Review Reports - Using Machine Learning Algorithms to Investigate the Impact of Temperature Treatment and Salt Stress on Four Forage Peas (<i>Pisum sativum</i> var. <i>arvense</i> L.)

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The measurements are well done and interesting. The main problem is that authors talk about "heat stress", but expose the plants to low temperatures (10 and 15 deg C). The maximal temperature used, 20 deg C, tends to be the optimal temperature for many plants. Why not go to 30 deg C or higher, which might well happen in the region of interest with global warming? The study will then make sense.

The use of machine learning algorithms is interesting, once the above problem is sorted.

The figures are too crowded and the labels are illegible.

There are many abbreviations and a list should be included.

Comments on the Quality of English Language

The English language seems mainly fine.

Author Response

General Response:

First, we thank the potential reviewer for her/his valuable comments and suggestions. In this step of revision, we have tried to respond to all comments and addressed all questions. We hope the revised version of manuscript gets positive feedback from you and will be acceptable for publication in the Horticulturae journal.

Sincerely,

Dr. Fatih Demirel

Comment1# The measurements are well done and interesting. The main problem is that authors talk about "heat stress", but expose the plants to low temperatures (10 and 15 deg C). The maximal temperature used, 20 deg C, tends to be the optimal temperature for many plants. Why not go to 30 deg C or higher, which might well happen in the region of interest with global warming? The study will then make sense.

The use of machine learning algorithms is interesting, once the above problem is sorted.

Response to comment1#

Temperature is an important environmental factor affecting germination and early seedling development. In general, temperatures below the optimum germination temperature adversely affect germination and seedling development. The optimum germination temperature for pea is reported to be 20 C. Therefore, in this study, the responses of forage pea genotypes to salt stress at temperatures below the optimum germination temperature were investigated. In addition, I would like to state that it will not be possible to try other temperatures in the near future, but we will take into account your recommendations that higher temperatures can be tested in our next study. Additionally, the sentences containing the expression heat stress in the manuscript were corrected as temperature treatment.

Comment2# The figures are too crowded and the labels are illegible.

Response to comment2#

The figures were re-corrected so that they are more visible and the labels are readable.

Comment3# There are many abbreviations and a list should be included.

Response to comment3#

All abbreviations were listed as suplementary.

Revisions are shown with a yellow background color.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The authors applied three algorithm models to validate the germination and growth responses of pea plants to various temperature and salinity treatments. While the authors emphasized high temperature as a primary environmental concern, and concluded that heat stress constrains were examined, the experimental design used temperature treatments (10, 15, and 20oC) within the optimal ranges for pea plants, which did not impose any heat stress effect. Adding temperature treatments in the supra-optimal range (25, 30, 35oC) are required in order to generate a full set of data for proper predictions on the temperature effects with machine learning strategies.

L19, various temperatures

L23, Cultivar ‘Tore’ had the best….

L83-84, “Another advantage of these approaches is that they are available.” the sentence is unclear on what additional information it provides. Please specify.

L124-128, revise to make a complete sentence

L177, the temperature treatments (10, 15 and 20oC) are in the optimum range for pea plant germination, which do not impose heat stress injury. That’s the reason why the growth variables increase when the temperature increases in this range.

L368-369, the conclusion that “temperature conditions rise, the cultivars become less sensitive to salt stress in terms of germination characteristics” is only tested when the temperature stays in the optimum range, which cannot be applied to heat stress conditions.

Comments on the Quality of English Language

Minor editing of the English language is needed.

Author Response

General Response:

Sincerely,

Dr. Fatih Demirel

Comment1# The authors applied three algorithm models to validate the germination and growth responses of pea plants to various temperature and salinity treatments. While the authors emphasized high temperature as a primary environmental concern, and concluded that heat stress constrains were examined, the experimental design used temperature treatments (10, 15, and 20^oC) within the optimal ranges for pea plants, which did not impose any heat stress effect. Adding temperature treatments in the supra-optimal range (25, 30, 35^oC) are required in order to generate a full set of data for proper predictions on the temperature effects with machine learning strategies.

Response to comment1#

Comment2# L19, various temperatures

Response to comment2#

The word error was corrected.

Comment3# L23, Cultivar ‘Tore’ had the best….

Response to comment3#

The word error was corrected.

Comment4# L83-84, “Another advantage of these approaches is that they are available.” the sentence is unclear on what additional information it provides. Please specify.

Response to comment4#

The sentence “These approaches are advantageous because they are easily accessible and can be im-plemented without requiring extensive resources or specialized equipment” was added instead of “Another advantage of these approaches is that they are available.”

