Physiological Responses, Cadmium Partitioning, and Mineral Nutrient Disruption in Brassicaceae Crops Exposed to Cadmium Stress

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

In his manuscript, Samet investigates the physiological responses, cadmium (Cd) uptake and partitioning, oxidative stress responses, and mineral nutrient disruption in four Brassicaceae crops exposed to increasing Cd concentrations under soil-based greenhouse conditions. The work addresses an important topic related to food safety, plant stress physiology, and heavy metal accumulation in edible crops. The study is particularly relevant in the context of increasing soil contamination and the need to identify crops with lower risks of Cd entry into the food chain. The integration of physiological, biochemical, and ionomic parameters represents a strong point of the study and provides a relatively comprehensive dataset. 

Overall, the manuscript is scientifically sound and based on a well-designed pot experiment. The results are generally consistent with current knowledge regarding Cd toxicity, oxidative stress induction, and nutrient interaction mechanisms. The comparative approach across multiple Brassicaceae species under identical soil conditions is valuable and increases the agronomic relevance of the findings. However, the manuscript is mainly descriptive and would benefit from additional mechanistic insight and deeper interpretation of some physiological responses

The AUthor should investigate Cd uptake and transport at the molecular level. Differences in Cd accumulation between species are likely linked to variation in metal transporter expression, such as ZIP, NRAMP, HMA, or ABC transporters. Measuring expression levels of key Cd transporter genes in roots and shoots under different Cd treatments would help explain why certain species show higher translocation or root retention capacity. Even targeted qPCR analysis of a few known transporters would substantially increase mechanistic depth.

he current results suggest strong root retention in some species and enhanced shoot translocation in others. However, it remains unclear whether tolerance is linked to vacuolar sequestration, cell wall binding, or cytosolic detoxification. Fractionation experiments or imaging approaches such as synchrotron-based X-ray fluorescence mapping or histochemical localization could help clarify Cd storage patterns within tissues.

The oxidative stress data are informative, but it would be beneficial to expand antioxidant system analysis. Measuring antioxidant enzyme activities such as superoxide dismutase, catalase, and peroxidase would provide stronger support for the interpretation of ROS-related stress responses. This would help explain why some species maintain lower oxidative damage despite high Cd accumulation.

Because nutrient imbalance appears to be an important outcome of Cd exposure, further investigation into nutrient uptake mechanisms would also strengthen the manuscript. For example, measuring expression or activity of nutrient transporters for potassium, calcium, or zinc could clarify whether nutrient disruption results primarily from competition at transport sites or from root damage and metabolic dysfunction.

Finally, root morphological analysis could provide useful complementary information. Cd tolerance often correlates with root architecture traits such as root length, surface area, or root hair density. Including root imaging or morphometric analysis could help link physiological tolerance to structural adaptation strategies.

 

Author Response

Responses to Reviewer 1

I sincerely thank you for the thorough evaluation of my manuscript and for the constructive and insightful comments provided. I appreciate the positive assessment of the experimental design, data presentation, and statistical analyses, as well as the recognition of the contribution of this study to advancing understanding of Cd stress tolerance and metal partitioning mechanisms in Brassicaceae crops. In response to the comments, I have carefully revised the manuscript to strengthen the scientific contextualization of my findings, expand the mechanistic interpretation, clarify methodological and analytical details, and more explicitly highlight the novelty and integrative nature of my species-dependent approach. All revisions have been incorporated directly into the manuscript, and the corresponding changes can be found in the indicated sections. Detailed point-by-point responses to each comment are provided below.

 

Comments 1: [Overall, the manuscript is scientifically sound and based on a well-designed pot experiment. The results are generally consistent with current knowledge regarding Cd toxicity, oxidative stress induction, and nutrient interaction mechanisms. The comparative approach across multiple Brassicaceae species under identical soil conditions is valuable and increases the agronomic relevance of the findings. However, the manuscript is mainly descriptive and would benefit from additional mechanistic insight and deeper interpretation of some physiological responses.]

Response: Thank you for the positive evaluation of our experimental design and the comparative approach adopted in this study. I appreciate the suggestion to strengthen the mechanistic interpretation and provide deeper physiological insights.

In response to this valuable comment, I have substantially revised the Discussion section to improve the mechanistic interpretation of the results. Specifically:

A new integrative paragraph has been added at the end of the Discussion section to provide a mechanistic synthesis linking Cd uptake, oxidative stress responses, photosynthetic pigment destabilization, nutrient imbalance (K, Ca, and Zn interactions), and growth inhibition.

The interpretation of correlation matrix analyses has been expanded to emphasize causal relationships between Cd accumulation, ROS-mediated damage, and physiological responses rather than descriptive associations.

Species-specific Cd-handling strategies among the studied Brassicaceae crops have been clarified, highlighting differences in sensitivity, buffering capacity, and Cd translocation behavior.

Additionally, a conceptual Cd-response model has been introduced to integrate the observed physiological responses into a unified framework, thereby enhancing the mechanistic understanding of Cd stress responses.

These revisions aim to move beyond descriptive reporting and provide a more integrative and mechanistic interpretation of the physiological responses observed under Cd stress. The relevant changes have been incorporated at the end of Section 4 (Discussion), immediately before the Conclusion section.

The revisions given below can be found in the indicated sections (Lines 1074-1089).

“Collectively, the present findings support an integrative physiological framework in which cadmium (Cd) toxicity is governed by the interaction between metal uptake, redox imbalance, and disruption of mineral nutrient homeostasis. Increased Cd accumulation and internal mobility were closely associated with elevated oxidative stress, suggesting that enhanced Cd translocation amplifies reactive oxygen species (ROS) generation, leading to membrane destabilization, pigment degradation, and growth inhibition. The consistent disruption of potassium homeostasis highlights the role of altered ion transport in amplifying Cd toxicity, whereas calcium may contribute to compensatory signaling and membrane stabilization under stress conditions. Species-specific patterns revealed distinct Cd-handling strategies, with cress and broccoli exhibiting high sensitivity, watercress showing partial physiological buffering, and white cabbage maintaining biomass despite efficient Cd transfer to edible tissues. Importantly, Cd-induced nutrient imbalance, together with enhanced Cd accumulation in aboveground tissues, represents a dual challenge by simultaneously reducing crop nutritional quality and increasing potential risks for human health. These findings emphasize the importance of species selection and management strategies aimed at limiting Cd uptake and translocation to safeguard both crop productivity and food safety.

Based on the integrated analysis, a conceptual response model can be proposed in which Cd uptake initiates ionic imbalance and oxidative stress signaling, triggering coordinated physiological adjustments that include pigment destabilization, growth inhibition, and species-dependent modulation of Cd partitioning between roots and shoots, ultimately determining both tolerance capacity and potential food safety risk.”

Comments 2: [The author should investigate Cd uptake and transport at the molecular level. Differences in Cd accumulation between species are likely linked to variation in metal transporter expression, such as ZIP, NRAMP, HMA, or ABC transporters. Measuring expression levels of key Cd transporter genes in roots and shoots under different Cd treatments would help explain why certain species show higher translocation or root retention capacity. Even targeted qPCR analysis of a few known transporters would substantially increase mechanistic depth.]

Response:  Thank you for this valuable suggestion, and I fully agree that molecular-level investigation of metal transporter systems (e.g., ZIP, NRAMP, HMA, and ABC transporters) would provide deeper insight into species-specific Cd uptake and translocation mechanisms.

While gene expression analysis was beyond the scope of the present study, I have strengthened the Discussion section by incorporating mechanistic interpretation supported by current literature regarding transporter-mediated Cd uptake and distribution. Specifically, I added a new discussion paragraph addressing how differences in Cd accumulation and translocation among species may be linked to variation in transporter activity and regulation, including ZIP and NRAMP transporters involved in metal uptake, HMA transporters associated with xylem loading and root-to-shoot translocation, and ABC transporters implicated in intracellular sequestration and detoxification.

These additions provide a mechanistic framework that complements our physiological findings and helps explain species-specific Cd partitioning patterns observed in this study. I also acknowledge that future studies integrating targeted gene expression analyses (e.g., qPCR-based approaches) would further clarify the molecular basis of Cd tolerance and transport.

The relevant additions have been incorporated into Section 4 (Discussion), particularly within the mechanistic interpretation of Cd accumulation and translocation processes.

The revisions given below can be found in the indicated sections (Lines 1051-1073).

“Species-specific differences in Cd accumulation and translocation observed in this study may also be interpreted in the context of transporter-mediated metal uptake and intracellular handling mechanisms. Cadmium uptake in plants is largely facilitated by broad-spectrum divalent metal transporters, including members of the ZIP (ZRT/IRT-like Protein) and NRAMP (Natural Resistance-Associated Macrophage Protein) families, which primarily mediate Zn²⁺ and Fe²⁺ transport but can inadvertently transport Cd²⁺ due to chemical similarity. Furthermore, heavy metal ATPases (HMAs) are known to play key roles in xylem loading and root-to-shoot translocation, potentially explaining the strong association between Cd accumulation and translocation factors observed in certain species. In addition, ATP-binding cassette (ABC) transporters contribute to intracellular sequestration and detoxification by facilitating vacuolar compartmentalization of Cd complexes. Variation in the regulation or activity of these transporter systems may underlie the contrasting Cd-handling strategies identified among species, where higher translocation efficiency or stronger root retention likely reflects differential transporter expression or activity. Although gene expression analyses were beyond the scope of the present study, the observed physiological patterns are consistent with transporter-mediated mechanisms widely reported in Brassicaceae and other plant species exposed to Cd stress.

Notably, the strong correlations observed between Cd accumulation, translocation indices, oxidative stress markers, and nutrient imbalance may indirectly reflect differential regulation or activity of metal transporter systems (e.g., ZIP, NRAMP, HMA, and ABC transporters), suggesting that species-specific physiological responses identified in this study are likely underpinned by transporter-mediated control of Cd uptake, redistribution, and intracellular sequestration [10, 13, 48].”

