Interannual Variation in Gas Exchange and Leaf Anatomy in Cenostigma pyramidale Is Exacerbated through El Niño and La Niña Climate Events

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The study presented by Pompelli et al. provides interesting data on physiological and anatomical responses of a shrub/tree (belonging to the Fabaceae) on water shortage. The study is expected to be of interest particularly to trait-based research and plasticity responses.

 There are various recommendations to be made. Generally, the materials and methods section is incomplete. Parameters appear somewhere in the manuscript (results or discussion) which were not introduced in the materials and methods section. A careful list should be made listing all measured parameters. Also, all measurement methods for all parameters have to be provided. Gas exchange measurements are related to leaf dry weight only. These are interesting data, but not standard. Area-based results should also be provided. Area-based and dry-weight based gas exchange values both comprise important information. Just to show dry-weight based values delivers an incomplete picture of the ecophysiological profile of the plants. See Osnas et al. (2018) Proceedings of the National Academy of Sciences, 115(21), 5480-5485 and Onoda et al. (2017). New Phytologist, 214(4), 1447-1463.

 Also the considered species should be better introduced in the materials section. In that section, it is barely mentioned.

 The discussion is too lengthy. It contains a lot of redundancies, quite common musings and also speculations, together with confusing sections. It should be shortened considerably, made much more concise and concentrate more on the findings of the study.

 In the following, some specific comments are listed.

 P.1: “ Industrial emissions of greenhouse gases lead to the buildup of atmospheric pollutants, contributing to global warming.” Which pollutants are meant? CO2, for instance, is a greenhouse gas, but not necessarily a “pollutant”.

P. 3: “A simple rise in stomatal CO2 absorption doesn't automatically lead to an increase in gas exchange.”I find this sentence odd. Stomatal CO2 uptake is a part of leaf gas exchange. How should stomatal CO2 uptake increase gas exchange? Or do the authors mean an increase in stomatal conductance which would increase CO2 diffusion into the leaf?

P. 3: “specific characteristics [29,30]. 33Leaf architecture plays a crucial role in facilitating CO2” Typing error: 33

 P. 5: “…old plants as described before [36].” Possibly, by [36] ?

 P. 5: “For each sample, 100 images were captured by a digital camera (Mikrosysteme Vertrieb GmbH, model ICC50 HD; Wetzlar, Germany)” 100 images were produced from one sample, what do these 100 images show?

 P. 5: “…where Kp is the potential specific stem conductivity,” As far as I understood the text, leaf anatomy was assessed. To which anatomical structure does Kp refer? Midrib, petiole, or entire vasculature on leaf cross section? Later, in table 4, however, Kp is termes “stem conductivity”. This all is quite confusing.

 P. 6: “Plasticity index” Here, it should be listed which and how traits were measured to obtain a plasticity index. This information is missing so far.

 P. 6: “Table 1, confirmed by Table 2, shows…” I would suggest: “Table 1 and Table 2 show that….”

 P.7: “Therefore, presenting gas exchange data per unit of leaf area would not be the best option. So, in this study, we will present gas exchange data on a gram basis of leaf tissue and not by metric area.”

 Gas exchange data per unit leaf area or per gram dry leaf tissue are just two approaches to describe leaf economy, and it cannot be said that one is the “best” or “not the best”. There are also more factors contributing to SLA than just photosynthetic tissue. Obtaining both of these parameters, gas exchange based on area as well as dry weight, provides more comprehensive ecophysiological information (see general comments above)

 P.7: “3.2 Water potential“ How was water potential measured? This description is completely missing from the materials and methods section.

 P. 7, legend to Figure 3: I find the description of the statistical results as shown in the Figure confusing.

 P. 7: A decrease in gas exchange from morning to afternoon is common (sometimes a small peak in the afternoon occurs). This decrease is practically absent for the 2019 data, and particularly so for the March 2019 data. I recommend to shorten this chapter (which contains repetitions of the data shown in Figure. 4) and to make it more concise. I would also strongly recommend to include the area-based measurements (at least showing them in a supplement, see general comments above).

 P. 9: “The correlations (Supplementary data file) of both gs and T could be responsive to…” What is meant with “T”? I suppose, from Fig. 6, that transpiration is meant. However, transpiration is termed “E” in Figure 6.

Also: What do the authors mean with “responsive”?

P. 9: “The correlations (Supplementary data file) of both gs and T could be responsive to modulation of PN, since correlations between PN and gs or PN and T were, respectively, 0.860 and 0.908 (both P < 0.00001). Therefore, to confirm that gs is not the preponderant factor mediating photosynthesis, we show in Figure 6A-B a regression between PN and gs and PN and T. The way of interpretation was season-dependent to contribute to correlations. In this case, we verify that all regressions between PN and T (Fig. 6B) return higher regression coefficients than regressions between PN and gs (Fig. 6A).”

