Rapeseed Supports Hairy Vetch in Intercropping, Enhancing Root and Stem Morphology, Nitrogen Metabolism, Photosynthesis, and Forage Yield
, Bo Wang 1,2, Guangsheng Zhou 1,2 and Jie Zhao 1,2,*
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsDear Authors,
After reading the manuscript, in my opinion, it deals with a rather interesting topic which is intercropping. The authors mainly focus on crop lodging and the impact on yield. However, in my opinion, attention should also be paid to other mechanisms of intercropping that may have interacted to affect the results obtained. The manuscript lacks line numbers so it is difficult to provide suggestions.
Abstract
however, the underlying mechanisms of overyielding in forage crop intercropping systems remain unclear - I do not fully agree with this statement there are many manuscripts describing the mechanisms of yield increase in intercropping. I suggest deleting.
thereby boosting forage biomass and quality - I suggest shortening to improved the analyzed crop parameters.
Introduction
In the introduction, the authors focus primarily on crop lodging and the links to it. I also suggest describing the benefits of intercropping. What are their limitations and why it was decided to grow rapeseed and vetch for forage reasons.
However, the mechanisms through which rapeseed-hairy vetch intercropping affects canopy structure and light distribution remain unclear - And how does the literature portray light distribution in other intercropping crops?
Materials and methods
2.1
and available potassium, 141.7 mg kg - Why are soil parameters from the start of the second year of field research given? Was the crop grown in two years on the same object? What were the pre-crops for the crop? In addition, the soil parameters are exactly the same as in the manuscript cited later (Wang et al. 2024) except that the field experiment there was started much earlier. Please clarify this.
2.2
Each treatment was replicated three times, and each plot measured 20 m in length and 2 m in width (Wang et al., 2024b). - In the literature cited (if you are referring to Wang et al. 2024), the research was done in different years so what does the citation refer to?
Rapeseed thinning was performed after emergence, and seedlings were fixed at the 3-5 leaf stage to ensure uni-form plant density. - Was inoculation of vetch plants used?
2.3
Forage fresh weight was measured by randomly harvesting three replicates of 1 m2 from each plot. - At what stage was the biomass harvested?
2.4
Six representative rapeseed plants, along with their surrounding soil cubes (30 cm × 30 cm × 30 cm), were carefully excavated from each plot during the flowering stage. - Was the flowering stage the biomass harvesting stage?
Results
In my opinion, the results are correctly recorded. What puzzles me is why the traits described in subsections 3.2; 3.3 and 3.4 were analyzed only for rapeseed, when all others were analyzed for both plants. In addition, I suggest completing the description of what the letters above the bars in the figures mean along the lines of the description under the table. The drawings should be able to function without the manuscript text.
Discussion
In the rapeseed-vetch intercropping system, the improved growing environment for vetch may stimulate cellulose and lignin synthesis in vetch stems, fundamentally enhancing their mechanical strength and reducing lodging risks (Raza et al., 2023). - What do the authors mean by the statement improved environment?
This improvement in light interception throughout the crop growth period may be a key factor contributing to increased biomass production. - In my opinion, not key but one of the key factors. There are many more mechanisms causing increased biomass production in intercropping that the authors do not mention.
Detailed anatomical analysis revealed that intercropping enhances cortex area, cortex thickness, vascular bundle number, vascular bundle length, and vascular bundle area in rapeseed stems, explaining the observed improvements in stem strength (Tian et al., 2023.) - ). The authors relate improvements in stem strength to increases in stem parameters (cortex thickness, etc.) but do not attempt to explain the putative mechanisms for the improvements in these parameters.
Efficient nutrient absorption enhances biomass and crude protein yield, as well as yield stability under stress conditions (Bargaz et al., 2017; Borden et al., 2020; Yin et al., 2024). - I suggest supplementing with an explanation of the mechanism of why intercropping with legumes in general absorbs more nutrients increasing protein yield, biomass and yield under abiotic stresses.
In addition, the authors in this section undertake a discussion of the results in another of their manuscripts which I consider incorrect.
Conclusions
The section is correctly written. I only suggest an addendum on the possible prospect of further research in this area and possible limitations observed in field research.
References
The first citations in the manuscript (page 1) [1-3] are typical of the Publisher subsequent citations are in a different form. This should be corrected. Additionally, e.g. Wang et al. 2024b does not appear in the References section.
Author Response
Responses to Reviewer #1:
Abstract
Q1: However, the underlying mechanisms of overyielding in forage crop intercropping systems remain unclear - I do not fully agree with this statement there are many manuscripts describing the mechanisms of yield increase in intercropping. I suggest deleting.