 

Comments 3: [The current results suggest strong root retention in some species and enhanced shoot translocation in others. However, it remains unclear whether tolerance is linked to vacuolar sequestration, cell wall binding, or cytosolic detoxification. Fractionation experiments or imaging approaches such as synchrotron-based X-ray fluorescence mapping or histochemical localization could help clarify Cd storage patterns within tissues.]

Response: Thank you for this insightful suggestion, and fully agree that intracellular compartmentation mechanisms, including vacuolar sequestration, cell wall binding, and cytosolic detoxification processes, play key roles in determining Cd tolerance and partitioning patterns.

While advanced imaging or fractionation approaches were beyond the scope of the present physiological study, I have strengthened the Discussion section by incorporating mechanistic interpretation supported by relevant literature. Specifically, I added a paragraph addressing how species-specific differences in Cd retention or translocation may be associated with differential compartmentation strategies, including vacuolar sequestration mediated by transporters, cell wall immobilization of Cd ions, and intracellular detoxification pathways. I also acknowledge that future studies employing synchrotron-based imaging or histochemical localization techniques would provide valuable insights into subcellular Cd distribution.

These additions aim to clarify the physiological patterns observed in this study within a broader mechanistic framework.

The revisions given below can be found in the indicated sections (Lines 891-904).

“Differences in root retention and shoot translocation observed among species may also reflect variation in intracellular Cd compartmentation strategies. Cadmium tolerance in plants is frequently associated with sequestration mechanisms that limit cytosolic Cd toxicity, including vacuolar storage mediated by tonoplast transporters, binding to cell wall components, and chelation with thiol-rich ligands followed by intracellular detoxification. Enhanced root retention may indicate efficient immobilization through cell wall binding or vacuolar sequestration, thereby restricting Cd mobility, whereas increased shoot translocation could reflect more active xylem loading or reduced sequestration capacity in root tissues. Although subcellular localization analyses were not performed in the present study, the physiological patterns observed are consistent with compartmentation-based tolerance mechanisms widely reported in Cd-exposed plants [13, 32, 33]. Future investigations using imaging techniques such as synchrotron-based X-ray fluorescence mapping or histochemical localization would provide valuable insight into tissue-specific Cd storage patterns.”

 

Comments 4: [The oxidative stress data are informative, but it would be beneficial to expand antioxidant system analysis. Measuring antioxidant enzyme activities such as superoxide dismutase, catalase, and peroxidase would provide stronger support for the interpretation of ROS-related stress responses. This would help explain why some species maintain lower oxidative damage despite high Cd accumulation.]

Response:  Thank you for this valuable suggestion, and agree that antioxidant enzyme analyses (e.g., superoxide dismutase, catalase, and peroxidases) would provide additional insight into ROS-scavenging mechanisms under Cd stress.

While enzymatic antioxidant measurements were beyond the scope of the present study, I have expanded the Discussion section to incorporate a mechanistic interpretation of oxidative stress responses supported by relevant literature. Specifically, I added a paragraph discussing how differences in oxidative damage among species may reflect variation in antioxidant defense capacity, including enzymatic ROS-scavenging systems. This addition helps explain why certain species exhibited lower oxidative damage despite elevated Cd accumulation, suggesting that tolerance may be associated with more efficient antioxidant regulation rather than reduced Cd uptake alone.

These revisions strengthen the physiological interpretation of ROS-related stress responses without altering the experimental design.

The revisions given below can be found in the indicated sections (Lines 812-822).

“Differences in oxidative damage among species despite comparable or elevated Cd accumulation may reflect variation in antioxidant defense capacity. Cadmium-induced oxidative stress is commonly mitigated through enzymatic ROS-scavenging systems, including superoxide dismutase (SOD), catalase (CAT), and various peroxidases, which collectively regulate cellular redox balance. Species exhibiting lower levels of lipid peroxidation and membrane damage may possess more efficient antioxidant responses that limit ROS propagation, thereby preserving cellular integrity even under high Cd exposure. Although antioxidant enzyme activities were not directly measured in the present study, the observed physiological patterns are consistent with previous reports demonstrating that enhanced antioxidant capacity contributes significantly to Cd tolerance by reducing oxidative damage rather than preventing Cd uptake itself [17, 43, 44].”

 

Comments 5: [Because nutrient imbalance appears to be an important outcome of Cd exposure, further investigation into nutrient uptake mechanisms would also strengthen the manuscript. For example, measuring the expression or activity of nutrient transporters for potassium, calcium, or zinc could clarify whether nutrient disruption results primarily from competition at transport sites or from root damage and metabolic dysfunction.]

Response: Thank you very much for this insightful suggestion and fully agree that nutrient transporter dynamics play a critical role in understanding Cd-induced nutrient imbalance. While molecular analysis of nutrient transporter expression was beyond the scope of the present physiological study, I have strengthened the Discussion section by incorporating mechanistic interpretation supported by relevant literature.

Specifically, I added a paragraph discussing how Cd-induced nutrient disruption may arise from multiple interacting mechanisms, including competition at shared transport pathways (particularly for divalent cations such as Zn²⁺), membrane depolarization affecting K⁺ uptake systems, and indirect effects resulting from oxidative damage and root metabolic impairment. This expanded discussion provides a mechanistic framework explaining how the nutrient imbalance observed in our study may reflect both transporter-level competition and stress-induced physiological dysfunction.

These additions enhance the mechanistic depth of the manuscript while maintaining the original experimental scope.

The revisions given below can be found in the indicated sections (Lines 997-1008).

“The nutrient imbalance observed under Cd exposure may arise from multiple interacting mechanisms involving both transporter-level competition and stress-induced physiological disruption. Cadmium can interfere with potassium uptake by altering membrane potential and inhibiting plasma membrane transport systems, thereby reducing K⁺ acquisition efficiency. Similarly, chemical similarity between Cd²⁺ and essential divalent cations such as Zn²⁺ may result in competition for shared transport pathways, including ZIP-family transporters. In addition, Cd-induced oxidative stress and root metabolic impairment may indirectly affect nutrient uptake capacity by damaging membrane integrity and disrupting energy-dependent transport processes. These combined mechanisms likely contribute to the differential nutrient profiles observed among species, suggesting that nutrient imbalance reflects both direct transporter competition and broader physiological stress responses [13, 50.”

 

Comments 6: [Finally, root morphological analysis could provide useful complementary information. Cd tolerance often correlates with root architecture traits such as root length, surface area, or root hair density. Including root imaging or morphometric analysis could help link physiological tolerance to structural adaptation strategies.]

Response: Thank you for your valuable suggestion, and agree that root morphological traits represent an important component of plant responses to Cd stress. Root architecture can influence metal uptake, retention, and tolerance by modifying the absorptive surface area and spatial distribution of transport activity.

Although detailed root imaging or morphometric analyses were beyond the scope of the present study, I have strengthened the Discussion section by incorporating an interpretation of how differences in root architecture may contribute to species-specific Cd uptake and tolerance strategies. Specifically, I added a paragraph discussing the potential role of root length, surface area, and root structural adaptations in modulating Cd acquisition and internal distribution.

These additions provide a broader integrative context linking physiological responses to potential structural adaptation mechanisms.

The revisions given below can be found in the indicated sections (Lines 857-868).

“Species-specific differences in Cd accumulation and tolerance may also be influenced by root architectural traits that determine the extent and spatial pattern of metal uptake. Root length, surface area, and root hair development can significantly affect absorptive capacity and the interaction between roots and contaminated soil environments. Plants with more extensive root systems may exhibit increased Cd uptake due to larger contact areas, whereas structural adaptations such as altered root morphology or localized retention mechanisms may contribute to limiting Cd translocation to shoots. Previous studies have suggested that Cd tolerance may be partially associated with root structural plasticity, enabling plants to balance nutrient acquisition with stress avoidance strategies. Although root morphometric analyses were not conducted in this study, the observed physiological differences among species may reflect underlying variations in root architecture that warrant further investigation [28, 32].”

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The paper entitled “Physiological Responses, Cadmium Partitioning, and Mineral Nutrient Disruption in Brassicaceae Crops Exposed to Cadmium Stress” conducts a comparative analysis of the response characteristics of four Brassicaceae crops (cress, watercress, broccoli, and white cabbage) regarding their physiological and biochemical changes, cadmium (Cd) partitioning patterns, and mineral nutrient uptake under Cd stress with different concentration gradients. The experimental design is relatively rational, with abundant data support, and the conclusions hold certain practical significance for food safety risk assessment in Cd-contaminated agricultural soils. That being said, several key details require further refinement and optimization to enhance the overall scientific rigor and comprehensiveness of the study prior to its progression to the next review stage.

 

In Abstract of the manuscript:

At Lines 19-21, the authors stated that "………root retention capacity declined at higher Cd levels, resulting in enhanced Cd translocation to shoots………". However, the term "higher Cd levels" is ambiguous as it does not specify the exact concentration gradient (e.g., 50 mg/kg Cd) corresponding to the experimental design. To improve the precision and reproducibility of the results, it is recommended that the authors clarify the specific range or threshold of Cd concentrations defined as "higher Cd levels" based on the data presented in the study.

 

In Keywords of the manuscript:

Keywords should be refined to fewer than five terms.

 

In Introduction of the manuscript:

In the Introduction section, it is recommended that the authors provide background information on cadmium (Cd) contamination in agricultural soils worldwide or in the target region, including typical Cd concentration ranges in slightly, moderately, and heavily polluted soils, as well as the main sources contributing to soil Cd accumulation. This background context is essential to justify the scientific rationale for the Cd concentration gradient (0, 5, 10, 20, and 50 mg/kg) employed in the study, helping readers understand how the experimental design aligns with real-world soil pollution scenarios and enhancing the relevance and persuasiveness of the research findings.

