Seed Priming with PEG 6000 and Silicic Acid Enhances Drought Tolerance in Cowpea by Modulating Physiological Responses

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript entitled: "Seed priming with polyethylene glycol 6000 and silicic acid can modulate cowpea physiology and enhance its tolerance to water stress" presents a study on the effects of seed priming with polyethylene glycol 6000 (PEG 6000) and silicic acid on cowpea's physiological responses to water stress. The research addresses a relevant issue in agricultural science, particularly in the context of climate change and food security. The study contributes to the ongoing search for sustainable agricultural practices that enhance crop resilience to water stress, a critical challenge for global food security. The manuscript is well-structured, with logical progression from introduction to conclusion. The writing is generally clear and concise.
Suggested revisions in the manuscript:
1) Consider rewording the title for conciseness while maintaining clarity, e.g., "Seed priming with PEG 6000 and silicic acid enhances cowpea drought tolerance by modulating physiological responses."
2) L9-L17: Missing email addresses.
3) L18: Clarify the meaning of "Tel. ???".
4) Please arrange keywords in alphabetical order.
5) The Introduction would benefit from a more extensive presentation of previous studies on the benefits of seed conditioning in other legumes or related crops.
6) L69-70: Provide a more detailed description of the research objectives and explicitly state the research hypothesis. There is only a brief mention of the hypothesis in L343-344.
7) L80: Specify the plant species along with its scientific name - Cowpea (Vigna unguiculata L.). Additionally, include "Plant Material" in the subheading title.
8) L84: Describe the drying method and temperature used for seeds after sterilization.
9) L86: Indicate the seed moisture content prior to the seed priming treatment.
10) L97-101: The terms "Stage I and II" and "Stage 1-5" are misleading. Replace "Stage I and II" with "Step I and II" or "Phase I and II" for clarity.
11) L107: Justify the use of a high temperature (40°C) during the seed priming process. Why were the seeds dried at such a high temperature? Specify the drying duration and the moisture content of the seeds after drying.
12) L115-116: According to ISTA, the optimal germination temperature for Vigna unguiculata is either a fluctuating regime of 20°C for 16 hours and 30°C for 8 hours per day or a constant temperature of 25°C. Explain why the experiment used a temperature regime of 28/32°C (12h/12h). Since soil drought was the studied stress factor, all other experimental conditions should be optimal for seed germination and seedling development.
13) L123-129: This section appears to be a TEMPLATE. Ensure that the methodology covers all analyzed parameters in the experiment, including photosynthetic rate, transpiration rate, stomatal conductance, intercellular CO₂ concentration, instantaneous water use efficiency, and instantaneous carboxylation efficiency.
14) L220: Replace "us" with "use".
15) DISCUSSION: The study suggests that specific priming treatments improve cowpea drought tolerance. However, an additional discussion on the feasibility of implementing these treatments in field conditions, including economic and logistical considerations, would enhance the applicability of the findings.
16) Figure 8: Add explanations for the abbreviations used in the figure caption.
17) In the Conclusions section, additional information on the practical applications of the research findings should be included. Furthermore, future research directions in this field should be outlined.
18) L349: Specify the species of the cultivar mentioned.
19) L353: Remove "more".
20) L356-366: Format according to the required template.
21) FIGURES: Replace commas with periods in values on the x-axis. Provide clarification on the symbols representing whiskers in the graphs (e.g., "standard error bars"?).
22) TEMPLATE: Ensure that the entire manuscript fully complies with the formatting requirements of Horticulture journal.
Overall, this manuscript presents a valuable contribution to the field of plant physiology and stress tolerance in legumes. The manuscript has potential for publication.

Comments on the Quality of English Language

I do not feel competent to assess the correctness of the English language. This should be done by language specialists.

Author Response

REVIEWER 1

Dear Reviewer, our greetings.

We appreciate your kind approach and suggestions regarding our manuscript, which have made the article more robust.

We have addressed the points raised and corrected the highlighted issues.

