The OJIP Kinetics Analysis Reveals Differential Thermal Tolerance Responses in Photosystem II of Coffea canephora Clones After Two Recurrent Cycles of Water Deficit

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Ranking of the manuscript:  MINOR REVISION

Formal problems:

From Abstract, it is not clear how old the experimental plants were at the time of treatment/measurements. Please clarify, whether they were 6 months old or not. In other words, were they grown in optimal water conditions from seed?

 

line 68 Please reprase a bit vague statement. Instead of ´ ...and compromises the photosynthetic assimilation rate´, I would suggest ´... limits/inhibits the photosynthetic...´

 

Lines 92-95 should be rejected. They state very basic and generally known facts about the OJIPs.

 

Line 96 I suggest better phrasing:

instead of ´...OJIP transient and its changes, it is possible to analyze the chain of biophysical events´ , the following should be used: ´... OJIP transient and its changes, it is possible to evaluate particular PSII-related biophysical events´.  MOreover, you measured and evaluated Ĺ´band  (Fig. 4), therefore it must be mentioned in Introduction.

 

I suggest a slight change in the title, since OJIP ChlF measurements are typically associated with PSII rather than whole photosynthetic apparatus considering chloroplastic compartments and even biochemistry of photosynthesis. Therefore, I suggest to change the title to

The OJIP Kinetics Analysis Reveals Differential Thermal Tolerance Responses in PSII of Coffea canephora Clones After Two Recurrent Cycles of Soil Water Deficit. 

 

Page 390-391

I suggest to specify what physiological parameters you have in mind, e.g.

... greater sensitivity in physiological parameters related to stomatal conductance...

 

Specific problems

Your statement (lines 109-113) is oversimplifying. You should mention also ´L ´ band and the mechanism behind, especially when K, L band occurence is indicative of a short-term stress effect.

 

In Discussion. You should bring some explanation why the ´K´ and ´L´band(s) are achieved in shorther times in plants exposed to 50, 55 degC than in other treatments. In other words, the effect of a short-term high temperature treatment on amplitude, and the time when the amplitude is achieved should be added in details.

 

I do appreciate that in Fig.8 the responses of φDo and DIo/CS are of similar pattern. My question is whether or not DIo/CSo is positively/negatively correlated with ABS/CSo. If yes, it should be discussed in terms of a short-term effect on structure and function on PSII.

 

Line 419-421. Please check whether your statement about altered LHC function at high temperature is supported by the changes in Fo and/or Fo/Fp in response to increasing experimental temperature.

 

Line 450-451 . Is your statement abour limited photosynthetic trasport supported by decrease ETo/CSo (in absolute values)? Positive K-band is exclusively about OEC alteration, not ET.

 

In Discussion/Conclusions

You did not measure gas exchange which is a pity since if you did so, you could relate the high-temperature induced negative changes in PSII functioning to Pn, T, and WUE. However, based on your previous paper of similar design (Baroni, D.F.; De Souza, G.A.R.; Bernado, W.D.P.; Santos, A.R.; Barcellos, L.C.D.S.; Barcelos, L.F.T.; Correia, L.Z.; De Almeida, 757
C.M.; Verdin Filho, A.C.; Rodrigues, W.P.; et al. Stomatal and Non-Stomatal Leaf Responses during Two Sequential Water Stress Cycles in Young Coffea Canephora Plants) were there found inhibitions of Gs, Pn that could be related to e.g. Performance index. If yes, please add some lines into Discussion on the Performance index.

 

 

Comments on the Quality of English Language

Some sentences are too wordy and might be shortened (in Results) undoubedly. This could be done either by the authors or during editorial processing.

Author Response

Reviewer #1

Open Review

(x) I would not like to sign my review report
( ) I would like to sign my review report

Quality of English Language

(x) The English could be improved to more clearly express the research.

We passed the whole manuscript to improve the clearness of English expressions.

( ) The English is fine and does not require any improvement.

