Leaf to Root Morphological and Anatomical Indicators of Drought Resistance in Coffea canephora After Two Stress Cycles

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript “Leaf to Root Morphological and Anatomical Indicators of Drought Resistance in Coffea canephora After Two Stress Cycles” by de Souza et al. investigates the drought tolerance in two Coffea canephora genotypes (3V and A1), by exposing them to two cycles of water stress. The results showed that 3V was more sensitive to water stress in terms of xylem vessel density in branches and xylem vessel area in roots. Both genotypes increased bulk elastic modulus under water stress, but A1 exhibited higher relative water content in leaves at the point of turgor loss. No significant differences were observed between genotypes in root mass distribution in the upper soil layers, but 3V showed higher root mass in deeper layers. The study concluded that A1 was more conservative in water use, while 3V was more drought tolerant, and suggested that physiological, morphological, and anatomical traits of coffee were coordinated along unique axes of resource acquisition/conservation under water stress.

While the article presented holds promise, aspects of it would benefit from additional refinement to enhance their robustness and rigor. Specific areas for improvement are outlined below:

Introduction issues:

Page 2: “Gene expression profile and the content of diverse molecules are greatly and differentially altered among coffee species, namely those associated with protective mechanisms against drought and other stress conditions, and they are even more complex under the exposure of multiple stressors”. At this point the authors could add a brief description of the main genes or metabolic pathways involved in drought resistance in coffee.

Methodology issues:

Page 15: “After this initial period, seven plants of each clone were sub-jected to soil water stress (WS), while the remaining seven were kept in well–watered (WW) conditions as control”. It would be recommended to justify the choice of sample size.

Page 15: “Between the two WS cycles, one period of an additional 19 days was established, when all plants were maintained well-watered”. Please, specify watering conditions in this sentence.

Pages 15-16: “The dynamics of leaf turgor and tissue water relations were assessed through pres-sure–volume (P–V) curve analysis [52, 53], using a Schölander–type [54] pressure chamber (model 3005; Soil Moisture Equipment Corporation, Santa Barbara CA, USA) and follow-ing the free transpiration method”. The authors could indicate why this method was used.

Page 16: “Those leafy axes were kept covered with a dark plastic bag in the labor-atory to ensure stomatal closure and complete rehydration. Fully expanded leaves (devel-oped during the two water stress cycles) were collected from the rehydrated branches and gradually dehydrated on the bench”. Indicate the time range in which these two rehydration processes were achieved.

Page 16: “For each plant (n = 7), the averages were taken for branch and root cuts, from six and eleven fields of view, respectively”. Please, indicate why these numbers of evaluated fields of view were chosen.

Page 16: “After initiating soil water stress cycles (WS–1 and WS–2), young leaves were selected to monitor the leaf expansion rate based on meas-urements of central vein length (CVL) taken at six–day intervals in four time–points for each of the two stress cycles”. The authors could indicate what criteria were used to consider young leaves.

Page 17: “The separated plant parts (orthotropic and plagiotropic branches, leaves, and roots) were dried in a forced–air oven model 320/5 (Eletrolab., São Paulo SP, Brazil) at 65 °C for 72 hours to determine the dry mass allocation in leaves (LDM), plagiotropic branches (PBDM), orthotropic branches (OBDM), and roots (RDM)”. Please, indicate how these parts of the plants were separated.

Discussion issues:

The authors could include a section addressing some limitations of the study, such as the use of young greenhouse-grown plants, which could limit the generalizability of the findings to field conditions in coffee.

Comments on the Quality of English Language

The manuscript requires revision by a native language specialist to address grammatical errors and improve paragraph structure.

Author Response

Responses to reviewer#1

Manuscript ID: agriculture-3479873

Type of manuscript: Article

Title: Leaf to Root Morphological and Anatomical Indicators of Drought Resistance in Coffea canephora After Two Stress Cycles

 The manuscript “Leaf to Root Morphological and Anatomical Indicators of Drought Resistance in Coffea canephora After Two Stress Cycles” by de Souza et al. investigates the drought tolerance in two Coffea canephora genotypes (3V and A1), by exposing them to two cycles of water stress. The results showed that 3V was more sensitive to water stress in terms of xylem vessel density in branches and xylem vessel area in roots. Both genotypes increased bulk elastic modulus under water stress, but A1 exhibited higher relative water content in leaves at the point of turgor loss. No significant differences were observed between genotypes in root mass distribution in the upper soil layers, but 3V showed higher root mass in deeper layers. The study concluded that A1 was more conservative in water use, while 3V was more drought tolerant, and suggested that physiological, morphological, and anatomical traits of coffee were coordinated along unique axes of resource acquisition/conservation under water stress.

