The Effect and Potential Mechanism of Fulvic Acid on Flavonoids in Lemon Leaves
Round 1
Reviewer 1 Report
Comments and Suggestions for Authors- Why this study did not also involved the C. limon fruits?
- Please italicize C. limon throughout the manuscript
- Please clarify in section 2.2 what was irrigated: the C. lemon leaves or the soil.
- Line 142 should be section 2.3 over section 2.2
- Line 144 should be Table 3 over Table 2
- Lines 154, 158, 160, 164, 166 and 171 should be section 2.3…
- There are some errors throughout the manuscript in terms such as “sample” and “from”- lines 237, 244, 245, 301
- Title of Table 1 should be renamed to “primer sequences used in this study”. Also, the genes listed in Table 1 should be formatted in italics.
- Why did the authors not perform a Pearson correlation instead of OPLS-DA? Did their data exhibit a normal distribution? Which variables are correlated; the ELISA or the qPCR results? Please clarify this point in section 2.8.
- Please format Table 2
- Authors say: “The results demonstrated that, compared to the control group, FA treatment led to an increase in the vitexin-2 content at two collection points, and an increase in the isoorientin content at five collection points. Additionally, the contents of eriocitrin, vitexin, and rutin were consistently elevated across all six collection points. The content of narcissoside and diosmin increased at three collection points, while the effect on hesperidin content was found to be insignificant (Figure 3).”
However, I can not see the content of any flavonoid, the Figure 3 is too small. Please improve it.
- Title of Figure 4 should be renamed to “The activities of five enzymes involved in flavonoid biosynthesis in samples extracted from C. lemon leaves treated with FA and untreated, over a six-month period”
- Title of Figure 5. Idem. The term biosynthetase does not fit here.
- Authors say: “The biosynthesis of flavonoid compounds in plants is primarily mediated by two pathways: the phenylalanine pathway and the coumarin pathway. The phenylalanine pathway serves as the main route for flavonoid biosynthesis, with the majority of flavonoids and dihydroflavonoids being synthesized through this pathway. On the other hand, the phenylalanine metabolic pathway offers an alternate route for the biosynthesis of specific isoflavonoids and flavonol compounds [19,20].”
However, flavonoids are synthesized through the phenylpropanoid pathway, transforming phenylalanine into 4-coumaroyl-CoA, which finally enters the flavonoid biosynthesis pathway. Please see reference https://doi.org/10.3389/fpls.2012.00222
- Line 360 replace humic acid with fulvic acid
Author Response
Why this study did not also involved the C. limon fruits?
Response: Thank you for your comment. To assess the impact of fulvic acid (FA) on flavonoids in lemon, we planned to collect C. limon samples monthly based on the FA irrigation method. However, due to the growth of C. limon fruits, it is not feasible to collect them every month. Nonetheless, it is worth noting that the leaves of C. limon are rich in flavonoids and hold potential for industrial applications. Additionally, we acknowledge the necessity of evaluating the effect of FA on flavonoids in C. limon fruits, given its relevance to FA application and lemon cultivation. Consequently, we intend to conduct this experiment in our future studies.
Please italicize C. limon throughout the manuscript.
Response: Done as suggested.
Please clarify in section 2.2 what was irrigated: the C. lemon leaves or the soil.
Response: Thank you for your comment. Fulvic acid was applied to the soil of Citrus lemon trees through irrigation. We have clarified it in section 2.2.
Line 142 should be section 2.3 over section 2.2.
Response: Thank you for your comment. We have revised it.
Line 144 should be Table 3 over Table 2.
Response: Thank you for your comment. We have revised it.
Lines 154, 158, 160, 164, 166 and 171 should be section 2.3.
Response: Thank you for your comment. We have revised them.
There are some errors throughout the manuscript in terms such as “sample” and “from”- lines 237, 244, 245, 301
Response: Thank you for your comment. We are very sorry to make these mistakes. We have revised them in our revised manuscript.
Title of Table 1 should be renamed to “primer sequences used in this study”. Also, the genes listed in Table 1 should be formatted in italics.
Response: Thank you for your comment. We have revised them in our revised manuscript.
Why did the authors not perform a Pearson correlation instead of OPLS-DA? Did their data exhibit a normal distribution? Which variables are correlated; the ELISA or the qPCR results? Please clarify this point in section 2.8.
Response: Thank you for your comment. Firstly, OPLS-DA is a multivariate statistical method that can identify potential synergistic relationships among multiple variables and detect correlated patterns in the data through modeling. In contrast, Pearson correlation analysis can only detect linear relationships between two variables. Therefore, to explore complex associations among multiple variables (flavonoid contents, groups, synthetases), OPLS-DA is a more appropriate method. Secondly, OPLS-DA can be used for classification and predictive analysis, which is also beneficial for the objectives of our study and could provide some evidence of the harvesting period's impact on flavonoid content and biosynthetic enzyme activity.
Regarding the normal distribution of the data, we did not conduct tests for the normality of the data distribution. This is another reason we did not use Pearson correlation analysis. If the data deviate from a normal distribution, this may impact the suitability of using Pearson correlation analysis.
For the OPLS-DA analysis, the variables considered were flavonoid content detected by HPLC-DAD and biosynthetic enzyme activity determined by ELISA. In our study, we observed that not every treatment group subjected to different harvesting periods exhibited higher flavonoid content compared to the corresponding control group. Similarly, for the ELISA results, it was also observed that not every treatment group exhibited higher biosynthetic enzyme activity than the control group. However, based on the q-PCR results, every treatment group exhibited higher biosynthetic enzyme activity than the control group. Therefore, biosynthetic enzyme activity determined by ELISA was selected as one of the variables analyzed using OPLS-DA.