Additionally, it is recommended that the authors introduce the current status of Cd contamination in agricultural soils in Turkey by citing relevant recent studies (e.g., published between 2021and 2026). This should include key information such as typical Cd concentration ranges in local contaminated soils, major pollution sources (e.g., industrial activities, agricultural practices, or atmospheric deposition), and regional variations in contamination levels. Incorporating this local context will not only justify the relevance of the study to Turkey’s agricultural and environmental challenges but also enhance the practical implications of the findings for local crop cultivation, food safety management, and soil pollution mitigation strategies.

 

In Materials and Methods of the manuscript:

In the Materials and Methods section, it is recommended that the authors briefly introduce the characteristics of the research location, including specific geographical coordinates (latitude and longitude), local climate type, average annual temperature and precipitation, as well as seasonal variation patterns of rainfall and humidity. This contextual information is crucial for readers to understand the natural environmental background of the greenhouse experiment and to assess the potential impacts of regional environmental factors on the stability of controlled greenhouse conditions. Additionally, it will enhance the reproducibility of the study and provide a reference for future research conducted in different ecological regions.

At Lines 132-138, the authors stated that "soils were thoroughly mixed to ensure homogeneous distribution of Cd and nutrients" after spraying the CdCl₂ solution, but the specific method used to achieve thorough mixing was not described in detail. In addition, the rationale for selecting a 24-hour equilibration period between Cd application and seed sowing was not provided. Clarifying these methodological details is crucial to ensure the uniformity of Cd distribution across pots and the reliability of the experimental treatments, thereby enhancing the reproducibility of the study.

 

In Results and Discussion of the manuscript:

In Section 3.4 (Oxidative stress indicators), a paradoxical response pattern was observed: under the 50 mg/kg Cd treatment, malondialdehyde (MDA) content was significantly lower than that in the control group, which directly contrasted with the elevated hydrogen peroxide (H₂O₂) levels (a key reactive oxygen species, ROS) under the same Cd concentration. However, the authors merely described this phenomenon without providing a clear mechanistic explanation for the inverse relationship between MDA and H₂O₂). It is recommended that the authors supplement an in-depth analysis of this abnormal response by citing relevant studies. This supplementary analysis will help clarify the uniqueness of the oxidative stress response in the studied Brassicaceae crops under high Cd concentrations and improve the scientific rigor of the results.

 

In the Conclusions of the manuscript:

In the Conclusion section, it is recommended that the authors summarize the limitations of this study. Specifically, the current research was conducted under controlled greenhouse pot conditions, which did not fully consider the impacts of complex actual field environmental factors (e.g., soil texture heterogeneity, soil microbial communities). Additionally, the experiment only involved a short-term Cd exposure period (six weeks), lacking long-term monitoring data that can reflect the dynamic responses of crops to Cd stress and the cumulative risks of Cd in the soil-plant system under natural field conditions.

 

In the References of the manuscript:

The formatting of the reference list requires rigorous standardization in strict accordance with the author guidelines of the journal Plants. Specifically, inconsistencies have been identified in the formatting of several references, including Reference 1, 10, 11, 15, 27, and 47.

Author Response

Responses to Reviewer 2

I sincerely thank you for the thorough evaluation of my manuscript and for the constructive and insightful comments provided. I appreciate the positive assessment of the experimental design, data presentation, and statistical analyses, as well as the recognition of the contribution of this study to advancing understanding of Cd stress tolerance and metal partitioning mechanisms in Brassicaceae crops. In response to the comments, I have carefully revised the manuscript to strengthen the scientific contextualization of my findings, expand the mechanistic interpretation, clarify methodological and analytical details, and more explicitly highlight the novelty and integrative nature of my species-dependent approach. All revisions have been incorporated directly into the manuscript, and the corresponding changes can be found in the indicated sections. Detailed point-by-point responses to each comment are provided below.

 

Comments 1: [ In Abstract of the manuscript: At Lines 19-21, the authors stated that "…root retention capacity declined at higher Cd levels, resulting in enhanced Cd translocation to shoots…". However, the term "higher Cd levels" is ambiguous because it does not specify the exact concentration gradient (e.g., 50 mg/kg Cd) used in the experimental design. To improve the precision and reproducibility of the results, the authors are recommended to clarify the specific Cd concentration range or threshold defined as "higher Cd levels" based on the data presented in the study.]

Response: Thank you for this valuable comment, and agree that greater precision is needed in the "Abstract." To address this concern, I have revised the relevant sentence to explicitly define the Cd concentration range corresponding to “higher Cd levels,” based on our experimental design and results.

Specifically, the term “higher Cd levels” has been replaced with a quantitative description referring to elevated Cd treatments (≥20 mg kg⁻¹ soil, particularly 50 mg kg⁻¹), where a marked decline in root retention capacity and enhanced Cd translocation to shoots were observed. This revision improves clarity, reproducibility, and alignment with the experimental data. The abstract has been revised accordingly.

The revisions given below can be found in the indicated sections (Lines 23-24).

"...however, root retention capacity declined at elevated Cd concentrations (20–50 mg kg⁻¹ soil), leading to greater Cd translocation to shoots."

Comments 2: [ In Keywords of the manuscript: Keywords should be refined to fewer than five terms.]

Response: Thank you for this suggestion. The keywords have been revised and reduced to fewer than five terms to comply with the journal's guidelines while maintaining the study's core thematic focus. The revised keywords emphasize cadmium stress, metal partitioning, physiological responses, and nutrient interactions relevant to Brassicaceae crops.

The revisions given below can be found in the indicated sections (Lines 38-39).

Keywords: Cadmium stress; Brassicaceae; metal partitioning; nutrient imbalance; oxidative stress

 

Comments 3: [In Introduction of the manuscript: [In the Introduction section, it is recommended that the authors provide background information on cadmium (Cd) contamination in agricultural soils worldwide or in the target region, including typical Cd concentration ranges in slightly, moderately, and heavily polluted soils, as well as the main sources contributing to soil Cd accumulation. This background context is essential to justify the scientific rationale for the Cd concentration gradient (0, 5, 10, 20, and 50 mg/kg) employed in the study, helping readers understand how the experimental design aligns with real-world soil pollution scenarios and enhancing the relevance and persuasiveness of the research findings.]

Response: Thank you for this valuable and constructive suggestion. In response, I have revised the Introduction section to include additional background information on global cadmium contamination in agricultural soils, including typical Cd concentration ranges reported for uncontaminated, slightly contaminated, moderately polluted, and heavily contaminated soils. I also added a discussion of major anthropogenic sources contributing to Cd accumulation, such as phosphate fertilizer application, industrial emissions, wastewater irrigation, and atmospheric deposition. These additions clarify that the selected Cd treatment levels (0, 5, 10, 20, and 50 mg kg⁻¹ soil) represent environmentally relevant contamination gradients and strengthen the scientific rationale by aligning the experimental design with realistic soil pollution scenarios.

 

 

The revisions given below can be found in the indicated sections (Lines 71-81).

“Cd contamination in agricultural soils has become an increasing global concern due to its persistence, mobility, and potential entry into the food chain. Background Cd concentrations in uncontaminated soils typically range between 0.1 and 0.5 mg kg⁻¹, whereas slightly contaminated agricultural soils may contain approximately 1–3 mg kg⁻¹ Cd. Moderately polluted soils often exhibit concentrations between 3 and 10 mg kg⁻¹, while heavily contaminated soils influenced by industrial activities, mining, or long-term phosphate fertilizer application may exceed 20–50 mg kg⁻¹ Cd. Major sources of Cd accumulation include phosphate fertilizers, industrial emissions, wastewater irrigation, and atmospheric deposition [2, 4, 5]. Therefore, evaluating plant responses across a gradient representing low to highly contaminated conditions is essential for understanding crop performance and food safety risks under realistic environmental scenarios [2]”.

The revisions given below can be found in the indicated sections (Lines 665-676).

“The severity of Cd-induced damage varies widely among plant species and is influenced by differences in uptake efficiency, internal distribution, detoxification capacity, and stress response mechanisms. Importantly, Cd tolerance does not necessarily correlate with reduced Cd accumulation in edible tissues, emphasizing the need for integrated evaluation of physiological responses together with metal partitioning when assessing crop performance and food safety risks under Cd stress [2]. The Cd concentrations applied in this study represent environmentally relevant contamination levels ranging from background conditions to heavily polluted agricultural soils, allowing species-specific responses to be interpreted within realistic soil pollution scenarios. Within this framework, the following sections discuss the effects of Cd exposure on growth performance, physiological adjustments, metal uptake and translocation dynamics, and nutrient homeostasis in Brassicaceae crops.”

The revisions given below can be found in the indicated sections (Lines 688-692).

“Taken together, the coordinated patterns observed among Cd accumulation, oxidative stress responses, nutrient imbalance, and growth inhibition suggest that species-specific tolerance is governed not by a single trait but by the integrated regulation of metal uptake, internal partitioning, and physiological adjustment mechanisms, as widely reported in Cd stress studies [2, 13, 28].”

 

Comments 4: [Additionally, it is recommended that the authors introduce the current status of Cd contamination in agricultural soils in Turkey by citing relevant recent studies (e.g., published between 2021and 2026). This should include key information such as typical Cd concentration ranges in local contaminated soils, major pollution sources (e.g., industrial activities, agricultural practices, or atmospheric deposition), and regional variations in contamination levels. Incorporating this local context will not only justify the relevance of the study to Turkey’s agricultural and environmental challenges but also enhance the practical implications of the findings for local crop cultivation, food safety management, and soil pollution mitigation strategies.]

