Cowpea Ecophysiological Responses to Accumulated Water Deficiency during the Reproductive Phase in Northeastern Pará, Brazil

Round 1

Reviewer 1 Report

Authors of the manuscript "Cowpea Ecophysiological Responses to Accumulated Water Deficiency during the Reproductive Phase in Northeastern Pará, Brazil" try to evaluate impact of water deficit on cowpea productivity.
The overall idea of the study is not new, impact of water deficit on crop productivity at various growth stages is generally known. However the main finding that the level of water stress equivalent to leaf water potential 0.88MPa is characteristic and critical value is interesting and valuable. However it is not clear if obtained results clearly reflects the findings it should be indicated.
In my opinion the manuscript should not be published in current form, it needs strong improvement.
Below some of the most important comments:
1.    It is not so often that I see a study base on such inaccessible literature - more than a half of references are not available as they are not in English. It has to be changed, unless it is directed to Brazilian readers, but I do not think so...
2.    Authors attribute characteristic value of 0.88MPa to climate (in abstract), I would expect that measurement of leaf potential allow for obtaining specific plant (cultivar) response to water deficit without strong links to the soil and climate. I suggest to describe the finding linking it to cultivar not to the soil and climate.
3.    Authors mix various descriptors of available soil moisture, that are not well described : accumulated water deficit, water depth, volumetric water content – it should be unified.
4.    Were the mobile covers used only during the day – as it is written? Why?
5.    There are no weather condition during the crop growth provided, at the same time air temperature and humidity were not controlled during the measurements of gas exchange. It is important flaw of experimental design. Potentially the drop occurring at 0.88MPa could result from the change in external conditions like air temperature and humidity, not only water availability. 
6.    Table 2 “water depth applied” there is sth wrong with the style (it includes rainfalls), please correct it.  
7.    The text bellow Fig. 1 states that T4 reached 0.117m3m-3, it should be pointed out that it dropped to much lower values than PWP as it can be seen from Fig.1. This concerns mostly 2016, the water availability in 2015 was much higher. This may suggest that differential response at 0.88 could be due to grouping of the measuring points (Fig. 4) from different years on the left and right form 0.88. It should be verified and clearly written that such effect do not occur (if occur then the main conclusion is not valid).
8.    Figure 2 various equations are used without proper justification, probably just to obtain the highest R2. I suggest to unify equations just to linear or exponential, then their coefficient could be compared indicating differences in the response t water deficit.
9.    Figure 4 – data on leaf temperature – it is difficult to evaluate if there is not provided nor controlled (in the cuvette) air temperature
10.    Please provide error bars to figure 5
11.    “ostiole” – why such term is used?

Author Response

All recommendations and corrections from all reviewers were accepted and inserted.

However, due to the short time to work on the corrections, we were unable to include the chow test, as requested by one of the reviewers. However, we are already working on the application of this test so that it is possible to include it in a 2nd round of corrections, if allowed.

In addition, I request permission to include one more author in the article because we had to seek help from a colleague from the university who is a specialist in plant physiology, and who helped us deeply in this phase of the work. This is Prof. Hugo Alves Pinheiro.

 

All corrections are in the body of the article and were highlighted in red

 

1st reviewer

 

Authors of the manuscript "Cowpea Ecophysiological Responses to Accumulated Water Deficiency during the Reproductive Phase in Northeastern Pará, Brazil" try to evaluate impact of water deficit on cowpea productivity.

 

The overall idea of the study is not new, impact of water deficit on crop productivity at various growth stages is generally known. However the main finding that the level of water stress equivalent to leaf water potential 0.88MPa is characteristic and critical value is interesting and valuable. However it is not clear if obtained results clearly reflects the findings it should be indicated.

 

In my opinion the manuscript should not be published in current form, it needs strong improvement.

 

Below some of the most important comments:

1.    It is not so often that I see a study base on such inaccessible literature - more than a half of references are not available as they are not in English. It has to be changed, unless it is directed to Brazilian readers, but I do not think so.

 

          R: all references in Portuguese were replaced by similar articles in English whose themes and results were similar and whose substitution would not cause a problem to the context.