We have clarified this point in section 2.8
Please format Table 2.
Response: Done as suggested.
Authors say: “The results demonstrated that, compared to the control group, FA treatment led to an increase in the vitexin-2 content at two collection points, and an increase in the isoorientin content at five collection points. Additionally, the contents of eriocitrin, vitexin, and rutin were consistently elevated across all six collection points. The content of narcissoside and diosmin increased at three collection points, while the effect on hesperidin content was found to be insignificant (Figure 3).”
However, I can not see the content of any flavonoid, Figure 3 is too small. Please improve it.
Response: Thanks for your comments. We have added a supplementary table, Table S1, in the supporting materials. This table includes the content of each flavonoid measured in our study.
Title of Figure 4 should be renamed to “The activities of five enzymes involved in flavonoid biosynthesis in samples extracted from C. lemon leaves treated with FA and untreated, over a six-month period”
Response: Done as suggested.
Title of Figure 5. Idem. The term biosynthetase does not fit here.
Response: Thanks for your comments. We have renamed it to “The gene expression levels of five enzymes involved in flavonoid biosynthesis in samples extracted form C. limon leaves treated with FA and untreated, over a six-month period determined by q-PCR.”.
Authors say: “The biosynthesis of flavonoid compounds in plants is primarily mediated by two pathways: the phenylalanine pathway and the coumarin pathway. The phenylalanine pathway serves as the main route for flavonoid biosynthesis, with the majority of flavonoids and dihydroflavonoids being synthesized through this pathway. On the other hand, the phenylalanine metabolic pathway offers an alternate route for the biosynthesis of specific isoflavonoids and flavonol compounds [19,20].”
However, flavonoids are synthesized through the phenylpropanoid pathway, transforming phenylalanine into 4-coumaroyl-CoA, which finally enters the flavonoid biosynthesis pathway. Please see reference https://doi.org/10.3389/fpls.2012.00222
Response: Thank you for your comment. We apologize for the confusion here. Firstly, we would like to correct our previous statement. "The coumarin pathway" should be revised to "the cinnamic acid pathway." The cinnamic acid pathway serves as the route for biosynthesis of coumarin compounds. It initiates with the precursor molecule phenylalanine. This pathway also leads to the synthesis of flavonoid glycosides and isoflavonoid compounds. It is important to note that the biosynthesis of coumarin compounds in plants is primarily mediated by the phenylalanine pathway. We have revised it and deleted the “the coumarin pathway” in our revised manuscript.
Line 360 replace humic acid with fulvic acid.
Response: Thank you for your comment. We have revised it in our revised manuscript.
Reviewer 2 Report
Comments and Suggestions for AuthorsPapew is well written presenting interesting data of effect of fulvic acid on flavonoids in Lemon leaves.
Major remark: way selected 8 flavonoids where chosen? According other authors, e.g. https://doi.org/10.1016/j.foodchem.2006.01.032
predominated flavonoids in lemon peel are rutin (listed in paper) and , naringin, quercetin, naringenin and others.
Some minor remarks:
1. Remove CAS and catalog nr for standards, presented in 2.1 subchapter;
2. After MeOH extraction, authors wrote:
" After evaporation of the solvent, a total flavonoid extract was obtained. 0.2 g of the total flavonoid extract..."
According to Fig. 3, investigated flavonoids were <20 mg/100g so they were a minority in extract obtained. I suggest to re-name extract into "exctract containing polar compounds" instead of "flavonoid extract"
3. Move Table 3 to supplementary materials, as not crucial part of manuscript;
4. It's not clear to me, why authors used HPLC–MS. Quantification was based on HPLC as I understood;
5. Add to "equiped with G1315D-DAD" detector;
6. Move Fig. 2 to the supplementary data as not key for manuscript information
Author Response
Way selected 8 flavonoids where chosen? According other authors, e.g. https://doi.org/10.1016/j.foodchem.2006.01.032. predominated flavonoids in lemon peel are rutin (listed in paper) and naringin, quercetin, naringenin and others.
Response: Thank you for your comment. We greatly appreciate you providing the reference, which we have cited in the introduction section. The selection of the 8 flavonoid compounds that we detected was based on the results of HPLC-MS identification. These compounds were consistently found in every collected sample and exhibited variations in their content.
Remove CAS and catalog no. for standards, presented in 2.1 subchapter.
Response: Done as your suggestion.
After MeOH extraction, authors wrote:" After evaporation of the solvent, a total flavonoid extract was obtained. 0.2 g of the total flavonoid extract..." According to Fig. 3, investigated flavonoids were <20 mg/100g so they were a minority in extract obtained. I suggest to re-name extract into "exctract containing polar compounds" instead of "flavonoid extract".
Response: Thank you for your valuable comment. We have revised it to "exctract" instead of "flavonoid extract".
Move Table 3 to supplementary materials, as not crucial part of manuscript.
Response: Done as your suggestion.
It's not clear to me, why authors used HPLC–MS. Quantification was based on HPLC as I understood.
Response: Thank you for your comment. In our study, we first utilized HPLC-DAD/MS technology to identify the flavonoids present in the samples. Once the structures of the flavonoids were confirmed, we proceeded to quantify their contents in the samples using the HPLC-DAD method.
Add to "equiped with G1315D-DAD" detector.
Response: Done as your suggestion.
Move Fig. 2 to the supplementary data as not key for manuscript information
Response: Done as your suggestion.
Round 2
Reviewer 2 Report
Comments and Suggestions for AuthorsI agree with improvements performed by authors. I strongly recommend increasing quality of Figure 1 and re-formatting both scales to be more clearly visible.
