Rice plants reduce methane emissions in high-emitting paddies

Background: Rice is understood to enhance methane emissions from paddy fields in IPCC guidelines. However, rice actually has two opposite functions related to methane: i) emission enhancement, such as by providing emission pathways (aerenchyma) and methanogenetic substrates; and ii) emission suppression by providing oxygen pathways, which suppress methanogenesis or enhance methane oxidation. The overall role of rice is thus determined by the balance between its enhancing and suppressing functions. Although previous studies have suggested that rice enhances total methane emissions, we aimed to demonstrate in high-emitting paddy fields that the overall methane emission is decreased by rice plants. Methods: We compared methane emissions with and without rice plants in triple cropping rice paddy fields in the Mekong Delta, Vietnam. The gas samples are collected using chamber method and ware analyzed by gas chromatography. Results: We found that rice, in fact, suppressed overall methane emissions in high-emitting paddies. The emission reductions increased with the growth of rice to the maximum tillering stage, then decreased after the heading stage, and finally recovered. Discussion: Our result indicates that the overall methane emission is larger than that of rice planted area. In addition, although many studies in standard-emitting paddies have found that the contribution of soil organic matter to methanogenesis is small, prior studies in high-emitting paddies suggest that methanogenesis depended mainly on soil organic matter accumulated from past crops. The higher the methane emission level, the lower the contribution of the rice-derived substrate; conversely, the higher the contribution of the rice providing oxygen. Finally, rice plants reduce methane emissions in high-emitting paddies. Conclusion: The present study demonstrates that during the growing season, rice is suppressing methane emissions in high-emitting paddies. This means the significance of using the rice variety which has high suppressing performance in high-emitting paddies.


Amendments from Version 2 Introduction
The role of rice in methane (CH 4 ) emissions changes according to emission levels. Because, rice performs three key functions related to CH 4 emissions: i) providing a CH 4 pathway through a well-developed system of intercellular air spaces (aerenchyma), ii) providing a substrate for methanogenesis, and iii) oxidizing CH 4 in rhizosphere by supporting O 2 countertransport through aerenchyma system 1-6 . The level of contribution of these functions varies with the overall emissions, and the total amount of CH 4 emitted to the atmosphere is thus a balance between CH 4 production and oxidation 6 . To the best of our knowledge, rice enhances overall CH 4 emissions from paddy fields. In addition, previous studies have mostly disregarded a potential impact of overall emission levels on the role of rice in enhancing or suppressing CH 4 emissions.
Cicerone and Shetter measured CH 4 emissions with a closed chamber on the water surface of a paddy field, and found that 4 hours after starting measurement, CH 4 concentration was 290 ppm over rice plants and only 4 ppm over open water 1 . Further studies have revealed that CH 4 produced under methanogenesis diffuses through the soil, which is oxidized by the surface barrier before reaching the atmosphere 2,7 . Rice absorbs diffused CH 4 from its roots and emits CH 4 through aerenchyma 3,5,7 . These facts suggest that CH 4 is not emitted from the soil without rice. Other recent studies have provided additional evidence that the primary source of CH 4 is currentseason photosynthates-specifically, root exudates or decaying tissues [8][9][10][11] . This results in CH 4 emissions that peak during the late stage of rice growth. Thus, the presence of rice plants has been determined to be the cause of CH 4 emissions in paddy fields.
Wassmann et al. 12 measured CH 4 emissions on the water surface of a paddy field amended with organic matter. They found that organic matter incorporation increased total CH 4 emission levels from 27-90 to 160-240 kg CH 4 ha −1 crop −1 , and the direct emission from soil by ebullition increased from 15-23% to 35-62%, respectively 12 . Since it is known that organic matter incorporation causes CH 4 emissions to peak during the early stages of rice growth 12 , when the rice is still small and the aerenchyma is not well developed, the results of Wassmann et al. 12 should be closely examined to determine whether ebullition increased with total emissions. According to the 2006 Intergovernmental Panel on Climate Change (IPCC) guidelines, CH 4 emissions for 100 days of rice cropping are 130 kg CH 4 ha −1 crop −1 ; however, emissions were observed at almost twice this value by Wassmann et al. 12 Average CH 4 emissions from rice paddies in Asia without and with organic matter incorporation ranged from 16 to 200 and 250 to 500, respectively 13 . Although ebullition has been little studied 14 , we think ebullition must occur at the high emission levels. Furthermore, in the study by Wassmann et al. 12 , twin CH 4 emissions peaks appeared, with an early peak corresponding to the organic matter amendment and a later peak corresponding to rice-originated substrate 12 . An alternate interpretation of these results is that the twin peaks could reflect the oxidation performance of rice, since CH 4 oxidation is known to increase with rice growth, up to the maximum tillering stage, and then decrease 15 .
We monitored CH 4 emissions in the paddy fields for 5 years (total 15 crops) in triple rice cropping fields in the Mekong Delta, Vietnam. The CH 4 emission level was an order of magnitude higher than IPCC standards. These high CH 4 emissions suggest that ebullition must have been occurring. We hypothesized that rice plants decrease overall methane emission based on the extremely high emission levels. It will be epochal if present the fact that rice plants decrease overall methane emission in paddy fields; because, rice is believed enhancing overall CH 4 emissions from paddy fields.

