Stem Coloration in Alfalfa: Anthocyanin Accumulation Patterns and Nutrient Profiles of Red- and Green-Stemmed Variants

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript is clearly written. It provides insights into the anthocyanin profile of red- and green-stemmed Medicago sativa. It also examines how certain parameters (acid detergent fiber, crude fat, Cu, Fe, Zn) vary depending on the variety. My comments are given below:

Lines 13-14. “Seven anthocyanin glycosides, including Cyanidin, Peonidin, and Malvidin, were identified”. Cyanidin, peonidin, and malvidin are not glycosides. Additionally, their first letters should not be capitalized.

Line 15. “types of anthocyanins”. Could the authors clarify what they mean by “types”?

Lines 21-22. “while zinc and crude fat synergistically enhance anthocyanin accumulation”. This statement implies a causal relationship, although the study examined correlation. The same issue applies to the sentence in the conclusion section (lines 419-421).

Line 36. The authors should consider using the term “anthocyanidin” instead of “anthocyanins,” as “anthocyanins” is a broader term that includes both glycosylated and aglycone forms.

Line 174. Please verify whether the reported number of quantified anthocyanins and flavonoids is accurate.

Line 177. Please check the accuracy of the given numbers. Specifically, a relatively high mass of plant material (50 mg) was extracted with a relatively low volume of solvent (0.5 mL). Since plant material is typically voluminous, this extraction method seems impractical. Additionally, in the context of powdered plant material, I suggest using the term “extracted” rather than “dissolved.”

Could the authors provide more details on the process of identifying anthocyanins and flavonoids? Was the identification performed by comparison with reference compounds?

Lines 184-185. Which standards were used to prepare the “standard curve equations”?

Line 277. The manuscript refers to “quercetin 3-O-β-glucuronide,” whereas Supplementary Material 3 mentions “quercetin-3-O-glucoside.” Please clarify which of the two is correct.

Table 3. “Cyanidin” refers to a specific compound. Therefore, I suggest using the term “cyanidin glycosides” instead. The same applies to other classes, except for flavonoids and procyanidins.

Author Response

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Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Dear Authors,

First of all, I would like to thank you for the opportunity to participate in the review of the manuscript "Anthocyanin-Nutrient Interactions in Alfalfa: The Key Role of Cyanidin in Stem Coloration".

The manuscript deals with an important and interesting topic, the quantitative analysis of a group of flavonoid compounds present in relatively small amounts in alfalfa plants.

I suggest the following changes to the manuscript and have the following comments:

I propose a change in the title, where I suggest the omission of anthocyanin and nutrient interactions from the title. In my opinion, although a correlation between nutrient accumulation and anthocyanin synthesis can be demonstrated mathematically, such a correlation is not plausible from a physiological point of view. I would, of course, omit the relevant findings from the rest of the manuscript. The differences in nutrient accumulation capacity between the two types of alfalfa are not necessarily due to differences in anthocyanin content.

In the Abstract section, the abbreviation NDF should be spelt out as it is mentioned here for the first time.

In the Introduction section, I suggest citing publications that investigate the nutrient content of different alfalfa varieties. If there is data on red and green stem varieties, this would be particularly valuable.

In the Materials and methods section, the number of samples and replicates used in the studies should be given. The type and amount of growing medium, the size of the pots and the number of plants grown per pot should also be reported.

In the Results section, the size of Figures 1, 2, 3, 4 and 6 and the size of the captions within the figures should be increased for clarity.

The unit of measurement is missing in Table 3. It needs to be provided.

Author Response

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Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

Comments to the manuscript agronomy-3534344 "Anthocyanin-Nutrient Interactions in Alfalfa: The Key Role of Cyanidin in Stem Coloration".

Authors propose the report of an experiment aimed to compare the growing performance of two alfalfa genotypes characterized by red or green stem. The role of the anthocyanins in the higher nutritional quality of the red-stem genotype was also investigated, as well as the interactions of this kind of compounds with the fiber and minerals content of the biomass yielded.

The manuscript is generally well written, with a clear introduction containing a complete state of the art and a clear statement of the research objectives. The materials and methods are sufficiently well written, while the results presentation  needs some integrations. The discussion is a little synthetic and may be improveded by adding more citations and comparisons with other findings of the scientific literature.

The manuscript represents a good contribute to the field of study and may be published after some minor changes.

The most critical point of the results section is the readability of the Figures number 1, 2, 3, 4, and 6. The size of the figures should be at least doubled in order to increase the size of the characters associated. The quality of the pictures in the Figure 3 sould be also improved.

The discussion of the relative lower content of magnesium in the red-stem genotype should be made considering also the possibility to have a higher chlorophyll content in the green-stem type. 

Author Response

Please see the attachment.

Author Response File: Author Response.pdf