Water Deficit Effects on Soybean Root Morphology and Early-Season Vigor
Round 1
Reviewer 1 Report
This manuscript presents a study on root development with the objective to evaluate the effects of several intensities of soil water deficit treatments on a number of aspects of soybean root development. This study was on root development during the early stages of plant growth in a pot study.
There are several problems with the manuscript.
(1) Classic studies on soybean root development in response to soil water were done many years ago by Howard Taylor, Betty Klepper, and Morris Huck. Considerable insight about soybean root development was published from these eariler studies, none of which were referenced in the current manuscript. Recognition of these previous studies would surely have influenced the approach of the current study.
(2) The study of root development has matured to the state where well-described hypothesis can be explored about specific mechanisms influencing root extension and water & nutrient uptake. Instead of investigating hypotheses, this study returned to the 'exploration approach' to root study. The need for stepping back to an exploration approach is not justified. Instead, many plant traits were measured after imposing water deficit treatments and statistically analyzed to find an empirical relationship with 'environmental productivity index' across treatments. Not surprisingly, there were very few statistically significant results for nearly all traits (Table 3). This empirical approach gave little new insight about water deficit and root development on plant productivity.
(3) A basic premise of the study was there were some root characteristics that influence water and nutrient uptake. Without a hypothesis there was no focused study on the impact of specific mechanisms and how they influence plant growth. Water uptake is now very well established to be tied to root length density. Amazingly, this study offered no results about root length density. Instead, root characteristics that have fairly minor impact on uptake were examined. The parameters studied seems to indicate a failure to recognize the current understanding about root uptake of water and nutrients.
(4) No details about pot volume or dimensions are presented. It is not possible to appreciate the quality of a root experiment unless there is a full description of the pot environment in which the experiment was done. The photographs in Figure 2, especially, seem to indicate a major impact of the pot size on the pattern of rooting. That is, these 30 d plants could not express continual increases in depth of rooting due to pot height.
(5) No details are given on how the root architecture (Figure 1 & 2) were preserved during the harvest and cleaning of the root system. That is, how were the roots positioned with respect to each other and the original dimensions of the pot to represent the architecture shown in these figures?
(6) The very high sand content of the soil results can result in a very unusual soil water extraction response (e.g. Sinclair et al., Agron. J. 90:363). On what basis was this particular sandy soil mixture created to undertake these water deficit studies?
(7) There are problems with Table 1. The caption indicates that day and night values are being presented. These data are not in the table. There must be an error in the VPD. It seems extremely unlikely that the air in a SPAR unit can be maintained at a VPD of more than 5 kPa at the temperatures of this experiment. Further, was each water-deficit treatment done in a separate individual SPAR chamber? This hardly seems the appropriate way to statistically lay out this experiment.
Author Response
This manuscript presents a study on root development with the objective to evaluate the effects of several intensities of soil water deficit treatments on a number of aspects of soybean root development. This study was on root development during the early stages of plant growth in a pot study.
There are several problems with the manuscript.
(1) Classic studies on soybean root development in response to soil water were done many years ago by Howard Taylor, Betty Klepper, and Morris Huck. Considerable insight about soybean root development was published from these earlier studies, none of which were referenced in the current manuscript. Recognition of these previous studies would surely have influenced the approach of the current study.
The authors appreciate the valuable comments and suggestions. A couple of references were added recognizing the work of previous studies.
(2) The study of root development has matured to the state where a well-described hypothesis can be explored about specific mechanisms influencing root extension and water & nutrient uptake. Instead of investigating hypotheses, this study returned to the 'exploration approach' to root study. The need for stepping back to an exploration approach is not justified. Instead, many plant traits were measured after imposing water deficit treatments and statistically analyzed to find an empirical relationship with the 'environmental productivity index' across treatments. Not surprisingly, there were very few statistically significant results for nearly all traits (Table 3). This empirical approach gave little new insight about water deficit and root development on plant productivity.
The major objective of this study was to understand the water stress effects on soybean root morphology and early vigor. This includes quantifying various growth and developmental traits including some physiological components. Soil moisture stress treatment effects were significant on most of the measured traits and cultivar effect also in some of the traits. However, treatment by cultivar effect was not significant for all most all the traits. Our intention of this study extended more towards quantification approach of the different shoot and root characteristics that could be used as response functions to predict crop responses under varying water stress levels, rather than describing the mechanisms affecting extensive root system and water and nutrient uptake. Justification for our approach has been given at the end of the introduction section.
(3) A basic premise of the study was there were some root characteristics that influence water and nutrient uptake. Without a hypothesis, there was no focused study on the impact of specific mechanisms and how they influence plant growth. Water uptake is now very well established to be tied to root length density. Amazingly, this study offered no results about root length density. Instead, root characteristics that have fairly minor impact on uptake were examined. The parameters studied seem to indicate a failure to recognize the current understanding about root uptake of water and nutrients.