While the article presented holds promise, aspects of it would benefit from additional refinement to enhance their robustness and rigor. Specific areas for improvement are outlined below:

Authors: We thank the reviewer for his/her considerations for improving the manuscript.

Introduction issues:

Page 2: “Gene expression profile and the content of diverse molecules are greatly and differentially altered among coffee species, namely those associated with protective mechanisms against drought and other stress conditions, and they are even more complex under the exposure of multiple stressors”. At this point the authors could add a brief description of the main genes or metabolic pathways involved in drought resistance in coffee.

Authors: Thank you for the suggestion.

We are looking for some genes and metabolic pathways activated in Coffea ssp genotypes and that are somehow related to drought tolerance in these genotypes. Some examples were added between lines 82 and 89.

It has been reported that the homeobox gene CaHB12 [12] enhances survival rates and regulates drought tolerance by modulating stomatal conductance and antioxidant activity. The genes CcNCED3, CcPYL7, and CcPP2C-1 are involved in ABA biosynthesis, perception, and signal transduction [13]. Additionally, the accumulation of metabolites such as carbohydrates (maltose, glucose, galactose, and lactose) and amino acids (tryptophan, L-cysteine, methionine, lysine, leucine, etc.) may contribute to increased tolerance to water stress conditions in coffee plants [14].

Methodology issues:

Page 15: “After this initial period, seven plants of each clone were subjected to soil water stress (WS), while the remaining seven were kept in well–watered (WW) conditions as control”. It would be recommended to justify the choice of sample size.

Authors: Thank you for your consideration.

This study is a continuation of the work published in the journal Stresses (available at https://doi.org/10.3390/stresses4030037). The experimental design was carefully planned to evaluate all physiological characteristics, particularly gas exchange, within a maximum measurement window of two hours. This approach was adopted to minimize the influence of drastic fluctuations in temperature and radiation on the quality of the data, especially for plants under water deficit conditions. Given a stabilization period for gas exchange measurements ranging between 120 and 300 seconds, and with 28 experimental units (plants), we determined that seven replicates would be a reasonable and practical number for our experimental conditions.

Page 15: “Between the two WS cycles, one period of an additional 19 days was established, when all plants were maintained well-watered”. Please, specify watering conditions in this sentence.

Authors: Thank you for your comment.

Therefore, we have added information (in lines 527 and 528) related to the system flow rate and daily irrigation time:

The irrigation system (flow rate ≈ 0.05 L min^-1 plant^-1) was automatically activated for 30 minutes twice a day (06:00 h and 18:00 h).

Pages 15-16: “The dynamics of leaf turgor and tissue water relations were assessed through pres-sure–volume (P–V) curve analysis [52, 53], using a Schölander–type [54] pressure chamber (model 3005; Soil Moisture Equipment Corporation, Santa Barbara CA, USA) and follow-ing the free transpiration method”. The authors could indicate why this method was used.

Authors: Thank you for your suggestion.

This method is widely used and, due to its ease of application to a large number of simultaneous samples, we believe it is the most suitable approach for our specific conditions.

We indicate the reason for choosing the method between lines 544 and 545:

because it was simpler and allowed the simultaneous analysis of a greater number of samples [59]

Page 16: “Those leafy axes were kept covered with a dark plastic bag in the labor-atory to ensure stomatal closure and complete rehydration. Fully expanded leaves (devel-oped during the two water stress cycles) were collected from the rehydrated branches and gradually dehydrated on the bench”. Indicate the time range in which these two rehydration processes were achieved.

Authors: Thank you for your suggestion.

As suggested, we have added to the text the approximate time during which the plants were kept under overnight rehydration (line 550) and under bench dehydration (lines 552 and 553).

Page 16: “For each plant (n = 7), the averages were taken for branch and root cuts, from six and eleven fields of view, respectively”. Please, indicate why these numbers of evaluated fields of view were chosen.