 

2.    Authors attribute characteristic value of 0.88MPa to climate (in abstract), I would expect that measurement of leaf potential allow for obtaining specific plant (cultivar) response to water deficit without strong links to the soil and climate. I suggest to describe the finding linking it to cultivar not to the soil and climate.

          R: the text in the abstract was corrected, emphasizing the fact that the threshold of the water potential found was due to the plant's response to environmental conditions and not due to the climate and soil.

 

3.    Authors mix various descriptors of available soil moisture, that are not well described : accumulated water deficit, water depth, volumetric water content – it should be unified.

          R: Texts have been adjusted as far as possible, but some of them are known terms and their respective definitions without the need to make distinctions between them. For this reason, we justify their permanence.

 

4.    Were the mobile covers used only during the day – as it is written? Why?

          R: We have included a text with a better explanation

5.    There are no weather condition during the crop growth provided, at the same time air temperature and humidity were not controlled during the measurements of gas exchange. It is important flaw of experimental design.

Potentially the drop occurring at 0.88MPa could result from the change in external conditions like air temperature and humidity, not only water availability.

          R: A figure with meteorological conditions during the two experiments was inserted.

          The methodology was adjusted to better clarify the objective of the work in respect of ecophysiological measurements.

In this research, as in countless others on ecophysiological responses, the only variables that were kept fixed were the flow of photosynthetically active radiation and the flow of CO2 concentration, considering that the measurements were carried out between 8 and 11 am.

The other meteorological variables were considered to be those of the environment itself, since the answer we were looking for was exactly the plant-atmosphere interactions under conditions of water limitation in the soil.

We also included in the methodology average values of air temperature during the ecophysiological measurement interval to demonstrate that the possible variability questioned in air temperature and RH were insignificant, and that therefore the answer we were looking for could not be influenced by the conditions of the atmosphere. . According to the results presented, the standard error of estimation of the air temperature and relative humidity between 8 am and 11 am were less than 0.3 oC and 2%.

 

6.    Table 2 “water depth applied” there is sth wrong with the style (it includes rainfalls), please correct it.

          R: there is no error....total water depth (blade) applied include rain and irrigation once the experimente was not carried out inside greenhouse or laboratory. The rain had to be count on. For this reason, the experiment was initiated after the rainy season, in order to avoid rain events as much as possible.

 

7.    The text bellow Fig. 1 states that T4 reached 0.117m3m-3, it should be pointed out that it dropped to much lower values than PWP as it can be seen from Fig.1. This concerns mostly 2016, the water availability in 2015 was much higher. This may suggest that differential response at 0.88 could be due to grouping of the measuring points (Fig. 4) from different years on the left and right form 0.88. It should be verified and clearly written that such effect do not occur (if occur then the main conclusion is not valid).

          R: the data in figure 4 were plotted with the years indicated separately (blue and red symbols) to confirm that the response was observed in both experiments.

 

8.    Figure 2 various equations are used without proper justification, probably just to obtain the highest R2. I suggest to unify equations just to linear or exponential, then their coefficient could be compared indicating differences in the response t water deficit.

          R: we decided to adjust only exponential equations to be able to compare R2 between the figures

9.    Figure 4 – data on leaf temperature – it is difficult to evaluate if there is not provided nor controlled (in the cuvette) air temperature

          R: considering the air temperature values informed during the measurement period, and considering that only photosynthetically active radiation and CO2 were fixed, leaving the other environmental conditions according to their natural viability (air temperature and relative humidity), and judging by the leaf temperature values found for the hours from 8 am to 11 am, which were slightly above the air temperature, therefore being consistent with the values of the ambient air temperature, attesting to the quality of the data.

 

10.    Please provide error bars to figure 5

          R: standard estimate error inserted in the figure

11.    “ostiole” – why such term is used?

          R: term replaced by “Stomatal opening”

 

 

Author Response File: Author Response.docx

Reviewer 2 Report

The work presents an interesting comprehensive study of the effect of the degree of water deficiency on the photosynthesis and productivity of cowpea (Vigna unguiculata (L.) Walp.) during maturation. 6 physiological characteristics of the leaf at the physiological stages R5, R7, R8 and R9 and the final productivity were studied. The threshold value of the water potential of the leaves -0.88 MPa was revealed, after which the rate of photosynthesis significantly decreases. The theme is important for the cultivation of cowpea in Brazil and for understanding common physiological patterns of photosynthesis. However, there are a number of issues that require clarification.