In this study, we utilized one of the efficient methods of examining the root morphological traits under controlled environments. This technique provides data for around ten root traits including cumulative root length, surface area, volume, diameter, no. of root tips, forks, and crossings. Basically, lateral roots have a significant role in water and nutrient uptake (Lynch, 1995). Also, depending on the genotype, plants vary in response to water stress conditions. If a genotype possesses increased no. of tips, forks, and crossings, its root system has the potential to enhance penetration through soil layers, resulting in a positive effect on plant nutrient uptake.
(4) No details about pot volume or dimensions are presented. It is not possible to appreciate the quality of a root experiment unless there is a full description of the pot environment in which the experiment was done. The photographs in Figure 2, especially, seem to indicate a major impact of the pot size on the pattern of rooting. That is, these 30 d plants could not express continual increases in depth of rooting due to pot height.
Thank you. The pot dimensions were given.
(5) No details are given on how the root architecture (Figure 1 & 2) were preserved during the harvest and cleaning of the root system. That is, how were the roots positioned with respect to each other and the original dimensions of the pot to represent the architecture shown in these figures?
The procedure of root washing, cleaning, and image acquisition have been described in detail in the previous studies. We have cited all the previous studies which described this method in a broader way. However, we provided a brief summary describing the way how we captured the scanned root image. In summary, we floated the cleaned individual root system in 5 mm of water in a 0.3-by 0.2 m Plexiglas tray and used a paintbrush to untangle and separate the root system to minimize root overlap. All the pots were the same in size, thus any differences in root morphology are related to genotype by treatment effect.
(6) The very high sand content of the soil results can result in a very unusual soil water extraction response (e.g. Sinclair et al., Agron. J. 90:363). On what basis was this particular sandy soil mixture created to undertake these water deficit studies?
As described in many previous root studies (we already cited all those studies in our manuscript), this medium is widely used to get a more descriptive root image. This medium consists of 87% sand, 2% clay, and 11% silt which defined as sandy loam. If we use more clay loam type soil, it would make so hard to get a clean root system while cleaning and washing steps without disturbing its original architecture or the morphology. After a thorough wash, we would lose many fine roots which can give a false description of the root’s original architecture. Therefore, the sandy loam medium has widely been recognized as a good soil medium to conduct root studies under controlled environments. However, in this study, we used decagon soil moisture sensors and collected daily soil moisture data to make sure all the pots had set treatment levels.
(7) There are problems with Table 1. The caption indicates that day and night values are being presented. These data are not on the table. There must be an error in the VPD. It seems extremely unlikely that the air in a SPAR unit can be maintained at a VPD of more than 5 kPa at the temperatures of this experiment. Further, was each water-deficit treatment done in a separate individual SPAR chamber? This hardly seems the appropriate way to statistically layout this experiment.
Thank you for bringing this into our attention. We have corrected the table heading for Table 1. All the data presented in this table are the average data for both day and night during the experimental period. Each water stress treatment was conducted in an individual SPAR chamber and the pots were organized in a completely randomized design in six rows with three pots per row with 9 replications per cultivar.
Reviewer 2 Report
This manuscript are clearly written and conclusions are clearly presented, which provide useful information for future research on drought stress on soybean root architecture and growth and vigor indices. My comments and questions for authors are as follow:
Line 15: in experiment 1 and 2 or in experiment I and II
Line 23: drought stress
Line 129: 2.4. Data Analysis
Line 219: 3.4. Photosynthesis and Fluorescence Parameters
Line 241: (Figure 3)
On page 5, 6 and 7 and throughout the manuscript please edit the way that you reference to the figures in the text unless the current format is according to the journal instruction.
Example: (FigureS5B) should be (Figure S5B)
Line 373: Please include the limitations of this study and further experiments to answer important questions resulting from this study.
Line 386 or 391: Are there any implications for future research? Please add it to the end of conclusions
Please prepare the references according to the Agronomy Journal instruction:
Journal Articles:
1. Author 1, A.B.; Author 2, C.D. Title of the article. Abbreviated Journal Name Year, Volume, page range.
Example Line 426-429:
Dai, A. Increasing drought under global warming in observations and models. Nature Cli. Cha. 2013, 3, 52- 58.
Yu, X.; Yang, A; James, A.T. Characterization of root architecture in an applied core collection for 428 phosphorus efficiency of soybean germplasm. Chin. Sci. Bull. 2017, 49, 1611-1620.
Comments for author File: 
Comments.pdf
Author Response
This manuscript is clearly written, and conclusions are clearly presented, which provide useful information for future research on drought stress on soybean root architecture and growth and vigor indices. My comments and questions for authors are as follow:
The authors appreciate the reviewer’s valuable comments and suggestions. The manuscript has been further revised based on the comments.
Line 15: in experiment 1 and 2 or in experiment I and II
Made the correction as suggested.
Line 23: drought stress
The change was made.
Line 129: 2.4. Data Analysis
Made the correction.
Line 219: 3.4. Photosynthesis and Fluorescence Parameters
Made the correction.
Line 241: (Figure 3)
Thank you. Made the change.
On pages 5, 6 and 7 and throughout the manuscript please edit the way that you refer to the figures in the text unless the current format is according to the journal instruction.