Response: I appreciate this valuable recommendation and have expanded the Introduction section to include recent studies addressing cadmium contamination in agricultural soils in Turkey. The revised text now summarizes reported Cd concentration ranges in different regions, highlights major pollution sources such as industrial activities, agricultural practices, and atmospheric deposition, and discusses regional variability in contamination levels. Incorporating this local context strengthens the scientific rationale of the study and emphasizes its relevance to Turkish agricultural systems, food safety considerations, and soil management strategies.

The revisions given below can be found in the indicated sections (Lines 117-129).

“Recent studies have highlighted increasing concern regarding Cd contamination in agricultural soils in various regions of Turkey, driven by industrial emissions, long-term fertilizer use, irrigation practices, and atmospheric deposition. Reported Cd concentrations in Turkish agricultural soils vary depending on regional characteristics, ranging from near-background levels in relatively uncontaminated areas to elevated concentrations in industrially influenced or intensively cultivated zones [20, 21]. Similar patterns of spatial variability and anthropogenic influence have been reported globally, emphasizing the need to evaluate crop responses across environmentally relevant contamination gradients [2]. Therefore, the Cd concentrations applied in this study (0, 5, 10, 20, and 50 mg kg⁻¹ soil) were selected to represent a gradient from background to heavily contaminated agricultural soils, enabling interpretation of species-specific responses under realistic environmental scenarios and strengthening the relevance of the findings for crop management and food safety risk assessment.”

 

Comments 5: [In the Materials and Methods section, it is recommended that the authors briefly introduce the characteristics of the research location, including specific geographical coordinates (latitude and longitude), local climate type, average annual temperature and precipitation, as well as seasonal variation patterns of rainfall and humidity. This contextual information is crucial for readers to understand the natural environmental background of the greenhouse experiment and to assess the potential impacts of regional environmental factors on the stability of controlled greenhouse conditions. Additionally, it will enhance the reproducibility of the study and provide a reference for future research conducted in different ecological regions.]

Response: Thank you for your suggestions. In response, I added a description of the research location to the Materials and Methods section, including geographical coordinates, regional climate characteristics, and general environmental conditions. Although the experiment was conducted under controlled greenhouse conditions, providing a regional climatic context improves reproducibility and facilitates comparison with studies performed in different ecological regions.

The revisions given below can be found in the indicated sections (Lines 166-174).

“The experiment was conducted during the 2024 summer season in the experimental greenhouses of the Faculty of Agriculture, Kocaeli University, Türkiye (40°40′47"N, 30°01′37"E). The study area is located within the Marmara transitional climatic zone, characterized by a humid temperate climate with moderate seasonal variability. Greenhouse conditions were maintained at an average daytime temperature of 27°C and a nighttime temperature of 17°C, with relative humidity ranging from 48% to 76%. Although environmental parameters were controlled inside the greenhouse, providing a regional climatic context improves reproducibility and facilitates comparison with similar studies conducted under different ecological conditions.”

 

Comments 6: [At Lines 132-138, the authors stated that "soils were thoroughly mixed to ensure homogeneous distribution of Cd and nutrients" after spraying the CdCl₂ solution, but the specific method used to achieve thorough mixing was not described in detail. In addition, the rationale for selecting a 24-hour equilibration period between Cd application and seed sowing was not provided. Clarifying these methodological details is crucial to ensure the uniformity of Cd distribution across pots and the reliability of the experimental treatments, thereby enhancing the reproducibility of the study.]

Response: Thank you for this important methodological comment. In response, I revised the Materials and Methods section to provide additional details on the soil preparation procedure. Specifically, I clarified that CdCl₂ and nutrient solutions were evenly sprayed onto the soil and thoroughly mixed manually using repeated turning and blending to ensure homogeneous distribution. I also added justification for the 24-hour equilibration period, explaining that this step was included to allow stabilization of Cd–soil interactions and adsorption processes, thereby improving treatment uniformity and experimental reproducibility.

The revisions given below can be found in the indicated sections (Lines 176-191).

“Cd treatments were prepared as aqueous solutions of CdCl₂ and incorporated into the soil by spraying before seed sowing. Fertilizer solutions were applied simultaneously with Cd treatments to ensure homogeneous nutrient availability across all pots. After application, soils were thoroughly mixed by repeated manual turning and blending to achieve a uniform distribution of Cd and nutrients. The amended soils were allowed to equilibrate for 24 h before sowing to stabilize metal–soil interactions. This equilibration period was implemented to facilitate stabilization of Cd–soil interactions, allowing adsorption processes and ionic distribution within the soil matrix to reach equilibrium and thereby improving treatment consistency and reproducibility.

Cd levels were applied once at the beginning of the experiment before sowing, and no additional Cd applications were performed during the experimental period. This single-dose approach was selected to simulate a stable soil contamination scenario rather than repeated exposure conditions. During the experiment, irrigation was maintained at approximately 70% of soil water-holding capacity (field capacity), and no leaching or drainage losses from pots were observed, ensuring stable Cd availability throughout the experiment.”

 

Comments 7: [In Results and Discussion of the manuscript: In Section 3.4 (Oxidative stress indicators), a paradoxical response pattern was observed: under the 50 mg/kg Cd treatment, malondialdehyde (MDA) content was significantly lower than that in the control group, which directly contrasted with the elevated hydrogen peroxide (H₂O₂) levels (a key reactive oxygen species, ROS) under the same Cd concentration. However, the authors merely described this phenomenon without providing a clear mechanistic explanation for the inverse relationship between MDA and H₂O₂). It is recommended that the authors supplement an in-depth analysis of this abnormal response by citing relevant studies. This supplementary analysis will help clarify the uniqueness of the oxidative stress response in the studied Brassicaceae crops under high Cd concentrations and improve the scientific rigor of the results.]

 

 

Response: I sincerely thank you for this insightful observation. In response, I expanded the Discussion section to provide a mechanistic interpretation of the apparent inverse relationship between H₂O₂ accumulation and MDA levels under the highest Cd treatment. The revised text discusses possible explanations, including activation of protective antioxidant mechanisms, membrane lipid remodeling, metabolic suppression under severe stress, and differential regulation of oxidative signaling versus lipid peroxidation processes. Supporting literature has been added to demonstrate that similar non-linear oxidative stress responses have been reported in Cd-stressed plants. These additions improve the interpretation of oxidative stress dynamics and strengthen the scientific rigor of the manuscript.

The revisions given below can be found in the indicated sections (Lines 799-811).

“Interestingly, under the highest Cd treatment (50 mg kg⁻¹), MDA levels decreased despite elevated H₂O₂ accumulation, indicating a non-linear oxidative stress response. Although increased ROS production typically promotes lipid peroxidation, reduced MDA levels under severe stress conditions have been reported in several plant species and may reflect activation of protective mechanisms rather than reduced oxidative pressure. High Cd exposure can induce metabolic adjustments such as enhanced antioxidant enzyme activity, membrane lipid remodeling, or selective degradation of peroxidized lipids, leading to stabilization of membrane integrity despite elevated ROS levels. Additionally, severe stress may suppress growth and metabolic activity, thereby limiting substrate availability for lipid peroxidation processes. Similar decoupling between ROS accumulation and MDA levels has been reported in Cd-stressed plants, where oxidative signaling and damage responses are differentially regulated depending on stress intensity and species-specific tolerance strategies [17, 43, 44].”

 

Comments 8: [In the Conclusions of the manuscript: In the Conclusion section, it is recommended that the authors summarize the limitations of this study. Specifically, the current research was conducted under controlled greenhouse pot conditions, which did not fully consider the impacts of complex actual field environmental factors (e.g., soil texture heterogeneity, soil microbial communities). Additionally, the experiment only involved a short-term Cd exposure period (six weeks), lacking long-term monitoring data that can reflect the dynamic responses of crops to Cd stress and the cumulative risks of Cd in the soil-plant system under natural field conditions.]

Response: I sincerely thank the reviewer for this valuable suggestion. In response, I have revised the Conclusion section to explicitly acknowledge the limitations of the study. I clarified that the experiment was conducted under controlled greenhouse pot conditions and that complex field-related factors such as soil heterogeneity, microbial interactions, and fluctuating environmental variables were not included. I also noted that the six-week exposure period reflects short-term Cd responses and does not capture long-term dynamics of Cd accumulation and crop adaptation under natural field conditions. I believe this clarification improves the scientific transparency and contextual interpretation of our findings.

These additions have been incorporated in the revised manuscript (Lines 1116-1125).

“This study was conducted under controlled greenhouse pot conditions, which allowed precise regulation of Cd levels and environmental parameters but may not fully reflect the complexity of field conditions. In natural agricultural systems, factors such as soil heterogeneity, microbial communities, fluctuating climatic variables, and long-term metal–soil interactions can influence Cd bioavailability, plant uptake dynamics, and cumulative risk patterns. Furthermore, the six-week exposure period represents short-term Cd stress responses and does not capture potential long-term adaptive mechanisms or progressive Cd accumulation in the soil–plant system. Therefore, future studies incorporating field-based experiments and long-term monitoring are necessary to validate and extend the present findings under realistic agronomic conditions.”

 

Comments 9: [In the References of the manuscript: The formatting of the reference list requires rigorous standardization in strict accordance with the author guidelines of the journal Plants. Specifically, inconsistencies have been identified in the formatting of several references, including Reference 1, 10, 11, 15, 27, and 47.]

Response: Thank you for pointing out the inconsistencies in the reference formatting. In response, I carefully reviewed and standardized the entire reference list in strict accordance with the author guidelines of Plants. The formatting of all references, including References 1, 10, 11, 15, 27, and 47, has been revised to ensure consistency in journal style, including author names, journal titles, volume numbers, page ranges, and DOIs where applicable. These corrections have improved the overall consistency and compliance of the manuscript.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

The title can be revised for clarity.