A1: Thank you for your valuable suggestion. The sentence has been revised to address your concern.
“However, the underlying mechanisms of overyielding in forage rapeseed–hairy vetch intercropping systems remain unclear.” (Line15)
Q2: thereby boosting forage biomass and quality - I suggest shortening to improved the analyzed crop parameters.
A2: Thank you for your valuable suggestion. The sentence emphasizes how intercropping improves population canopy light distribution, root and stem morphology, and photosynthesis-related parameters, ultimately leading to increased yield and quality. We believe that the original phrasing provides a clearer logical flow and better reflects the mechanisms involved. Therefore, we have decided to retain the current version of the sentence.
Introduction
Q3: In the introduction, the authors focus primarily on crop lodging and the links to it. I also suggest describing the benefits of intercropping. What are their limitations and why it was decided to grow rapeseed and vetch for forage reasons.
A3: Thank you for your insightful suggestion. Additional details about the benefits of forage rapeseed and hairy vetch intercropping have been included in the introduction section (Lines 47-50).
“Forage rapeseed and hairy vetch are valuable forage resources due to their high biomass and crude protein content. Preliminary findings from our research suggest that rape-seed-hairy vetch intercropping represents a promising system for producing high-biomass and high-protein forage”
Q4: However, the mechanisms through which rapeseed-hairy vetch intercropping affects canopy structure and light distribution remain unclear - And how does the literature portray light distribution in other intercropping crops?
A4: Thank you for your valuable suggestion. Additional explanation regarding light distribution in other intercropping systems has been included (Lines 67-68).
“Light interception at different spatial levels is commonly used to evaluate light distri-bution in intercropping crop populations”
Materials and Methods
Q5: 2.1 and available potassium, 141.7 mg kg - Why are soil parameters from the start of the second year of field research given? Was the crop grown in two years on the same object? What were the pre-crops for the crop? In addition, the soil parameters are exactly the same as in the manuscript cited later (Wang et al. 2024) except that the field experiment there was started much earlier. Please clarify this.
A5: Thank you for pointing out this issue. We apologize for the confusion caused by our oversight. This study is based on a long-term field experiment that began in 2017. As a result, the initial soil parameters are the same as those reported in our previously published manuscript. The experimental conditions have been clarified and revised in the manuscript (Lines 96–98) as follows:
“This study was conducted based on long-term field experiments in the experi-mental field of Huazhong Agricultural University, Wuhan, China (30.52°N, 114.31°E). The data presented in this article were collected from 2020 to 2022. Prior to planting in 2017, the initial soil nutrient contents were as follows: total nitrogen, 0.88 g kg−1; total phosphorus, 0.48 g kg−1; total potassium, 16.09 g kg−1; organic matter, 11.62 g kg−1; al-kalihydrolyzable nitrogen, 96.6 mg kg−1; available phosphorus, 14.2 mg kg−1; and available potassium, 141.7 mg kg−1.”
Q6: 2.2 Each treatment was replicated three times, and each plot measured 20 m in length and 2 m in width (Wang et al., 2024b). - In the literature cited (if you are referring to Wang et al. 2024), the research was done in different years so what does the citation refer to? Rapeseed thinning was performed after emergence, and seedlings were fixed at the 3-5 leaf stage to ensure uniform plant density. - Was inoculation of vetch plants used?
A6: Thank you for your thoughtful comments. The citation refers to the similar experimental design and planting density control used in this study, as the long-term field experiment followed a consistent protocol over the years. To clarify, the density of vetch plants was adjusted after germination to ensure uniformity (Lines 121-122).
“Rapeseed thinning was performed after emergence, and seedlings were fixed at the 3–5 leaf stage to ensure uniform plant density; hairy vetch seedlings were thinned to achieve the designed density after germination.”
Q7: 2.3 Forage fresh weight was measured by randomly harvesting three replicates of 1 m2 from each plot. - At what stage was the biomass harvested? 2.4 Six representative rapeseed plants, along with their surrounding soil cubes (30 cm × 30 cm × 30 cm), were carefully excavated from each plot during the flowering stage. - Was the flowering stage the biomass harvesting stage?
A7: The forage crops were harvested at the final flowering stage of rapeseed (Line 125). We specifically chose the flowering stage to measure the root morphology of rapeseed, as roots typically reach their highest biomass at this stage. Additionally, other traits were also evaluated during this stage.