 

Reviewer

1) Consider rewording the title for conciseness while maintaining clarity, e.g., "Seed priming with PEG 6000 and silicic acid enhances cowpea drought tolerance by modulating physiological responses.

Author: The title was adjusted as suggested. (Lines: 2-3)

 

Reviewer

2) L9-L17: Missing email adresses

Author: Adjustments were made to the authorship of the work. (Lines: 4-20)

 

Reviewer:

3) L18: Clarify the meaning of "Tel. ???

Author: The term “Tel.” has been corrected. (Line: 20)

 

Reviewer:

4) Please arrange keywords in alphabetical order

Author: The keywords were organized in alphabetical order, as suggested: gas exchange; silicon; photosynthesis; Vigna unguiculata (L.). (Line: 36)

Reviewer:

5) The Introduction would benefit from a more extensive presentation of previous studies on the benefits of seed conditioning in other legumes or related crops

 

Author: Information about other results in other cultures has been added to the introduction as suggested.

Among the various available technologies, seed priming stands out for inducing tolerance to deleterious biotic and abiotic effects in plants through stress memory imposed during the seed conditioning process. This process results in physiological and metabolic improvements in the resulting plants, leading to individuals with greater vigour and resistance to stress [Jatana et al., 2024; Diya et al., 2024]. Studies indicate that the seed priming technique has shown promising results in various crops such as maize [Kakar et al., 2023], wheat [Alzoubi et al., 2025], soybean [Vanitha et al., 2024], faba bean [Aboellail et al., 2023], and cowpea [12,13]. In general, the beneficial effects observed in these species are associated with increased regulation of primary and secondary metabolites, greater antioxidant capacity, and activation of osmoprotective and osmoregulatory mechanisms, providing plants with greater resistance to environmental stresses.

In addition to the aforementioned information, it is noteworthy that various inducing agents can be used in seed priming, such as silicon [14], spectral light radiation [15], and polyethylene glycol 6000 (PEG 6000) [16]. However, information on the combination of these factors in the osmotic conditioning of cowpea seeds is still scarce, and further studies are needed to elucidate the effectiveness of this type of treatment. In this context, the hypothesis is that physiological seed conditioning can mitigate the effects of drought in cowpea plants by promoting improvements in physiological responses. Therefore, the aim of this study was to induce drought tolerance in cowpea through physiological seed conditioning using polyethylene glycol 6000 and silicic acid.

 

Reviewer:

6) L69-70: Provide a more detailed description of the research objectives and explicitly state the research hypothesis. There is only a brief mention of the hypothesis in L343-344.

Author: A better description of the scientific hypothesis has been added, in addition to adjustments to the text's coherence, as suggested:

L68-77. In addition to the aforementioned information, it is worth noting that several inducing agents can be used in seed priming, with silicon [14], spectral light radiation [15], and polyethylene glycol 6000 (PEG 6000) [16] being particularly noteworthy. However, information on the combination of these factors in the priming of cowpea seeds remains scarce, and further studies are required to elucidate the effectiveness of this type of treatment.

In this context, the hypothesis is that seed priming may mitigate the effects of drought in cowpea plants by promoting improvements in physiological responses. Therefore, the aim of this study was to induce drought tolerance in cowpea through physiological seed conditioning with polyethylene glycol 6000 and silicic acid.

 

Reviewer:

7) L80: Specify the plant species along with its scientific name - Cowpea (Vigna unguiculata L.). Additionally, include "Plant Material" in the subheading title.

Author: The plant species was included in L87 along with its scientific name:

L87. Cowpea seeds [Vigna unguiculata (L.) Walp.] of the cultivar BRS Pingo de Ouro.

 

Reviewer:

8) L84: Describe the drying method and temperature used for seeds after sterilization.

Author: The initial drying method was best described as suggested:

L92. The seeds were then briefly dried at room temperature using sterilized paper [17].

L94. Immediately after this process, 50 seeds with a moisture content of approximately15% were placed in Gerbox®-type plastic boxes...