The abstract is fairly comprehensive but the overall structure should be improved. Also provide comparative results in %increase or decrease.

The introduction does not provide sufficient background. There should be narrative links between sentences and paragraphs so that the reader is able to follow your argument. Aims of the study not well written.

Line 132: Cd treatments were prepared as aqueous solutions of CdCl₂ and incorporated into the soil by spraying before seed sowing. Please explain the application method and provide more details in the M&M section.

Also, how Cd levels (0, 5, 10, 20, and 50 mg kg⁻¹ soil) were prepared (amount of salt, solubility etc.). How many times the Cd treatment was applied during experimentation? 

Line 133: Please add doses and timing details of fertilizer solutions (NH₄NO₃, KH₂PO₄, and 133 K₂SO₄) applied. 

Subheading 2.7. Oxidative stress parameters lacks details of measurements processes. 

Weight of sample for each measurement ? Also, specify either fresh or dry matter used, where needed.

Add details of whole experiment period?

One way or two way ANOVA?

How many biological and technical replicates were taken? How many plants per pot? How many plants per treatment? Age of plants at the time of Cd application and harvesting?

Please mention the specific methods used for measuring Cd levels in plant tissues and root rhizosphere. 

The discussion mostly just repeats the results without enough explanation or interpretation.

Moreover, elaborating more on how these processes such as (Cd accumulation and oxidative stress impact photosynthetic pigments) might be interconnected with other metabolic pathways in different Brassicaceae species could provide deeper insights.

Comments on the Quality of English Language

There a minor grammatical errors, awkward phrasing, repetitive information, etc. that need to be addressed to enhance the clarity, readability, and overall quality of the manuscript.

Author Response

Responses to Reviewer 3

I sincerely thank you for the thorough evaluation of my manuscript and for the constructive and insightful comments provided. I appreciate the positive assessment of the experimental design, data presentation, and statistical analyses, as well as the recognition of the contribution of this study to advancing understanding of Cd stress tolerance and metal partitioning mechanisms in Brassicaceae crops. In response to the comments, I have carefully revised the manuscript to strengthen the scientific contextualization of my findings, expand the mechanistic interpretation, clarify methodological and analytical details, and more explicitly highlight the novelty and integrative nature of my species-dependent approach. All revisions have been incorporated directly into the manuscript, and the corresponding changes can be found in the indicated sections. Detailed point-by-point responses to each comment are provided below.

 

Comments 1: [The abstract is fairly comprehensive but the overall structure should be improved. Also provide comparative results in %increase or decrease.]

Response: Thank you for this helpful suggestion. In response, the "Abstract" has been revised to improve clarity and structural flow. Quantitative comparisons have been incorporated by presenting key findings as percentage increases or decreases across treatments and species. These additions enhance readability, highlight comparative trends among Brassicaceae crops, and improve the impact and interpretability of the results.

The revisions given below can be found in the indicated sections (Lines 15-22).

“Cd exposure significantly affected growth and physiological responses in a species-dependent manner. Compared to the control, shoot dry weight decreased by up to 66.4% in broccoli and 51.7% in cress at the highest Cd level, while white cabbage exhibited comparatively greater tolerance. Oxidative stress indicators showed contrasting patterns, with hydrogen peroxide (H₂O₂) increasing by up to 8.8-fold, whereas proline and membrane permeability (MP) responses varied among species. Photosynthetic pigments declined in cress but increased in broccoli under high Cd conditions, suggesting differential adaptive strategies.”

Comments 2: [The introduction does not provide sufficient background. There should be narrative links between sentences and paragraphs so that the reader is able to follow your argument. Aims of the study not well written.]

Response: Thank you very much for this valuable suggestion. In response, I revised the Introduction section to improve narrative continuity by adding linking sentences between paragraphs and strengthening the logical progression from global Cd contamination to species-specific research gaps. Additionally, the study objectives were rewritten to provide a clearer focus and to better highlight the scientific rationale, emphasizing species comparison, Cd partitioning, and food safety relevance. These revisions improve readability and enhance the overall clarity of the "Introduction."

The revisions given below can be found in the indicated sections (Lines 81-84).

“However, despite extensive research on Cd toxicity, species-specific differences in Cd accumulation, partitioning, and physiological responses remain insufficiently understood, particularly among Brassicaceae crops widely consumed as vegetables.”

The revisions given below can be found in the indicated sections (Lines 134-143).

“In this study, I investigated the responses of four Brassicaceae crops—white cabbage (Brassica oleracea var. capitata), broccoli (Brassica oleracea var. italica), cress (Lepidium sativum), and watercress (Eruca vesicaria)—to increasing soil Cd levels (0, 5, 10, 20, and 50 mg kg⁻¹ soil supplied as CdCl₂) under greenhouse conditions. The study aimed to (i) quantify species-specific growth and physiological responses to graded Cd stress, (ii) evaluate Cd uptake, accumulation, and partitioning between roots and shoots, and (iii) assess nutrient imbalance and oxidative stress dynamics in relation to potential food safety risks. By integrating physiological traits with Cd distribution patterns, this work seeks to provide comparative insights into tolerance mechanisms and crop suitability under Cd-contaminated conditions.”

 

Comments 3: [Line 132: Cd treatments were prepared as aqueous solutions of CdCl₂ and incorporated into the soil by spraying before seed sowing. Please explain the application method and provide more details in the M&M section.]

Response: Thank you for your helpful suggestion. In response, I revised the Materials and Methods section to provide additional details regarding the Cd application procedure. Specifically, I clarified that CdCl₂ solutions were evenly applied using a hand sprayer, followed by thorough manual mixing through repeated turning and blending to ensure homogeneous distribution of Cd within the soil. These additions improve clarity and enhance reproducibility of the experimental methodology. Therefore, a comparative framework linking physiological tolerance with Cd partitioning behavior is needed to better understand crop suitability and potential dietary risks under Cd-contaminated conditions.

Additionally, a sentence highlighting the knowledge gap and novelty of the study was incorporated into the "Introduction" to improve narrative flow and clarify the scientific contribution.

The revisions given below can be found in the indicated sections (Lines 176-191).

“Cd treatments were prepared as aqueous solutions of CdCl₂ and incorporated into the soil by spraying before seed sowing. Fertilizer solutions were applied simultaneously with Cd treatments to ensure homogeneous nutrient availability across all pots. After application, soils were thoroughly mixed by repeated manual turning and blending to achieve a uniform distribution of Cd and nutrients. The amended soils were maintained under moist conditions and allowed to equilibrate for 24 h before sowing. This equilibration period was implemented to facilitate stabilization of Cd–soil interactions, allowing adsorption processes and ionic distribution within the soil matrix to reach equilibrium and thereby improving treatment consistency and reproducibility. During the experiment, irrigation was maintained at approximately 70% of soil water-holding capacity (field capacity), and no leaching or drainage losses from pots were observed, ensuring stable Cd availability throughout the experiment.”

The revisions given below can be found in the indicated sections (Lines 130-133)

“Despite extensive studies on cadmium toxicity, comparative evaluations integrating physiological responses, Cd partitioning dynamics, and nutrient imbalance across multiple Brassicaceae crops under identical soil conditions remain limited, particularly from a food safety perspective”.

 

Comments 4: [Also, how Cd levels (0, 5, 10, 20, and 50 mg kg⁻¹ soil) were prepared (amount of salt, solubility etc.). How many times the Cd treatment was applied during experimentation?]

Response: Thank you for this valuable comment. In response, I expanded the Materials and Methods section to clarify how Cd treatments were prepared and applied. Cadmium treatments (0, 5, 10, 20, and 50 mg kg⁻¹ soil) were prepared using aqueous solutions of CdCl₂. The required amount of CdCl₂ was calculated based on soil mass and target Cd concentration, dissolved in distilled water, and uniformly applied to the soil using a hand sprayer to ensure even distribution. Following application, soils were thoroughly mixed by repeated manual turning and blending to achieve homogeneous Cd distribution across pots. Cd treatment was applied once at the beginning of the experiment before sowing, and no additional Cd applications were performed during the experimental period. This single-dose approach was selected to simulate a stable soil contamination scenario rather than repeated exposure conditions. During the experiment, irrigation was maintained at approximately 70% of soil water-holding capacity (field capacity), and no leaching or drainage losses from pots were observed, ensuring stable Cd availability throughout the experiment.

The revisions given below can be found in the indicated sections (Lines 185-191).

“Cd levels were applied once at the beginning of the experiment before sowing, and no additional Cd applications were performed during the experimental period. This single-dose approach was selected to simulate a stable soil contamination scenario rather than repeated exposure conditions. During the experiment, irrigation was maintained at approximately 70% of soil water-holding capacity (field capacity), and no leaching or drainage losses from pots were observed, ensuring stable Cd availability throughout the experiment.”

 

Comments 5: [Line 133: Please add doses and timing details of fertilizer solutions (NH₄NO₃, KH₂PO₄, and 133 K₂SO₄) applied.]

Response: Thank you for this helpful suggestion. In response, I revised the Materials and Methods section to clarify fertilizer application rates and timing. Basal fertilization was applied at rates equivalent to 150 mg N kg⁻¹, 75 mg P kg⁻¹, and 150 mg K kg⁻¹ soil, supplied as NH₄NO₃, KH₂PO₄, and K₂SO₄, respectively. Basal fertilizer solutions were prepared in distilled water and applied simultaneously with the Cd treatments before sowing to ensure uniform nutrient availability across treatments. This single basal application was selected to provide consistent nutrient conditions throughout the experiment and to minimize variability related to nutrient supply during plant growth.

 

The revisions given below can be found in the indicated sections (Lines 158-164).

