Photosynthetic and Antioxidant Responses of Gymnocarpos przewalskii to Simulated Rainfall Changes

Round 1

Reviewer 1 Report

This is a well-written and structured article, relevant to the ecology of Gymnocarpos przewalskiiis and other desert species. Due to the large volume of results, I recommend that a multivariate analysis of the data should be carried out first and then the analysis of variance of the most responsive variables. Overall, I understand the relevance and novelty of the manuscript presented.

Author Response

 

Dear Editors and Reviewers:

Thank you for your letter and for the reviewers’ comments concerning our manuscript entitled “Photosynthetic and antioxidant responses of Gymnocarpos przewalskii to simulated rainfall changes” (Manuscript ID: 2252769). Those comments are all valuable and very helpful for revising and improving our paper, as well as the important guiding significance to our research. We have studied the comments carefully and have made a correction which we hope meets with approval. Revised portions of the paper are marked with the “Track Changes” function in Microsoft Word. The main corrections in the paper and the responses to the reviewer’s comments are as follows:

Responds to reviewer 1’s comments

Comment: This is a well-written and structured article, relevant to the ecology of Gymnocarpos przewalskiiis and other desert species. Due to the large volume of results, I recommend that a multivariate analysis of the data should be carried out first and then the analysis of variance of the most responsive variables. Overall, I understand the relevance and novelty of the manuscript presented.

Response : Thank you for your constructive and helpful suggestion. We learn that multivariate analysis is commonly used in drought resistance of plants includes regression anaylsis, canonical correlation analysis, principle component analysis, cluster analysis, etc, which can be used to study relaion among parameters, screen key indicators, carry out comprehensive analysis, aiming at evaluating drought resistance (Parchin et al., 2013; Saeidi et al., 2017), selecting drought-resistant variety (Shamin et al., 2014; Ghanbari and Javan, 2015). In addition, multivariate analysis is usually applied to various species (varieties, genotypes, hybrids).

 

[1] Saeidi, K.; Zare, N .;Baghizadeh, A.; Zakaria, R. A. Differences in antioxidant, morphological and biochemical responses to drought stress in different cultivars of common bean (Phaseolus vulgaris L.). Imam Khomeini International University and Iranian Biotechnology Society, 2017, 6, 36-47.

http:// doi.org/10.30479/IJGPB.2017.1489

[2] Parchin, R.A.;  Najaphy, A.;Farshadfar, E.; Hokmalipour, S. Assessment of drought tolerance in genotypes of wheat by multivariate analysis. World Appl.Sci. J. 2013, 22, 594-600.

https:// doi.org/10.5829/idosi.wasj.2013.22.04.1915

[3] Shamin, F.; Saqlan, S.M.; Athar, H.U.R.; Waheed, A. Screening and selection of tomato genotypes/cultivars for drought tolerance using multivariate analysis. Pak. J.Bot.2014, 46,1165-1178.

[4] Ghanbari, M.; Javan, S.M. Study the response of mung bean genotypes to drought stress by multivariate analysis. Int. J. Agric. Innovations Res. 2015, 3, 2319-1473.

 

Thank you again for your valuable and instructive comments and suggestions.

Sincerely yours,

Haixia Huang

Author Response File: Author Response.docx

Reviewer 2 Report

1.      Introduction:

 There are very long paragraphs.

Second paragraph, first phrase:

All stressful situations can increase the production of ROS. Here, the authors must explore the relationship between water stress and physiological responses.

 Is Salaola collina or Salsola collina?

 

2.      Materials and methods:

2.2 Plant material and test design:

How many plants per treatment were evaluated? Was the sampling defined by individuals or by planting area? What is the difference between the planting area (40cm X 40) cm and the test plots (1m x 1m)? The irrigation volume was defined for which of the two areas, 40cm x40cm or 1m x 1m? This information needs to be clearer for us to understand the experimental conditions.

In addition to irrigation every five days, did the plants receive rainwater? Wouldn't this lead the plants in treatment of water restriction to less stressful conditions than expected?

 

2.3.  Measurement of gas exchange parameters

In Farquhar and Sharkey 1982, the formula is 1- (A/Ao), not 1- (Ci/Ca). The A and Ao data are obtained from the A-Ci curve. Make sure you have complete information to calculate stomatal limitations.