 

	Yes	Can be improved	Must be improved	Not applicable
Does the introduction provide sufficient background and include all relevant references?	( )	(x)	( )	( )
Is the research design appropriate?	(x)	( )	( )	( )
Are the methods adequately described?	(x)	( )	( )	( )
Are the results clearly presented?	( )	(x)	( )	( )
Are the conclusions supported by the results?	( )	(x)	( )	( )
Are all figures and tables clear and well-presented?	(x)	( )	( )	( )

Comments and Suggestions for Authors

Ranking of the manuscript:  MINOR REVISION

Thank you for your suggestions and comments, and we stay open to any new suggestion that can appear. The inclusions in the manuscript that are related to your observations are marked in red. Please see the attachment.

Formal problems:

From Abstract, it is not clear how old the experimental plants were at the time of treatment/measurements. Please clarify, whether they were 6 months old or not. In other words, were they grown in optimal water conditions from seed?

Authors: We added such information in abstract – lines 31-32:

… non-limited water conditions for seven months (Ψ_mSoil > -20 kPa),…

and in M&M - lines 585-586:

…seedlings grown under non-limited water conditions…

Line 68 Please rephrase a bit vague statement. Instead of ´ ...and compromises the photosynthetic assimilation rate´, I would suggest ´... limits/inhibits the photosynthetic...´

Authors: Thank you, it was substituted (Line 66).

Lines 92-95 should be rejected. They state very basic and generally known facts about the OJIPs.

Authors: Thank you for this suggestion. Those lines were rewritten, but maintained for large public in ecophysiology, where not all researcher are familiar with OJIP curves.

Line 96 I suggest better phrasing:

instead of ´...OJIP transient and its changes, it is possible to analyze the chain of biophysical events´ , the following should be used: ´... OJIP transient and its changes, it is possible to evaluate particular PSII-related biophysical events´.  Moreover, you measured and evaluated Ĺ´band  (Fig. 4), therefore it must be mentioned in Introduction.

Authors: Thank you for this suggestion, which really introduced into the problem. The text was changed as you suggested (Lines 102-105):

… and we added: ‘The pick that appears around 300 µs between the O and K steps (L-band) indicates decreased energy transfer between PSII antenna complexes, suggesting that the system is vulnerable to damage, serving as indicator of short-term stress [37 ̶ 39].’

I suggest a slight change in the title, since OJIP ChlF measurements are typically associated with PSII rather than whole photosynthetic apparatus considering chloroplast compartments and even biochemistry of photosynthesis. Therefore, I suggest changing the title to

The OJIP Kinetics Analysis Reveals Differential Thermal Tolerance Responses in Photosystem II of Coffea canephora Clones After Two Recurrent Cycles of Water Deficit. 

Authors: Thank you for this suggestion (Line 3), we made such substitution in the title to make it as direct as possible.

Page 390-391

I suggest to specify what physiological parameters you have in mind, e.g.

... greater sensitivity in physiological parameters related to stomatal conductance...

Authors: Thank you for this suggestion. We improved the text (Lines 380-386):

Previous findings indicated that clone '3V' exhibited drought tolerance following two cycles of water deficit, being less influenced on leaf net CO₂ assimilation rate, effective quantum yield in PSII photochemistry, photochemical quenching, linear electron transport rate, and photochemical reflectance index [6], which was supported by some morphological traits - particularly the root growth [45]. In contrast, clone 'A1' displayed greater sensitivity in the above-mentioned physiological parameters [6], but demonstrated a more conservative water-use strategy for growth [45].  

 

Specific problems

Your statement (lines 109-113) is oversimplifying. You should mention also ´L ´ band and the mechanism behind, especially when K, L band occurrence is indicative of a short-term stress effect.