Results and discussion
We compared CH 4 emissions with (Rice) and without rice (No Rice) on the water (soil) surface of triple cropping rice paddy fields in the Mekong Delta. Our results showed that rice presence decreased CH 4 emissions by half of No Rice. The effect of rice was large even in the early growth stage because of the high plant density (230 kg ha −1 in dry weight; approximately 3 cm interval) and the rapid growth in the tropical climate (see Figure 1, Figure 2). Although the high CH 4 emission is shown in the first week, it is considered to be caused by the erratic character of ebullition. In fact, there was no difference in the emission between Rice and No Rice on both the 7th and 9th day (see Figure 1). The difference of the total emissions between the treatments is significant (p=0.013; one-sided Welch's t-test). More importantly, the treatments have formed each group ( Figure 2). This supports the difference caused by not the locations but the treatments. The p-value is 0.013 if the difference made by location. Complete, unprocessed data are available on figshare 16 . There was no marked CH 4 emissions peak in the late-stage of the rice mentioned in previous studies [8][9][10][11] . This suggests that the amount of methanogenesis from the ricederived substrate is relatively small. Note the high emission levels the N 2 atmosphere technique, CH 4 oxidation ratios have been found to be around 40% on average and were relatively stable throughout the rice growing season 17 . However, genuine CH 4 oxidation, as measured using inhibitors, tend to decrease with rice growth, and the reduction rate for total CH 4 emissions can reach up to 20% 17 . Although, most of those studies assume plant-mediated transportation 17 , in general, our results roughly matched the results using the N 2 atmosphere technique. This suggests that the reduction in CH 4 emissions is not due to genuine oxidation and is more likely to be due to methanogenesis inhibition by oxygen from aerenchyma, which lasts until harvesting 18 .
We found high CH 4 emissions in unplanted paddy fields of which not incorporate organic materials. Despite this lower input of methanogenesis substrate, CH 4 emission levels were 12 times higher than the IPCC guidelines. The emission levels remained almost stable after reaching a maximum. This suggests that  (500-1400 kg CH 4 ha −1 crop −1 ). These findings suggest that total CH 4 emissions are reduced by oxidation or methanogenesis inhibition associated with growing the rice plant.
We also found that the reduction rate of CH 4 emissions increased with the growth of the rice plant. The CH 4 reduction rate was calculated using a moving average of five values by the following formula. reduction rate = (No Rice -Rice)/No Rice The rate peaked at maximum tillering stage, then bottomed at after heading stage, and then recovered (see Figure 3). The decrease around the heading stage was caused partially by an increase of emissions in rice-planted areas, and mainly by a decrease of emissions in the unplanted area.
No consensus has yet been reached on the extent to which methanotrophs or rice roots attenuate CH 4 emissions 17 . Using methanogenesis at our site mainly depends on soil organic matter that has been accumulated from past rice crops. Prior studies have suggested that the contribution of soil organic matter to methanogenesis is small; however, these studies also indicated that higher emission levels tend to be associated with higher contribution rates of soil organic matter [8][9][10][11]19 . Furthermore, a recent study found that large rice plants reduce CH 4 emissions compared to small rice plants in paddy fields with high soil C contents; instead, they show the opposite effect in paddies with low soil C contents 20 . Thus, our results are consistent with prior studies that assume that emission levels are proportional to the amount of soil organic matter which can be a methanogenesis substrate.
High-emitting paddies of CH 4 emissions, which exist widely across tropical Asia 13 , would have substantial soil organic matter stock formed by sequential rice cropping under flooded conditions 21 . For instance, the use of a rice variety which has better performance of methanogenesis inhibition in high-emitting paddies is very effective; the 10 % reduction is equivalent to 100% of methane emission in standard paddy fields. On the other hand, without rice plants, the methane emission from the existing soil organic matter stock will double by ebullition; the 100% increase is equivalent to 1000% of the standard paddies. This suggests that the future study for the soil organic matter stock map is critical.