Results
Q8: In my opinion, the results are correctly recorded. What puzzles me is why the traits described in subsections 3.2; 3.3 and 3.4 were analyzed only for rapeseed, when all others were analyzed for both plants. In addition, I suggest completing the description of what the letters above the bars in the figures mean along the lines of the description under the table. The drawings should be able to function without the manuscript text.
A8: Thank you for your valuable feedback. In this study, we focused on exploring the importance of rapeseed stem lodging resistance and root-related traits within the intercropping system. Since hairy vetch has a soft stem structure, it was not feasible to measure its stem strength, and therefore these parameters were not analyzed for vetch. Additionally, the description of the letters above the bars in each figure has been added to ensure the figures can be interpreted independently of the manuscript text.
“Different letters indicate significant differences between treatments according to Duncan’s multiple range test (DMRT) at p < 0.05.”
Discussion
Q9: In the rapeseed-vetch intercropping system, the improved growing environment for vetch may stimulate cellulose and lignin synthesis in vetch stems, fundamentally enhancing their mechanical strength and reducing lodging risks (Raza et al., 2023). - What do the authors mean by the statement improved environment?
A9: Thank you for pointing out the need for clarification. The sentence (Line 387) has been revised to specify what is meant by the "improved environment."
“Cellulose and lignin are key components of stem strength [30]. In the rapeseed-vetch intercropping system, the improved light irradiance for vetch may stimulate cellulose and lignin synthesis in vetch stems, fundamentally enhancing their mechanical strength and reducing lodging risks”
Q10: This improvement in light interception throughout the crop growth period may be a key factor contributing to increased biomass production. - In my opinion, not key but one of the key factors. There are many more mechanisms causing increased biomass production in intercropping that the authors do not mention.
A10: Thank you for your insightful suggestion. The sentence has been revised to reflect your point. (Lines 407–409)
“This improvement in light interception throughout the crop growth period is likely one of the key factors driving the increase in biomass production.”
Q11: Detailed anatomical analysis revealed that intercropping enhances cortex area, cortex thickness, vascular bundle number, vascular bundle length, and vascular bundle area in rapeseed stems, explaining the observed improvements in stem strength (Tian et al., 2023.) - ). The authors relate improvements in stem strength to increases in stem parameters (cortex thickness, etc.) but do not attempt to explain the putative mechanisms for the improvements in these parameters.
A11: Thank you for your valuable comment. We have expanded the discussion to include possible mechanisms underlying the improvements in rapeseed stem anatomical traits within the intercropping system. The following text has been added to the discussion section (Lines 467-471).
“Stem anatomical traits were significantly enhanced by the application of N and P ferti-lizers [31,55]. In the rapeseed-hairy vetch intercropping system, the N and P concen-tration in rapeseed were significantly improved, which may be one of the key factors contributing to the improvement of rapeseed stem anatomical traits under intercrop-ping conditions ”
Q12: Efficient nutrient absorption enhances biomass and crude protein yield, as well as yield stability under stress conditions (Bargaz et al., 2017; Borden et al., 2020; Yin et al., 2024). - I suggest supplementing with an explanation of the mechanism of why intercropping with legumes in general absorbs more nutrients increasing protein yield, biomass and yield under abiotic stresses.
In addition, the authors in this section undertake a discussion of the results in another of their manuscripts which I consider incorrect.
A12: Thank you for your insightful feedback. We acknowledge that there was an over-discussion of intercropping effects on NPK absorption in this section, which referred to results from another manuscript. To address this, we have deleted the related part of the discussion to ensure the content is focused solely on this study. Additionally, we have supplemented the discussion with the possible mechanisms by which legume intercropping improves nutrient uptake. (Lines 477-479).
“Moreover, under conditions of soil nutrition deficiency, intercropping stimulates mi-crobial diversity and root biomass allocation, particularly the distribution of fine roots. This further improves nutrient absorption, enhances biomass and crude protein yield, and contributes to yield stability”
Q13: The section is correctly written. I only suggest an addendum on the possible prospect of further research in this area and possible limitations observed in field research.
A13: Thank you for your thoughtful suggestion. In the main text, we have included a discussion on potential areas for future research in the conclusion section (Lines 499-502):
Q14: The first citations in the manuscript (page 1) [1-3] are typical of the Publisher subsequent citations are in a different form. This should be corrected. Additionally, e.g. Wang et al. 2024b does not appear in the References section.
A14: We have corrected the inconsistency in citation formatting to ensure uniformity throughout the manuscript. Additionally, the incorrect reference to Wang et al. 2024b has been removed.