Authors: Thank you. We added (Lines 99-105):

‘By analyzing the kinetics of the OJIP transient and its changes, it is possible to evaluate particular PSII-related biophysical events that describe the energy flow from light energy absorption and electron transport in the PSII reaction centers (RCs) to the reduction of the final PSI electron acceptors. The pick that appears around 300 µs between the O and K steps (L-band) indicates decreased energy transfer between PSII antenna complexes, suggesting that the system is vulnerable to damage, serving as indicator of short-term stress [37 ̶ 39].’

In Discussion. You should bring some explanation why the ´K´ and ´L´ band(s) are achieved in shorther times in plants exposed to 50, 55 degC than in other treatments. In other words, the effect of a short-term high temperature treatment on amplitude, and the time when the amplitude is achieved should be added in details.

Authors: Thank you. The L and K bands do not appear 'faster' in high-temperature treatments; they appeared at approximately 150 µs and approximately 300 µs, respectively. It was added (see lines 451-456):

 ‘At the highest observed temperatures (50 and 55 °C), the limitations in the electron donor side (OEC, K-band) and the energy dysconnectivity between the PSII components (L-band) occurred. At such state, alternative electron donors briefly reduced P680⁺ and Q_A, but due to inability to maintain electron flow towards Q_B, the P680⁺ accumulation was induced and fluorescence decreased from 300 µs onwards, making the K̶ point apparent [57].’

I do appreciate that in Fig. 8 the responses of φDo and DIo/CS are of similar pattern. My question is whether or not DIo/CSo is positively/negatively correlated with ABS/CSo. If yes, it should be discussed in terms of a short-term effect on structure and function on PSII.

Authors: There is a positive correlation between ABS/CSo and DI/CSo. This correlation occurs due to the inactivation of reaction centers caused by high temperatures. Since some of the RCs become inactivated, but the antenna complex continues to receive light energy, much of this energy is "forced" to be absorbed by the functional PSII complexes (increased ABS/CSo), causing an overload on the RCs, which decreases the Capture (TRo/CSo) and Transport (ETo/CSo) of energy, and results in greater energy dissipation (observed by the increased DI/CSo). We reported such relations in lines 524-531:

The progressive increase in supra-optimal temperatures triggers the inactivation of PSII RCs [11] and can be monitored through RC/CS₀ parameter (Figure 8, Side B; Table S3; Figure 10, step 2). It describes the density of the active RCs able to reduce Q_A, so its decrease is related to greater inactivation of RCs [61,72,73]. In C. canephora exposed to elevated temperatures, lower inactivation of RCs was observed in ‘A1’ (especially after water deficit experience - ‘A1’-WS) than in ‘3V’ (Figure 8, Side B; Table S3). This reduced inactivation of RCs is directly related to greater efficiency in energy capture and transport, and lower non-photochemical dissipation [61,73], as observed in our results (Figure 8, Side B; Table S3).

Line 419-421. Please check whether your statement about altered LHC function at high temperature is supported by the changes in Fo and/or Fo/Fp in response to increasing experimental temperature.

Authors: Yes, our data confirmed the increase in Fo and φDo (synonymous of Fo/Fp or Fo/Fm). Higher temperatures caused an increase in Fo due to the dissociation of PSII components and caused an increase in Fo/Fp due to the inactivation of RCs and the inability of electron acceptors to reduce, resulting in greater energy dissipation. Following text was added (see lines 432-439):

‘In ChlF, the dissociation between LHCII and PSII was interpreted from the increase in F₀ or the O step in the OJIP transient (Figure 10, step 1), associated with increased ChlF emission by the antenna complexes and reduced excitation energy transfer to PSII [33,41,55]. Our results showed an increase in F₀ or the O level (Figure 1) at temperatures above 45 °C; however, at 55 °C, clone ‘3V’ was shown to be more sensitive, due to significant increase in F₀, compared to the clone ‘A1’. Additionally, lower values of F₀ were observed in the ‘3V’-WS treatments at 50 °C and in ‘A1’-WS at 55 °C.’