 

Reviewer:

9) L86: Indicate the seed moisture content prior to the seed priming treatment.

Author: The moisture content was inserted and the text was adjusted for better understanding, as suggested.

L111-121. Seed conditioning (E) was carried out using combinations of three water potentials induced by polyethylene glycol 6000 (PEG 6000): no water deficit (Ψw = 0 MPa), moderate deficit (Ψw = –0.4 MPa), and severe deficit (Ψw = –0.8 MPa), along with two concentrations of silicic acid (a commercial product containing 42% silicon): 0 and 200 mg L⁻¹. The conditioning was conducted under high light intensity conditions, using red light with a wavelength between 600 and 680 nm (RL) and a constant temperature of 40 °C. All these conditions were employed to activate defence responses in the seeds, as described by Araújo et al. [2018], Costa et al. [15], and Alencar et al. [17] (Table 1). After the treatment, the seeds were dried under the same light and temperature conditions until they returned to their original dry weight, with moisture content ranging from 12% to 15%.

 

Reviewer:

10) L97-101: The terms "Stage I and II" and "Stage 1-5" are misleading. Replace "Stage I and II" with "Step I and II" or "Phase I and II" for clarity.

Author: The terms were replaced in the text and for better understanding the adjustments were adopted, as suggested:

L130-135. The seeds obtained from the priming in Stage I were sown in polyethylene pots with a capacity of 0.8 L, filled with 1 kg of substrate consisting of 75% homogenised soil and 25% cattle manure. After sowing, the pots were transferred to a Fitotron-type growth chamber, with the temperature set to 32 °C during the day and 28 °C at night, relative humidity maintained at 60%, and a 12-hour photoperiod, aiming to simulate conditions similar to those found in the semi-arid region of Paraíba.

Reviewer

11) L107: Justify the use of a high temperature (40°C) during the seed priming process. Why were the seeds dried at such a high temperature? Specify the drying duration and the moisture content of the seeds after drying..

Author: The idea was to create a stress memory as described by Costa et al. [15] and Alencar et al. [17]

Reviewer:

12) L115-116: According to ISTA, the optimal germination temperature for Vigna unguiculata is either a fluctuating regime of 20°C for 16 hours and 30°C for 8 hours per day or a constant temperature of 25°C. Explain why the experiment used a temperature regime of 28/32°C (12h/12h). Since soil drought was the studied stress factor, all other experimental conditions should be optimal for seed germination and seedling development.

Author: We have adjusted the text to clarify the observations of the Reviewer:

L130-135. The seeds obtained from the priming in Stage I were sown in polyethylene pots with a capacity of 0.8 L, filled with 1 kg of substrate consisting of 75% homogenised soil and 25% cattle manure. After sowing, the pots were transferred to a Fitotron-type growth chamber, with the temperature set to 32 °C during the day and 28 °C at night, relative humidity maintained at 60%, and a 12-hour photoperiod, aiming to simulate conditions similar to those found in the semi-arid region of Paraíba.

 

Reviewer

13) L123-129: This section appears to be a TEMPLATE. Ensure that the methodology covers all analyzed parameters in the experiment, including photosynthetic rate, transpiration rate, stomatal conductance, intercellular CO₂ concentration, instantaneous water use efficiency, and instantaneous carboxylation efficiency.

Author: We adjusted the text as suggested:

L141-157. The gas exchange assessments were conducted using an LI-6800 Portable Photo-synthesis System (LI-COR Inc., Lincoln, Nebraska, USA). Throughout the evaluations, a constant photon flux density of 1200 μmol m^-2 s^-1 of actinic light was employed. The chamber's relative humidity was maintained at 60%, and the CO₂ concentration at 400 μmol mol^-1. The air temperature within the chamber was monitored using a thermoe-lectric sensor located at its base, and it was set at 27 °C. Plants were evaluated for the following parameters: photosynthetic rate (A) (μmol CO₂ m^-2 s^-1), transpiration rate (E) (mmol H₂O m^-2 s^-1), stomatal conductance (gs) (mmol m^-2 s^-1), and intercellular CO₂ concentration (Ci) (μmol CO₂ m^-2 s^-1). These variables were measured utilizing the third fully expanded leaf, counted from the apex of the main branch, during the morning hours (between 7 and 11 am).