If you performed the A-Ci curves, please include the procedures in the methodology and the data among the results. This information will make your work more robust.

 

2.4. Measurement of chlorophyll fluorescence parameters

Insert the references for chlorophyll fluorescence parameters. There are no references for these methods in this section.

 

3.      Results

3.1. Effect of rainfall change on photosynthetic parameters.

The water supply is not just rain, right? Increase or decrease of water availability would be more appropriate.

 

3.2. Effect of rainfall change on light response parameters.

“The P_n was saturated at the PAR of approximately 1200 μmol m^-2 s^-1 under R-30 and R-15 and decreased when PAR was higher than 1500 μmol m^-2 s^-1.”

This is different of the result in table 2. The saturation point was at 1200 or above 1400 as the table says?

 

4.      Discussion

 

“Light response parameters can reflect the plant photosynthetic potential (Ye 2007), and P_nmax can reflect the maximal photosynthetic capacity of plants under certain conditions.”

 

 These phrases are well-established concepts in the literature and not the discussion of their results.

 

The correct is photoinhibition, not photo-inhibition.

 

5.      Conclusion

About photosynthetic limitations, check if the methodological procedures allow reaching this conclusion.

How to differentiate if the damage to the PSII was caused by the decrease in the pigment content or by the action of ROS?

 

 

 

 

 

 

 

 

 

 

 

 

Author Response

Dear Editors and Reviewers:

    Thank you for your letter and for the reviewers’ comments concerning our manuscript entitled “Photosynthetic and antioxidant responses of Gymnocarpos przewalskii to simulated rainfall changes” (Manuscript ID: 2252769). Those comments are all valuable and very helpful for revising and improving our paper, as well as the important guiding significance to our research. We have studied the comments carefully and have made a correction which we hope meets with approval. Revised portions of the paper are marked with the “Track Changes” function in Microsoft Word. The main corrections in the paper and the responses to the reviewer’s comments are as follows:

Responds to reviewer 2’s comments

Comments 1:   Introduction:

There are very long paragraphs.

Second paragraph, first phrase:

All stressful situations can increase the production of ROS. Here, the authors must explore the relationship between water stress and physiological responses.

Response: Thank you for your constructive and helpful suggestion.In the second paragraph we have added some information on the relationship between water stress and physiological responses in the introduction (Revised version, lines 63-68). The content is as follows:

Water stress often causes partial stomatal closure to conserve water (Song et al., 2016), simultaneously limits the CO₂ entry, reduces carboxylation efficiency of RuBisCO, the electron in electron transport chain are accepted by O₂, accordingly enhances oxygenation and photorespiration thereby increasing ROS production (Noctor et al., 2002). In addition, excess light energy in the PSII causes changes in photochemistry chloroplasts of leaves subjected to drought stress, leading to excess accumulation of ROS(Peltzer et al., 2002).

    [1] Song, X.L.; Wang, Y.H.; Lv, X.M. Responses of plant biomass, photosynthesis and lipid     peroxidation to warming and precipitation change in two dominant species (Stipa grandis and Leymus chinensis) from north China grasslands. Ecol. Evol. 2016, 6, 1871–1882. http://doi.org/10.1002/ece3.1982

[2] Noctor, G.; Veljovic-Jovanovic, S.; Driscoll, S.; Novitskaya, L.; Foyer, C.H. Drought and oxidative load in the leaves of C₃ plants: a predominant role for photorespiration? Ann. Bot. 2002, 89, 841–850. http://doi.org/10.1093/aob/mcf096

[3] Peltzer, D.; Dreyer, E.; Polle, A. Differential temperature dependencies of antioxidative enzymes in two contrasting species: Fagus sylvatica and Coleus blumei. Plant Physiol. Biochem. 2002, 40, 141–150. http:// doi.org/10.1016/S0981-9428(01)01352-3

Comments 2:  Is Salaola collina or Salsola collina?

Response: Thank you for your valuable query. The correct writing should be Salsola collina, we have corrected in revised version (Line 68).

Comments 3:  2. Materials and methods:

2.2 Plant material and test design:

How many plants per treatment were evaluated? Was the sampling defined by individuals or by planting area? What is the difference between the planting area (40cm X 40) cm and the test plots (1m x 1m)? The irrigation volume was defined for which of the two areas, 40cm x40cm or 1m x 1m? This information needs to be clearer for us to understand the experimental conditions.

