Brassinosteroids Benefit Plants Performance by Augmenting Arbuscular Mycorrhizal Symbiosis

ABSTRACT Arbuscular mycorrhizal (AM) play an important role in improving plant growth and development. The interaction between phytohormones and AM symbiosis is gradually revealed. Here we examined the effect of Brassinosteroids (BR) on AM symbiosis and discussed the synergistic promotion of plant growth by BR and AM symbiosis. The xylophyta Eucalyptus grandis Hill (E. grandis) was inoculated with AM fungi Rhizoglomus irregularis R197198 (R. irregularis) and treated with different concentrations (0, 1, 10, and 100 nM) of 24-epibrassinolide (24-epiBL) for 6 weeks. With the increase of 24-epiBL concentration, E. grandis growth was firstly promoted and then inhibited, but inoculation with AM fungi alleviated this inhibition. 24-epiBL and R. irregularis colonization significantly improved E. grandis growth and antioxidant system response, and the synergistic effect was the best. Compared with the control group, 24-epiBL treatment significantly increased the mycorrhizal colonization and arbuscular abundance of AM fungi R. irregular in E. grandis roots. The expression of AM symbiosis maker genes was significantly increased by 24-epiBL treatment. Both 24-epiBL treatment and AM colonization upregulated gibberellins (GA) synthesis genes, but no inhibition caused by GA levels was found. 24-epiBL is a kind of synthetic highly active BR. Based on the results of 24-epiBL treatment, we hypothesized that BR actively regulates AM symbiosis regulates AM symbiosis without affecting GA-INSENSITIVE DWARF1 (GID1)-DELLA expression. The synergistic treatment of BR and AM symbiosis can significantly promote the growth and development of plants. IMPORTANCE Brassinosteroids (BR) and Arbuscular mycorrhizas (AM) symbiosis play an important role in improving plant growth and development. Previous studies have shown that there is a complex regulatory network between phytohormones and AM symbiosis. However, the interactions of BR-signaling and AM symbiosis are still poorly understood. Our results suggest that BR actively regulates the colonization and development of AM fungi, and AM fungal colonization can alleviate the inhibition of plant growth caused by excessive BR. In addition, BR actively regulates AM symbiosis, but does not primarily mediate gibberellins-DELLA interaction. The synergistic treatment of BR and AM symbiosis can significantly promote the growth and development of plants. The conclusions of this study provide a reference for phytohormones–AM symbiosis interaction.


Revise
Line 109 More details are needed; sample preparation? Sample volumes? Line 118 Primer sequences can be moved to supplementary material Figure 4J -I believe this figure was not refereed to in the text Reviewer #2 (Comments for the Author): 1. The authors lead some conclusions depending on the date that are not statistically significant. For example, the title says "Brassinosteroids promote plant performance by augmenting arbuscular mycorrhizal"; however, there is no statistically significant differences in the fresh weight of roots, the taproot length, and the plant height between AM-inoculated and uninoculated plants treated with the same concentration of 24-epiBL ( Fig. 1A, C, D). Although the fresh weight of shoots of AMinoculated plants was increased compared to uninoculated plants only when 100 nM of 24-epiBL was treated, it was not statistically significantly increased when 10 nM of 24-epiBL was treated, which induced the same level of AM colonization as 100 nM of 24-epiBL treatment (Fig. 1B, Fig. 2). Similarly, many discussions about Figs.1, 3, and 4 were based on statistically nonsignificant data. 2. The authors argue that "both BR and AM fungi colonization can promote the response of the plant's antioxidant system and alleviate oxidative stress, and the combination of the two treatments has the best effect" (lines 177-170), based on the results that AM inoculation and BL treatment increased the antioxidant enzyme activities (Fig. 3). However, it is possible that these enzymes were activated as a result of increased ROS levels by AM inoculation and BL treatment. Therefore, it is unclear whether AM inoculation and BL treatment are good for plants to prevent oxidative stress. 3. The authors argue that "BR mainly regulates AM symbiosis through GA-independent pathways" (lines 242), based on the results that BL treatment did not affect the expression levels of DELLA and GID1 (Fig. 3). However, it is possible that BL regulates AM symbiosis through GA related factors other than the expression levels of DELLA and GID1. To conclude the GAindependent BL functions, other experiments such as BL treatment on GA-related mutants are required.