With this data, the instantaneous efficiency of water use (WUEi) [(μmol CO₂ m^-2 s^-1) (mmol H₂O m^-2 s^-1)^-1] was quantified. Through the ratio between photosynthetic rate (A) and transpiration rate (E) and instantaneous carboxylation efficiency (iCE) [(μmol CO₂ m^-2 s^-1) (μmol CO₂ m^-2 s^-1)^-1], through the ratio between photosynthetic rate (A) and intercellular CO₂ concentration (Ci).

Reviewer

14) L220: Replace "us" with "use".

Author: was replaced “us” with “use”

 

Reviewer

15) DISCUSSION: The study suggests that specific priming treatments improve cowpea drought tolerance. However, an additional discussion on the feasibility of implementing these treatments in field conditions, including economic and logistical considerations, would enhance the applicability of the findings.

Author: Information regarding economic and logistical aspects has been added, as suggested.

However, little is known about the combination of PEG 6000 and silicon in seed conditioning. Although the action of silicon in this type of treatment was observed by Özdemir [35] in purple maize, promoting improvements in gas exchange, especially in net carbon assimilation and transpiration rate, further studies are still needed. Bourioug et al. [36] also reported an increase in photosynthesis and transpiration in sunflower plants subjected to water stress after conditioning with PEG 6000.

These results reinforce the hypothesis that these agents may be used to induce drought stress tolerance. Furthermore, other factors may enhance the beneficial effects of osmotic seed conditioning, such as light conditions, as demonstrated in studies with Moringa oleifera Lam. [15]. However, studies addressing the combined effects of these factors in the seed treatment of cowpea are scarce.

Another relevant aspect is described by Saha et al. [2022], in which the researchers discuss different seed priming techniques and their positive outcomes in inducing stress tolerance under field conditions, further supporting the potential of this technique in the present study. Moreover, seed priming is characterised by low application costs and high adaptive potential, being recommended not only for the physiological benefits it promotes in plants but also for its economic feasibility. Thus, the application of this technique to cowpea cultivation at field level is plausible, both due to the promising results and the economic and logistical aspects involved.

 

Reviewer

16) Figure 8: Add explanations for the abbreviations used in the figure caption..

Author: An explanation has been inserted. As suggested:

Figure 8. W100 Control: represents the values obtained under normal irrigation conditions (100% field capacity); W50 Control: represents the values obtained under limited irrigation conditions (50% field capacity), without seed priming application; W50 Seed Priming: presents the minimum and maximum values of the variables photosynthetic rate (A), transpiration rate (E), stomatal conductance (gs), intercellular CO₂ concentration (Ci), instantaneous water use efficiency (iWUE), and instantaneous carboxylation efficiency (iCE), regardless of the treatment, under water deficit conditions.

Reviewer

17) In the Conclusions section, additional information on the practical applications of the research findings should be included. Furthermore, future research directions in this field should be outlined.

Author: We have made adjustments to the text to meet the Reviewer's suggestion:

Given the above, it is concluded that the osmotic conditioning of seeds with PEG 6000 and silicic acid in Vigna unguiculata L. Walp., BRS Pingo de Ouro cultivar, can promote increases in photosynthetic rates, even under water restriction, mainly through the induction of greater instantaneous water use efficiency and carboxylation efficiency. Osmotic treatments 2 (40 °C + Ψw 0 MPa + 200 mg L-1 Si + RL), 3 (40 °C + Ψw -0,4 MPa + 0 mg L-1 Si + RL), and 4 (40 °C + Ψw -0,4 MPa + 200 mg L-1 Si + RL) sto-od out for their greater efficiency in improving gas exchange. These findings suggest a highly effective and promising treatment combination for enabling cowpea cultivation in water-restricted environments, contributing to food security.