 I cannot understand this part of the manuscript. Both assimilation P and transpiration T depend on stomatal conductance. Therefore, P correlates with T via gs. This text part leaves the impression that photosynthesis should depend on transpiration. The authors should make more clear what is meant here or this part should be deleted. (as explained in the preceding comment, I assume that T means transpiration)

Also, the following detailed regressions between Ci and P are, in my opinion, not suitable to obtain detailed information on the photosynthesis process with respect to the different conditions. For that, it would be necessary to produce a complete A/Ci curve to obtain Vcmax and other parameters of photosynthesis.

 P. 9: “The vapor pressure deficit (VPD; Fig. 8) also reflects the effect of air temperature” VPD is an environmental parameter which should be separated from the physiological results and shown together with the other climate parameters.

P. 12: “In comparison to the values presented in March 2019, the values presented in August 2019 increased by 10.9%, 16.5%, 2.2%, 12.1%, 2.6%, 14.9%, respectively in total leaf thickness (TT), ADAE, ABAE, PP, midrib thickness (MT), midrib length (ML), and midrib area (MA). In another way, the TT, spongy mesophyll thickness (SP), midrib xylem thickness (MXT), midrib xylem area (MXA), vessel area (VA), number of vessels (NV), and potential specific stem conductivity (Kp), respectively decreased in August 2019 in 3.5%, 26.2%, 9%, 4.2%, 11.2%, 4.1%, and 10.8%.”

Something is wrong here: TT increases first from March to August 2019 and then decreases from March to August 2019.

P. 14: The PCA analysis requires more explanation.

P. 15: The color scale does not fit to the values in the text, or (I guess) does “1” mean “100 %”?  And what to the asterisks mean? Probably statistically significant?

P. 15: Plasticity does not only mean the variability (or phenotypic response) to a hostile environment, but to environmental conditions in general.  Also, the text describing phenotypic plasticity results is confusing and should be completely overhauled. I am not sure whether it makes sense to pool plasticity of the various traits.

P. 17: I suggest to skip the first 8 lines of the discussion and to start directly with the findings of this study. The leaf phenology of C. pyramidale should be explained in the Introduction. Additionally, there was no mention of leaf phenology so far, neither in the materials and methods section nor in the results. If this study includes research on leaf longevity in C. pyramidale, this should be mentioned from the start.

P. 17/18: “The discrepancy in this concept also is exacerbated by Santos, et al. [2], which suspended 100% of total irrigation of C. pyramidale seedlings by 5 days, but the plants still produce photosynthesis, a strict evidence of the presence of leaves in extremely dehydrated plants.” I cannot understand this sentence

P. 18: “It is worth noting that SLA is influenced not only by leaf area but also by leaf density and thickness” Here, a citation would be appropriate, for instance Onoda et al. mentioned above.

P. 19: “It is worth noting that in this study PN was not influenced by gs, E (Fig. 6), or VPD (Fig. 8).” How should PN not be influenced by gs? (it is of course not directly related to E or VPD…) Furthermore, some sentences later the authors describe that “According to Pinho-Pessoa et al. [36], C. pyramidale displays lower values of PN mediated by stomatal closure.”

Stomatal closure regulates gs! The next sentences also make little sense and this whole text part should be overhauled.

P. 19: “This finding contradicts our results because the net photosynthesis evaluated during water stress and lower leaf water potential was insignificant if comparable to real possibilities to realize gas exchange.” To put it shortly, in the study, a distinct reduction of photosynthesis was found in the dry year compared to a year with a normal rainy season.

P. 19: “…a legitimate error of estimation or data interpretation. Thus, we can infer that the difference between our results and re- sults described by Falcão, et al. [52] is merely speculative and decisions cannot be made about different analysis methodologies, especially when there is doubt regarding its analysis and the data interpretation. However, the net photosynthesis does not show a constant or fixed net value, instead ranging across the measurement of time.” What do the authors mean here? This is all quite unclear and confusing and should be either largely shortened or deleted.

Possibly, some discrepancies between the data of this study and other studies may be explained expressing photosynthesis by area vs. dry weight.

The entire discussion section is much too lengthy and includes many general musings about plant responses to environment. See general comments.

Comments on the Quality of English Language

There are some problems with the English text here and there, and some moderate editing would be beneficial.

Author Response

Please see attached letter

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The topic selection of the article has important practical significance. The overall content of the manuscript is not a big problem, and the language is also smooth. There are a few small suggestions: 1. The research period is from 2019 to 2020, why did it not start submitting until the end of 2023? Can you explain the reason? 2.How to prove that the difference in indicators is caused by interannual water differences, rather than plant growth or sampling? 3. The image is somewhat blurry, and the clarity should be further increased.4. The multiplication sign in the formula should be used ×， Using x can easily be misunderstood by people.

Comments on the Quality of English Language

The language is also smooth.

Author Response

Please see attached letter

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript was substantially improved. I have no further issues to discuss.