“Basal fertilization was applied uniformly to all pots at rates of 150 mg kg⁻¹ nitrogen (N), 75 mg kg⁻¹ phosphorus (P), and 150 mg kg⁻¹ K using ammonium nitrate (NH₄NO₃), mono-potassium phosphate (KH₂PO₄), and potassium sulfate (K₂SO₄), respectively. Cd treatments were applied once prior to sowing by incorporating aqueous CdCl₂ solutions into the soil. Plants were grown under controlled greenhouse conditions, and plant material within each pot was averaged to obtain one representative value per biological replicate in subsequent analyses.

 

Comments 6: [Subheading 2.7. Oxidative stress parameters lacks details of measurements processes. Weight of sample for each measurement ? Also, specify either fresh or dry matter used, where needed. Add details of whole experiment period?]

Response:

Thank you for this valuable comment. In response, I revised Section 2.7 (Oxidative stress parameters) to include detailed descriptions of the measurement procedures, including extraction methods, assay principles, absorbance wavelengths, and relevant literature references for H₂O₂, MDA, and membrane permeability analyses. These additions improve methodological transparency and reproducibility. Additionally, I have revised Section 2.4 (Harvest and sample preparation) to clarify the experimental timeline, sample handling procedures, and material type used for subsequent analyses.

Specifically:

I clarified that plants were harvested after six weeks of Cd exposure to ensure cumulative physiological and biochemical responses were evaluated.

I specified that fresh tissues designated for biochemical analyses were processed immediately after harvest to avoid metabolic degradation.

I further clarified that shoot and root samples used for elemental and Cd analyses were oven-dried at 65°C until constant weight, and dry biomass was determined before grinding.

Additional methodological details regarding root washing with aerated 0.5 mM CaCl₂ solution were included to explain the removal of loosely bound apoplastic ions and improve reproducibility.

These revisions improve methodological transparency and ensure a clearer distinction between fresh and dry sample usage throughout the study.

The revisions given below can be found in the indicated sections (Lines 225-247).

“2.7. Oxidative stress parameters

Hydrogen peroxide (H₂O₂), malondialdehyde (MDA), proline content, and membrane permeability (MP) were determined as indicators of oxidative stress using established spectrophotometric methods. All biochemical analyses were performed using fresh leaf tissue. For each measurement, 0.25 g of fresh plant material was homogenized in appropriate extraction buffers under cold conditions to prevent biochemical degradation.

H₂O₂ content was measured following the method of Mukherjee and Choudhuri [24], based on the spectrophotometric detection of hydrogen peroxide after reaction with potassium iodide. Absorbance was recorded using a UV–Vis spectrophotometer, and H₂O₂ concentration was calculated according to the standard procedure described in the original method.

Lipid peroxidation was evaluated by determining malondialdehyde (MDA) content using the thiobarbituric acid (TBA) reaction according to Hodges et al. [25]. The absorbance of the reaction mixture was measured spectrophotometrically, and MDA concentration was calculated after correction for non-specific turbidity.

Proline content was determined using the ninhydrin-based colorimetric assay described by Bates et al. [26], where absorbance was measured after reaction with acid ninhydrin reagent.

Membrane permeability (MP) was assessed by measuring electrolyte leakage following Yan et al. [27]. Electrical conductivity was measured before and after boiling, and MP was calculated using the following equation:

MP (EC, %) = (C₁ / C₂) × 100

where C₁ and C₂ represent electrolyte conductivity measured before and after boiling, respectively.”

 

The revisions given below can be found in the indicated sections (Lines 193-206).

“Plants were grown from sowing until harvest under controlled greenhouse conditions, and physiological and biochemical measurements were performed at the final harvest stage to assess cumulative responses to Cd exposure. After six weeks of Cd exposure, plants were harvested.

Shoots were cut at the soil surface, washed with running tap water, and rinsed three times with deionized water to remove adhering soil particles. Roots were carefully separated from the soil and immersed in an aerated 0.5 mM CaCl₂ solution for 15 min to desorb loosely bound apoplastic ions, followed by thorough rinsing with deionized water.

Fresh plant tissues designated for biochemical analyses were processed immediately after harvest to avoid metabolic degradation.

Shoot and root samples intended for elemental and Cd analyses were oven-dried at 65°C for 48 h until constant weight and weighed to determine dry biomass. Dried samples were ground to a fine powder using a laboratory mill for subsequent mineral nutrient and Cd concentration analyses.”

 

Comments 7: [One-way or two-way ANOVA?]

Response: Thank you for this helpful comment. In response, I have clarified the statistical analysis section to describe the experimental design and analytical approach better.

Specifically, the experiment was arranged as a factorial design (Cd level × species) in a completely randomized design with three replicates (pots) per treatment. Each pot was considered the experimental unit to avoid pseudoreplication. Accordingly, data were analyzed using two-way analysis of variance (ANOVA) with Cd level and species as fixed factors, including their interaction (Cd×species), thereby allowing evaluation of both individual and interactive effects of the treatments.

I also clarified that plant material within each pot was averaged to obtain a single value per experimental unit. The revised statistical description has been incorporated into the Materials and Methods section (Statistical Analysis subsection) to improve clarity and reproducibility.

The revisions given below can be found in the indicated sections (Lines 249-261).

“The experiment was arranged as a factorial design (Cd level × species) in a completely randomized design with three replicates (pots) per treatment. Each pot was considered the experimental unit. For each parameter, plant material within a pot was averaged to obtain a single value per experimental unit, thereby avoiding pseudo replication.

Data normality was verified using the Shapiro–Wilk test prior to statistical analyses. Data were analyzed using two-way analysis of variance (ANOVA) with Cd level and species as fixed factors, including their interaction (Cd × species), using MINITAB software (Version 16; Minitab Inc., State College, PA, USA).

Pearson’s correlation coefficients (r) were calculated to evaluate relationships among physiological and biochemical parameters in shoot and root tissues, and correlation matrices were generated using XLSTAT 2023. Mean comparisons were performed using Duncan’s Multiple Range Test (DMRT) at a significance level of α = 0.05. Statistical significance was defined as p < 0.05 (*), p < 0.01 (**), p < 0.001 (***), and ns (not significant).”

 

Comments 8: [How many biological and technical replicates were taken? How many plants per pot? How many plants per treatment? Age of plants at the time of Cd application and harvesting?]

Response: Thank you for this important comment. I have revised the Materials and Methods section to clarify the experimental replication and sampling strategy.

The experiment was conducted using a completely randomized factorial design with three biological replicates (pots) per treatment. Each pot represented one experimental unit. After seedling emergence, plants were thinned to maintain 12 uniform plants per pot for Brassica species (white cabbage and broccoli) and 5 plants per pot for Lepidium sativum (cress) and Eruca vesicaria (watercress) to ensure comparable growth density among species.

Cadmium treatments were applied once prior to sowing by incorporating CdCl₂ solutions into the soil. Plants were grown under controlled greenhouse conditions and harvested after six weeks of growth (six weeks of Cd exposure). For biochemical and physiological analyses, plant material within each pot was averaged to obtain one value per biological replicate.

These details have now been added to the revised Materials and Methods section to improve clarity and reproducibility.

The revisions given below can be found in the indicated sections (Lines 146-164).

“The experiment was conducted as a factorial pot experiment with two factors: Cd levels and Brassicaceae crop species. Five Cd levels (0, 5, 10, 20, and 50 mg kg⁻¹ soil), supplied as cadmium chloride (CdCl₂), and four Brassicaceae crops were evaluated. The experiment was arranged in a completely randomized design with three biological replicates (pots) per treatment. Each experimental unit consisted of one pot, and treatments were randomly distributed within the greenhouse to minimize positional effects.

Seeds of four crops, cress (Lepidium sativum), watercress (Eruca vesicaria), broccoli (Brassica oleracea var. italica), and white cabbage (Brassica oleracea var. capitata), were sown in 15-cm-diameter plastic pots filled with 4 kg of soil. After seedling emergence, plants were thinned to maintain 12 uniform plants per pot for Brassica species (white cabbage and broccoli) and 5 uniform plants per pot for cress and watercress to ensure comparable plant density among species. The physicochemical properties of the experimental soil are presented in Table 1.

Basal fertilization was applied uniformly to all pots at rates of 150 mg kg⁻¹ nitrogen (N), 75 mg kg⁻¹ phosphorus (P), and 150 mg kg⁻¹ K using ammonium nitrate (NH₄NO₃), mono-potassium phosphate (KH₂PO₄), and potassium sulfate (K₂SO₄), respectively. Cadmium treatments were applied once prior to sowing by incorporating aqueous CdCl₂ solutions into the soil. Plants were grown under controlled greenhouse conditions, and plant material within each pot was averaged to obtain one representative value per biological replicate in subsequent analyses.”

 

Comments 9: [Please mention the specific methods used for measuring Cd levels in plant tissues and root rhizosphere.]

Response: Thank you for this helpful suggestion. I revised the Materials and Methods section to provide detailed information regarding Cd determination. Specifically, I added a description of the dry-ashing digestion procedure, sample preparation steps, and analytical measurement using ICP-OES (Perkin Elmer Optima 2100 DV). These additions improve the methodological clarity and reproducibility of the study.

The revisions given below can be found in the indicated sections (Lines 215-219).

“For Cd determination, 0.5 g of oven-dried shoot or root material was subjected to dry ashing at 500 °C for 6 h in a muffle furnace. The resulting ash was dissolved in 5 mL of 0.1 M HCl and filtered before analysis. Cadmium concentrations were quantified using inductively coupled plasma optical emission spectrometry (ICP-OES; Perkin Elmer Optima 2100 DV, Waltham, MA, USA) following the procedure described by Miller [23].”

 

Comments 10: [The discussion mostly just repeats the results without enough explanation or interpretation. Moreover, elaborating more on how these processes such as (Cd accumulation and oxidative stress impact photosynthetic pigments) might be interconnected with other metabolic pathways in different Brassicaceae species could provide deeper insights.]