Conclusion
To our knowledge, most studies of CH 4 emissions in paddy fields have been conducted in fields with low overall emission levels. Since the role of rice in CH 4 emissions varies according to the overall emission levels, these results cannot be appropriately generalized to rice paddies with high emission levels. The results of our study suggest that rice reduces emissions in high-emitting paddies. This means the significance of using the rice variety which has high suppressing performance in high-emitting paddies. The emission levels are related to the amount of soil organic matter which can be a methanogenesis substrate; this suggests that the future study for the soil organic matter stock map is critical.

Study site
Experimental fields were in Tan Loi 2 Hamlet, Thuan Hung village, Thot Not district, Can Tho city, Vietnam. Farmers conduct triple rice cropping by direct seeding and full flooding. This district receives almost 2 months of a flood annually from the Mekong River. The flood decomposes rice straw underwater to the extent that it is no obstacle for seeding. Therefore, farmers start the rice cropping by leveling the fields, without incorporating rice straw. We observed CH 4 emissions in three paddy fields (26 × 17 m each) under conventional conditions for 5 years from September 2011. A preliminary study was conducted with the rice variety OM501 (suitable for the season) in the Summer-Autumn season of 2016 by the same methods and paddies of the present study (see Figure 4). In the present study, we used the three fields for replication. We conducted the present study after the annual flood (4 November 2016-12 February 2017); these fields did not incorporate rice straw because of the period was after the annual flood.

Treatment
We compared CH 4 emissions with (Rice) and without rice (No Rice). We set 2 × 2 m squares of plastic films on a part of each three fields just before seeding, then carefully removed them with seeds on the films immediately after seeding. In other points, there was no difference with farmers' conventional rice-growing procedures. Farmers scattered 230 kg ha −1 (in dry weight) of germinated rice seed (variety Jasmine) on drained wet paddy fields' surfaces on 5th November. This wet condition was maintained for 7 days, then the fields were kept flooded until 89 days after seeding (DAS), and the rice harvested on 100 DAS. The farmers applied fertilizer, which included 76 kg of urea on 12 November, 53 kg each of urea and NPKS (16-16-8-13), diammonium phosphate on 19 November, and 53 kg each of urea and NPKS on 15 December. The daily average water levels were monitored with water level loggers (HOBO U20; Onset Computer Corporation, Bourne, Massachusetts) at the corner of the fields, and the average levels were 2.0 cm (−0.6 to 6.1 cm) until drained.

Measurement of CH 4 emissions
We set an approximately 2 m long and 0.5 m wide ladders from the center of the shorter bund to allow measurement of CH 4 without touching the paddy soil surface. Those ladders were on the borders of the non-planted areas in each field. We set PVC chamber bases on the paddy fields of both sides of the ladders to avoid measurement perturbation. Chambers (60 × 80 cm and 100 cm high, transparent acryl) were set on a watertight chamber bases for every measurement. Measurements were taken at 8 a.m. because previous research has indicated that emissions at this time have a high correlation (ca. 90% of average emission) with average daily emissions 22 . We mixed the air in the chamber with a fan for 5 min after setting the chamber, then sampled the first gas, then sampled the second gas 20 min later. We conducted the measurements once a week throughout the rice growing stage, but every 3 days for 2 weeks after seeding, heading stage, and around draining. The samples were analyzed by gas chromatography (GC-14B, Shimazu, Kyoto). The cumulative CH 4 emissions were calculated by linear interpolation.

Ethics statement
This study was conducted with the approval of the farmer.

Statistical analysis
Data were processed using Microsoft Excel 2016.

Data availability
Raw data of this article is available from figshare: https://doi.org/10.6084/m9.figshare.6916277.v1 16 . Data are available under the terms of the Creative Commons Zero "No rights reserved" data waiver (CC0 1.0 Public domain dedication).

Grant information
The author(s) declared that no grants were involved in supporting this work. 2.

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.
Version 2 09 July 2019 Reviewer Report https://doi.org/10.5256/f1000research.21593.r50479 © 2019 Tran D. This is an open access peer review report distributed under the terms of the Creative Commons , which permits unrestricted use, distribution, and reproduction in any medium, provided the original Attribution Licence work is properly cited.

Center of Water Management and Climate Change (WACC), Vietnam National University -Ho Chi Minh City (VNU -HCM), Ho Chi Minh City, Vietnam
The manuscript has been improved based on the comments. However, I recommended the authors to consider the following 2 points before approval for indexing: The authors replied: "We corrected the method section and clarified the details. For the site location map, we think it can be found on the internet nowadays.". I do not agree, please add the Figure because the common audience doesn't have much time to find the location.
Conclusion is not quite sufficient in terms of relating to previous findings.
No competing interests were disclosed. Competing Interests:

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.
Author Response 22 Jul 2019 , Japan International Research Center for Agricultural Sciences, Tsukuba, Japan

Masato Oda
Thank you for the comment.