Response: Thank you for your constructive and helpful suggestion. Eighteen plants per treatment were included. Sampling was defined by planting area, three plots per treatment were included, and each included six plants. 40cm × 40 cm refers to row spacing, and 1m × 1m stands for the area of each plot. The irrigation volume was defined for the test plot (1m × 1m).

Comments 4: In addition to irrigation every five days, did the plants receive rainwater? Wouldn't this lead the plants in treatment of water restriction to less stressful conditions than expected?

Response: Thank you for your constructive and helpful suggestion.The natural rainfall was sheltered by awning. We opened the awning on cloudy and rainy days to avoid the impact of rainwater, so the plants did not receive rainwater. In this way, the simulated rainfall for each treatment was controlled by irrigation.

Comments 5:  2.3.  Measurement of gas exchange parameters

In Farquhar and Sharkey 1982, the formula is 1- (A/Ao), not 1- (Ci/Ca). The A and Ao data are obtained from the A-Ci curve. Make sure you have complete information to calculate stomatal limitations.

If you performed the A-Ci curves, please include the procedures in the methodology and the data among the results. This information will make your work more robust.

Response: Thank you for your constructive and helpful suggestion. In our study, we did not perform the A-C_i curves. We cited the literature from the other literature, and we promise not to make similar mistakes in the future. The formula of L_s (L_s=1-(C_i/C_a)) was referred to the following literature.

Yin, C.Y.; Berninger, F.; Li, C.Y. Photosynthetic responses of Populus przewalski subjected to drought stress. Photosynthetica 2006, 44, 62–68. http://doi.org/10.1007/s11099-005-0159-y

Comments 6: 2.4. Measurement of chlorophyll fluorescence parameters

Insert the references for chlorophyll fluorescence parameters. There are no references for these methods in this section.

Response: Thank you for your constructive and helpful suggestion. We have inserted the references for chlorophyll fluorescence parameters in the revised version (line 165). The references are as follows:

[1] Baker, N.R. Chlorophyll fluorescence: A probe of photosynthesis in vivo. Annu. Rev. Plant Biol. 2008, 59, 89–113. http://doi.org/10.1146/annurev.arplant.59.032607.092759

[2] Roháček, K. Chlorophyll fluorescence parameters: the definitions, photosynthetic meaning, and mutual relationships. Photosynthetica, 2002, 40, 13–29. http://doi.org/10.1023/A:1020125719386

Comments 7: 3. Results

3.1. Effect of rainfall change on photosynthetic parameters.

The water supply is not just rain, right? Increase or decrease of water availability would be more appropriate.

Response: Thank you for your constructive and helpful suggestion. In the title (Effect of rainfall change on photosynthetic parameters), rainfall refers to the simulated rainfall, we wanted to analyze how the increase or decrease of simulated rainall affected the photosynthetic parameters.

Comments 8:  3.2. Effect of rainfall change on light response parameters.

“The P_n was saturated at the PAR of approximately 1200 μmol m^-2 s^-1 under R_-30 and R_-15 and decreased when PAR was higher than 1500 μmol m^-2 s^-1.”

This is different of the result in table 2. The saturation point was at 1200 or above 1400 as the table says?

Response: Thank you for your constructive and helpful suggestion. P_n-PAR curve was drawn according to the observed P_n at PAR gradient desinged, from the change curve, The P_n was nearly saturated at the PAR of approximately 1400 μmol m^-2 s^-1 under R_-30 and R_-15 and decreased when PAR was higher than 1500 μmol m^-2 s^-1. The photoresponse parameters were estimated according to the model proposed by Ye et al.(2013), so the L_sp under R_-30 and R_-15 were below 1500 μmol m^-2 s^-1, which may be caused by estimate error.

Ye, Z.P.; Suggett, J.D.; Robakowski, P.; Kang, H.J. A mechanistic model for the photosynthesis- light response based on the photosynthetic electron transport of PSII in C₃ and C₄ species. New Phytol. 2013, 152, 1251–1262. http://doi.org/10.1111/nph.12242

Comments 9:  4. Discussion

    “Light response parameters can reflect the plant photosynthetic potential (Ye 2007), and P_nmax can reflect the maximal photosynthetic capacity of plants under certain conditions.”