Authors: Thank you for your suggestion.

Because the cuts are made freehand, especially in cross-sections of more lignified branches, which are more resistant, the quality of the cuts is lower. Therefore, to avoid errors due to tissue overlap, we chose to take measurements in a smaller number of fields of view. Added between lines 582 and 584

Due to the resistance of the material and the quality of the sections, fewer fields of view were obtained from the branches.

 Page 16: “After initiating soil water stress cycles (WS–1 and WS–2), young leaves were selected to monitor the leaf expansion rate based on measurements of central vein length (CVL) taken at six–day intervals in four time–points for each of the two stress cycles”. The authors could indicate what criteria were used to consider young leaves.

Authors: We appreciate the suggestion.

Monitoring leaf expansion using central vein length (CVL) was performed on leaves with CVL > 1 cm (indicated in line 589), where it was possible to use the ruler with less risk of leaf damage.

Page 17: “The separated plant parts (orthotropic and plagiotropic branches, leaves, and roots) were dried in a forced–air oven model 320/5 (Eletrolab., São Paulo SP, Brazil) at 65 °C for 72 hours to determine the dry mass allocation in leaves (LDM), plagiotropic branches (PBDM), orthotropic branches (OBDM), and roots (RDM)”. Please, indicate how these parts of the plants were separated.

Authors: Thank you for your suggestion.

We have added the detail between lines 600 and 602:

Branches and leaves were separated using pruning shears. To divide the root layers, the pots were cut laterally and each soil layer with its root portion was cut and washed separately.

 Discussion issues:

The authors could include a section addressing some limitations of the study, such as the use of young greenhouse-grown plants, which could limit the generalizability of the findings to field conditions in coffee.

Authors: Thank you for your consideration.

We have added to the text, briefly between lines 477 and 482, the main points that we consider relevant and that may result in differential responses between field and greenhouse experiments when working with water stress:

However, we believe that under field conditions, these responses may be significantly influenced by resource competition between aboveground and belowground plant components [50]. Additionally, the severity of water stress and the frequency of drought events can be modulated by physical soil characteristics [51] and soil cover dynamics [52]. These factors may lead to varied stress responses, highlighting the complexity of plant adaptation in natural environments.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Report on "Leaf to Root Morphological and Anatomical Indicators of Drought Resistance in Coffea canephora After Two Stress Cycles"

The study conducted the drought experiments, and compared the morphological and anatomical responses in the leaves and roots of two coffea genotypes. 

Drought is a globally significant event that can cause significant agricultural damage, and quantitative research into the stress response of crops is an important topic.

In this study, the authors evaluated the morphological and physiological responses of coffee genotypes in drought-treated class that included two periods of restricted irrigation, and in moist class that were fully watered. The study design and analysis approach seemed well established. The results present a wide range of key data, and the arguments derived from the results are generally considered to be valid.

I would like to present only minor comments.

The analysis was done using two-way or three-way analysis of variance. I think that approach is reasonable. However, in Figures 1 and 3, only the P value is shown. It would be more academic to show the F statistic as well.

Also, in some figures (e.g. Figure 3a), the "=" sign appears as a minus sign, which may be due to the resolution.

In the third paragraph of the discussion,

the effect of plant density and competition is described, but since the density effect was not examined in this study, I think this is an over-discussion.

The manuscript is slightly long, so I recommend the authors to reduce redundant descriptions.

Author Response

Responses to reviewer#2

Manuscript ID: agriculture-3479873

Type of manuscript: Article

Title: Leaf to Root Morphological and Anatomical Indicators of Drought Resistance in Coffea canephora After Two Stress Cycles

Comments and Suggestions for Authors

Report on "Leaf to Root Morphological and Anatomical Indicators of Drought Resistance in Coffea canephora After Two Stress Cycles"

The study conducted the drought experiments, and compared the morphological and anatomical responses in the leaves and roots of two coffea genotypes. 

Drought is a globally significant event that can cause significant agricultural damage, and quantitative research into the stress response of crops is an important topic.

In this study, the authors evaluated the morphological and physiological responses of coffee genotypes in drought-treated class that included two periods of restricted irrigation, and in moist class that were fully watered. The study design and analysis approach seemed well established. The results present a wide range of key data, and the arguments derived from the results are generally considered to be valid.