Response: Thank you for this constructive suggestion. I revised the Discussion section to strengthen the mechanistic interpretation of the results. Specifically, I expanded the discussion linking Cd accumulation, oxidative stress responses, and photosynthetic pigment alterations with underlying metabolic processes, including ROS-mediated damage, nutrient homeostasis, and species-specific detoxification strategies. These additions provide a deeper physiological context and improve integration of the findings with known mechanisms of Cd stress in Brassicaceae species.

The revisions given below can be found in the indicated sections (Lines 840-848).

Beyond the descriptive relationships observed in the present study, the strong association between Cd accumulation, oxidative stress markers, and pigment degradation suggests interconnected metabolic disruptions affecting multiple physiological pathways. Cadmium exposure is known to impair photosynthetic machinery both directly and indirectly by inducing excessive production of ROS, which can damage chloroplast membranes, disrupt thylakoid ultrastructure, and inhibit chlorophyll biosynthesis [15, 17. Elevated H₂O₂ levels observed in several species indicate enhanced oxidative pressure, which may accelerate lipid peroxidation and promote chlorophyll degradation through membrane destabilization and enzyme inactivation [14, 43].

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The Author replies to all comments.

Author Response

I sincerely thank the reviewer for the overall evaluation and constructive suggestions. In response to the comments, I implemented several targeted improvements throughout the manuscript to enhance clarity, strengthen the connection between results and conclusions, and improve the analytical depth of the "Discussion". Following both the reviewer’s recommendations and additional feedback, I refined the presentation of key results by incorporating comparative percentage changes where appropriate, strengthened mechanistic interpretations to better explain the observed physiological responses, and added integrative statements to explicitly link the findings with the proposed conceptual framework.

These revisions were made while preserving the original structure of the manuscript to maintain coherence, and I believe they significantly improve the clarity and consistency of the study.

 

Comments 1: [Are the results clearly presented? (Can be improved).]

Response:

Thank you for this constructive suggestion. In the revised manuscript, several targeted improvements were implemented to enhance clarity and presentation of the results. In particular, additional quantitative comparisons (percentage increases/decreases) were incorporated into the Discussion to improve interpretability (Lines 693–695; Lines 721–724; Lines 760–767; Lines 778–782; Lines 830–832). Furthermore, mechanistic explanations were strengthened, and certain descriptive passages were refined or condensed following the recommendations, which improved the overall analytical clarity and readability of the results and their interpretation (Lines 976–998; Lines 1007–1008).

These revisions were made while preserving the original structure of the manuscript, and we believe they significantly improve the clarity of result presentation.

(Sentences highlighted in yellow within the text.)

 

Comments 2: Are the conclusions supported by the results? (Can be improved)

Response:

Thank you for this helpful evaluation. In the revised manuscript, I strengthened the linkage between the Discussion and the Conclusion sections to ensure that the conclusions are directly supported by the presented findings. Specifically, I incorporated additional quantitative comparisons in the Discussion, clarified the mechanistic interpretation of Cd-induced physiological responses, and added an integrative synthesis sentence in the Conclusion to explicitly connect biomass reduction, pigment modulation, oxidative stress responses, Cd partitioning, and nutrient imbalance with the proposed conceptual framework (Lines 1137–1140).

These targeted refinements improve the coherence between the results, discussion, and conclusions while preserving the original structure of the manuscript.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

The author's response on my previous comment no. 4 provides more detail, but it still does not fully answer my questions—specifically regarding solubility, the amount of salt used, and whether CdCl₂ mass was calculated based on the elemental Cd target or the salt itself.

Please specify the actual amount of salt added for each treatment level. Readers need to know whether "50 mg kg⁻¹ Cd" means 50 mg of Cd ions or 50 mg of CdCl₂ (which contains less Cd).

Was the CdCl₂ fully soluble at the applied concentrations? Please confirm that no precipitation occurred during preparation or application.

A critical limitation is the single Cd application via hand sprayer before sowing, as this may not reflect real-world conditions where metals accumulate over time or are introduced through repeated events (e.g., irrigation with contaminated water, atmospheric deposition). Moreover, without an equilibration period, the initial bioavailability may be higher than in aged contaminated soils, potentially overestimating toxicity.

Did the author determine the studied soil's Cd concentrations after the experiment?

Lines 748-759, please provide comparative results (% increase or decrease).

The current discussion part is too lengthy and lacks mechanistic depth. In this part author should address how and why the observed effects occurred. For example, if enzyme activities changed, the regulatory pathways involved should be discussed; if nutrient uptake was affected, transporter competition or signaling crosstalk should be considered. 

Author Response

I sincerely thank you for the thorough and constructive evaluation of our manuscript. The detailed comments and suggestions were highly valuable in improving the clarity, methodological transparency, and mechanistic interpretation of the study. In response, I carefully revised the manuscript by incorporating targeted improvements throughout the Methods and Discussion sections, strengthening mechanistic explanations, refining quantitative comparisons, and enhancing the linkage between results and conclusions, while preserving the overall structure of the manuscript.

 

Comment 1: [The author's response to my previous comment no. 4 provides more detail, but it still does not fully answer my questions—specifically regarding solubility, the amount of salt used, and whether CdCl₂ mass was calculated based on the elemental Cd target or the salt itself.

Please specify the actual amount of salt added for each treatment level. Readers need to know whether "50 mg kg⁻¹ Cd" means 50 mg of Cd ions or 50 mg of CdCl₂ (which contains less Cd).]

Response:

Thank you for this important clarification request. The “Materials and Methods” section has been revised to explicitly clarify the preparation of Cd treatments and the calculation procedure.

Cadmium treatments were applied using cadmium chloride monohydrate (CdCl₂·H₂O) as the Cd source. The target concentrations (0, 5, 10, 20, and 50 mg kg⁻¹ soil) were calculated based on elemental Cd (Cd²⁺) rather than the total mass of the salt. Accordingly, the required amount of CdCl₂·H₂O was determined by converting the desired elemental Cd concentration into the equivalent mass of the salt based on its molecular weight.

For the application, CdCl₂·H₂O was first completely dissolved in distilled water to ensure full solubility and homogeneous distribution. The resulting solution was then uniformly incorporated into the soil before sowing.

These clarifications have been added to the revised manuscript to improve methodological transparency and reproducibility (Lines 161–164).

 

Comment 2: [Was the CdCl₂ fully soluble at the applied concentrations? Please confirm that no precipitation occurred during preparation or application.]

Response:

Thank you for this important clarification. Cd treatments were prepared using CdCl₂·H₂O, which was fully dissolved in distilled water before application. To ensure complete solubility, the solution was gently heated during preparation. The solution was visually inspected before application, and no precipitation was observed. The dissolved Cd solution was then uniformly incorporated into the soil to achieve homogeneous distribution among treatments. This clarification has been added to the "Materials and Methods" section (Lines 164–171).

 

 

Comment 3: [A critical limitation is the single Cd application via hand sprayer before sowing, as this may not reflect real-world conditions where metals accumulate over time or are introduced through repeated events. Moreover, without an equilibration period, the initial bioavailability may be higher than in aged contaminated soils.]

Response:

Thank you for this valuable and insightful comment. The experimental design was intentionally based on a single Cd application using a well-characterized, uncontaminated agricultural soil to ensure precise control over Cd exposure levels and to allow clear interpretation of species-specific physiological responses. Before the experiment, soil samples were collected from a non-contaminated, uncultivated area and subjected to detailed physicochemical analysis, including baseline Cd concentration, as presented in Table 1.

The use of uncontaminated soil enabled us to minimize background variability and to directly evaluate the effects of defined Cd treatments, thereby improving experimental reproducibility and interpretation of the proposed hypothesis. While we acknowledge that repeated or long-term Cd inputs may better reflect certain field conditions, the present controlled design aimed to isolate short-term physiological responses under defined Cd exposure scenarios.

Additionally, as clarified in the revised manuscript, treated soils were allowed to equilibrate for 24 h after Cd application before sowing to promote stabilization of Cd–soil interactions and reduce variability in initial bioavailability (Lines 164–171).

I have added text in the “Conclusion” section to acknowledge this limitation and to emphasize that future field-based or long-term studies would be valuable to validate the findings under more complex environmental conditions (Lines 1155–1160).

 

Comment 4: [Did the author determine the studied soil's Cd concentrations after the experiment?]

Response:

Thank you for this important question. Soil Cd concentration was determined before the experiment as part of the initial soil characterization (Table 1). Post-experimental soil Cd analysis was not performed, as the primary objective of the study was to evaluate plant physiological responses and Cd accumulation patterns under defined exposure levels rather than to quantify changes in soil Cd pools over time.

Given the relatively short experimental duration and controlled pot conditions, Cd availability was assumed to remain within the intended treatment ranges. We acknowledge that post-harvest soil analysis could provide additional insight into Cd redistribution and residual availability, and this has now been mentioned as a consideration for future studies (Lines 1155–1160).

 

Comment 5: [Lines 748-759, please provide comparative results (% increase or decrease).]

Response:

Thank you for this helpful suggestion. Comparative percentage changes relative to the control have now been included in the Discussion (Lines 760–767) to better quantify the magnitude of pigment responses under Cd stress. In addition, we strengthened the interpretation by explicitly linking pigment changes with biomass responses to clarify their functional relevance and improve mechanistic interpretation (Lines 778–782). These revisions enhance clarity while preserving the overall structure of the "Discussion."

 

Comments 6: [The current discussion part is too lengthy and lacks mechanistic depth. In this part, the author should address how and why the observed effects occurred. For example, if enzyme activities changed, the regulatory pathways involved should be discussed; if nutrient uptake was affected, transporter competition or signaling crosstalk should be considered.]