These phrases are well-established concepts in the literature and not the discussion of their results.

The correct is photoinhibition, not photo-inhibition.

Response: Thank you for your constructive and helpful suggestion. We have revised similar problems in the discussion, please check in revised version. Thank you for correcting the mistake, photo-inhibition has been corrected to photoinhibition in the revison.

Comments 10:  5. Conclusion

About photosynthetic limitations, check if the methodological procedures allow reaching this conclusion.

Response: Thank you for your constructive and helpful suggestion. In this study, gas exchange parameters were measured, and the result showed that the photosynthetic rate under R_-30 and R_-15 decreased significantly compared with R₀, which indicated the photosynthesis was limited. Based on statements on stomatal limitation or non-stomatal limitation to photosynthesis (Zhang et al., 2010; Liang et al., 2020), we inferred that stomatal limitation was responsible for the P_n decrease in Gymnocarpos przewalskii under R_-15, while the decrease in P_n mainly due to non-stomatal limitation under R_-30. In the future, we should follow your suggestion to research the relationship between rate of CO₂ assimilation (A) and intercellur CO₂ concentration (C_i), perform the A-Ci curves to make the conclusion more robust.   

The statements on stomatal limitation or non-stomatal limitation (Zhang et al., 2010; Liang et al., 2020 ) to photosynthesis are as follows:

The changing direction of C_i and L_s are the standards to judge the stomatal- and nonstomatal limits. When P_n and G_s fall, C_i falls and L_s rises, and thus, a stomatal limit is found out. When C_i rises and L_s falls, the nonstomatal limit is found (Zhang et al., 2010).

Zhang, S.Y.; Zhang, G.C.; Gu, S.Y.; Xia, J.B.; Zhao, J.K. Critical responses of photosynthetic     efficiency of goldspur apple tree to soil water variation in semiarid loess hilly area. Photosynthetica 2010, 48, 589–595. http://doi.org/10.1007/s11099-010-0076-6

When decreased P_n as a result of drought stress is accompanied by increased (or unchanged) C_i and decreased L_s, non-stomatal factors are the main cause of reduced photosynthetic rate. In contrast, when decreased P_n is accompanied by unchanged or increased L_s, while C_i is unchanged or decreased, stomatal factors are the main cause (Liang et al., 2020).

Liang, G.T.; Liu, J.H.; Zhang, J.M. Effects of drought stress on photosynthetic and physiological parameters of tomato. J. Am. Soc. Hortic. Sci. 2020, 145, 1–6. http://doi.org/10.21273/JASHS04725-19

Comments 11: How to differentiate if the damage to the PSII was caused by the decrease in the pigment content or by the action of ROS?

Response: Thank you for your constructive and helpful suggestion. In our study, F_v/F_m and QY_Lss under R_-30 significantly decreased compared with that under R₀, which indicated that photoinhibition occurred in PSII, the excitation engergy was excess in PSII. The decrease in the pigment content leads to reduction in light capture in PSII centre, reduces the damage accordingly (Ma et al., 2006). While Imbalance between light capture and its utilization in the PSII causes changes in photochemistry of chloroplasts in drought stressed leaves (Foyer and Noctor 2000), resulting in excess production of highly reactive and dangerous ROS species (Peltzer et al., 2002 ) , aggravates membrane lipid peroxidation. ROS-mediated inactivation is thought to be one of the primary causes of reversible loss of photosynthetic activity commonly termed “photoinhibition”( Logan et al., 2006). Based on the above, we infer that the damage to the PSII was mainly caused by the action of ROS.

[1] Ma, X.L.; Wang ,Y. J.; XIE, S. L.; Li, F.; Wang, W. Glycinebetaine applied through roots protects the photosynthetic apparatus of tobacco seedlings under water stress. J.Plant Physiol. Mol.Biol. 2006, 32, 465-472. (In Chinese with English summary)

[4] Logan, B.A.; Kornyeyev, D.; Hardison, J.; Scott Holaday, A. The role of antioxidant enzymes in photoprotection. Photosynth.res. 2006, 88, 119-132. https://doi.org/10.1007/s11120-006-9043-2

 

Thank you again for your valuable and instructive comments and suggestions.

