly insufficient and requires additional molecular verification, such as JA content measurement, verification of related protein interactions, and analysis of downstream gene expression changes.

Response1: In both wild-type and transgenic Arabidopsis lines under salt treatment, we also examined the expression level of the membrane protein AtNHX1, which can regulate cytosolic sodium ion concentration under high-salinity stress to maintain vacuolar ion homeostasis, thereby mitigating salt-induced damage to plants. Therefore, we hypothesize that the proteins encoded by SpJAZ1/5/7 may physically interact with the protein encoded by SpNHX1, implying that JAZ proteins could play a role in modulating the activity of the SpNHX1 membrane transporter. To further explore the SpJAZ7 interaction network, we performed a yeast two-hybrid library screen and identified potential SpJAZ7-interacting proteins. Sequencing analysis of the positive clones resulted in 61 high-confidence protein matches. The sequencing results are provided in an accompanying Excel file. We have also evaluated SpJAZ7 for self-activation and toxicity in the yeast system. Currently, we are validating the interactions between SpJAZ7 and these 61 candidate proteins through independent yeast two-hybrid assays. Additionally, we are currently employing a yeast-based screening approach to identify proteins that interact with SpJAZ1, SpJAZ5, and SpJAZ7. We are confident that our ongoing work will further elucidate the mechanisms by which SpJAZ1, SpJAZ5, and SpJAZ7 contributes to salt and Cd tolerance.

Comments1: Only heterologous overexpression verification experiments were carried out, so it is impossible to fully reflect the true function of the gene in the natural host.

Response1: We fully acknowledge the reviewer's point regarding the inherent limitations of heterologous expression systems in fully replicating the native cellular context of the gene donor organism. The primary objective of employing heterologous overexpression in this study was to validate the functional role of the JAZ genes in salt and Cd tolerance within a controlled and genetically tractable background. Numerous studies have demonstrated the utility of such systems for the initial screening and functional characterization of key genes, particularly when genetic transformation of the native host remains challenging.

Indeed, establishing a stable transgenic system for S. portulacastrum has proven exceptionally difficult. In our preliminary efforts, we attempted to silence the candidate genes using Virus-Induced Gene Silencing (VIGS) method. Targeting a conserved region shared among SpJAZ1, SpJAZ5, and SpJAZ7, we achieved successful SpJAZ-like gene silencing in a limited number of trials. Following one week of salt treatment, plants with silenced SpJAZ-like exhibited pronounced wilting in a significant number of leaves and generally poorer growth compared to the two control groups. These VIGS results are provided in the supplementary figure. Noting the recent success of the Cut-Dip-Budding (CDB) delivery technique in transforming herbaceous and succulent plants, we are currently adapting this method for S. portulacastrum, with the aim of finally establishing a reliable transgenic platform for functional validation within its native genetic background.

We believe the current experimental evidence, derived from both heterologous expression and preliminary native host silencing, strongly suggests a potential role for these genes in salt and Cd stress responses. Subsequent studies will focus on verifying the detailed functional network of these genes through approaches such as gene editing and overexpression in the native host. The present findings provide crucial preliminary evidence and establish a foundational basis for future cross-species mechanistic exploration.

Comments1: The potential for environmental remediation, mentioned in the research background, was not revisited in subsequent experiments. Specifically, there were no experiments examining the synergistic effects of salt and Cd, and the conclusions did not address the plant’s potential role in environmental remediation.

Response1: Thank you for raising this insightful and valuable critique. We fully concur with the reviewer's perspective. In the revised version, we have refined the focus of the introduction and conclusion to more closely align with the core research content of this study. We acknowledge that the ultimate environmental challenge, particularly in saline soils co-contaminated with heavy metals such as Cd, involves concurrent exposure. Establishing direct experimental links to combined stress and definitive remediation metrics represents an essential next phase of research. Our current work provides the fundamental molecular and physiological groundwork for this subsequent stage. We are committed to completing and extending our follow-up studies to render this translational pathway clearer and more compelling. We are confident this will establish a more satisfactory connection between the promising background, the mechanistic discoveries, and their future environmental applications.

Comments1: Were statistical significance tests (e.g., p-values) performed? Error bars and significance markers are not indicated in the figures.

Response1: All data presented in this study have undergone appropriate statistical significance testing. In the revised manuscript, significant results are explicitly indicated both within the figures and their corresponding legends.

Comments1: The image is relatively blurry and the text labels are not clear, especially in Figure 3.

Response1: We have carefully reviewed and updated all figures and textual labels within the manuscript to ensure that readers can quickly and accurately extract the key information presented in each figure.

 

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Dear authors,

your manuscript dedicated to the analysis of the transcriptome and functional validation of JAZ subfamily genes in S. portulacastrum presents an extensive study of differentially expressed genes under salt and cadmium stress.