Reviewer:

18) L349: Specify the species of the cultivar mentioned.

Author: Species was mentioned:

Moringa oleifera Lam.

Reviewer:

19) L353: Remove "more".

Author: “more” has been removed from the text.

Reviewer:

20) L356-366: Format according to the required template.

Author: Adjusted.

Reviewer:

21) FIGURES: Replace commas with periods in values on the x-axis. Provide clarification on the symbols representing whiskers in the graphs (e.g., "standard error bars"?).

Author: The figures and captions were adjusted as suggested.

Reviewer:

22) TEMPLATE: Ensure that the entire manuscript fully complies with the formatting requirements of Horticulture journal.

Author: The manuscript was fully revised to meet Horticulturae standards.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The article investigates how seed priming with polyethylene glycol 6000 and silicic acid influences cowpea physiology and enhances its tolerance to water stress. The authors conducted a controlled experiment to test whether pre-treatment of seeds could mitigate the negative effects of water scarcity. The article’s topic is not novel, but the specific combination of treatments and detailed physiological evaluations in cowpea brings the element of novelty. The article seems valuable for practical agricultural application and it is contributing to existing scientific literature.

The introduction is clear and gives a relevant context to the study.

The Materials and Methods section is well structured in most parts. Some areas need clarifications and changes.

Line 92: was there a specific reason for this period of time? Specify or cite the methodology.

Lines 123-129 This paragraph need to be completed as at the moment there is a template instead. It seems like this part of methodology is missing.

The Results section mostly presents the findings clearly. The figures are complete, easy to understand and well described, but the readability of axis labels and legends could be better (maybe a larger or different font would be better).

Lines 147-154: This fragment needs to be rewritten for clarity, it’s a bit chaotic and hard to understand.

Line 149: Explain here and in all other parts of the results from what baseline or control these reductions were calculated.

Line 170: Explain why were they all notably clearer.
Line 191: Explanations better fit in the discussion section, as they are interpretations rather than results.

Lines 203, 221: Definitions and explanations included here should be moved to Methods or Discussion as Results should present data only.

The Discussion section provides an interpretation of the study's results, connecting them to existing literature. However, it should be reorganised. For example: some subsections start with “Also” or “However” suggesting continuation of a thought rather than a new subject. Perhaps they could be presented without being divided to give it continuity.

More direct comparative references to studies examining PEG and silicon specifically could be added.

Some parts of the discussion belong in the Results or Introduction. For example:

Descriptions of detailed percentage changes are better in Results.

Fundamental explanations (such as "EiUA is a measure that relates carbon assimilation to transpiration") could be moved to the Introduction.

The Conclusion summarizes key findings, states the practical significance of the study and identifies the most promising treatments. However, it restates multiple priming combinations without clearly specifying which treatment is the single most recommended or practically viable. Suggestions for follow-up studies could be added as well.

Line 353: "This may reveal a possible ideal combination" sounds a bit informal, consider rephrasing it, for example: "These findings suggest a highly effective treatment combination".

The used literature is appropriate. 

 

Comments on the Quality of English Language

The manuscript is written well using appropriate scientific vocabulary. However, there are several minor issues, mainly related to grammar and spacing (extra or missing spaces between words and punctuation) that should be corrected. The manuscript should be carefully proofread, perhaps by professional service or a native speaker.

Author Response

REVIEWER 2

Dear Reviewer, our greetings.

We appreciate your kind approach and suggestions regarding our manuscript, which have made the article more robust.

We have addressed the points raised and corrected the highlighted issues.

Reviewer: The introduction is clear and gives a relevant context to the study.

Author: Thanks for the observation.

Reviewer: The Materials and Methods section is well structured in most parts. Some areas need clarification and changes.

Author: We have made adjustments to the text for better clarification.

Reviewer: Line 92 was there a specific reason for this period of time? Specify or cite the methodology.