The following statement raises the most questions: Line 273-274: “When the leaf water potential reached -0.88 MPa (Figure 4), the analyzed variables significantly decreased” – 1. The significance of the decrease is not confirmed statistically, although it is possible to compare the averages before and after -0.88 MPa using ANOVA. Besides, the presence of a break point in a linear function can be checked by Chow test. 2. All graphs in Fig. 4 seems to be approximated by a smooth nonlinear function, as in Fig. 2-3, and without a break point. 3. The physiological parameters change under the several factors – water deficiency, phase of development. Is it the possibility that the decline in parameters is related to the developmental phase, and not to the water potential? 4. The nonlinear approximations at Figure 4a b are unconvincing. By what method are they built? There is no a-d transcript in the figure caption.

There is no table with the initial experimental data for the days of measurements, which can be given as a supplement.

References must be numbered in order of appearance in the text.

The designations of physiological parameters are introduced in pages 134-141. And then they are entered repeatedly in the text, which is excessive.

The footnote titles to the figures are marked with small or large letters a-d, A-D; sometimes in brackets, sometimes without; not everywhere these letters are in the title.

Line 67-68:”(1°19'24"S Latitude, 47°57'38" W Longitude, 41 m Altitude). The climate of the experimental area is defined as Am according to Köppen's climatic classification” – may be it is Af (tropical rainforest) with driest month precipitation more than 60 mm? At previous article [10] if was Af.

Line 115, 138: “using Time Domain Reflectometer (TDR) sensors installed in each treatment”;  “photon flow density (PPFD)” –  the abbreviations are not needed, because they are not used further.

Line 159: “However, the daily averages of meteorological data observed during 2015 and 2016” – average for what period? We can see the significant differences in the years in the moisture regime during the growing season. Data on temperature, insolation, etc. for the growing season or maturation time are of interest.

Line 173: Table 2. In title number of irrigations (NI), in the column name - IN.

Line 190: Figure 1. We need to decrypt FC, RAW, and PWP. Why does the water content in the soil differ before 36 DAS? In the caption for Figure 2015 (a) and 2016 (b), and the figures are marked A, B. The name of the right axis  should start with a capital letter.

Line 202: “The results of leaf water potential (Ψwf) – maybe "ΨWf measurement results"? The designation leaf water potential (ΨWf) was introduced earlier (Line 141), here it is repeated.

Line 224:” m-2 s-1 in 2015 and 82.79 mmol H2O m-2 s-1 in 2016, at the end of the cowpea (Figure 2B)” - what does “the end of the cowpea“ mean?

Lines 245-247: In the two years of experimental conduction, for all evaluations, the values of internal CO2 concentration (Ci) responded exponentially”  – in Fig. 2D we can see polynomial dependence, not exponential. Interesting, but not explained, is the increase in CO2 with an increase in water deficiency above 80 mm.

Lines 246, 336: “CO2 concentration (Ci)”– The designation was introduced earlier (Line 135), here it is repeated.

Line 263, fig. 3 – why are these figures shown separately from Figure 2?

277-278: «Similar results were found by Nascimento et al. [22], studying the tolerance of cowpea to water deficit, where by reducing …» - what does it mean “similar"? The presence of a breaking point? The value of ΨWf break point is the same?

Line 294-295: why is the yield in 2016 higher than in 2015, if the meteorological conditions are the same (Lines 159-162) and the time to beginning of ripening was the same?