Reviewer 2 Report
Comments and Suggestions for AuthorsAs authors concluded in 5. Conclusion, I understand well that the intercropping of rapeseed with vetch significantly enhances rapeseed stem strength and root anchoring capacity, providing physical support for vetch and thereby reducing lodging risk. This intercropping system improves stomatal structure, and leaf photosynthesis, optimizing the growing environment for both crops. However, I wonder that these conclusions were sole from the research results obtained in the present study or closely linked with those in the previous article (Wang et al., 2024).
I would like to ask the authors to revise the present manuscript followed by the comments below:
Review report to Agronomy-3414958
Jianli Ji, et al. “Rapeseed supports hairy vetch in intercropping, enhancing nitrogen metabolism, photosynthesis, and forage yield”
General comments: As authors concluded in 5. Conclusion, I understand well that the intercropping of rapeseed with vetch significantly enhances rapeseed stem strength and root anchoring capacity, providing physical support for vetch and thereby reducing lodging risk. This intercropping system improves stomatal structure, and leaf photosynthesis, optimizing the growing environment for both crops. However, I wonder that these conclusions were sole from the research results obtained in the present study or closely linked with those in the previous article (Wang et al., 2024).
I would like to ask the authors to revise the present manuscript followed by the comments as follows:
1. Title: In the present manuscript, authors showed the results of morphological changes (improvements) in the intercropping of rapeseed and hairy vetch in Figure 1 (d), Figures 2, 3, 4, 5 and Table 1. Therefore, I recommend revising the title to incorporate “enhancing stem and root morphological traits, nitrogen metabolism, ---“.
2. Abstract: L6–7, were investigated to assess the effects of intercropping on population lodging resistance, canopy light distribution, and photosynthetic physiology. However, in the results section, there was no data to directly determine lodging resistance, light distribution pattern in the canopy, except for supplementary Figure S2, nor photosynthetic physiology, such as Rubisco activity. I recommend that you should present the concrete traits you observed in the present manuscript. I also recommend that if you refer to “Intercropping also optimized vertical canopy light distribution,” then, you should transfer Figure S2 to the figure in the main text.
3. Materials and Methods: In 2.2., “Rapeseed thinning was performed after emergence, and seedlings were fixed at the 3–5 leaf stage to ensure uniform plant density.” ← How did you fix the seedling density of rapeseed in the present study? For forage production, it is unusual to do thinning of seedlings, different from food crop production. How was the thinning of hairy vetch seedlings? Fertilization level was so high at 9 g N m-2 at the basal and 5 g N m-2 at the additional fertilization. Therefore, the objective of the present study is not aimed for the intercropping of hairy vetch with rapeseed to assist the biological nitrogen fixation of legume species. How do you think of the level of fertilization in the present study?
4. In 2.6.1., Light interception rate was measured by canopy analyzer (L9 on Page 4). However, no data was present in the results section.
5. In 2.6.2., Photosynthetic gas exchange was measured. However, the replication of measurements and time of the day to measure the photosynthetic gas exchange were not described. Therefore, I cannot understand the significant difference in the traits shown in Table 2. In addition, dismal letters should be unified to 252 → 252.0, 0.13 → 0.130, 0.44 → 0.440, 0.005 → 0.0050.
6. Results: In Figure 1(a), (b), (c), there are white circles with arrows in the figure, however, no comments on the meaning of these symbols in the figure legends nor in the main text. I wonder why the authors put these symbols.
7. In Figure 1. The legend (a) and (b) should be reversed. Please check it.
8. In the general sense, crop lodging is related to the strength of aboveground parts such as stem breaking strength and the strength of underground root parts such as pulling resistance. In the present determined traits, can authors conclude these relationships related to lodging resistance of both crops?
9. 3.5. Gas exchange. In rapeseed with IR, the change in net photosynthetic rate (A) was not correlated with the changes in stomatal conductance (gs), but almost correlated with those in transpiration rate (E) in both 2021 and 2022. Therefore, intercellular CO2 concentration (Ci) did not drop even under the increase of A in IR. I think that the measurement was conducted under the chamber at the relative humidity of 60%, therefore, stomata can open at the measurement. However, the transpiration rate cannot match with the degree of the stomatal opening especially in 2022 when rainfall was almost nil from February to April (Fig. S1). How do you think of these changes in the traits related to leaf photosynthesis? Did you measure the soil moisture content at the measurements of photosynthesis?
10. In Figure 7, the antioxidant system enzyme and ROS content were only decreased with the intercropping of rapeseed and hairy vetch. And the meaning of these decreases appeared first in the conclusion section as “delays plant senescence and enhances the activities of nitrogen metabolism enzymes in Page 16. I think that this sentence should be dictated in the Discussion section.