Author: The text was adjusted, in short, this period was necessary for the seeds to be soaked without the roots being emitted.

Reviewer: Lines 123-129 This paragraph need to be completed as at the moment there is a template instead. It seems like this part of methodology is missing.

Author: Thank you for your observation, the paragraph has been adjusted and is complete.

 Reviewer: The Results section mostly presents the findings clearly. The figures are complete, easy to understand and well described, but the readability of axis labels and legends could be better (maybe a larger or different font would be better).

Author: We appreciate the suggestions. However, we are unable to modify the font or increase the size of the axis labels in the figures, as they are already set at size 11. Enlarging them further could lead to disproportionate and misaligned axes.

Reviewer:  Lines 147-154: This fragment needs to be rewritten for clarity, it’s a bit chaotic and hard to understand.

Author: The paragraph has been adjusted to make the text flow more smoothly.

Reviewer: Line 149: Explain here and in all other parts of the results from what baseline or control these reductions were calculated.

Author: It was better detailed throughout the results from which control the reductions were calculated, as suggested.

Reviewer: Linha 170: Explique por que todas elas foram notavelmente mais claras.

Author: Adjustments were made throughout the text.

Reviewer:  Line 191 Explanations better fit in the discussion section, as they are interpretations rather than results.

Author: Changes were made to the Results section to avoid misinterpretation and ensure a more objective presentation of the data.

Reviewer: Lines 203, 221: Definitions and explanations included here should be moved to Methods or Discussion as Results should present data only.

Author: This adjustment was made to the results section, as suggested.

Reviewer:  The Discussion section provides an interpretation of the study's results, connecting them to existing literature. However, it should be reorganised. For example: some subsections start with “Also” or “However” suggesting continuation of a thought rather than a new subject. Perhaps they could be presented without being divided to give it continuity.

Author: Adjustments were made to the Discussion with a focus on improving textual fluidity and argumentative coherence, as recommended.

Reviewer: More direct comparative references to studies examining PEG and silicon specifically could be added.

Author: Some references have been added throughout the text that examine these two agents in seed priming.

Reviewer:  Some parts of the discussion belong in the Results or Introduction. For example:

Descriptions of detailed percentage changes are better in Results.

Fundamental explanations (such as "EiUA is a measure that relates carbon assimilation to transpiration") could be moved to the Introduction.

Author: Adjustments were made to the Discussion with a focus on improving textual fluidity and argumentative coherence, as recommended.

Reviewer:  The Conclusion summarizes key findings, states the practical significance of the study and identifies the most promising treatments. However, it restates multiple priming combinations without clearly specifying which treatment is the single most recommended or practically viable. Suggestions for follow-up studies could be added as well.

Author: The conclusion was better described, giving the text better fluidity and objectivity.

Reviewer:  Line 353: "This may reveal a possible ideal combination" sounds a bit informal, consider rephrasing it, for example: "These findings suggest a highly effective treatment combination".

Author: The adjustment was made as suggested.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Dear Authors,

After carefully reviewing the revised version of the manuscript entitled "Seed priming with PEG 6000 and silicic acid enhances drought tolerance in cowpea by modulating physiological responses", I acknowledge that the authors have satisfactorily addressed my previous comments.

I have only a few minor suggestions that I would like to bring to your attention:

1) The keywords are still not listed in alphabetical order.

 2) In line with my earlier suggestions, the authors have appropriately changed "Stage I" and "Stage II" to "Step I" and "Step II". However, they also replaced "Stage 1...Stage 6" with "Step 1...Step 6", which I believe was unnecessary. In my opinion, the original "Stage 1...Stage 6" should be retained to maintain a clear distinction.

Author Response

Dear Reviewer

The new suggested adjustments have been made.

Best regards,

Authors

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Than you for you reply and all changes made. In my opinion the manuscript has been improved and it can be consider for publication in the current form.

Author Response

Dear Reviewer,

Thank you for your feedback and kindness.
Best regards,
Authors

Author Response File: Author Response.pdf