Line 450-451. Is your statement about limited photosynthetic transport supported by decrease ETo/CSo (in absolute values)? Positive K-band is exclusively about OEC alteration, not ET.

Authors: The statement is correct. The variables describe distinct but correlated processes. Lines  The positive amplitude of the K-band is related to the inactivation of OEC, which limits the supply of electrons to ETC, consequently resulting in a reduction in ETo/CSo values. We rewrote (lines 461-464):

‘The double normalization W_OJ and ΔW_OJ (Figure 3) revealed the appearance of the K-band, and its positive amplitude was associated with greater inactivation of the OEC (Figure 10, step 3) which limits the supply of electrons to ETC [37].’

In Discussion/Conclusions

You did not measure gas exchange, which is a pity since if you did so, you could relate the high-temperature induced negative changes in PSII functioning to Pn, T, and WUE. However, based on your previous paper of similar design (Baroni, D.F.; De Souza, G.A.R.; Bernado, W.D.P.; Santos, A.R.; Barcellos, L.C.D.S.; Barcelos, L.F.T.; Correia, L.Z.; De Almeida, C.M.; Verdin Filho, A.C.; Rodrigues, W.P.; et al. Stomatal and Non-Stomatal Leaf Responses during Two Sequential Water Stress Cycles in Young Coffea Canephora Plants) were there found inhibitions of Gs, Pn that could be related to e.g. Performance index. If yes, please add some lines into Discussion on the Performance index.

Authors: Thank you. We added (lines 540-541):

The exposure of C. canephora plants to recurrent cycles of water deficit may result in a possible mitigation of the effects of heat stress, especially for clone ‘A1’, supported by previously observed PI_abs responses, which indicated drought acclimation in the second drought event [6].

Comments on the Quality of English Language

Some sentences are too wordy and might be shortened (in Results) undoubedly. This could be done either by the authors or during editorial processing.

Authors: Thank you, we meticulously passed over the references and reformatted what was not previously well written. Such parts of text are marked in violet color.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

This tudy investigates the priming effect of recurrent drought on the heat tolerance of two Coffea canephoraclones. It is structurally sound but requires significant improvements.
1. As mentioned, why 'A1' develops a positive stress memory while '3V' does not is superficial. The authors should explore inherent differences between the clones, such as root architecture, hormonal signaling, or gene expression patterns.
2. Why use 50°C and 55°C as the extreme temperature during heat waves?
3. Whether leaf age is controlled, or it may influence thermal stress responses?
4. The sample size (n=5) is relatively small, it could increase the biological replicates to improve statistical power.
5. The discussion remains largely descriptive of the fluorescence parameter changes. It should delve deeper into the potential physiological and molecular mechanisms underlying this differential "stress memory" hypotheses regarding antioxidant systems, heat shock protein expression, osmotic adjustment, or epigenetic modifications.
6. The reference needs to be meticulously formatted according to the target journal's specific guidelines (journal name abbreviations, author et al).

Author Response

Open Review

(x) I would not like to sign my review report
( ) I would like to sign my review report

Quality of English Language

(x) The English could be improved to more clearly express the research.

We passed the whole manuscript to improve the clearness of English expressions.

( ) The English is fine and does not require any improvement.

 

	Yes	Can be improved	Must be improved	Not applicable
Does the introduction provide sufficient background and include all relevant references?	( )	(x)	( )	( )
Is the research design appropriate?	( )	(x)	( )	( )
Are the methods adequately described?	( )	(x)	( )	( )
Are the results clearly presented?	( )	(x)	( )	( )
Are the conclusions supported by the results?	( )	(x)	( )	( )
Are all figures and tables clear and well-presented?	( )	(x)	( )	( )

Comments and Suggestions for Authors

This study investigates the priming effect of recurrent drought on the heat tolerance of two Coffea canephora clones. It is structurally sound but requires significant improvements.

Thank you for your suggestions and comments, and we stay open to further recommendations. The inclusions in the manuscript that are related to your observations are marked in blue. Please see the attachment.