11. In Figure 8(b), stem breaking strength of rapeseed was the principal factor to determine nitrogen content, photosynthetic rate of hairy vetch and biomass yield of intercrops. It is difficult to understand for the principal position of rapeseed stem breaking strength affecting hairy vetch nitrogen photosynthetic metabolism. How did the authors calculate this Figure 8, since this was not sectioned in Materials and Methods, except for 2.8. Data analysis in SEM (which was so simple and difficult to understand the calculation methods)?
12. In supplementary files, figure legends need to be revised in the English grammatical points.
For example, Fig. S3. Comparison the Radiation interception ratio between different cropping pattern at varied height.
→ Comparisons of the radiation interception ratios between different cropping patterns at varied heights.
Sincerely,
Author Response
Responses to Reviewer #2:
Q1: General comments: As authors concluded in 5. Conclusion, I understand well that the intercropping of rapeseed with vetch significantly enhances rapeseed stem strength and root anchoring capacity, providing physical support for vetch and thereby reducing lodging risk. This intercropping system improves stomatal structure, and leaf photosynthesis, optimizing the growing environment for both crops. However, I wonder that these conclusions were sole from the research results obtained in the present study or closely linked with those in the previous article (Wang et al., 2024).
A1: Thank you for your thoughtful comment. This manuscript was based on our long-term field experiments, and the results presented here were derived from data collected independently for this study. To clarify this point, we have added more details to the methodology section (Lines 96-98).
“This study was conducted based on long-term field experiments in the experi-mental field of Huazhong Agricultural University, Wuhan, China (30.52°N, 114.31°E). The data presented in this article were collected from 2020 to 2022. Prior to planting in 2017, the initial soil nutrient contents were as follows: total nitrogen, 0.88 g kg−1; total phosphorus, 0.48 g kg−1; total potassium, 16.09 g kg−1; organic matter, 11.62 g kg−1; al-kalihydrolyzable nitrogen, 96.6 mg kg−1; available phosphorus, 14.2 mg kg−1; and available potassium, 141.7 mg kg−1.”
Title
Q2: In the present manuscript, authors showed the results of morphological changes (improvements) in the intercropping of rapeseed and hairy vetch in Figure 1 (d), Figures 2, 3, 4, 5 and Table 1. Therefore, I recommend revising the title to incorporate “enhancing stem and root morphological traits, nitrogen metabolism, ---“.
A2: Thank you for your valuable suggestion. We have revised the title to better reflect the focus and findings of the manuscript.
“Rapeseed supports hairy vetch in intercropping, enhancing root and stem morphology, nitrogen metabolism, photosynthesis, and forage yield”
Abstract
Q3: Abstract: L6–7, were investigated to assess the effects of intercropping on population lodging resistance, canopy light distribution, and photosynthetic physiology. However, in the results section, there was no data to directly determine lodging resistance, light distribution pattern in the canopy, except for supplementary Figure S2, nor photosynthetic physiology, such as Rubisco activity. I recommend that you should present the concrete traits you observed in the present manuscript. I also recommend that if you refer to “Intercropping also optimized vertical canopy light distribution,” then, you should transfer Figure S2 to the figure in the main text.
A3: Thank you for your detailed and constructive feedback. We have revised the abstract to better reflect the specific traits observed in this study. The updated sentence now reads (Lines 17–18):
“Over two years of field experiments, three cropping systems—rapeseed sole cropping, hairy vetch sole cropping, and rapeseed-hairy vetch intercropping—were investigated to assess the effects of intercropping on root and stem morphology, canopy light distribution, photosynthetic, and nitro-gen metabolism. ”
Additionally, we have moved Supplementary Figure S2 to the main text as recommended, to provide direct evidence for the statement regarding the optimization of vertical canopy light distribution. This change ensures that the results discussed in the manuscript are better supported by the main figures. We appreciate your suggestions, which have helped to improve the clarity and accuracy of the abstract and the overall presentation of the manuscript.
Materials and Methods
Q4: Materials and Methods: In 2.2., “Rapeseed thinning was performed after emergence, and seedlings were fixed at the 3–5 leaf stage to ensure uniform plant density.” ← How did you fix the seedling density of rapeseed in the present study? For forage production, it is unusual to do thinning of seedlings, different from food crop production. How was the thinning of hairy vetch seedlings? Fertilization level was so high at 9 g N m-2 at the basal and 5 g N m-2 at the additional fertilization. Therefore, the objective of the present study is not aimed for the intercropping of hairy vetch with rapeseed to assist the biological nitrogen fixation of legume species. How do you think of the level of fertilization in the present study?
