Efficacy of Lippia alba Essential Oil in Alleviating Osmotic and Oxidative Stress in Salt-Affected Bean Plants
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsThe manuscript entitled : "The efficacy of Lippia alba essential oil in mitigating osmotic and oxidative stress in salt-affected bean plants" presents an interesting and timely study on the use of Lippia alba essential oil as a biostimulant to mitigate the effects of salt-induced osmotic and oxidative stress in Phaseolus acutifolius plants. The topic is highly relevant given the increasing salinization of soils and the urgent need forsustainable strategies to enhance crop resilience under abiotic stress conditions.
The authors demonstrate that seed priming with L. alba essential oil improves various physiological and biochemical parameters associated with salt stress tolerance, including water uptake, chlorophyll content, proline accumulation, and antioxidant enzyme activity.
Comments and suggested revisions :
Title - consider slightly rephrasing the title for improved clarity. A suggested alternative could be: “Efficacy of Lippia alba essential oilin alleviating osmotic and oxidative stress in salt-stressed bean plants.”
Keywords - please revise the keywords so that they differ from the terms used in thetitle. Additionally, ensure that the keywords are listed in alphabetical order.
Introduction: The research objectives and hypothesis should be clearly and concisely stated in the final paragraph of the Introduction. Therefore, I recommend merging lines 77–82 and 112–114 in a cohesive manner.
Materials and Methods
The methodology section requires more detailed and transparent information regarding the number of biological replicates and sample sizes foreach measured parameter. Additionally, the germination and salt stress protocols need further clarification:
Please describe the seed priming procedure in detail.
Specify the type and quality of filter paper used.
Indicate the temperature conditions during both priming and germination.
In line 151, clarify the initial seed moisture content.
What was the initial germination capacity of the seeds?
Was germination capacity evaluated after seed priming and again after the halotreatment?
Figure 2 - in the figure legend please provide an explanation for panel (b) to ensure clarity for the reader.
Conclusion - the first two sentences of the conclusion are redundant and can be omitted. Instead, this section should:
- succinctly summarize the key findings of the study,
- clearly state whether the research objective was met and how
- suggest potential directions for future research,
- highlight how the results may contribute to the development of adaptive strategies for crop cultivation under saline conditions.
The manuscript should be carefully proofread by a native English speaker or a professional editor. There are occasional issues with grammar and syntax that may affect clarity.
Author Response
We thank the referee for the comments and suggestions. The modifications were included and highlighted in the manuscript and the responses to the referee are reported below.
The manuscript entitled: "The efficacy of Lippia alba essential oil in mitigating osmotic and oxidative stress in salt-affected bean plants" presents an interesting and timely study on the use of Lippia alba essential oil as a biostimulant to mitigate the effects of salt-induced osmotic and oxidative stress in Phaseolus acutifolius plants. The topic is highly relevant given the increasing salinization of soils and the urgent need for sustainable strategies to enhance crop resilience under abiotic stress conditions.
The authors demonstrate that seed priming with L. alba essential oil improves various physiological and biochemical parameters associated with salt stress tolerance, including water uptake, chlorophyll content, proline accumulation, and antioxidant enzyme activity.
Comments and suggested revisions:
Comment 1: Title - consider slightly rephrasing the title for improved clarity. A suggested alternative could be: “Efficacy of Lippia alba essential oil in alleviating osmotic and oxidative stress in salt-stressed bean plants.”
Response 1: the title was modified accordingly (lines 2-3)
Comment 2: Keywords - please revise the keywords so that they differ from the terms used in the title. Additionally, ensure that the keywords are listed in alphabetical order.
Response 2: the keywords were revised (lines 32-33)
Comment 3: Introduction: The research objectives and hypothesis should be clearly and concisely stated in the final paragraph of the Introduction. Therefore, I recommend merging lines 77–82 and 112–114 in a cohesive manner.
Response 3: the two paragraphs were merged and added at the end of the introduction (lines 105-113)
Materials and Methods
Comment 4: The methodology section requires more detailed and transparent information regarding the number of biological replicates and sample sizes for each measured parameter.
Response 4: the number of biological replicates was added (lines 154, 159, 160). The sample sizes (n) used for the physiological determinations are already reported in the capture of each table and figure
Comment 5: Please describe the seed priming procedure in detail.