1. As mentioned, why 'A1' develops a positive stress memory while '3V' does not is superficial. The authors should explore inherent differences between the clones, such as root architecture, hormonal signaling, or gene expression patterns.

Authors: We thank you for this recommendations. The root architecture is explored in Rakocevic et al. 2023 and De Souza et al., 2025 (see lines 579-581):

‘The ‘3V’ clone was previously characterized to have deeper root growth [45] root architecture over soil layers [76] and tolerance to water deficit, considering the dynamics of some physiological [6], anatomical and morphological [45] parameters.’

We did not have the opportunity, up today, to enter into gene expression and hormonal signaling, but we are making some analysis under the scope of one new project, to better understand such mechanisms.

2. Why use 50°C and 55°C as the extreme temperature during heat waves?

Authors: In order to respond, we added lines 635-642:

‘Short-stress exposure to temperatures starting from 35 °C was chosen, considering that C. canephora is not so robust as thought, being highly sensitive to temperature, surviving the mean maximum temperature of ~30 during vegetative growth and flowering, which strongly impact on final yield [78]. Furthermore, in our experiment, plants were grown under mean and maximum temperatures ranged between 30 and 40 °C, respectively [6]. Even at 50 and 55 °C, short-stress exposure of 15 minutes does not cause total loss of PSII functionality, considering some JIP_Test parameters in Carica papaya [61].’

3. Whether leaf age is controlled, or it may influence thermal stress responses?

Authors: Leaf age was highly controlled, and leaves were the last fully expanded, due to experimental procedure, see lines 629-631:

‘In coffee plants, younger leaves are more sensitive to temperature increases than older ones [15]. Therefore, fully expanded leaves that had developed during the two successive cycles of soil drying and rehydration were collected at predawn.’

4. The sample size (n=5) is relatively small, it could increase the biological replicates to improve statistical power.

Authors: To defend our approach, we added (see Lines 689-690):

‘The sample size was considered satisfactory, due to meticulous collection of the same leaf age and position on plagiotropic axes.’

5. The discussion remains largely descriptive of the fluorescence parameter changes. It should delve deeper into the potential physiological and molecular mechanisms underlying this differential "stress memory" hypotheses regarding antioxidant systems, heat shock protein expression, osmotic adjustment, or epigenetic modifications.

Authors: Thank you for your suggestion. We developed discussion on "stress memory" (see lines 562-573):

We would like to highlight that our work had more phenomenological than mechanistic proposition, discovering changes in ChF patterns related to "stress memory" phenomena in coffee plants. Despite this, our results indicated potential for further exploration of the mechanisms involved in acquiring tolerance in different genotypes, and for deciphering the possibilities of improving Coffea spp. priming techniques. Therefore, we are concluding that the acclimation of PSII in clone ‘A1’ under water deficit conditions ("stress memory") was the main factor underlying its greater thermotolerance. The deep confirmation of "stress memory" mechanisms involves alterations in antioxidant metabolism and transcriptional memory, mainly related to the expression of genes linked to ABA synthesis, the encoding of MYB signaling proteins and miRNAs [75]. Additionally, lipid metabolism and expression of genes linked to aquaporins, chaperonins, 70 kDa heat shock proteins, and antioxidant enzymes are also related to this phenomena [46].

Additional explanations to reviewer #2: Our work had a more phenomenological than mechanistic focus by considering the changes in ChF patterns. Through it, we sought to propose that ChF tools can be used for the possible detection of "stress memory" in coffee plants. In this phenomenological approach, our question is: How can ChF tools be used to detect possible thermotolerance responses induced after recurrent cycles of water deficit? In a mechanistic approach, our question would be: How do stress memory mechanisms alter thermotolerance in coffee leaves?

6. The reference needs to be meticulously formatted according to the target journal's specific guidelines (journal name abbreviations, author et al).