 Authors: We thank the reviewer for his/her considerations for improving the manuscript.

I would like to present only minor comments.

The analysis was done using two-way or three-way analysis of variance. I think that approach is reasonable. However, in Figures 1 and 3, only the P value is shown. It would be more academic to show the F statistic as well.

 Authors: Thank you for your consideration.

We believe that the F statistic is the way to calculate the P-value, and that in our case, it was sufficient to demonstrate the statistical significance in the figures mentioned, as well as the others in the body of the manuscript.

Also, in some figures (e.g. Figure 3a), the "=" sign appears as a minus sign, which may be due to the resolution.

 Authors: Thank you for your observation.

In our manuscript, the resolution of the images does not appear to compromise the visualization of the data. In any case, we are sending high-resolution images to the journal, which we believe will not impact the reading and interpretation of the data.

In the third paragraph of the discussion,

the effect of plant density and competition is described, but since the density effect was not examined in this study, I think this is an over-discussion.

Authors: Thank you for your observation.

Indeed, self-thinning responses are observed under conditions of competition between plants. Therefore, as suggested, we have removed this approach from the text.

Text excluded between lines 395 and 404.

The manuscript is slightly long, so I recommend the authors to reduce redundant descriptions.

Authors: Thank you for your observation.

We tried to look for observations that were redundant as considered, but we considered the information important for a better understanding of the text. Other reviewers also suggested the addition of new information to improve our approach to the topic studied.

We look forward to your contribution in this regard.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript analyzes the interrelated physio-morphological and anatomical responses of two genotypes of Coffea canephora ('3V' and 'A1'), from leaves to branches and root organs, thus revealing their drought tolerance strategies during two consecutive drought events. The findings demonstrated distinct drought adaptation mechanisms between the genotypes, with 'A1' genotype exhibiting a more conservative and less plastic response, while '3V' genotype demonstrated enhanced drought tolerance potential through root depth modification. Some suggestions and advice were provided to enhance the level of the manuscript.

1. In the abstract, the author should summarize the study in one concise sentence.
2. The significance of the study should be stated at the end of the abstract.
3. Does "Potted plants" in the abstract refer to "Coffea canephora"? Please rewrite the sentence: "Potted plants from the two genotypes cultivated under well-watered (WW) conditions were subjected to water-stress (WS) conditions."
4. To enhance readability, Details information about "the soil pH and chemical properties are available" should be incorporated into the "Materials and Methods" section of the manuscript.
5. In the "Results" section, data shown in the manuscript should be described, and data that is not shown should not be described. Please add the data of "strong leaf shed" in 2.4 or delete it.
6. Figure 5c illustrates the leaf number. In this study, is the variation in "leaf number" equivalent to "leaf shed"? If not, why does the discussion include the statement "Therefore, leaf shed (Figure 5c),…"? Please check carefully and make changes.
7. To emphasize the different strategies adopted by the "A1" and "3V" genotypes in response to drought, the comparative analysis of the adaptation measures adopted by each genotype under drought conditions should be reinforced in the discussion section.
8. The scale should be clearly visible in Figure 2, please re-upload the image.

Author Response

Responses to reviewer#3

Manuscript ID: agriculture-3479873

Type of manuscript: Article

Title: Leaf to Root Morphological and Anatomical Indicators of Drought Resistance in Coffea canephora After Two Stress Cycles

The manuscript analyzes the interrelated physio-morphological and anatomical responses of two genotypes of Coffea canephora ('3V' and 'A1'), from leaves to branches and root organs, thus revealing their drought tolerance strategies during two consecutive drought events. The findings demonstrated distinct drought adaptation mechanisms between the genotypes, with 'A1' genotype exhibiting a more conservative and less plastic response, while '3V' genotype demonstrated enhanced drought tolerance potential through root depth modification. Some suggestions and advice were provided to enhance the level of the manuscript.

Authors: We thank the reviewer for his/her considerations for improving the manuscript.

1. In the abstract, the author should summarize the study in one concise sentence.

Authors: Thank you for your consideration.