A4: Thank you for your insightful comments and questions.
Seedling thinning for uniform density: In our study, thinning was performed to achieve precise and uniform plant densities for both rapeseed and hairy vetch. This step was essential to minimize potential data variability caused by uneven plant distribution, ensuring reliable and reproducible results. Specifically, rapeseed thinning was conducted at the 3–5 leaf stage, while hairy vetch thinning was carried out shortly after germination. Although thinning is less common in forage production, it was necessary in our experimental design to maintain consistent planting density across all treatments.
Fertilization levels: The nitrogen application rates used in this study (9 g N m⁻² as basal and 5 g N m⁻² as additional fertilization) reflect the standard fertilization practices in rapeseed production regions of China. The primary objective of this study was not to investigate the role of biological nitrogen fixation by the legume species, but rather to assess the effects of intercropping on root and stem morphology, photosynthesis, and nitrogen metabolism under typical field conditions. The chosen fertilization levels ensured optimal crop growth and allowed us to focus on the intercropping effects under realistic agricultural management practices.
Q5: In 2.6.1., Light interception rate was measured by canopy analyzer (L9 on Page 4). However, no data was present in the results section.
A5: Thank you for pointing this out. We have now added the data on light interception rates to the Results section (Lines 226-239).
“Intercropping rapeseed with vetch significantly influenced light interception at various growth stages and vertical canopy heights compared to sole cropping. Inter-cropping significantly increased the canopy height compared to hairy vetch sole cropping, while no significant differences were observed compared to rapeseed sole crop-ping after the seedling stage. The canopy height of both sole-cropped and intercropped rapeseed populations increased rapidly from the seedling stage to the flowering stage and then stabilized, while the canopy height of vetch showed minimal change after entering the bolting stage. Compared to light interception rate of sole cropped rapeseed and hairy vetch at the same vertical canopy height, intercropping increased the light interception rate of rapeseed and hairy vetch by 5.9%-39.4% and 71.6%, respectively, at seedling stage, by 10.6%-144.4% and 5.6%-71.1% at the bolting stage, by 3.6%-129.9% and 57.7%-670.9% at the flowering stage; and by 14.6%-174.2% and 1.4-41 times at the final flowering stage (Figure 2).”
Q6: In 2.6.2., Photosynthetic gas exchange was measured. However, the replication of measurements and time of the day to measure the photosynthetic gas exchange were not described. Therefore, I cannot understand the significant difference in the traits shown in Table 2. In addition, dismal letters should be unified to 252 → 252.0, 0.13 → 0.130, 0.44 → 0.440, 0.005 → 0.0050.
A6: Details of photosynthetic gas exchange measurements: We have added detailed information on the replication and timing of photosynthetic gas exchange measurements to the Materials and Methods section (Lines161-166). The decimal formatting in Table 2 has been revised as suggested to maintain consistency and improve readability.
“During the flowering stage, under clear-sky conditions between 10:00 and 15:00, the net photosynthetic rate, transpiration rate, stomatal conductance, and intercellular CO2 concentration were measured. Six fully expanded third leaves from the apex of rapeseed plants and functional hairy vetch leaves from the middle canopy of each plot were selected for measurements using a Li-Cor 6800 portable photosynthesis system (LI-COR Inc., Lincoln, NE, USA).”
Results
Q7: Results: In Figure 1(a), (b), (c), there are white circles with arrows in the figure, however, no comments on the meaning of these symbols in the figure legends nor in the main text. I wonder why the authors put these symbols.
A7: Thank you for bringing this to our attention. We have added a description of the white circles and arrows below Figure 1 to clarify their meaning (Lines 223-224).
“The white circles and arrows indicate the magnified area depicted in sub-figures a, b, and c.”
Q8: In Figure 1. The legend (a) and (b) should be reversed. Please check it.
A8: Thanks, done.
Discussion
Q9: In the general sense, crop lodging is related to the strength of aboveground parts such as stem breaking strength and the strength of underground root parts such as pulling resistance. In the present determined traits, can authors conclude these relationships related to lodging resistance of both crops?
A9: Thank you for your insightful comment. In our study, we measured the canopy height under different cropping patterns. The results showed that intercropping significantly increased the canopy height of hairy vetch compared to sole cropping (lines 228–229). Additionally, from the light distribution data, we observed that hairy vetch plants grew spatially, not just along the ground. This indicates that the physical support provided by rapeseed plants in the intercropping system improved the overall lodging resistance of the intercropping population.