Authors: Thank you, we meticulously passed over the references and reformatted what was not previously well written. Such parts of text are marked in violet color.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

1. The current title is slightly verbose; it is recommended to fine-tune it to better highlight the core findings.

2. It is advised to explicitly state the agronomic or breeding significance at the end of the abstract. For example: "This study suggests that selecting drought-resistant varieties should take into account their subsequent response to high-temperature stress to avoid cross-sensitivity caused by selecting for a single trait."

3. Introduction: The research gap is not clearly defined. It is necessary to clearly explain the theoretical basis for the hypothesis that "recurrent water deficit induces heat tolerance." It is recommended to supplement the discussion with relevant research progress on cross-stress responses in Coffea species to strengthen the study's innovativeness.

4. Introduction: The hypothesis needs refinement. The original hypothesis states "water deficit may enhance PSII stability" but does not distinguish the pre-experimental basis for the different clones. It is suggested to add information on the prior phenotypic differences between '3V' (drought-tolerant) and 'A1' (drought-sensitive) to set the stage for the "genotype-dependent" conclusion in the results.

5. The explanation for the enhanced heat tolerance in clone 'A1' after water deficit, attributing it merely to "improved energy dissipation efficiency," is insufficient. It is recommended to discuss potential physiological mechanisms in conjunction with the literature (e.g., changes in thylakoid membrane lipid composition, activation of the antioxidant system, etc.).

6. The reason for the increased heat sensitivity in clone '3V' after water deficit is not explicitly explained. Supplementary discussion is needed, such as proposing a hypothesis regarding the coordinated regulation between its root system and the photosynthetic apparatus.

7. The conclusion "heat tolerance is genotype-dependent" is too general. It is recommended to supplement it with specific differences in key parameters (e.g., at 55°C, the PIabs of 'A1-WS' was 2.3 times higher than that of '3V-WS') to strengthen the persuasiveness of the conclusion.

8. Throughout the manuscript, unify the translation of "soil water deficit" and "drought stress" (it is recommended to consistently use "water deficit" for both), and avoid mixing terms like "drought stress" and "water stress."

9. The overall language is fluent, but some sentences are overly long. It is recommended to break them down for improved readability (e.g., lines 94–112 on page 3).

10. A more systematic comparison with previous studies on "drought-induced heat tolerance" in coffee or other crops is needed to better highlight the innovative aspects or contradictions of this study.

Author Response

Reviewer #3

(x) I would not like to sign my review report

( ) I would like to sign my review report

Quality of English Language

(x) The English could be improved to more clearly express the research.

We passed the whole manuscript to improve the clearness of English expressions. We meticulously passed over the references and reformatted what was not previously well written. Such parts of text are marked in violet color.

( ) The English is fine and does not require any improvement.

	Yes	Can be improved	Must be improved	Not applicable
Does the introduction provide sufficient background and include all relevant references?	( )	(x)	( )	( )
Is the research design appropriate?	( )	(x)	( )	( )
Are the methods adequately described?	( )	(x)	( )	( )
Are the results clearly presented?	( )	(x)	( )	( )
Are the conclusions supported by the results?	( )	(x)	( )	( )
Are all figures and tables clear and well-presented?	( )	(x)	( )	( )

Thank you for your suggestions and comments, and we stay open to any new suggestion that can appear. The inclusions in the manuscript that are related to your observations are marked in green. Please see the attachment.

 

Comments and Suggestions for Authors

1. The current title is slightly verbose; it is recommended to fine-tune it to better highlight the core findings.

Authors: Thank you. We changed the title slightly to highlight the core findings (line 3):

The OJIP Kinetics Analysis Reveals Differential Thermal Tolerance Responses in Photosystem II of Coffea canephora Clones After Two Recurrent Cycles of Water Deficit

1. It is advised to explicitly state the agronomic or breeding significance at the end of the abstract. For example: "This study suggests that selecting drought-resistant varieties should take into account their subsequent response to high-temperature stress to avoid cross-sensitivity caused by selecting for a single trait."