Our summary was indeed very long and not as suggested by the journal. Therefore, as suggested, we tried to summarize it in short and objective sentences. It can be seen between lines 25 and 39:

Coffea canephora genotypes adopt distinct strategies to cope with drought and rehydration. We hypothesized that the greater drought tolerance of genotype ‘3V’ compared to ‘A1’, previously reflected in physiological and anatomical leaf traits after two water stress (WS) cycles, could also be observed in P–V curve responses, root and branch anatomy, leaf midrib elongation (CVL), and root distribution. The ‘3V’ and ‘A1’ plants were grown under well-watered (WW) conditions and two cycles of water stress (WS). ‘3V’ was more sensitive to WS, with reduced branch xylem vessel density (BXVD), while ‘A1’ demonstrated increased BXVD. Root xylem vessel area (RXVA) decreased to a greater extent in ‘3V’ than in ‘A1’, and both genotypes showed increased bulk elastic modulus. Regardless of water conditions, ‘A1’ maintained a higher relative leaf water content at the turgor loss point (RWC_TLP). Morphological acclimation did not occur in the second WS cycle. ‘3V’ developed greater root mass in deeper soil layers than ‘A1’ under the WS condition. These findings suggest that ‘A1’ follows a conservative drought-avoidance strategy with lower physio-morphological plasticity, while ‘3V’ exhibits greater drought tolerance. Such responses highlighted coordinated physiological, morphological, and anatomical adaptations of above- and below-ground organs for resource acquisition and conservation under WS.

1. The significance of the study should be stated at the end of the abstract.

Authors: Thank you for your consideration.

We believe that the different strategies of clones to deal with drought and how they coordinate along a single growth axis were the most relevant results, as shown in the new abstract.

1. Does "Potted plants" in the abstract refer to "Coffea canephora"? Please rewrite the sentence: "Potted plants from the two genotypes cultivated under well-watered (WW) conditions were subjected to water-stress (WS) conditions."

Authors: Thank you for the suggestion.

We have rewritten the new Abstract, indicating that plants of the genotypes 'A1' and '3V' were grown in pots.

1. To enhance readability, Details information about "the soil pH and chemical properties are available" should be incorporated into the "Materials and Methods" section of the manuscript.

Authors: Thank you for your suggestion.

We chose to keep some details of the chemical analysis of the soil, such as nutrient content, in the supplementary material. However, as suggested, we show between lines 516 and 518 the main characteristics that can limit the absorption of nutrients by plants. However, we emphasize that after the chemical analysis was performed, the soil had its pH corrected and was fertilized according to regional recommendations for the crop, with the aim of not generating a new stressor for the plants.

Briefly, the soil pH was 5.6, hydrogen + aluminum was 1.1 cmol_c dm^-3, organic matter 1%, sum of bases at 4.0 cmol_c dm^-3, CEC at 5.1 cmol_c dm^-3, effective CEC at 4.0 cmol_c dm^-3, base saturation at 78.3%. More nutritional details are shown in [33].

1. In the "Results" section, data shown in the manuscript should be described, and data that is not shown should not be described. Please add the data of "strong leaf shed" in 2.4 or delete it.

Authors: Thank you for the suggestion.

As suggested, we have removed from the text supplementary data not presented in the text or in the supplementary materials file.

1. Figure 5c illustrates the leaf number. In this study, is the variation in "leaf number" equivalent to "leaf shed"? If not, why does the discussion include the statement "Therefore, leaf shed (Figure 5c),…"? Please check carefully and make changes.

Authors: Thank you for your observation.

Indeed, the figure is related to the leaf number, which was lower in WS. This resulted from leaf fall that was not accounted for in this experiment.

1. To emphasize the different strategies adopted by the "A1" and "3V" genotypes in response to drought, the comparative analysis of the adaptation measures adopted by each genotype under drought conditions should be reinforced in the discussion section.

Authors: Thank you for the suggestion.

We have modified the last paragraph of the discussion as a conclusion, where we address these comparative results that reinforce our discussion.

1. The scale should be clearly visible in Figure 2, please re-upload the image.

Authors: Thank you for the suggestion.

In fact, the scales generated by the image capture software were not very clear, so we tried to improve the quality of this scale manually, increasing the thickness of the lines and using a color with greater contrast. However, due to the size of the images on the panel, the quality of the scales was not excellent.

Author Response File: Author Response.pdf