Example: (FigureS5B) should be (Figure S5B)
Thank you. The manuscript has been revised based on the suggestions.
Line 373: Please include the limitations of this study and further experiments to answer important questions resulting from this study.
A few limitations have been discussed.
Line 386 or 391: Are there any implications for future research? Please add it to the end of conclusions
Future implications have been discussed in the discussion section.
Please prepare the references according to the Agronomy Journal instruction:
Journal Articles:
1. Author 1, A.B.; Author 2, C.D. Title of the article. Abbreviated Journal Name Year, Volume, page range.
Example Line 426-429:
Dai, A. Increasing drought under global warming in observations and models. Nature Cli. Cha. 2013, 3, 52- 58.
Yu, X.; Yang, A; James, A.T. Characterization of root architecture in an applied core collection for 428 phosphorus efficiency of soybean germplasm. Chin. Sci. Bull. 2017, 49, 1611-1620.
Thank you. The reference section has been revised based on comments and suggestions.
Reviewer 3 Report
This paper has potentially useful data for readers of “Agronomy”, but I have some concerns described below. Major revisions should be done before the publication.
The purpose of this paper was to investigate the difference of response to drought stress between indeterminate and determinate soybean cultivars. However, the manuscript does not provide the reasons why determinate and indeterminate cultivars were compared and the discussion regarding their difference. The comparison between two genotypes was not enough to clarify the difference between two types, because the genotypes seems to have different traits other than stem determinacy. The authors should use many genotypes or near isogenic line of Dt1/dt1 locus, which is well known to control the stem determinacy.
In this paper, the two-way ANOVA does not detect any interaction of genotypes with drought treatment, which indicates statistically that there is no difference of drought response between two genotypes. However, the authors argued the difference between the genotypes. Many readers will not accept this argument.
Minor comments
L80. Please provide the soil water content of soil used in this study at field capacity and wilting points if possible.
L124 Fluorescence → fluorescence yield of opened photosystem II (PSII)
Author Response
This paper has potentially useful data for readers of “Agronomy”, but I have some concerns described below. Major revisions should be done before the publication.
The authors appreciate the reviewer’s valuable comments and suggestions. The manuscript has been further revised based on the comments.
The purpose of this paper was to investigate the difference of response to drought stress between indeterminate and determinate soybean cultivars. However, the manuscript does not provide the reasons why determinate and indeterminate cultivars were compared and the discussion regarding their difference. The comparison between two genotypes was not enough to clarify the difference between two types, because the genotypes seem to have different traits other than stem determinacy. The authors should use many genotypes or near-isogenic line of Dt1/dt1 locus, which is well known to control the stem determinacy.
Thank you. We do agree with the reviewer. The reason for choosing these two cultivars in the present study is those two were the most popular and commonly grown cultivars in the US mid-south region. We have discussed the limitations of this study and future implications at the end of the discussion section which provides more room for future research.
In this paper, the two-way ANOVA does not detect any interaction of genotypes with drought treatment, which indicates statistically that there is no difference in drought response between two genotypes. However, the authors argued the difference between the genotypes. Many readers will not accept this argument.
Analysis of variance was used to determine crop parameter response to soil moisture stress. Means among treatments were compared using the least significant difference at P<0.05 probability.
Soil moisture stress treatment effects were significant on most of the measured traits and cultivar effect also in some of the traits. However, treatment by cultivar effect was not significant for all most all the traits. Since there was no interaction effect, the environmental productivity index was used to quantify the soil moisture deficit effects on soybean growth and developmental parameters.
Minor comments
L80. Please provide the soil water content of soil used in this study at field capacity and wilting points if possible.
The soil moisture stress treatments included five levels of irrigation, 100, 80, 60, 40, and 20%, which were maintained based on percent evapotranspiration (ET) values recorded on the previous day. Each SPAR unit was set at the given soil moisture stress treatment. All treatments were irrigated with the same water volume as in the 100% ET treatment until the time that each treatment was imposed. The ET measured on a ground area basis (L d−1) throughout the treatment period as the rate at which condensate was removed by the cooling coils at 900-s intervals (Reddy et al. 2001; Timlin et al. 2007) by measuring the mass of water in collecting devices connected to a calibrated pressure transducer. The plants were grown in cylindrical PVC pots (15.2 cm diameter by 30.5 cm high) in which soil was packed at a high bulk density (>1 Mg/m3). The control treatment was maintained at 100% field capacity in which the recorded soil moisture reading was 0.15 m3 m-3 based on the Theta-probe decagon soil moisture sensor. In order to maintain that moisture reading at a constant level, we irrigated the control plants 3 times per day. Other treatments (80, 60, 40, and 20%) were maintained by providing irrigation 80, 60, 40, and 20% of the control treatment. The amount and timing of the irrigation were adjusted based on the evapotranspiration of each sunlit growth chamber.
L124 Fluorescence → fluorescence yield of opened photosystem II (PSII)
Thank you. The correction was made.
Round 2
Reviewer 3 Report
The manuscript is in nice condition now for publication.