Authors: Thank you for this valuable conclusion. We included the suggested text in lines 45-47:

This study suggests that selecting drought-resistant varieties should consider their subsequent response to short high-temperature stress to avoid cross-sensitivity caused by selecting for a single environmental factor.

1. Introduction: The research gap is not clearly defined. It is necessary to clearly explain the theoretical basis for the hypothesis that "recurrent water deficit induces heat tolerance."

Authors: Thank you. We included the following text in lines 81-89:

‘In crop species, such as common bean [25], wheat [26], or maize [27], as well as in species from natural environments, such are tropical forest trees [28], or alpine grasses [29], plants frequently exposed to drought may develop enhanced thermotolerance induced by drought events, characterized by “stress memory” responses. Such phenomena occurs because the signaling pathways underlying drought and heat stress responses are shared, as these conditions commonly occur simultaneously [30]. It was shown that the PSII stability increased after previous exposure to environmental pressures [31,32]. However, studies demonstrating the acquisition of drought-induced thermotolerance remain scarce for most agricultural crops.’

 

…and in lines 120-122:

‘We hypothesized that C. canephora, commonly cultivated in hot regions and frequently exposed to drought events [3], may exhibit “stress memory” responses associated to the acquisition of thermotolerance.’

 

It is recommended to supplement the discussion with relevant research progress on cross-stress responses in Coffea species to strengthen the study's innovativeness.

 

Authors: Thank you. We included the following text in lines 386-394:

‘Recent studies in Coffea species have demonstrated a complex cross-interaction between drought and heat stress events, generally in a genotype-dependent manner [5,17]. They indicate that, in some cases, the simultaneous or successive occurrence of these stress events can trigger protective responses in PSII and in the photosynthetic apparatus. Such protective responses are commonly associated with the activity of photoprotective compounds, such as zeaxanthin, and antioxidant enzymes, as well as with the synthesis of non-structural carbohydrates, alterations in fatty acid profiles, modulation of gene expression, and the activity of heat shock proteins [46].’

 

1. Introduction: The hypothesis needs refinement. The original hypothesis states "water deficit may enhance PSII stability" but does not distinguish the pre-experimental basis for the different clones.

Authors: Thank you. We included  the following text in lines 88-90:

‘It was shown that the PSII stability increased after previous exposure to environmental pressures [31,32].’

 

It is suggested to add information on the prior phenotypic differences between '3V' (drought-tolerant) and 'A1' (drought-sensitive) to set the stage for the "genotype-dependent" conclusion in the results.

Authors: Thank you. Following text was added in lines 379-393, as was also suggested by reviewer #1:

‘Previous findings indicated that clone '3V' exhibited drought tolerance following two cycles of water deficit, being less influenced on leaf net CO₂ assimilation rate, effective quantum yield in PSII photochemistry, photochemical quenching, linear electron transport rate, and photochemical reflectance index [6], which was supported by some morphological traits - particularly the root growth [45]. In contrast, clone 'A1' displayed greater sensitivity in the above-mentioned physiological parameters [6], but demonstrated a more conservative water-use strategy for growth [45]. Recent studies in Coffea species have demonstrated a complex cross-interaction between drought and heat stress events, generally in a genotype-dependent manner [5,17]. They indicate that, in some cases, the simultaneous or successive occurrence of these stress events can trigger protective responses in PSII and in the photosynthetic apparatus. Such protective responses are commonly associated with the activity of photoprotective compounds, such as zeaxanthin, and antioxidant enzymes, as well as with the synthesis of non-structural carbohydrates, alterations in fatty acid profiles, modulation of gene expression, and the activity of heat shock proteins [46].’  

The explanation for the enhanced heat tolerance in clone 'A1' after water deficit, attributing it merely to "improved energy dissipation efficiency," is insufficient. It is recommended to discuss potential physiological mechanisms in conjunction with the literature (e.g., changes in thylakoid membrane lipid composition, activation of the antioxidant system, etc.).

