Exogenous Proline Application Mitigates Salt Stress in Physalis ixocarpa Brot.: Morphophysiological, Spectroscopic, and Metabolomic Evidence

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript entitled “ Exogenous Proline Application Mitigates Salt Stress in  Physalis ixocarpa Brot.: Morphophysiological, Spectroscopic,  and Metabolomic Evidenceis of significant relevance to agricultural science, particularly for improving crop resilience in salinized soils. The experimental design is robust, the methodology is sound and well-described, and the results are compelling and clearly presented. The manuscript is generally well-written. I recommend publication after minor revisions to address the following points:

1. The finding that phenolic antioxidants (catechol) disappear under salt stress and are not restored by proline is intriguing. The authors' hypothesis that proline provides prevantative protection, reducing the need for energy-costly phenolic synthesis, is plausible. However, this conclusion could be strengthened by:Directly measuring oxidative stress markers (MDA for lipid peroxidation, H₂O₂ levels) to confirm that oxidative damage is indeed lower in the proline-treated, salt-stressed plants despite the lack of phenolics and Briefly discussing alternative explanations, such as the potential proline-induced activation of other, non-phenolic antioxidant systems (like SOD, CAT, APX).
2. Add paragraph on the economic importance of P. ixocarpa in introduction section.
3. The manuscript uses both “Physalis izocarpa” (in the title, abstract, and Table 1) and “Physalis ixocarpa” (in the introduction, methods, and elsewhere). The accepted spelling should be confirmed and used consistently throughout the manuscript, including in tables and figures.
4. Please clarify the timeline. It states two independent experimental repeats (first repeat in September 2024, second repeat in September-October 2024)". This seems to indicate the second repeat spanned two months, which is unusually long for a germination assay. Please clarify the duration of each repeat.

 

 

 

 

 

Author Response

Q1. The finding that phenolic antioxidants (catechol) disappear under salt stress and are not restored by proline is intriguing. The authors' hypothesis that proline provides preventative protection, reducing the need for energy-costly phenolic synthesis, is plausible. However, this conclusion could be strengthened by: Directly measuring oxidative stress markers (MDA for lipid peroxidation, H₂O₂ levels) to confirm that oxidative damage is indeed lower in the proline-treated, salt-stressed plants despite the lack of phenolics and briefly discussing alternative explanations, such as the potential proline-induced activation of other, non-phenolic antioxidant systems (like SOD, CAT, APX).

Response: We thank the reviewer for this valuable insight. We acknowledge that direct measurement of oxidative stress markers would strengthen our interpretation. While such measurements are beyond the scope of the current study, we have enhanced our discussion to address both points raised. First, we now explicitly acknowledge this limitation and identify it as an important direction for future research. Second, we have added a comprehensive discussion of alternative mechanisms through which proline may provide protection without inducing phenolic synthesis, including the potential activation of enzymatic antioxidant systems (SOD, CAT, APX) and enhancement of the ascorbate-glutathione cycle. These additions provide a more nuanced interpretation of our findings while maintaining scientific rigor.

Changes made: New paragraph inserted in Discussion section 4.4 (GC-MS Metabolomic Analysis)

“[…] While our data strongly suggest that proline prevents oxidative damage through metabolic regulation and membrane stabilization, we acknowledge that direct measurement of oxidative stress markers would provide definitive evidence for this mechanism. Future studies should quantify malondialdehyde (MDA) and hydrogen peroxide (H₂O₂) levels to confirm whether proline-treated plants indeed experience lower oxidative damage despite reduced phenolic content.

Several alternative mechanisms may explain how proline provides protection without inducing phenolic synthesis. Exogenous proline has been shown to enhance enzymatic antioxidant systems, including superoxide dismutase (SOD), catalase (CAT), and ascorbate peroxidase (APX), which can compensate for reduced phenolic antioxidants (Nounjan et al., 2012; El Moukhtari et al., 2020). Additionally, proline strengthens the ascorbate-glutathione (AsA-GSH) cycle, improving H₂O₂ detoxification through enhanced activities of dehydroascorbate reductase and glutathione reductase while maintaining favorable AsA/DHA and GSH/GSSG ratios (Islam et al., 2009; Hasanuzzaman et al., 2019). Proline itself also functions as a direct radical scavenger and protein stabilizer, potentially reducing primary ROS generation in chloroplasts and mitochondria (Szabados & Savouré, 2010). These mechanisms collectively provide plausible explanations for the observed depletion of phenolic antioxidants that are not restored by exogenous proline treatment.”