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Dear editors and reviewers,
Thank you for your thorough review of our work and we are very grateful for you input. We have revised our manuscript thoroughly based on your comments. Details are as follows: Reviewer #1 Comment 1: Line 25, Please write 24-epiBL also in full.
Comment 2: Line 32-33, Authors should not generalize findings for a brassinosteroid to all brassinosteroids.
Response 2: Thanks for the reviewer's kind suggestion. As a kind of synthetic BR analogue, 24-epiBL is feasible to explore the physiological effects of BR in plants. However, there is no denying that its activity is not exactly the same as the various BRs found in plants. Your suggestion is very good, so we have revised our expressions in the text. I can revise it again if you think there is a more accurate statement. The revised details are as follows: Line 32-34, 24-epiBL is a kind of synthetic highly active BR. Based on the results of 24-epiBL treatment, we hypothesized that BR actively regulates AM symbiosis regulates AM symbiosis without affecting GID1-DELLA expression.
Comment 3: Line 53, Please revise; this section is in the past tense.
Response 3: Thanks for the reviewer's kind suggestion. We have corrected "After the AM fungal spores germinated in the soil, the hyphae continuously elongated and branched in search of a host" to "After the AM fungal spores germinate in the soil, the hyphae continuously elongate and branch in search of a host" according your comment. The revised details can be found in Line 53-54.
Comment 4: Line 88, Material and method -> Materials and methods.
Response 4: Thanks for the reviewer's kind suggestion. We have corrected "Material and method" to "Materials and methods" according your comment. The revised details can be found in Line 89.