Authors: Thank you. We would like to emphasize that the focus of our study is primarily phenomenological, aiming to interpret how ChlF can be used to monitor potential stress responses, rather than mechanistic, in the sense of explaining why such responses occurred. However, to clarify certain points, we have made the following improvements and additions in:

 

Lines 423-426:

‘Changes in fatty acid composition and the activity of heat shock proteins are associated with enhanced thermostability in plants growing in warm environments or undergoing long-term acclimation [50]. However, in our study, short-term heat shock was simulated, under which no adjustments in membrane thermostability were observed.’

 

and lines 513-517:

‘The increase in energy dissipation here observed is possibly associated with an enhancement of CEF reported in plants subjected to heat stress [68]. The CEF enhancement induces changes in the trans-thylakoid proton gradient [69] and, consequently, promotes the activation of the xanthophyll cycle, which plays a key role in the thermal dissipation of excess energy [70].’

1. The reason for the increased heat sensitivity in clone '3V' after water deficit is not explicitly explained. Supplementary discussion is needed, such as proposing a hypothesis regarding the coordinated regulation between its root system and the photosynthetic apparatus.

Authors: Thank you. We included the following text in lines 546-552:

‘Previous studies have shown that clone ‘3V’ exhibits greater stability of photochemical responses throughout two successive cycles of soil water deficit than ‘A1’, as well as a slower decline in leaf transpiration, thereby maintaining a higher leaf cooling capacity during the first stress cycle [6]. This is largely due to its greater root deepening capacity [45]. In contrast, during the first cycle of water deficit, clone ‘A1’ exhibits more severe photochemical damage, which later resembled the responses observed in ‘3V’, suggesting a possible acclimation of PSII to stress conditions [6].’

 

…and in lines 565-567:

Therefore, we are deducing that the acclimation of PSII in clone ‘A1’ under water deficit conditions ("stress memory") was the main factor underlying its greater thermotolerance.

 

1. The conclusion "heat tolerance is genotype-dependent" is too general. It is recommended to supplement it with specific differences in key parameters (e.g., at 55°C, the PIabs of 'A1-WS' was 2.3 times higher than that of '3V-WS') to strengthen the persuasiveness of the conclusion.

Authors: Thank you. We improved the Conclusions, see line 699:

… indicating damage to the photosynthetic apparatus, specifically in PSII.

 

…and lines 705-710:

‘Complete loss of the typical OJIP transient shape was observed in clone ‘3V’ after short-term heat shock at the highest temperature (55 °C), whereas clone ‘A1’ maintained the characteristic curve pattern, particularly after the occurrence of water deficit stress. These responses were reflected in JIP-test parameters such as ET₀/CS₀, RC/CS₀, and PI_abs, in which clone ‘A1’, especially after two water deficit cycles, maintained significantly higher values compared to the other treatments.’

7. Throughout the manuscript, unify the translation of "soil water deficit" and "drought stress" (it is recommended to consistently use "water deficit" for both), and avoid mixing terms like "drought stress" and "water stress."

 

Authors: Thank you. In the whole text we used only ‘water stress’, and such correction was performed even in figures.

1. The overall language is fluent, but some sentences are overly long. It is recommended to break them down for improved readability (e.g., lines 94–112 on page 3).

Authors: Thank you. We improved the text of whole manuscript. We broke the phrases. The text improvements are marked in violet color.

1. A more systematic comparison with previous studies on "drought-induced heat tolerance" in coffee or other crops is needed to better highlight the innovative aspects or contradictions of this study.

Authors: Thank you. One extensive text about such problems was included as previously mentioned and exposed (suggestions  under points 3 and 5).

 

 

 

Author Response File: Author Response.pdf

Round 2

Reviewer 3 Report

Comments and Suggestions for Authors

The author responded well to the comments and improved the manuscript.