Sincerely yours,

Haixia Huang

Reviewer 3 Report

 

In the manuscript, photosynthetic and antioxidant responses of Gymnocarpos przewalskii to simulated rainfall changes were studied with control test. I think that the study approach is solid and believe it can provide further insight into the response of G. przewalskii seedlings to different water conditions under global climate change. But I think the manuscript still needed some discussions including: 1) the difference and connection between the seedlings and the adult individuals to different water conditions. 2) what can be inferred from your results under more climate extremes in the future?

Author Response

Dear Editors and Reviewers:

Thank you for your letter and for the reviewers’ comments concerning our manuscript entitled “Photosynthetic and antioxidant responses of Gymnocarpos przewalskii to simulated rainfall changes” (Manuscript ID: 2252769). Those comments are all valuable and very helpful for revising and improving our paper, as well as the important guiding significance to our research. We have studied the comments carefully and have made a correction which we hope meets with approval. Revised portions of the paper are marked with the “Track Changes” function in Microsoft Word. The main corrections in the paper and the responses to the reviewer’s comments are as follows:

Responds to reviewer 3’s comments

Comment: In the manuscript, photosynthetic and antioxidant responses of Gymnocarpos przewalskii to simulated rainfall changes were studied with control test. I think that the study approach is solid and believe it can provide further insight into the response of G. przewalskii seedlings to different water conditions under global climate change. But I think the manuscript still needed some discussions including: 1) the difference and connection between the seedlings and the adult individuals to different water conditions. 2) what can be inferred from your results under more climate extremes in the future?

Response：Thank you for your constructive and helpful suggestion. We carry out some discussions on the difference and connection between the seedlings and the adult individuals to different water conditions. Based on the results we have inferred the response of G. przewalskii seedlings to more climate extremes in the future. Please check in revised version (Lines 506-531).

Comment 1: The difference and connection between the seedlings and the adult individuals to different water conditions.

Response : Thank you for your constructive and helpful suggestion. So far, we have not studied the response of seedlings and the adult individuals to different water conditions. The research showed that seedlings would face greater water stress versus the adult individuals, possiblly due to the small root system (Bragg et al., 1993), reduced photosynthetic physiological activity relative to older, larger individuals (Cui et al., 1991). With the increase of drought conditions, SOD and APX activity increased firstly then decreased in the adult individuals of G. przewalskii at different ages (about 15 a and 53 a ) , under severe drought CAT and POD activity significantly increased, and the older individuals have stronger drought resistance (Huang et al., 2020). Above results indicated that there are connection and difference between the seedlings and the adult individuals of G. przewalskii to different water conditions. So it is necessary to explore the response of seedlings and adult individuals of G. przewalskii to rainfall changes. It is helpful to fully understand the water adaptation mechanism.

 

[1] Bragg, W.K.; Knapp, A. K.; Briggs, J. M . Comparative water relations of seedling and adult Quercus species during gallery forest expansion in tallgrass prairie. For. Ecol. Manage. 1993, 56, 29-41. http://doi.org/10.1016/0378-1127(93)90101-R

[2] Cui, M.; Smith, W.K. Photosynthesis, water relations and mortality in Abies lasiocarpa seedlings during natural establishment. Tree Physiol.1991, 8, 37–46. http://doi.org/10.1093/treephys/8.1.37

[3] Huang, H.X.; Lian, Z.H.; Wang, L.; Yang, Q.Q.; Wei, Z.Y.; Ma, Y. J.; Zhang J X. Response of osmotic regulation substances and antioxidant enzyme activity in leaves of Gymnocarpos przewalskii to drought. Arid Zone Res. 2020, 37, 227–235. http://doi.org/10.13866/j.azr.2020.01.26 (In Chinese with English summary)

Comment 2: What can be inferred from your results under more climate extremes in the future?