Response 5: information was added (lines 134-139)
Comment 6: Specify the type and quality of filter paper used.
Response 6: information was added (lines 141, 152)
Comment 7: Indicate the temperature conditions during both priming and germination.
Response 7: the information is already reported in Materials and Methods (lines 143, 149, 154). All seeds were primed and then germinated at room temperature (RT)
Comment 8: In line 151, clarify the initial seed moisture content.
Response 8: the information was added (line 151)
Comment 9: What was the initial germination capacity of the seeds?
Response 9: we considered the initial germination capacity as germination rate (%) of not stressed (0 mM NaCl) control seeds; in this case the germination rate was 92% ± 4% (the data is reported in Table 1)
Comment 10: Was germination capacity evaluated after seed priming and again after the halotreatment?
Response 10: In table 1 are reported the data of the germination before and after seed priming
Comment 11: Figure 2 - in the figure legend please provide an explanation for panel (b) to ensure clarity for the reader.
Response 11: information was added (lines 348-351). The legend of the figure has been corrected
Comment 12: Conclusion - the first two sentences of the conclusion are redundant and can be omitted. Instead, this section should:
- succinctly summarize the key findings of the study,
- clearly state whether the research objective was met and how
- suggest potential directions for future research,
- highlight how the results may contribute to the development of adaptive strategies for crop cultivation under saline conditions.
Response 12: the Conclusion was rewritten (lines 449-460)
Comments on the Quality of English Language
Comments 13: The manuscript should be carefully proofread by a native English speaker or a professional editor. There are occasional issues with grammar and syntax that may affect clarity.
Response 13: for the entire manuscript, the English was checked and modified
Reviewer 2 Report
Comments and Suggestions for AuthorsThe manuscript title “The efficacy of Lippia alba essential oil in mitigating osmotic and oxidative stress in salt-affected bean plants” is a good manuscript but requires improvements and some clarification on figure 3 results. Lippia alba (Mill.) N.E.Br. ex Britton and P. Wilson is a native Colombian plant found throughout Central and South America, historically utilized by pre-Columbian populations for its numerous beneficial properties, including antifungal, antibacterial, antiviral, and anti-inflammatory activities. The essential oil of L. alba is rich in phytochemicals with antioxidant properties, making it potentially valuable for pharmacological and biotechnological applications, particularly in protecting horticultural crops from salinity stress.
The study suggests that L. alba essential oil could be effectively utilized in agriculture to enhance the resilience of crops against salinity stress, offering a promising avenue for improving crop productivity in saline environments.
This MS needs improvements and some clarification on figure 3 results. Comments for authors:
- Line 150: “L. alba” is a scientific name it should be italic throughout the manuscript.
- Line 180: in equation “] ∙ 100” here ∙ means multiply? I suggest its better to use multiply symbols. Same for the next equations.
- Line 199: “Data are expressed as µg Ca2+ × mg f.w.−1 .” any standard or standard was made? Standard information and concentration is missing.
- Line 206: “at 664.1 nm (chlorophyll a) and 648.6 nm 206 (chlorophyll b). The concentration of total photosynthetic pigments was calculated ac- 207 cording to Lichtenthaler [35] and expressed as µg · g f.w.−1” which equation was used to calculate chlorophyll contents?
- Line 218: “carbohydrates was evaluated” please add few more line to elaborate this protocol used in this study.
- Table 3: Soluble sugars results are presented but methods are not mentioned.
- Why do the proline results have too much difference in EO 40 and 80, 91.8 ±2 a*** 485.7 ± 41.3 b*** respectively. From 91.8 to 485.7? Is it too high? I suggest authors recheck the results.
- Figure 2: what SOD graph have no error bars?
- In figure 3: “. Significant differences to CTRL are reported as * p < 0.05; ** p < 356 0.01; *** p < 0.001.” authors have mentioned two different types of statistical symbols on the graphs but in the legends mentioned on “**” what does a,b,c means and why author used two different statistics methods? Some figures only have a,b? seems confusing…
- Usually after moderate salt stress the antioxidant enzymatic activities increase in plants, according to previous studies but in this study why control plants showed significantly lower activity of antioxidant enzymes after 40mM NaCl stress? It seems totally different to previously published reports. For 80mM stress, as it is high salt stress, it is okay that under high stress the control plants antioxidant defense collapsed.