The material presented in the manuscript and supplementary files is substantial and allows for a comprehensive discussion of how JAZ genes are involved in plant adaptation and interact with jasmonate signaling pathways. The work is undoubtedly interesting and novel. It employs various bioinformatic data analysis methods and methodological approaches – working with plant cuttings from the natural habitat, cultivating them under laboratory conditions, and using transgenic Arabidopsis plants with JAZ genes isolated from these plants.

However, the article appears very cumbersome. It could be made more elegant by clearly formulating the research objective, shortening the Introduction section (as many ideas from this section are later repeated in the Discussion), and possibly moving some figures to the Supplement.

There are some minor comments:

1. It is unclear what the indices Y and G signify in Figure 1 and in the text on page 5.

2. What means "different material" refer to Figure 5? Does it represent three different plants, or something else?

3. In Figures 7 and 8, the y-axis is labeled as "Reduction of chlorophyll," while the values are expressed in mg/g. Should the axis perhaps be labeled "Chlorophyll content"?

4. It is known that S. portulacastrum is a CAM-type photosynthesis plant and can conserve water under stress. It also has specific CO2 metabolism patterns at night and during the day. In this regard, a question arises: is the change in JAZ gene expression levels over time under stress is associated with the diurnal cycle of CO2 utilization mechanisms?

5. In the analysis of DEGs, it would be valuable to see genes encoding CAM photosynthesis enzymes.

Overall, the obtained data are very interesting and provide an opportunity to examine even more deeply and comprehensively the role of JAZ genes not only in tolerance to salinity and cadmium but possibly also in the regulation of plant water balance, stomatal movements, and CAM photosynthesis.

Minor text revisions would enhance your manuscript.

Author Response

Comments 1: It is unclear what the indices Y and G signify in Figure 1 and in the text on page 5.

Response 1: Thank you for your valuable feedback. We have clarified the terminology pertaining to the different transcriptomic groups in the legend of Figure 1 to eliminate any potential ambiguity.

Comments 2: What means "different material" refer to Figure 5? Does it represent three different plants, or something else?

Response 2: You are correct. Different materials refer to wild-type and transgenic Arabidopsis. We have revised the imprecise description in Figure 5 accordingly.

Comments 3: In Figures 7 and 8, the y-axis is labeled as "Reduction of chlorophyll," while the values are expressed in mg/g. Should the axis perhaps be labeled "Chlorophyll content"?

Response 3: The ordinate represents chlorophyll reduction. To prevent potential ambiguity, we have refined this description.

Comments 4: It is known that S. portulacastrum is a CAM-type photosynthesis plant and can conserve water under stress. It also has specific CO2 metabolism patterns at night and during the day. In this regard, a question arises: is the change in JAZ gene expression levels over time under stress is associated with the diurnal cycle of CO2 utilization mechanisms?

Response 4: This is a highly interesting question. Current research on S. portulacastrum has primarily focused on its salt-tolerance mechanisms and ecological remediation functions, such as sand stabilization, heavy metals adsorption, and water purification, whereas its photosynthetic type has not been unequivocally determined. Given its habitat and physiological characteristics, it is plausible that S. portulacastrum may possess a carbon fixation pathway analogous to CAM. However, this hypothesis remains to be empirically confirmed through further experimentation.

Comments 5: In the analysis of DEGs, it would be valuable to see genes encoding CAM photosynthesis enzymes.

Response 5: Thank you for your valuable suggestion. We fully concur with your perspective that focusing on genes encoding enzymes involved in the CAM pathway within the DEG analysis is of significant importance for gaining a deeper understanding of the stress adaptation mechanisms in S. portulacastrum, a coastal succulent plant. Among genes assoaciate with photosynthesis, we performed differential expression of two isoforms (PPC1 and PPC2) of the phosphoenolpyruvate carboxylase (PEPC) gene, both of which are associated with the CAM photosynthetic pathway. The expression profiles of PPC1 and PPC2 are presented in the attached figure. Based on these findings, we initially hypothesized that S. portulacastrum may exhibit certain characteristics of CAM photosynthetic plants.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

This study focuses on the salt and cadmium-tolerant coastal succulent plant, Sesuvium portulacastrum, which has significant practical and application value. The key function of the JAZ gene was analyzed, and the critical genes SpJAZ1/5/7 under salt stress were screened out. Through heterologous overexpression in Arabidopsis, the plants acquired tolerance to salt and cadmium stress. However, the study still has significant shortcomings in the depth of mechanism and insufficient experimental verification. Here, I express the main reasons for Major revision and suggest how the authors can improve it.

1. In terms of mechanism depth, the interaction network between the JAZ and the downstream stress resistance pathways has not been fully elucidated, and its function has only been verified through heterologous expression in Arabidopsis thaliana, without genetic manipulation verification in itself;
2. In terms of content presentation, although the evolutionary tree analysis and DEGs heat map are clearer than before, the analysis and explanation are relatively weak;
3. The stress treatments are all single stress treatments, and the response mechanism of the JAZ gene under salt-cadmium combined stress has not been explored, so relevant experiments need to be supplemented for in-depth exploration.

Author Response