Q2. Add paragraph on the economic importance of P. ixocarpa in introduction section.

Response: We appreciate this suggestion to strengthen the introduction. We have added a comprehensive paragraph detailing the economic importance of P. ixocarpa, including its cultivation area, production volumes, export value, and role in supporting smallholder farmers, particularly in Mexico where it ranks among the top vegetable crops.

Changes made: New paragraph inserted in the Introduction section, after the paragraph describing P. ixocarpa's nutritional and antimicrobial properties.

“Physalis ixocarpa holds considerable economic importance in Mexican agriculture, ranking among the country's five most economically significant vegetable crops (González-Pérez & Guerrero-Beltrán, 2021; Ramos-López et al., 2018). In 2020, Mexican tomatillo cultivation covered approximately 40,117 hectares across 30 states, with an average yield of 19.3 t/ha (Balbuena-Mascada et al., 2022). Production has grown substantially, reaching approximately 778,000 tons by 2018, representing 4.7% of national vegetable output (Mexico Business News, 2020). The leading production state, Sinaloa, generated 164,500 tons valued at MX$501 million (≈US$22.8 million), with Zacatecas and Jalisco as other major producers (Mexico Business News, 2020). The crop supports predominantly smallholder farmers, with most production occurring on plots smaller than one hectare (Ramos-López et al., 2018). Mexico's tomatillo exports have shown remarkable growth, increasing from US$26 million in 2009 to US$82 million in 2018, primarily to the United States, though markets in the UK, UAE, France, Canada, and Belize are expanding (Mexico Business News, 2020).”

Q3. The manuscript uses both “Physalis izocarpa” (in the title, abstract, and Table 1) and “Physalis ixocarpa” (in the introduction, methods, and elsewhere). The accepted spelling should be confirmed and used consistently throughout the manuscript, including in tables and figures.

Response: We have carefully reviewed the entire manuscript, including all tables and figures. The correct spelling "Physalis ixocarpa" is consistently used throughout. We have verified that all instances are properly italicized as required for species names. The reviewer may have encountered a display issue in the PDF version; we have ensured consistency in the final submission.

Q4. Please clarify the timeline. It states two independent experimental repeats (first repeat in September 2024, second repeat in September-October 2024)". This seems to indicate the second repeat spanned two months, which is unusually long for a germination assay. Please clarify the duration of each repeat

Response: We apologize for the ambiguity in our timeline description. Each germination experiment lasted exactly 15 days. The second repeat began on September 23 and concluded on October 7, 2024, spanning the transition between months but maintaining the standard 15-day duration. We have clarified this in the manuscript.

Changes made: "[…] (first repeat: September 2-16, 2024; second repeat: September 23 - October 7, 2024)"

Reviewer 2 Report

Comments and Suggestions for Authors

This study investigates the potential of exogenous proline to mitigate salt stress in Physalis ixocarpa (tomatillo). This is a well-designed and executed study that uses a multi-faceted approach to understand the effects of proline on tomatillo plants under salt stress. The authors use a combination of morphophysiological, spectroscopic, and metabolomic analyses to provide a comprehensive picture of the plant's response to proline treatment.

The manuscript is well-written and easy to follow.  I have a few minor comments that I believe would improve the manuscript.

• Provide picture of the plant to show visible changes before and after proline treatment.
• Proline Pretreatment Effects on Salt-Stressed Seeds: Enhance this section to mention the effect of different proline concentrations on seed germination and why at 10mM proline the germination rate is saturated/declining.
• In the methods section, add make and model information for the sonicator and other key instruments, along with supplier and grade for chemicals.
• In the results section, the authors state that the "most relevant changes" from the ATR-FTIR analysis are summarized in Table 4. provide a brief explanation of why these changes are considered the most relevant.
• In the discussion, the authors state that "proline's effectiveness in preventing oxidative stress formation through pre-emptive osmotic and membrane stabilization, thereby reducing the metabolic cost of phenolic antioxidant synthesis." This is an interesting hypothesis, but it would be helpful to provide more evidence to support it.

 

 

Author Response

Q1. Provide picture of the plant to show visible changes before and after proline treatment.

Response: We thank the reviewer for this suggestion. We have added a new figure showing representative plants from each treatment group after 30 days of growth, clearly illustrating the morphological differences between control, salt-stressed, and proline-treated plants under salt stress.