Author Response
We thank the referee for the comments and suggestions. The modifications have been included and highlighted in the manuscript and the responses are reported below.
The manuscript title “The efficacy of Lippia alba essential oil in mitigating osmotic and oxidative stress in salt-affected bean plants” is a good manuscript but requires improvements and some clarification on figure 3 results. Lippia alba (Mill.) N.E.Br. ex Britton and P. Wilson is a native Colombian plant found throughout Central and South America, historically utilized by pre-Columbian populations for its numerous beneficial properties, including antifungal, antibacterial, antiviral, and anti-inflammatory activities. The essential oil of L. alba is rich in phytochemicals with antioxidant properties, making it potentially valuable for pharmacological and biotechnological applications, particularly in protecting horticultural crops from salinity stress.
The study suggests that L. alba essential oil could be effectively utilized in agriculture to enhance the resilience of crops against salinity stress, offering a promising avenue for improving crop productivity in saline environments.
This MS needs improvements and some clarification on figure 3 results. Comments for authors:
Comment 1: Line 150: “L. alba” is a scientific name it should be italic throughout the manuscript.
Response 1: modified (line 149)
Comment 2: Line 180: in equation “] ∙ 100” here ∙ means multiply? I suggest it is better to use multiply symbols. Same for the next equations.
Response 2: done (lines 180, 186, 211)
Comment 3: Line 199: “Data are expressed as µg Ca2+ × mg f.w.−1.” any standard or standard was made? Standard information and concentration is missing.
Response 3: information was added (lines 197-198)
Comment 4: Line 206: “at 664.1 nm (chlorophyll a) and 648.6 nm 206 (chlorophyll b). The concentration of total photosynthetic pigments was calculated according to Lichtenthaler [35] and expressed as µg · g f.w.−1” which equation was used to calculate chlorophyll contents?
Response 4: the equation was added (lines 208-213)
Comment 5: Line 218: “carbohydrates was evaluated” please add few more line to elaborate this protocol used in this study.
Response 5: information was added (lines 224-230)
Comment 6: Table 3: Soluble sugars results are presented but methods are not mentioned.
Response 6: information was added (lines 224-230). For more clarity we also changed “soluble sugars” with “carbohydrates” in Table 3
Comment 7: Why do the proline results have too much difference in EO 40 and 80, 91.8 ± 2 a*** 485.7 ± 41.3 b*** respectively. From 91.8 to 485.7? Is it too high? I suggest authors recheck the results.
Response 7: we re-checked the proline results, but the data are correct. A marked enhancement of proline has been observed in some of our previous studies (Stassinos, P.M.; Rossi, M.; Borromeo, I.; Capo, C.; Beninati, S.; Forni, C. Enhancement of Brassica napustolerance to high saline conditions by seed priming. Plants 2021, 10, 403. https://doi.org/10.3390/plants10020403; Borromeo, I.; Domenici, F.; Del Gallo, M.; Forni, C. Role of polyamines in the response to salt stress of tomato. Plants 2023, 12, 1855. https://doi.org/10.3390/plants12091855; Borromeo, I.; Del Gallo, M.; Forni, C. Salt Stress and Tomato Resilience: From Somatic to Intergenerational Priming Memory. Horticulturae 2025, 11, 236. https://doi.org/10.3390/horticulturae11030236), where an increase (from 4 to 7 times) of the osmolyte was recorded in primed plants compared to controls. In this case, the increase is about 5 times, and is within the range previously observed in other species. The enhancement of proline would also be correlated with the strong reduction of osmotic stress in primed plants, induced by salt.
Comment 8: Figure 2: what SOD graph have no error bars?