It is possible to shorten the discussion, the processes of stomatal regulation of photosynthesis included in all plant physiology textbooks. For example: Lines 339-341: “Thus, stomatal limitation would be the main factor for the restriction of photosynthetic performance, because the greater the stomatal opening, the greater the CO2 diffusion to the substomatal chamber”. Lines 343-345: “When plants are submitted to water stress, there is usually a reduction in the stomatal opening as a protection mechanism, and consequently there is a reduction in the CO2 inflow into the substomatal chamber“. Lines 349-350 “when the water in the soil is less abundant, the stomata reduce its opening“. Lines 352-353: “The transpiration control by stomata is a mechanism used by many species to restrict water loss and overcome drought periods”. Lines 369-370: “The lower productivity was due to the water stress caused by treatments submitted to smaller irrigation depths, which reduced the soil water supply“. Lines 377-381: “According to Silva et al. [29], water deficiency makes several physiological and metabolic processes unfeasible in plants, causing decreased productivity, since water is one of the main responsible for the stomatal regulation. Such behavior can be explained as one of the drought resistance mechanisms used by cowpea plants, aiming at better conditions to overcome the lack of water, producing less pods [1]“. Lines 385-387: “an increase in leaf temperature and a reduction in photoassimilate production, leading to a reduction in production components and final productivity”. Lines 388-393: ”the reduction in leaf area of plants according to water deficiency limits productivity due to the decreased light interception and CO2 absorption, as found in the present study. Therefore, it is concluded that it is extremely important to adequately supply the water demand for cowpea, since it causes the maintenance of the flow of water and nutrients from the soil to the leaves [27], providing good conditions for the crop growth and development, leading to high productivity [33]”.

 

I hope the authors will easily correct the unclear phrases. Good luck!

Author Response

All recommendations and corrections from all reviewers were accepted and inserted.

However, due to the short time to work on the corrections, we were unable to include the chow test, as requested by one of the reviewers. However, we are already working on the application of this test so that it is possible to include it in a 2nd round of corrections, if allowed.

In addition, I request permission to include one more author in the article because we had to seek help from a colleague from the university who is a specialist in plant physiology, and who helped us deeply in this phase of the work. This is Prof. Hugo Alves Pinheiro.

 

All corrections are in the body of the article and were highlighted in red

 

2nd reviewer

The work presents an interesting comprehensive study of the effect of the degree of water deficiency on the photosynthesis and productivity of cowpea (Vigna unguiculata (L.) Walp.) during maturation. 6 physiological characteristics of the leaf at the physiological stages R5, R7, R8 and R9 and the final productivity were studied. The threshold value of the water potential of the leaves -0.88 MPa was revealed, after which the rate of photosynthesis significantly decreases. The theme is important for the cultivation of cowpea in Brazil and for understanding common physiological patterns of photosynthesis. However, there are a number of issues that require clarification.

The following statement raises the most questions:

Line 273-274: “When the leaf water potential reached -0.88 MPa (Figure 4), the analyzed variables significantly decreased” – 1. The significance of the decrease is not confirmed statistically, although it is possible to compare the averages before and after -0.88 MPa using ANOVA. Besides, the presence of a break point in a linear function can be checked by Chow test.

          R: a new figure was included with the ANOVA summary for each phenological phase and for each year.

In this figure, it is possible to identify the points where significant statistical difference occurs, as a way of showing that from a given treatment, which is associated with a respective water potential, ecophysiological responses present significant statistical differences. We imagine that with this figure it is possible to prove and justify the differences between the averages before and after the hypothetical value of water potential -0.88, which is associated with the T3 treatment.

Unfortunately, due to the short time for corrections, we were unable to apply the chow text to prove the break point in the linear function, but if we are given the opportunity, we are already working on it to include it in a 2nd round of correction of the article.

2. All graphs in Fig. 4 seems to be approximated by a smooth nonlinear function, as in Fig. 2-3, and without a break point.

          R: in fact the visual impression is that there is no break, but judging by the significant differences between T2 and T3 treatments in almost all phases for all ecophysiological variables, it is to be expected that there will be a breaking point in this correction, indicating a change in the mathematical function used. After applying the chow test, we will be able to confirm this.

3. The physiological parameters change under the several factors – water deficiency, phase of development. Is it the possibility that the decline in parameters is related to the developmental phase, and not to the water potential?

          R: The new figure included with the ANOVA summary for each phenological phase and for each year hlep us to prove that the most importante fator that cause decline in ecofisiological variables is water limitation instead of age of plant.

If we consider only the T1 treatment in which the water was not limited, it is clear that even with the advance of the phenological phases in the reproductive stage, the variables did not present significant differences when compared to the differences between the treatments. This alone reinforces the hypothesis that water was in fact the main limiting factor for the observed reductions.