Response: Thank you for this insightful and constructive comment. In line with your advice, I have carefully revised the "Discussion" section while preserving its overall structure. Rather than restructuring the section, I incorporated targeted mechanistic explanations across multiple subsections to clarify how and why the observed physiological and biochemical responses occurred (Lines 693–695; Lines 721–724; Lines 818–819).

Specifically, I strengthened the interpretation of Cd-induced oxidative stress by clarifying its indirect ROS-generating mechanisms and redox imbalance (Lines 830–832). In the sections addressing Cd uptake and translocation, I expanded the mechanistic discussion by referring to transporter-mediated pathways (e.g., ZIP, NRAMP, and HMA families) that regulate Cd mobility and partitioning (Lines 877–880; Lines 914–916).

In addition, within the nutrient imbalance subsection, I refined and condensed the mechanistic explanation of potassium (K⁺) homeostasis under Cd stress. The previously extended description of K⁺ channel modulation and membrane depolarization mechanisms was streamlined to improve clarity and conciseness while retaining the relevant references [44, 48, 51, 52] and preserving mechanistic depth (Lines 966–968; Lines 976–978; Lines 1007–1008). A conceptual sentence was also added to emphasize the regulatory role of K⁺ homeostasis in species-specific Cd tolerance.

These revisions enhance the analytical and mechanistic depth of the "Discussion" while maintaining coherence and avoiding unnecessary expansion of the manuscript.

Author Response File: Author Response.pdf

Round 3

Reviewer 3 Report

Comments and Suggestions for Authors

Based on the concerns detailed below, I cannot recommend acceptance of this manuscript in its current form.

I found author's response unsatisfactory to my questions regarding the amount of salt used, and whether CdCl₂ mass was calculated based on the elemental Cd target or the salt itself.

In my previous comments, I raised the following question: Without an equilibration period, how the targeted Cd concentration (0, 5, 10, 20, and 50 mg Cd kg⁻¹ soil), intended to induce stress, maintained in the soil throughout the experiment?

Were any antioxidant enzyme activities quantified in response to oxidative stress?

Could the author clarify the specific plant tissues (e.g., roots, shoots, leaves) in which oxidative stress parameters were measured and showed responses to Cd exposure across all Brassicaceae crops investigated?

Wavelengths for H₂O₂, MDA, proline content measurements?

Differentiate between the results presented in '3.1.3. Cadmium distribution in the shoots and roots' and '3.2. Cadmium concentration, accumulation, and translocation '

Author Response

Responses to Reviewer 3

Thank you very much for your careful evaluation of my manuscript and for the constructive and insightful comments provided. I sincerely appreciate the time and effort invested in reviewing our work. The suggestions offered by the reviewer have been extremely helpful in improving the clarity, methodological transparency, and scientific interpretation of the manuscript.

In response to the comments, I have carefully revised the manuscript and implemented several improvements, including clarification of methodological details, expansion of the discussion to provide deeper physiological interpretation, refinement of the oxidative stress analysis, and additional explanations regarding cadmium application procedures and experimental conditions. All relevant sections of the manuscript have been revised accordingly, and the specific changes are indicated in the responses below. The corresponding modifications can also be found in the revised manuscript at the indicated line numbers.

Below, we provide a detailed point-by-point response to each comment.

I hope that the revisions satisfactorily address the reviewer’s concerns and improve the overall quality of the manuscript.

Comment 1: [I found author's response unsatisfactory to my questions regarding the amount of salt used, and whether CdCl₂ mass was calculated based on the elemental Cd target or the salt itself.

In my previous comments, I raised the following question: Without an equilibration period, how the targeted Cd concentration (0, 5, 10, 20, and 50 mg Cd kg⁻¹ soil), intended to induce stress, maintained in the soil throughout the experiment?]

Response:

Thank you for this comment. The preparation of Cd treatments has been clarified in the revised manuscript. As stated in the Materials and Methods section, Cd treatments were applied using cadmium chloride monohydrate (CdCl₂·H₂O), and the required salt quantities were calculated based on target elemental Cd concentrations (0, 5, 10, 20, and 50 mg Cd kg⁻¹ soil). The corresponding CdCl₂·H₂O mass was determined from its molecular weight, and the salt was fully dissolved in distilled water before application (Lines 161-166).

"Cd treatments were applied once prior to sowing using cadmium chloride monohydrate (CdCl₂·H₂O) as the Cd source. Target concentrations (0, 5, 10, 20, and 50 mg Cd kg⁻¹) were calculated from elemental Cd, and the corresponding CdCl₂·H₂O mass was determined from the molecular weight to achieve the desired Cd levels. The CdCl₂·H₂O was fully dissolved in distilled water, gently heated to enhance solubility, and visually inspected to confirm complete dissolution and absence of precipitation before application."

In addition, I have now provided further clarification regarding the maintenance of target Cd concentrations during the experiment. To prevent potential Cd loss through leaching, plastic liners were placed inside each pot to ensure water impermeability. Soil water content was maintained close to field capacity using a gravimetric irrigation approach, where pots were periodically weighed, and water lost through plant uptake and evaporation was replenished accordingly. These measures minimized changes in soil solution volume and helped maintain the intended Cd exposure levels throughout the experimental period (Lines 192-203).

"Cd treatments were applied once at the beginning of the experiment before sowing using cadmium chloride monohydrate (CdCl₂·H₂O), and no additional Cd applications were performed during the experimental period. This single-dose approach was selected to simulate a stable soil contamination scenario rather than repeated exposure conditions. To prevent potential Cd loss through leaching and maintain stable Cd concentrations in the soil, plastic liners were placed inside each pot before filling with soil to ensure water impermeability. Soil water-holding capacity (field capacity) was determined before the experiment, and irrigation was managed using a gravimetric approach. Pots were regularly weighed, and water lost through plant uptake and surface evaporation was replenished with distilled water to maintain soil moisture close to field capacity. This approach minimized changes in soil solution volume and prevented Cd loss from the soil system, thereby helping maintain the intended Cd exposure levels throughout the experimental period."

Comment 2: [Were any antioxidant enzyme activities quantified in response to oxidative stress?

Could the author clarify the specific plant tissues (e.g., roots, shoots, leaves) in which oxidative stress parameters were measured and showed responses to Cd exposure across all Brassicaceae crops investigated?]

Response:

Thank you for this helpful question. In the present study, antioxidant enzyme activities were not quantified, as the focus was primarily on non-enzymatic oxidative stress indicators. Oxidative stress parameters, including H₂O₂, MDA, proline content, and membrane permeability (MP), were determined using fresh leaf tissues collected from the youngest fully expanded leaves immediately before harvest. These measurements were performed to evaluate Cd-induced oxidative responses in photosynthetically active tissues. This clarification has now been added to the Materials and Methods section (Lines 238–251).

"Oxidative stress parameters were determined using fresh leaf tissues collected from the youngest fully expanded leaves immediately before harvest. All biochemical analyses were performed using 0.25 g of fresh plant material homogenized in appropriate extraction buffers under cold conditions to prevent biochemical degradation. Non-enzymatic oxidative stress indicators, including hydrogen peroxide (H₂O₂), malondialdehyde (MDA), proline content, and membrane permeability (MP), were quantified using established spectrophotometric methods to evaluate Cd-induced oxidative responses in photosynthetically active tissues. Antioxidant enzyme activities were not included in the present study, as the focus was primarily on non-enzymatic oxidative stress indicators. H₂O₂ content was measured following the method of Mukherjee and Choudhuri [24], based on the spectrophotometric detection of hydrogen peroxide after reaction with potassium iodide. Absorbance was recorded using a UV–Vis spectrophotometer (Shimadzu UV-1201, Kyoto, Japan), and H₂O₂ concentration was calculated according to the standard procedure described in the original method."

 Comment 3: [Wavelengths for H₂O₂, MDA, proline content measurements?]

Response:

Thank you for pointing out this important methodological detail. In the revised manuscript, I have added the specific wavelengths used for spectrophotometric measurements. Absorbance was measured at 390 nm for H₂O₂, 532 nm (with correction at 600 nm) for MDA, and 520 nm for proline determination, following the respective standard protocols. This information has now been included in the Materials and Methods section (Lines 252-258).

"Hydrogen peroxide (H₂O₂), malondialdehyde (MDA), and proline contents were determined using established spectrophotometric methods. Absorbance values were measured at 390 nm for H₂O₂, 532 nm (with correction at 600 nm) for MDA, and 520 nm for proline determination according to the respective standard protocols. Lipid peroxidation was evaluated by determining MDA content using the thiobarbituric acid (TBA) reaction described by Hodges et al. [25], with turbidity correction applied at 600 nm. Proline content was quantified using the ninhydrin-based colorimetric assay of Bates et al. [26]."

 Comment 4: [Differentiate between the results presented in '3.1.3. Cadmium distribution in the shoots and roots' and '3.2. Cadmium concentration, accumulation, and translocation’]

Response:

Thank you for this helpful comment. I agree that the distinction between these two subsections needs clarification. In the revised manuscript, I have clarified that Section 3.1.3 focuses on the relative distribution of Cd between shoot and root tissues expressed as percentage partitioning (Lines 335–336), whereas Section 3.2 presents the absolute Cd concentrations in plant tissues together with derived accumulation and translocation indices (BCF, TF, and TAR). This clarification has been added to the introductory sentences of both sections to improve the logical distinction between distribution patterns and quantitative accumulation metrics (Lines 357–359).

"This section focuses on the relative distribution of Cd between shoot and root tissues, expressed as percentage partitioning within the plant."

"In contrast to the distribution analysis presented above, this section describes the absolute Cd concentrations in plant tissues and derived accumulation indices, including bio-concentration factor (BCF), translocation factor (TF), and total accumulation rate (TAR)." 

Author Response File: Author Response.pdf