Comment5# L124-128, revise to make a complete sentence

Response to comment5#

The sentence “Germination percentage (GP), root length (RL), shoot length (SL), fresh weight (FW), dry weight (DW), mean absolute deviation (MAD), mean absolute percentage error (MAPE), root mean square error (RMSE), and R-squared (R2), multivariate adaptive regress-sion spline (MARS), extreme gradient boosting (XGBoost), gaussian processes classifier (GPC)” was removed.

Comment6# L177, the temperature treatments (10, 15 and 20^oC) are in the optimum range for pea plant germination, which do not impose heat stress injury. That’s the reason why the growth variables increase when the temperature increases in this range.

Response to comment6#

Temperature is an important environmental factor affecting germination and early seedling development. In general, temperatures below the optimum germination temperature adversely affect germination and seedling development. The optimum germination temperature for pea is reported to be 20 ^oC. Therefore, in this study, the responses of forage pea genotypes to salt stress at temperatures below the optimum germination temperature were investigated. In addition, I would like to state that it will not be possible to try other temperatures in the near future, but we will take into account your recommendations that higher temperatures can be tested in our next study.

Comment7# L368-369, the conclusion that “temperature conditions rise, the cultivars become less sensitive to salt stress in terms of germination characteristics” is only tested when the temperature stays in the optimum range, which cannot be applied to heat stress conditions.

Response to comment7#

As you mentioned in your comment, the difference between situations where temperature conditions are within the optimum range and extreme temperature conditions should not be underestimated. However, the findings of our study indicate that temperature conditions (from 10 C to 20 C) decrease the sensitivity to salt stress. These results were obtained by conducting the tests within a certain temperature range, and it is a fact that these findings may differ under extreme temperature conditions. while the current findings indicate that increasing temperature reduces sensitivity to salt stress, further research under extreme temperature conditions will help us better understand the extent of this effect. therefore, as I mentioned before, I would like to state that we will take into account your suggestions that higher temperatures can be tested in our next study and we will make preparations for a new study on this subject.

Revisions are shown with a blue background color.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

The paper investigated the effects of temperature and salt stress on four forage peas. Although it’s an interesting paper, there are some weaknesses in the experimental design and data analysis.

1. Title: change ‘some’ to ‘four’.

2. Abstract: there was no results about the effects of combination stress on peas, and the conclusion should be revised.

3. Lines 29-31:what’s the meaning of figures in the brackets?

4. Introduction: the latest research of cold stress should be provided. What’s the hypothesis? The objectives need to be stated clearly.

5. Line 97: where did the authors get the four seeds?

6. Lines 100-106: how many treatments in total? Each treatment only repeated four times?

7. The trial only lasted 14 days?

8. Lines 118-170: it is not clear why the machine learning method adopted in this study ?

9. Table 1: for the fresh weight and dry weight, the unit should be g per plant.

10. The interactive effects of temperature and salinity stress should be provided.

Author Response

General Response:

Sincerely,

Dr. Fatih Demirel

Comment1# The paper investigated the effects of temperature and salt stress on four forage peas. Although it’s an interesting paper, there are some weaknesses in the experimental design and data analysis.

Response to comment1#

Comment2# Title: change ‘some’ to ‘four’.

Response to comment2#

The word “four” was added instead of “some”.

Comment3# Abstract: there was no results about the effects of combination stress on peas, and the conclusion should be revised.

Response to comment3#

The Abstract was revised

Comment4# Lines 29-31:what’s the meaning of figures in the brackets?

Response to comment4#

It refers to the best values within the goodness of fit criteria for the prediction of variables by the algorithms. The necessary correction has been made.

Comment5# Introduction: the latest research of cold stress should be provided. What’s the hypothesis? The objectives need to be stated clearly.

Response to comment5#

Line61-75: The sentences “Cold stress, also known as low temperature stress, encompasses the condition of chilling, which occurs within the temperature range of 0 to 15 °C [Aslam et al., 2022]. The productivity of plants is contingent upon their capacity to adapt, and the primary cause for a decrease in produce is the occurrence of abrupt cold [Jankovska-Bortkevič et al., 2023]. Like other severe climatic shifts, freezing temperatures often cause harm to cells and macromolecules such as proteins and nucleic acids. Additionally, low temperatures may lead to osmotic and oxidative stress [Niu et al., 2014]. As a result, it starts stress signalling, which means that different stress-related signaling pathways are turned on (phytohormones, secondary metabolite production, etc.). The goal is to restore balance by protecting and fixing proteins, nucleic acids, and membranes. For plants to be able to tolerate low temperatures, the steps listed above must work properly so that reactive oxygen species (ROS) like hydrogen peroxide, superoxide, hydroxyl radicals, singlet oxygen, and others don't build up too much and the plants can return to a state of balance. Besides ROS, calcium (Ca²⁺) signaling may also play a role in finetuning different biochemical processes, based on the quantity in the cell [Aslam et al., 2022].” were added.