Comment 5: Line 105, Revise
Response 5: Thanks for the reviewer's kind suggestion. According to his/her advices, we have added the operation details of the root pre-processing and rewritten the entire paragraph. The revised details are as follows: Line 107-113, Part of the fresh roots of each AM plant were randomly selected and immersed in 10% KOH (w /v) at 90°C for 8 h (replaced with fresh KOH solution every 2 h), neutralized with 2% HCL (w /v) for 20 min, washed 3 times with sterile water, and then stained with 5 mM wheat germ agglutinin Alexa Response 7: Thanks for the reviewer's kind suggestion. According to his/her advices, we have changed the primer sequence in the main manuscript to supplementary material and uploaded it. The revised details can be found in Line 127-128.
Comment 8: Figure 4J -I believe this figure was not refereed to in the text.
Response 8: Thanks for the reviewer's kind suggestion. " Fig. 4G, H&I" in the text actually refers to " Fig.   4H, I&J" marked on the image respectively, and we have corrected the numbers on the image. The details related to it can be found in Line208, and Line434.
Reviewer #2 : Comment 1: The authors lead some conclusions depending on the date that are not statistically significant.
For example, the title says "Brassinosteroids promote plant performance by augmenting arbuscular mycorrhizal"; however, there is no statistically significant differences in the fresh weight of roots, the taproot length, and the plant height between AM-inoculated and uninoculated plants treated with the same concentration of 24-epiBL (Fig. 1A, C, D). Although the fresh weight of shoots of AM-inoculated plants was increased compared to uninoculated plants only when 100 nM of 24-epiBL was treated, it was not statistically significantly increased when 10 nM of 24-epiBL was treated, which induced the same level of AM colonization as 100 nM of 24-epiBL treatment (Fig. 1B, Fig. 2). Similarly, many discussions about Figs.1, 3, and 4 were based on statistically non-significant data.
Response 1: Thanks for the reviewer's kind suggestion. As stated in the comments, the differences between AM plants, different concentrations 24-epiBL plants and the control group are not all statistically significant. However, AM plants and BR-treated plants performed better than the NM group. Even if they are not statistically significant, it can also indicate that AM and BR have an insignificant promoting effect on plant growth. Therefore, according to the suggestion, we have revised the title to more accurately reflect the results of our experiment. If there is a more accurate statement, we can modify it again. The revised details are as follows: Line 1, Brassinosteroids benefit plants performance by augmenting arbuscular mycorrhizal symbiosis Comment 2: The authors argue that "both BR and AM fungi colonization can promote the response of the plant's antioxidant system and alleviate oxidative stress, and the combination of the two treatments has the best effect" (lines 177-170), based on the results that AM inoculation and BL treatment increased the antioxidant enzyme activities (Fig. 3). However, it is possible that these enzymes were activated as a result of increased ROS levels by AM inoculation and BL treatment. Therefore, it is unclear whether AM inoculation and BL treatment are good for plants to prevent oxidative stress. Line 177-180, On the contrary, the contents of MDA, H 2 O 2 , and OFR decreased with the increase of 24-epiBL concentration (Fig. 3 D, E&F). The performance of antioxidant enzyme (CAT, SOD and POD) activity in AM E. grandis plants roots was higher than that of NM plants (Fig. 3 A, B&C). The contents of MDA, H 2 O 2 and OFR in AM roots were lower (Fig. 3 D, E&F). irregularis roots after 6 weeks of treatment (R. irregularis colonization and 24-epiBL application).
Comment 3: The authors argue that "BR mainly regulates AM symbiosis through GA-independent pathways" (lines 242), based on the results that BL treatment did not affect the expression levels of DELLA and GID1 (Fig. 3). However, it is possible that BL regulates AM symbiosis through GA related factors other than the expression levels of DELLA and GID1. To conclude the GA-independent BL functions, other experiments such as BL treatment on GA-related mutants are required.
Response 3: Thanks for the reviewer's kind suggestion. According to his/her advices, we have revised our presentation to reflect our results more clearly. In addition, it is our further research content to explore whether BR-signaling influences AM symbiosis to mediate GA pathway. This suggestion is of great importance to us. We will refine our next step by BL treatment on GA-related mutants. The revised details are as follows: Line 185, BR promotes the expression of AM symbiotic marker genes Line 245, BR regulates AM symbiosis without affecting GID1-DELLA expression Line 285-288, Taken together, we hypothesized that BR promotes AM symbiosis not by regulating the expression of GID1and DELLA. Whether BR mediates GA-signaling pathway to affect AM symbiosis needs to be further explored by relying on GA-related mutants.
All the changes we make in the manuscript are highlighted by coloured text.
If there are any further issues, please don't hesitate to tell us. Thank you for submitting your manuscript to Microbiology Spectrum. As you will see your paper is very close to acceptance. Thank you for carefully considering the comments of the reviewers and providing additional information. In my assessment of your responses there is just one more point that I would like to see addressed: similar to the points of reviewer #2, I think it is important to make clear that the effect of BR+AM on plant performance in general was quite small although the effect of BR on AM colonization was much more significant. As this was an experimental condition, it is possible that the impact on plant performance would be greater under other environmental conditions and pressures, but that would be an area for additional work. If you could address this point specifically in the conclusions I think it would be beneficial for those who are interested in this area of research and want to build on your results.
Please modify the manuscript along the lines I have recommended. As these revisions are quite minor, I expect that you should be able to turn in the revised paper in less than 30 days, if not sooner.
When submitting the revised version of your paper, please provide (1) point-by-point responses to the issues I raised in your cover letter, and (2) a PDF file that indicates the changes from the original submission (by highlighting or underlining the changes) as file type "Marked Up Manuscript -For Review Only". Please use this link to submit your revised manuscript. Detailed information on submitting your revised paper are below.

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Preparing Revision Guidelines
To submit your modified manuscript, log onto the eJP submission site at https://spectrum.msubmit.net/cgi-bin/main.plex. Go to Author Tasks and click the appropriate manuscript title to begin the revision process. The information that you entered when you first submitted the paper will be displayed. Please update the information as necessary. Here are a few examples of required updates that authors must address: • point-by-point responses to the issues I raised in your cover letter • Upload a compare copy of the manuscript (without figures) as a "Marked-Up Manuscript" file. • Each figure must be uploaded as a separate file, and any multipanel figures must be assembled into one file. Line 296-300, And the synergistic treatment of BR and AM colonization has quite a small effect on plant performance, although the effect of BR on AM colonization was much more significant. Moreover, it is possible that their effects on plant performance may be greater under other environmental conditions and stresses, but additional studies are needed to prove this.
All the changes we make in the manuscript are highlighted by coloured text.
If there are any further issues, please don't hesitate to tell us.
Best regards! Yours Ming Tang Your manuscript has been accepted, and I am forwarding it to the ASM Journals Department for publication. You will be notified when your proofs are ready to be viewed.
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