Response 8: SOD activity was assessed by N-PAGE gel. This is a qualitative (and not quantitative) analysis, thus the results are obtained basing on the elaboration of the pixels obtained from gel analyses, thus not requiring statistical elaboration. SOD activity was expressed in Arbitrary Units (A.U.) that corresponds to the pixel density of each lane obtained by the program Image J. Examples of this test are reported in other studies [Jaszek M, Kos K, Matuszewska A, GrÄ…z M, Stefaniuk D, OsiÅ„ska-Jaroszuk M, Prendecka M, Jóźwik E, Grzywnowicz K. Effective stimulation of the biotechnological potential of the medicinal white rot fungus: Phellinus pini by menadione-mediated oxidative stress. Appl Biochem Biotechnol. 2014 Sep;174(2):644-56 https://doi.org/10.1007/s12010-014-1064-2; Santangeli, M.; Capo, C.; Beninati, S.; Pietrini, F.; Forni, C. Gradual exposure to salinity improves tolerance to salt stress in rapeseed (Brassica napus L.). Water 2019, 11, 1667. https://doi.org/10.3390/w11081667;Stassinos P.M., Rossi M., Borromeo I., Capo C. Beninati S., Forni C. (2022). Amelioration of salt stress tolerance in rapeseed (Brassica napus) cultivars by seed inoculation with Arthrobacter globiformis. Plant Biosystems 156 (2): 370–383.https://doi.org/10.1080/112653504.2020.1857872]
Comment 9: In figure 3: “Significant differences to CTRL are reported as * p < 0.05; ** p < 356 0.01; *** p < 0.001.” authors have mentioned two different types of statistical symbols on the graphs but in the legends mentioned on “**” what does a,b,c means and why author used two different statistics methods? Some figures only have a,b? seems confusing…
Response 9: The different symbols are referred to two different statistical analyses applied, as described in the paragraphs 2.6 “Statistical analysis” (lines 261-268). The first one is an intragroup test (identified by different letters, i.e. a, b, c), which evaluates the effect of increasing salinity in the group of CTRLs and in primed group, separately. In this case, the factor investigated is only the salinity. The second statistical analysis is intergroup (identified by *, **, ***) and it is carried out by comparing the responses of the CTRLs with those of the primed plants. In this latter case, the factor investigated is the effect priming treatment alone, since the comparisons are performed under the same salinity conditions.
Comment 10: Usually after moderate salt stress the antioxidant enzymatic activities increase in plants, according to previous studies but in this study why control plants showed significantly lower activity of antioxidant enzymes after 40 mM NaCl stress? It seems totally different to previously published reports. For 80 mM stress, as it is high salt stress, it is okay that under high stress the control plants antioxidant defense collapsed.
Response 10: This trend was observed for SOD and APX enzymes, whereas for CAT, POD and PPO activities no change was found in moderately stressed CTRLs plants (40 mM NaCl). In CTRL exposed to moderate stress (40 mM NaCl), it is possible (see Fig 2b) that the decrease of SOD activity (about 50%) is compensated by a light enhancement of the activities of PPO, CAT and POD (+11%, +16%, +44% respectively). Further investigations are in progress to verify this theory. Regarding APX, this result is not unusual, since it is related to the salt sensitivity of the bean species of this study. In different works, it has been reported a significant difference between antioxidant responses to salt in tolerant and sensitive (or moderately sensitive) plant species; i.e., in tolerant crops, marked enhancements of enzymatic activities are very often observed, while in sensitive crops or even cultivars, moderately salt stress may reduce the antioxidant power of an enzyme (as observed in the case of APX). Examples of this trend have been detected in other works [Stassinos, P.M.; Rossi, M.; Borromeo, I.; Capo, C.; Beninati, S.; Forni, C. Enhancement of Brassica napus tolerance to high saline conditions by seed priming. Plants 2021, 10, 403. https://doi.org/10.3390/plants10020403; Borromeo, I.; Domenici, F.; Giordani, C.; Del Gallo, M.; Forni, C. Enhancing Bean (Phaseolus vulgaris L.) resilience: unveiling the role of halopriming against saltwater stress. Seeds 2024, 3, 228-250. https://doi.org/10.3390/seeds3020018; Min Zhang, Yanming Fang, Yonghua Ji, Zeping Jiang, Lei Wang. Effects of salt stress on ion content, antioxidant enzymes and protein profile in different tissues of Broussonetia papyrifera. South African Journal of Botany, Volume 85, 2013, Pages 1-9, ISSN 0254-6299, https://doi.org/10.1016/j.sajb.2012.11.005]
Round 2
Reviewer 1 Report
Comments and Suggestions for AuthorsNo comments.
Reviewer 2 Report
Comments and Suggestions for Authorsthe authors did sufficient revisions.