Line111-116: The purpose of the study was revised.

Comment6# Line 97: where did the authors get the four seeds?

Response to comment6#

Line 131-134: The sentence “Root length (RL) (cm), shoot length (SL) (cm), fresh weight (FW) (g), and dry weight (DW) (g) were measured by randomly selecting 10 seedlings per replication. Germina-tion percentage (GP), RL, SL, and FW values were recorded 14 th day.” was edited.

Comment7# Lines 100-106: how many treatments in total? Each treatment only repeated four times?

Response to comment7#

Line 134-136: Germination experiments were evaluated using analysis of variance (ANOVA) in accordance with a completely randomized trial design comprising a 4 (cultivar) × 3 (Temperature) × 5 (EC) factorial arrangement with four replicates.

Comment8# The trial only lasted 14 days?

Response to comment8#

Comment9# Lines 118-170: it is not clear why the machine learning method adopted in this study ?

Response to comment9#

Line 145-149: The sentence “These ML algorithms were chosen for their ability to handle complex and nonlinear relationships between variables, as well as their robustness in making accurate predictions based on multiple input factors, which is essential for optimizing the germination parameters under varying conditions of salt stress and temperature treatment.” was added

Comment10# Table 1: for the fresh weight and dry weight, the unit should be g per plant.

Response to comment10#

Comment11# The interactive effects of temperature and salinity stress should be provided.

Response to comment11#

Figures 1, 2, 3, and 4 were edited. Additionally, the results and grouping of interactions were presented in tables in the supplementary.

General Response:

Sincerely,

Dr. Fatih Demirel

Response to comment1#

Comment2# L19, various temperatures

Response to comment2#

The word error was corrected.

Comment3# L23, Cultivar ‘Tore’ had the best….

Response to comment3#

The word error was corrected.

Comment4# L83-84, “Another advantage of these approaches is that they are available.” the sentence is unclear on what additional information it provides. Please specify.

Response to comment4#

Comment5# L124-128, revise to make a complete sentence

Response to comment5#

Response to comment6#

Temperature is an important environmental factor affecting germination and early seedling development. In general, temperatures below the optimum germination temperature adversely affect germination and seedling development. The optimum germination temperature for pea is reported to be 20 ^oC. Therefore, in this study, the responses of forage pea genotypes to salt stress at temperatures below the optimum germination temperature were investigated. In addition, I would like to state that it will not be possible to try other temperatures in the near future, but we will take into account your recommendations that higher temperatures can be tested in our next study.

Response to comment7#

Revisions are shown with a green background color.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The MS is greatly improved and ready to be published.

Author Response

Thanks for accepting the article.

Reviewer 2 Report

Comments and Suggestions for Authors

The experimental design needs to include a bigger range of temperatures to examine either cold or heat stress effects. If the authors now use the same data based on 10-20 oC to address chilling stress effects (instead of heat stress in the initial version) on pea plants, lower temperatures (0 and 5 oC) needs to be included.

Author Response

General Response:

Sincerely,

Dr. Fatih Demirel

Comment1# The experimental design needs to include a bigger range of temperatures to examine either cold or heat stress effects. If the authors now use the same data based on 10-20 ^oC to address chilling stress effects (instead of heat stress in the initial version) on pea plants, lower temperatures (0 and 5 ^oC) needs to be included.

Response to comment1#

First of all, thank you for your detailed evaluation of our article. We made all the revisions you requested in the first round.. However, since the publication went to 2 other reviewers other than you, and they gave a “publishable” opinion, we cannot make some of your revisions in order not to compromise the integrity of the study.The ideal germination temperature for economical fodder pea cultivation is 20 degrees C(see: 10.29133/yyutbd.715245). This was also confirmed in our study. High germination percentage was obtained at this temperature. As the temperature drops, 0 ^oC is the freezing point and germination does not occur. In addition, in studies conducted in the literature (see: 10.15316/SJAFS.2022.057), germination is either absent or very low at 5 ^oC. As you will appreciate, economical cultivation cannot be done at this level. Therefore, we regret that we will not be able to fulfill your request as it would go beyond the purpose of the study.

Secondly, ML algorithms were chosen due to their ability to accurately predict outcomes by capturing complex and nonlinear relationships between variables. This predictive capability is crucial for optimizing germination parameters under varying conditions of salt stress and temperature treatment, enabling more effective agricultural practices. Based on the findings, the expected estimated values of temperature treatment can be estimated. For example, the predicted values created by the model for root length and temperature values applied below 10 ^oC showed that death could result at 1 ^oC or below Table 1 and Table 2.