While we did not directly measure traits such as stem breaking strength or root pulling resistance in this study, our findings suggest that the structural support of rapeseed plants contributed to reducing the lodging risk of hairy vetch. Future studies could focus on quantifying these mechanical properties to further explore their relationship with lodging resistance in intercropping systems.
Q10: 3.5. Gas exchange. In rapeseed with IR, the change in net photosynthetic rate (A) was not correlated with the changes in stomatal conductance (gs), but almost correlated with those in transpiration rate (E) in both 2021 and 2022. Therefore, intercellular CO2 concentration (Ci) did not drop even under the increase of A in IR. I think that the measurement was conducted under the chamber at the relative humidity of 60%, therefore, stomata can open at the measurement. However, the transpiration rate cannot match with the degree of the stomatal opening especially in 2022 when rainfall was almost nil from February to April (Fig. S1). How do you think of these changes in the traits related to leaf photosynthesis? Did you measure the soil moisture content at the measurements of photosynthesis?
A10: Thank you for your insightful question.
We did not measure soil moisture content during the photosynthesis measurements. However, based on our observations, the soil water content was sufficient to support crop growth under the experimental conditions. This is likely due to the low temperatures and high air humidity during the measurement period. The high air humidity contributed to a low vapour pressure deficit (VPD), which increases stomatal opening but restricts the transpiration rate of leaves. This phenomenon could explain why the transpiration rate did not align with the degree of stomatal opening, particularly in 2022. Our explanation is supported by previous findings that both atmospheric and soil water status influence photosynthesis and growth (Du et al., 2018) [1].
We appreciate your comments and suggestions, as they highlight important factors that could influence the observed gas exchange traits. In future studies, we plan to include soil moisture content measurements to further investigate the relationship between soil water availability, stomatal behavior, and photosynthesis.
[1] Du, Q.; Zhang, D.; Jiao, X.; Song, X.; Li, J. Effects of atmospheric and soil water status on photosynthesis and growth in tomato. Plant, Soil and Environment 2018, 64, 13-19.
Q11: In Figure 7, the antioxidant system enzyme and ROS content were only decreased with the intercropping of rapeseed and hairy vetch. And the meaning of these decreases appeared first in the conclusion section as “delays plant senescence and enhances the activities of nitrogen metabolism enzymes in Page 16. I think that this sentence should be dictated in the Discussion section.
A11: Thank you for your valuable suggestion. We have incorporated the explanation of ROS and antioxidant system enzyme activity into the Discussion section (Lines 438-451).
“When subjected to biotic or abiotic stress, plants accumulate ROS, which cause damage to cells and tissues. At the same time, antioxidant system enzymes (POD, CAT, SOD) are activated to degrade ROS [49,50]. In our study, compared to monocropping, both ROS levels and antioxidant system enzymes activities in the leaves of rapeseed and hairy vetch were reduced under intercropping. This suggests that the decrease in ROS content in the rapeseed-vetch intercropping system may not be solely due to the activity of antioxidant enzymes but rather due to the down-regulation of ROS produc-tion during the formation stage.”
Q12: In Figure 8(b), stem breaking strength of rapeseed was the principal factor to determine nitrogen content, photosynthetic rate of hairy vetch and biomass yield of intercrops. It is difficult to understand for the principal position of rapeseed stem breaking strength affecting hairy vetch nitrogen photosynthetic metabolism. How did the authors calculate this Figure 8, since this was not sectioned in Materials and Methods, except for 2.8. Data analysis in SEM (which was so simple and difficult to understand the calculation methods)?
A12: Thank you for your thoughtful comment.
We determined that rapeseed stem breaking strength plays a crucial role in supporting the entire intercropping population by improving light distribution and canopy structure. Based on these relationships, we used structural equation modeling (SEM) to explore how intercropping affects rapeseed stem breaking strength, nitrogen metabolism, photosynthesis, and biomass production of both crops.
To address your concern, we have added more detailed information about the SEM analysis to the “2.8 Data analysis” section (Lines 201-207):
“Structural equation modeling (SEM) was performed in AMOS 24.0 (SPSS Inc., Chicago, IL, USA) to explore the effects of intercropping on rapeseed stem breaking strength, nitrogen metabolism, net photosynthetic rates of both crops, and biomass. Path coeffi-cients were derived using maximum likelihood estimation. The SEM model fit was evaluated based on several indices, including the relative chi-square/degree of freedom (X2/df), goodness of fit index (GFI), comparative Fit Index (CFI), and root mean square error of approximation (RMSEA). ”
Q13: In supplementary files, figure legends need to be revised in the English grammatical points.