For the site local map:
We added the link to the Google map according to the article guidelines of F1000Research. The link takes no time for the reader of F1000Research, in addition, it's scalable.

Conclusion:
We added a conclusion which was presented in the abstract but not presented in Conclusion sections. The aim of the present study is the role of rice in the paddy field for methane emission in the historical context. Therefore, we mentioned it in the first sentence of the abstract as "Rice is understood to enhance methane emissions from paddy fields", then concluded it in the Conclusion the historical context. Therefore, we mentioned it in the first sentence of the abstract as "Rice is understood to enhance methane emissions from paddy fields", then concluded it in the Conclusion as "To our knowledge, most studies of CH 4 emissions in paddy fields have been conducted in fields with low overall emission levels. Since the role of rice in CH 4 emission varies according to the overall emission levels, these results cannot be appropriately generalized to rice paddies with high emission levels." No competing interests were disclosed. Competing Interests: 05  Thank you for reconsidering our manuscript. The methodology is described in the first sentence of the 'Results and discussion': "We compared CH emissions with (Rice) and without rice (No Rice) on the water (soil) surface of triple cropping rice paddy fields in the Mekong Delta". Our treatment is only "with" or "without" rice, the other condition is the same as farmers' conventional. See the second sentence in the 'treatment' of 'Methodology' section: "We set 2 × 2 m squares of plastic films on a part of each three fields just before seeding, then carefully removed them with seeds on the films immediately after seeding. In other points, there was no difference in farmers' conventional rice-growing procedures." Other responses are as follows:

Water management:
The water management is full flooding. See the second sentence of the 'Methods' section: "Farmers conduct triple rice cropping by direct seeding and full flooding."

Justification of the first-week emission:
4

Justification of the first-week emission:
See the third sentence of the 'Results and discussion': "Although the high CH emission is shown in the first week, it is considered to be caused by the erratic character of ebullition. In fact, there was no difference in the emission between Rice and No Rice on both the 7th and 9th day (see Figure 1)." Furthermore, we added the following sentence: "The difference of the total emissions between the treatments is significant (p=0.013; one-sided Welch's t-test). More importantly, the treatments have formed each group (Figure 2). This supports the difference caused by not the locations but the treatments. The p-value is 0.013 if the difference made by location." In addition, supplemental figure 4 is the supporting data.
Page 3, top of right column: "Since it is known that organic matter incorporation causes CH4 emissions to peak during the early stages of rice growth". Reference needed.
Page 3, right column: "Although ebullition has been little studied, ebullition must occur at high emission levels." Why? Please explain and provide references.
First sentence of the conclusion: "Most studies of CH4 emissions in paddy fields have been conducted in fields with low overall emission levels." Again, no reference provided. A reference to a review article showing that this is true would help.
The text is unclear in several places. For instance: Abstract: "our results in high-emitting paddies suggest that methanogenesis depended mainly on soil organic matter accumulated from past crops." How did you come to this conclusion?
Abstract: "The higher the methane emission level, the lower the contribution of the rice-derived substrate". How does this interpretation follow from your own results?
Last sentence of introduction: "It will be epochal if present the fact that rice plants decrease overall methane emission in paddy fields." The meaning of this sentence is unclear.
Page 4, left column, top paragraph: "The decrease around the heading stage was caused partially by an increase of emissions in rice-planted areas, and mainly by the erratic emissions in the unplanted area." If this decrease is erratic, then how can we be sure that the high emissions earlier in the growing season aren't erratic as well? How can we be sure that high emissions before planting in the unplanted plots are not erratic?
Page 5, top of right column: "We observed CH4 emissions in 18 paddy fields (26 × 17 m each) under several conditions for 5 years from September 2011." Where are these data reported? If you don't report them, then why mention this? The earliest data that are reported in this manuscript are from 2016.
Page 5, top of right column: "In the present study, we used three fields for replication." Are these fields the same fields that were described above (i.e. 26*17 m in size)? This is not clear.
The article still contains quite a few grammatical errors or poor structure. A few suggestions: Abstract, methods section: Delete "of" to write "methane emissions with and without rice plants".
Last sentence of intro: "We hypothesised that rice plants decrease overall methane emission based on the extremely high emission levels." First sentence of the results section would be a good place to mention study location.
2nd sentence of results: "Our results showed that rice presence decreased CH4 emissions by 2nd sentence of results: "Our results showed that rice presence decreased CH4 emissions by half." Page 4, right column: "This suggests that methanogenesis at our site mainly depends on soil organic matter that has been accumulated from past rice crops." Last paragraph of results section: Change "huge" to "large", or "substantial", and provide references to support this statement.
No competing interests were disclosed. Competing Interests: Reviewer Expertise: Agronomy, biogeochemistry, greenhouse gas fluxes.
I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.
Author Response 22 Jul 2019 , Japan International Research Center for Agricultural Sciences, Tsukuba, Japan