Response : Thank you for your constructive and helpful suggestion. Extreme precipitation has been determined as a potential threat to global ecosystem (Webster et al., 2021), wet areas are expected to be wetter and dry areas are predicted to be drier (Tremberth 2011). In more extreme climate, what will happen to physiological characteristics of G. przewalskii? Based on the change of indicators more associated with biomass production and resource utilization under simulated rainfall, it can be inferred that the photosynthesis (P_n, P_nmax) will be more sensitive to extremely increased rainfall extremely decreased rainfall, photosynthetic capacity will enhance significantly with increase of rainfall, while the photochemical efficiency of PSII (F_v/F_m, QY_Lss) will be more responsive to extremely decreased rainfall, will reduce with decrease of rainfll. Improvement in WUE will reduce under extremely decreased rainfall, and the range of light energy utilization (L_sp, L_cp) will narrow significantly. The reaction between atmospheric CO₂ concentration and soil moisture has significant effect on the growth, development , productivity of plants, and ultimately affects the function of terrestrial ecosystem (Chong et al., 2017; Guehl et al., 1994).To better understand the adaptation of desert plants to future climate change, it is necessary to study further the interactive effect of elevated CO₂ and precipitation change on biomass and physiological characteristics of G. przewalskii, designing extreme rainfall gradients.

 

[1] Webster, C.L.; Kilminster, K.L.; Sánchez Alarcón, M.; Bennett K.; Strydom S.; McNamara S.; Lavery P.S.; McMahon, K.M. Population-specific resilience of Halophila ovalis seagrass habitat to unseasonal rainfall, an extreme climate event in estuaries. J. Ecol. 2021, 109, 3260–3279.        http:// doi.org/10.1111/1365-2745.13648

[2] Trenberth, K.E. Changes in precipitation with climate change. Climate Research, 2011, 47, 123–138. http://doi.org/10.3354/cr00953

[3] Chong, P.F.; Ji, J.L.; Li, Y.; Shan, L.S.; Su, S.P. Photosynthetic physiology responses to elevated CO₂ concentration and changing precipitation in desert plant Reaumuria soongorica. J. Desert Res. 2017, 37, 714–723. http://doi.org/10.7552/j.issn.1000-694X.2016.00091 (in Chinese with English Summary)

[4] Guehl, J.M.; Picon, C.; Aussenac, G.; Gross, P. Interactive effects of elevated CO₂ and soil drought on growth and transpiration efficiency and its determinants in two European forest tree species. Tree Physiol.1994, 14, 707–724. http://doi.org/10.1093/treephys/14.7-8-9.707

 

Thank you again for your valuable and instructive comments and suggestions.

Sincerely yours,

Haixia Huang

Round 2

Reviewer 2 Report

The author's responses answer the questions raised. 

 

I suggest including the reference that supports the calculation of stomatal limitations:

 

 

Berry, J.A., Downton, W.J.S.: Environmental regulation of photosynthesis. - In: Govindjee (ed.): Photosynthesis. Vol. II. Pp. 263-343. Academic Press, New York - London - Paris - San Diego - San Francisco - Sao Paulo - Sydney - Tokyo - Toronto 1982.

 

 

 

Author Response

Dear Reviewer:

Thank you for your letter and for the reviewers’ comments concerning our manuscript entitled “Photosynthetic and antioxidant responses of Gymnocarpos przewalskii to simulated rainfall changes” (Manuscript ID: 2252769). The comments are valuable and very helpful for revising and improving our paper, as well as the important guiding significance to our research. We have studied the comment carefully and have made a correction which we hope meets with approval. Revised portions of the paper are marked with the “Track Changes” function in Microsoft Word. The main corrections in the paper and the responses to the reviewer’ s comments are as follows:

Response to reviewer’ comments

Comments:

I suggest including the reference that supports the calculation of stomatal limitations:

    Berry, J.A., Downton, W.J.S.: Environmental regulation of photosynthesis. - In: Govindjee (ed.): Photosynthesis. Vol. II. Pp. 263-343. Academic Press, New York - London - Paris - San Diego - San Francisco - Sao Paulo - Sydney - Tokyo - Toronto 1982.

Response:

Thank you for your constructive and helpful suggestion. We have taken your suggestion and added the above reference that supports the calculation of stomatal limitations. The reference number is 29 in the revised version, please check in the Line 154, Lines 632-633. We cite the reference as follows:

Berry, J.A.; Downton, W.J.S. Environmental regulation of photosynthesis. Photosynthesis. Academic Press, New York, 1982. ISBN: 0-12-294302-3

Thank you again for your valuable and instructive comments and suggestions.

Sincerely yours,

Haixia Huang

Author Response File: Author Response.docx