For example, Fig. S3. Comparison the Radiation interception ratio between different cropping pattern at varied height.
→ Comparisons of the radiation interception ratios between different cropping patterns at varied heights.
A13: Thank you for pointing this out. The figure legends in the supplementary files have been revised according to your suggestion.
Round 2
Reviewer 1 Report
Comments and Suggestions for AuthorsDear Authors,
Thank you for addressing the suggestions you sent. With regard to the current content of the manuscript, I have some additional suggestions:
L50 I suggest referring to the mechanisms of yield increase of intercropping (nitrogen fixation and making it available to non-leguminous crops, better use of environmental resources - crop complementarity, reduction of spread of diseases, reduction of weeds)
L96-102 From the answer I understand that soil samples were not taken before the start of each year of field research? The other questions were not addressed: What was the pre-crop in each year? Or was the field experiment conducted in the same area each year? Was inoculation of vetch plants used?
The References section still needs to be corrected. Species names are not in italics. There are manuscript titles in which each word is capitalized. In addition, I suggest references to manuscripts from the last 10 years.
Good luck
Author Response
Q1: L50 I suggest referring to the mechanisms of yield increase of intercropping (nitrogen fixation and making it available to non-leguminous crops, better use of environmental resources - crop complementarity, reduction of spread of diseases, reduction of weeds)
A1: Thank you for your valuable suggestion. We have revised the sentence to incorporate your feedback.
“The potential mechanisms underlying its benefits include nitrogen fixation by hairy vetch, which makes nitrogen available to non-leguminous crops, improved utilization of envi-ronmental resources due to crop complementarity, reduced spread of diseases, and effec-tive suppression of weeds ” (Lines 50-53)
Q2: L96-102 From the answer I understand that soil samples were not taken before the start of each year of field research? The other questions were not addressed: What was the pre-crop in each year? Or was the field experiment conducted in the same area each year? Was inoculation of vetch plants used?
A2: Thank you for your thoughtful questions. The winter forage crops–forage maize double-cropping long-term field experiments were conducted in the same field and location each year. Winter forage crops were cultivated from October to April, followed by silage maize from May to July. Soil samples were collected during each growing season after the harvest of winter crops and maize. In this manuscript, we did not focus on the effects of intercropping on soil quality, and the soil nutrient data presented only reflect the initial soil fertility of the experimental field. If you are interested in soil quality changes under rapeseed–hairy vetch intercropping systems, more detailed information can be found in our previous publications (Wang et al., 2024a; Wang et al., 2024b).
Regarding inoculation, vetch plants were grown without inoculation (Lines 125-126).
“Hairy vetch plants were grown without inoculation.”
Wang, Z., Wang, C., Tan, X., Gao, G., El-Badri, A.M., Batool, M., Li, Z., Ai, X., Kuai, J., Wang, J., Xu, Z., Wang, B., Zhou, G., Zhao, J., 2024a. Diversified spatial configuration of rapeseed-vetch intercropping benefits soil quality, radiation utilization, and forage production in the Yangtze River Basin. Field Crops Res. 318, 109587.
Wang, Z., Wang, C., Tan, X., Lou, H., Wang, X., Shao, D., Ning, N., Kuai, J., Wang, J., Xu, Z., Wang, B., Zhou, G., Jiang, D., Zhao, J., 2024b. Developing diversified forage cropping systems for synergistically enhancing yield, economic benefits, and soil quality in the Yangtze River Basin. Agric. Ecosyst. Environ. 365, 108929.
Q3: The References section still needs to be corrected. Species names are not in italics. There are manuscript titles in which each word is capitalized. In addition, I suggest references to manuscripts from the last 10 years
A3: Thank you for your valuable suggestion. We have carefully updated the References section, ensuring that species names are italicized and manuscript titles are formatted correctly. Additionally, older references have been replaced with more recent ones, focusing on publications from the last 10 years.
Reviewer 2 Report
Comments and Suggestions for AuthorsDear authors,
I understand that the authors revised properly based on comments by reviewers, except for Line 230.
Line 230: Intercropping significantly increased the canopy height of hairy vetch compared to the sole cropping of the legume species.
Sincerely,
Author Response
Q1: I understand that the authors revised properly based on comments by reviewers, except for Line 230. Line 230: Intercropping significantly increased the canopy height of hairy vetch compared to the sole cropping of the legume species.
A1: Thank you for your thorough and insightful review. We have removed the statement, as it was not supported by observational data.