As the work did not aim to compare the differences between phases, or even between the vegetative and reproductive phase, our hypothesis that the accumulated water deficiency during the reproductive phase would be the main limiting factor for the gas exchange processes, resulting in the yield gap observed.

4. The nonlinear approximations at Figure 4a b are unconvincing. By what method are they built? There is no a-d transcript in the figure caption.

          R: a linear function was adjusted for cases A and B. The dynamic fit curve procedure of Sigmaplot was used to adjust the functions in all figures.

There is no table with the initial experimental data for the days of measurements, which can be given as a supplement.

          R: the methodology was adjusted in order to improve the understanding of the experiment

References must be numbered in order of appearance in the text.

R: It was corrected and numbered according to the authors' guid

The designations of physiological parameters are introduced in pages 134-141. And then they are entered repeatedly in the text, which is excessive.

          R: terms have been corrected

The footnote titles to the figures are marked with small or large letters a-d, A-D; sometimes in brackets, sometimes without; not everywhere these letters are in the title.

          R: Corrected

Line 67-68:”(1°19'24"S Latitude, 47°57'38" W Longitude, 41 m Altitude). The climate of the experimental area is defined as Am according to Köppen's climatic classification” – may be it is Af (tropical rainforest) with driest month precipitation more than 60 mm? At previous article [10] if was Af.

          R: It was a mistake on previous article...the correct climate classification is Am...between august and november the month precipitation is below 60mm according to the climatological data (https://www.climatempo.com.br/climatologia/235/castanhal-pa)

Line 115, 138: “using Time Domain Reflectometer (TDR) sensors installed in each treatment”;  “photon flow density (PPFD)” –  the abbreviations are not needed, because they are not used further.

          R: Ok...corrected

Line 159: “However, the daily averages of meteorological data observed during 2015 and 2016” – average for what period? We can see the significant differences in the years in the moisture regime during the growing season. Data on temperature, insolation, etc. for the growing season or maturation time are of interest.

          R: text included to define the average period .... figure was included

Line 173: Table 2. In title number of irrigations (NI), in the column name - IN.

          R: corrected

Line 190: Figure 1. We need to decrypt FC, RAW, and PWP. Why does the water content in the soil differ before 36 DAS? In the caption for Figure 2015 (a) and 2016 (b), and the figures are marked A, B. The name of the right axis  should start with a capital letter.

          R: symbols were described in the figure, a text was inserted to justify the difference in humidity before the differentiation of the blades, corrected figure letters, corrected Y axis title

Line 202: “The results of leaf water potential (Ψwf) – maybe "ΨWf measurement results"? The designation leaf water potential (ΨWf) was introduced earlier (Line 141), here it is repeated.

          R: corrected  

Line 224:” m-2 s-1 in 2015 and 82.79 mmol H2O m-2 s-1 in 2016, at the end of the cowpea (Figure 2B)” - what does “the end of the cowpea“ mean?

          R: corrected....The text “cowpea crop cycle” was included

Lines 245-247: In the two years of experimental conduction, for all evaluations, the values of internal CO2 concentration (Ci) responded exponentially”  – in Fig. 2D we can see polynomial dependence, not exponential.

          R: figura foi ajustado para uma função exponencial conforme sugestão de outro revisor

Interesting, but not explained, is the increase in CO2 with an increase in water deficiency above 80 mm.

          R: a text with a reference was included

Lines 246, 336: “CO2 concentration (Ci)”– The designation was introduced earlier (Line 135), here it is repeated.

          R: corrected

Line 263, fig. 3 – why are these figures shown separately from Figure 2?

          R: it was just so that the figure didn’t get too big ... following recommendation, they were joined

277-278: «Similar results were found by Nascimento et al. [22], studying the tolerance of cowpea to water deficit, where by reducing …» - what does it mean “similar"? The presence of a breaking point? The value of ΨWf break point is the same?

          R: The text has been reorganized .. the similar term was associated with the answers obtained and not with the break point 0.88

Line 294-295: why is the yield in 2016 higher than in 2015, if the meteorological conditions are the same (Lines 159-162) and the time to beginning of ripening was the same?