As a result, one of the purposes of using ML is to produce predictions on unknown data using the performance of the trained model.

Author Response File: Author Response.docx

Reviewer 3 Report

Comments and Suggestions for Authors

The authors improved the MS according to my comments. The paper could be accepted.

Author Response

Thanks for accepting the article.

Round 3

Reviewer 2 Report

Comments and Suggestions for Authors

In the case that the data were based on a moderate range of temperature treatments (10-20 oC), my primary suggestion is that the introduction, results and discussion need to be organized more coherently.

The introduction could be reorganized to clarify that salt stress was the primary abiotic stress examined. The first 3 paragraphs on temperature stress (global warming, heat stress and cold stress) can be reduced as the mechanisms for heat or cold stress were not the focus of this manuscript.

For the results, could the cold stress predictions provided in the author's reply be included?

In the discussion about temperature effect (L339-353), cold stress was not discussed at all, though it was introduced. For the heat stress effect, could it be backed up with similar prediction data generated by the algorithms?

Author Response

General Response:

Sincerely,

Dr. Fatih Demirel

Comment1# In the case that the data were based on a moderate range of temperature treatments (10-20 oC), my primary suggestion is that the introduction, results and discussion need to be organized more coherently.

Response to comment1#

The manuscript was re-edited in accordance with the reviewer's suggestion.

Comment2# The introduction could be reorganized to clarify that salt stress was the primary abiotic stress examined. The first 3 paragraphs on temperature stress (global warming, heat stress and cold stress) can be reduced as the mechanisms for heat or cold stress were not the focus of this manuscript.

Response to comment2#

Line 47-61: The manuscript was re-edited in accordance with the reviewer's suggestion

Line 62-77: The sentences “The treatment of seeds with temperature may have a considerable impact on the germination and early development phases of seeds that have been subjected to salt stress [11, 12]. Elevated temperatures have the potential to compound the negative consequences of salinity by further reducing the osmotic potential and increasing the evaporation rate, which ultimately results in a more severe dehydration stress [13]. On the other hand, it has been discovered that that certain temperature regimes may mit-igate the negative consequences of salt stress [9]. An example of this would be that ap-propriate heat conditions have the potential to boost the enzyme activity associated with germination, such as α-amylase, which in turn promotes greater mobilization of stored reserves. Furthermore, temperature treatments have the potential to affect the production and activity of heat shock proteins (HSPs) and antioxidant enzymes. These proteins and enzymes play essential roles in reducing the oxidative damage caused by reactive oxygen species (ROS) when they are subjected to salt stress [14, 15]. In order to boost seedling vigor and overall plant resistance to salt, appropriate temperature treatments may be used. These treatments work by stabilizing cellular structures and increasing processes that allow plants to tolerate stress [16].” were added.

Comment3# For the results, could the cold stress predictions provided in the author's reply be included?

Response to comment3#

Line 313-321: The sentences “Using the predictive capabilities of machine learning algorithms, expected ger-mination parameters between 0 and 20 °C were predicted to optimize germination parameters under changing salt stress and temperature treatment conditions and to enable more effective agricultural practices (Supplementary Table S5). The aim was to predict the expected values of the temperature process on unobserved data based on the findings. According to the results, it was predicted that the application of machine learning algorithms might result in the failure of fodder pea plants to germinate at temperatures of 9 °C or lower for the SL parameter, 1 °C or lower for the RL parame-ter, 5 °C or lower for the FW parameter, and 1 °C or lower for the DW parameter.” were added.

Comment4# In the discussion about temperature effect (L339-353), cold stress was not discussed at all, though it was introduced. For the heat stress effect, could it be backed up with similar prediction data generated by the algorithms?

Response to comment4#

Line 345-350: The sentences “High ambient temperatures pose a substantial threat to agricultural productivity worldwide throughout plant growth and germination phases. Additionally, plants exposed to low temperature within the temperature range of 0 to 15 °C face challenges in productivity due to osmotic and oxidative stress, which necessitates activation of various stress-related signaling pathways to restore cellular balance” were added.

Line 403-409: The sentences “Based on the findings, the expected estimated values of the temperature process on unobserved data were determined. According to the results, it was predicted that ML algorithms at 9 degrees or less according to the SL parameter, 1 degree or less ac-cording to the RL parameter, 5 degrees or less according to the FW parameter, and 1 degree or less according to the DW parameter may result in the failure of fodder pea plants to germinate. When the temperature drops below the ideal range for germination, fodder pea plants either do not germinate at all or have a very poor germination rate.” were added.

Author Response File: Author Response.pdf