Changes made: Section 3.3.1

"The visual differences among treatments are evident in Figure 1, which shows representative plants after 30 days of in vitro culture. Control plants exhibited vigorous growth with expanded green leaves, while salt-stressed plants showed stunted growth and chlorotic symptoms. Proline treatment (particularly at 8 mM) partially restored plant vigor, with improved leaf coloration despite reduced root development."

"Figure 1. Physalis ixocarpa plants after 30 days of in vitro culture under different treatments. From left to right: control (no salt), 75 mM NaCl, and 75 mM NaCl supplemented with 4, 6, 8, or 10 mM proline."

Q2. Proline Pretreatment Effects on Salt-Stressed Seeds: Enhance this section to mention the effect of different proline concentrations on seed germination and why at 10mM proline the germination rate is saturated/declining.

Response: We have expanded our discussion of proline concentration effects, providing explanations for the optimal response at 8 mM and the reduced efficacy at 10 mM.

Changes made: Section 3.2

"Proline's protective effects showed clear concentration dependency. At 4 mM, germination improved modestly to 70.7%, while 6 mM achieved 76.7%. The optimal concentration was 8 mM, restoring germination to 78% and remarkably recovering fresh weight to near-control levels (1.322 vs. 1.354 g), indicating not just improved germination but enhanced seedling vigor. However, at 10 mM, both parameters declined (70.7% germination, 0.664 g fresh weight), suggesting a biphasic response typical of osmolyte applications."

Q3. In the methods section, add make and model information for the sonicator and other key instruments, along with supplier and grade for chemicals.

Response: We have verified that all requested information is already present in the manuscript. The sonicator model (UP200Ht, Hielscher Ultrasonics, Teltow, Germany) is specified in Section 2.6, chemical supplier (Merck KGaA, Darmstadt, Germany) in Section 2.1, and reagent grade ("All reagents were of analytical grade") is clearly stated. We have double-checked that all equipment specifications are complete throughout the methods section.

Q4. In the results section, the authors state that the "most relevant changes" from the ATR-FTIR analysis are summarized in Table 4. provide a brief explanation of why these changes are considered the most relevant.

Response: We appreciate this request for clarification. We have added an explanation of our selection criteria for identifying the most relevant spectral changes.

 Changes made: Section 3.4

"Table 4 summarizes the most relevant spectral changes, selected based on their magnitude and interpretative significance. We focused on bands that either appeared de novo, completely disappeared, or showed substantial shifts (≥5 cm⁻¹) between treatments, as these changes indicate significant molecular reorganization rather than minor conformational adjustments. Minor spectral variations (<5 cm⁻¹) were excluded as they likely represent subtle structural modifications without major functional implications."

Q5. In the discussion, the authors state that "proline's effectiveness in preventing oxidative stress formation through pre-emptive osmotic and membrane stabilization, thereby reducing the metabolic cost of phenolic antioxidant synthesis." This is an interesting hypothesis, but it would be helpful to provide more evidence to support it.

Response: We thank the reviewer for highlighting this important mechanistic aspect. We have expanded our discussion with additional evidence supporting the pre-emptive protection hypothesis, including proline's direct effects on membrane stability, protein protection, and metabolic efficiency. This expansion is coordinated with our response to Reviewer 1's Query 1 to provide a comprehensive treatment of this mechanism.

Changes made: Discussion section 4.4

“[…] The pre-emptive protection mechanism is supported by multiple lines of evidence. Proline functions as an osmoprotectant that maintains cellular turgor before dehydration-induced damage occurs, with cellular proline concentrations increasing up to 100-fold within hours of stress initiation (Szabados & Savouré, 2010; Verbruggen & Hermans, 2008). This rapid accumulation prevents the concentration of reactive species that would otherwise trigger oxidative cascades (Sharma & Dietz, 2006). Furthermore, proline directly stabilizes membrane phospholipids through hydrogen bonding with both phosphate groups and fatty acid chains, preventing lipid peroxidation at its source (Hoque et al., 2008; Naliwajski & Skłodowska, 2021). The metabolic cost analysis reveals that proline synthesis requires only 2 ATP and 2 NADPH per molecule via the P5CS-P5CR pathway (Fichman et al., 2015), whereas phenolic biosynthesis through the shikimate and phenylpropanoid pathways demands substantially higher energy investment—approximately 15 ATP and 10 NADPH per lignin monomer unit (Neilson et al., 2013; Erb & Kliebenstein, 2020). This 5-fold difference in energy requirements explains why proline-mediated protection represents an evolutionarily advantageous strategy, allowing plants to maintain protection while preserving resources for growth and development (Rajasheker et al., 2019)."