          R: despite the difference in productivity between years, there was no significant difference between years as assessed in previous work. Even so, a possible explanation for the higher productivity in 2016 may have been the largest total evapotranspirations of the culture, which were slightly higher in the year 2016 (Table 2)

It is possible to shorten the discussion, the processes of stomatal regulation of photosynthesis included in all plant physiology textbooks. For example:

          R: we follow the recomendation

Lines 339-341: “Thus, stomatal limitation would be the main factor for the restriction of photosynthetic performance, because the greater the stomatal opening, the greater the CO2 diffusion to the substomatal chamber”.

          R: Text was deleted as suggestedLines 343-345: “When plants are submitted to water stress, there is usually a reduction in the stomatal opening as a protection mechanism, and consequently there is a reduction in the CO2 inflow into the substomatal chamber“.

R: Text was deleted as suggested

 Lines 349-350 “when the water in the soil is less abundant, the stomata reduce its opening“.

          R: The text has been reorganized

Lines 352-353: “The transpiration control by stomata is a mechanism used by many species to restrict water loss and overcome drought periods”.

          R: Text was deleted as suggested

 Lines 369-370: “The lower productivity was due to the water stress caused by treatments submitted to smaller irrigation depths, which reduced the soil water supply“.

R: Text was deleted as suggested

Lines 377-381: “According to Silva et al. [29], water deficiency makes several physiological and metabolic processes unfeasible in plants, causing decreased productivity, since water is one of the main responsible for the stomatal regulation. Such behavior can be explained as one of the drought resistance mechanisms used by cowpea plants, aiming at better conditions to overcome the lack of water, producing less pods [1]“.

R: Text was deleted as suggested

Lines 385-387: “an increase in leaf temperature and a reduction in photoassimilate production, leading to a reduction in production components and final productivity”.

R: The text has been reorganized

Lines 388-393: ”the reduction in leaf area of plants according to water deficiency limits productivity due to the decreased light interception and CO2 absorption, as found in the present study. Therefore, it is concluded that it is extremely important to adequately supply the water demand for cowpea, since it causes the maintenance of the flow of water and nutrients from the soil to the leaves [27], providing good conditions for the crop growth and development, leading to high productivity [33]”.

R: The text has been reorganized

I hope the authors will easily correct the unclear phrases. Good luck!

 

Author Response File: Author Response.docx

Reviewer 3 Report

This manuscript describes 2-year trial of cowpea plants under different water depths, and it was found that cowpea was sensitive to water shortage and leaf water potential 0.88 MPa was suggested to be the value of cowpea to water stress. The experiment of this study is well designed and results were well illustrated, but there are still some things need to be improved.

1. there are problems with Table 1, the font size seems to be larger than the manuscript text, and this table should be a three-line table.
2. the text size of acknowledge and reference is not consistent with main body of the manuscript.

Author Response

All recommendations and corrections from all reviewers were accepted and inserted.

However, due to the short time to work on the corrections, we were unable to include the chow test, as requested by one of the reviewers. However, we are already working on the application of this test so that it is possible to include it in a 2nd round of corrections, if allowed.

In addition, I request permission to include one more author in the article because we had to seek help from a colleague from the university who is a specialist in plant physiology, and who helped us deeply in this phase of the work. This is Prof. Hugo Alves Pinheiro.

 

All corrections are in the body of the article and were highlighted in red

 

 

3rd reviewer

This manuscript describes 2-year trial of cowpea plants under different water depths, and it was found that cowpea was sensitive to water shortage and leaf water potential 0.88 MPa was suggested to be the value of cowpea to water stress. The experiment of this study is well designed and results were well illustrated, but there are still some things need to be improved.

1. there are problems with Table 1, the font size seems to be larger than the manuscript text, and this table should be a three-line table.

 

R: the table was corrected

1. the text size of acknowledge and reference is not consistent with main body of the manuscript.

 

R: ok...corrected

 

Author Response File: Author Response.docx

Round 2

Reviewer 2 Report

The article became much more interesting after editing. Unfortunately, we did not see any statistical evidence for the presence of a break point in Figure 5. We will treat its presence as a hypothesis.