Masato Oda
Thank you very much for your kind suggestion. Furthermore, thank you so much for correcting and improving the English. We completed the lack of references.
We revised the manuscript for the other points as follows:

Page 3: The explanation of "ebullition".
We added the words "the direct emission from soil by".

Introduction: The reason of epochal.
We added as follows: "; because, rice is believed enhancing overall CH emissions from paddy fields".

Abstract: The reason of the methanogenesis source.
We correct as follows: "our results" to "prior studies".

Abstract: "The higher…How does this interpretation...?"
Thank you for leading. We changed the last part as follows: "; conversely, the higher the contribution of the rice providing oxygen. Finally, rice plants reduce methane emissions in high-emitting paddies." 5. Results, line 9: Why is "ebullition" between quotation marks? We deleted.

Page 4, left column, top paragraph: The explanation of erratic.
We changed to "a decrease of".

Page 5, top of right column: The field size.
It is the same size because of the same fields. We think it is obvious by the above correction for the number of fields.

To mention the study location in the results section.
No competing interests were disclosed. The authors of "Rice cultivation reduces methane emissions in high-emitting paddies" present an interesting manuscript with great effort to demonstrate the balance between rice's emitting and suppressing function changes according to overall methane emission levels. In general, the manuscript is well-written. However, I recommend the authors to do a major revision to the manuscript before being potentially accepted for indexing by the Journal.
The Introduction section should add research objectives or in terms of research questions that help readers to promptly understand the paper's content.
In the Methods section, description and activities are needed in more detail; additionally a map of the case study would be useful.
In the Results and Discussion section, I expected more discussion that enhance more work on comparing findings with previous studies. In addition, the limitations and future outlooks need to be elaborated upon.
A Conclusion is important to add into the manuscript since it presents the real implication of the research based on the authors' perspective.

Is the study design appropriate and is the work technically sound? Partly
Are sufficient details of methods and analysis provided to allow replication by others? 1.

Is the study design appropriate and is the work technically sound? Yes
Are sufficient details of methods and analysis provided to allow replication by others? Yes

If applicable, is the statistical analysis and its interpretation appropriate? No
Are all the source data underlying the results available to ensure full reproducibility? Yes

Are the conclusions drawn adequately supported by the results? Partly
No competing interests were disclosed.
I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.
Author Response 14 Jun 2019 , Japan International Research Center for Agricultural Sciences, Tsukuba, Japan

Masato Oda
Thank you for your precious suggestion! We changed the word "cultivation" to "plants" in the title. We think the word is more suitable to the content. Thank you again. We aim to demonstrate rice plants are suppressing overall methane emission in high CH4 emission paddy fields. We changed the last sentence of the background as follows. "that the balance..." to "that the overall methane emission is decreased by rice plants." We expect that this change clarifies some other points that you indicate. In association with, we think the experiment in the low background is not necessary because they have been conducted by plenty of studies. We think it's obvious from Figure 1 and 2; for your information, the p-value by one-sided Welch's t-test for the total emissions is 0.013. Thank you very much for introducing the study to support our conclusion. We referred to that in the last paragraph of the discussion. We have referred to rather old-dated studies because we aim to mention the role of rice in the context of the history of the study for paddy fields CH4 emission. The sentence indicates common sense; therefore, we would like to change as follows: "Rice is understood to enhance methane emissions from paddy fields as mentioned in IPCC guidelines." We would like to change the sentence ", all studies ... these studies". to "rice enhances overall CH4 emissions from paddy fields. In addition, previous studies". We added the formula, and we clarified the sentences in the results section. 6. 7. 8.

9.
overall CH4 emissions from paddy fields. In addition, previous studies". We added the formula, and we clarified the sentences in the results section. We think the soil carbon contents is important; however, that is out of range of this study and we don't have the map. Thank you so much! We had used an English editing service, but that was not enough. We have revised the English.
No competing interests were disclosed. before starting on any article revisions.
No competing interests were disclosed.

Competing Interests:
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