Review Reports

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The Manuscript entitled ; Amino Acids as Multifunctional Molecules in Plants: From 2 Fundamental Metabolism to Precision Agriculture; provides sufficient information about amino acids and its biological functions. The current form of English need further improvement. Please check the citations throughout manuscript. Kindly revise accordingly to further improve the manuscript (PDF). Best wishes 

Comments for author File: Comments.pdf

Comments on the Quality of English Language

Kindly thoroughly revise whole manuscript to avoid grammatical mistakes 

Author Response

Comment：The Manuscript entitled ; Amino Acids as Multifunctional Molecules in Plants: From 2 Fundamental Metabolism to Precision Agriculture; provides sufficient information about amino acids and its biological functions. The current form of English need further improvement. Please check the citations throughout manuscript. Kindly revise accordingly to further improve the manuscript (PDF). Best wishes 

Response: We sincerely thank you for your valuable suggestions and the meticulous annotations provided in the PDF. These have greatly contributed to improving the quality of our manuscript. We have substantially revised the manuscript by incorporating the comments from both Reviewer 2 and Reviewer 3. In the revised version, we have made every effort to ensure that all assertions are supported by appropriate literature citations and have carefully checked the reference formatting throughout. All sections have been revised for clarity, grammar, spelling, and punctuation. In addition, we have corrected minor typographical errors and ensured consistent use of terminology throughout the manuscript. We believe the revised manuscript now meets the required standard of written English. We trust that these revisions fully address the reviewer's concerns. We are grateful for the time and expertise invested in evaluating our work, which has undoubtedly strengthened the manuscript.

                             

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

Plants April 2026

Amino Acids as Multifunctional Molecules in Plants: From Fundamental Metabolism to Precision Agriculture

 

The manuscript (plants: 4276775) has been reviewed. Some suggestions and queries for improvement are discussed.

The article provides insights into the multifunctional roles of amino acids in plants, beyond being “building blocks”, discussing the integral importance as key molecules in stress tolerance, nitrogen transport, and signaling molecules in plants. While researchers have largely focused on understanding the structural roles of amino acids in plants, other important yet less explored functions remain.

 The topic is of key scientific interest, and understanding the less-studied yet important functions of amino acids in plants would open new avenues across multiple fields, including agriculture.

 

Abstract, line 7-8, Amino acids are often…………………they do much more.

This sentence does not seem appropriate.

Amino acids are considered key building components in plant cells; therefore, they serve important functions. Please revise the sentence to begin with the structural roles of amino acids in plants and then discuss less-studied functions and their socio-economic importance.

 

 

Line 17-19, Understanding this hidden side of amino acids……………………..nano-based delivery systems.

This part talks about how the knowledge of key functions of amino acids would define interesting innovations in cultivating climate-resilient crops. It is important to briefly discuss 1-2 examples (case studies), challenges faced, and prioritize actionable goals.

Line 19-21: In short, amino acids are not just ……………..cope with stress.

How, discuss briefly with future implications of the study.

 

 

Line 75-79: Through a critical examination……………nutritional quality.

No information on how the literature review was carried out is mentioned anywhere in the text. The rationale and objective of the paper, the time period of the article, the literature selection criterion, the databases, the websites referred to, inclusion and exclusion basis of literature. It is necessary to briefly discuss how the study was performed and the factors considered for the study.

 

Figure 3. Translating amino acid research into sustainable agricultural practices.

Discuss the development of amino-acid-based biostimulants for the generation of stress-tolerant crops. What is the current progress/developments in this direction? Are there any commercially marketed formulations currently on the market? If yes, give examples.

 

Some minor corrections:

Please be consistent with the use of scientific names for biological species. Line 225, Arabidopsis can be either in normal font or italicized, likewise in the entire manuscript.

 

Moderate English revisions are required for sentence clarity, spelling, and punctuation.

 

Gene nomenclature: the complete name of the gene is usually written in normal font, while the abbreviation is written in italics.

 

The literature review includes a lot of information on the multiple structural and functional roles of different amino acids in plants. It is suggested to provide some information in a figure representation rather than an elaborate literature discussion.

 

Comments on the Quality of English Language

Moderate English revisions are required.

Author Response

We sincerely thank the reviewer for the careful evaluation and constructive suggestions. We have thoroughly revised the manuscript to address all points raised. Below, we provide a point-by-point response, indicating precisely where each change has been made (using line numbers from the revised manuscript) to facilitate the reviewer’s re-assessment.

Comment 1:“Abstract, line 7-8, Amino acids are often…………………they do much more. This sentence does not seem appropriate. Amino acids are considered key building components in plant cells; therefore, they serve important functions. Please revise the sentence to begin with the structural roles of amino acids in plants and then discuss less-studied functions and their socio-economic importance.”

Response:We fully agree. The Abstract’s opening has been rewritten to first highlight the fundamental structural role of amino acids as protein building blocks before moving to their other biological functions. The revised first sentence now reads (see Lines 11–12 of the revised Abstract):
“Amino acids are organic compounds that serve as the fundamental building blocks of proteins and are additionally responsible for a multitude of other biological functions.”
The socio‑economic importance is addressed later in the Abstract and is extensively discussed in Section 3.3.2, where we detail commercial biostimulants (e.g., Terra‑Sorb®, HICURE®) and their field‑proven benefits for stress‑tolerant crop production.

Comment 2:“Line 17-19, Understanding this hidden side of amino acids……………………..nano-based delivery systems. This part talks about how the knowledge of key functions of amino acids would define interesting innovations in cultivating climate-resilient crops. It is important to briefly discuss 1-2 examples (case studies), challenges faced, and prioritize actionable goals.“

Response:We appreciate this practical suggestion. We have now incorporated the requested elements into both the Abstract and the main text. In the Abstract (Lines 22–25), we added a concise statement of challenges and goals:
“We discussed how these insights could lead to smarter biostimulants, gene‑edited crops with better nutrient efficiency, and nano‑based delivery systems, although challenges in standardization and effective field translation remain.”
More substantively, in Section 3.3.2 (Lines 938–947), we substantially expanded the discussion of biostimulants. We provide two commercial case studies—Terra‑Sorb® (an enzymatic hydrolysate containing free L‑α‑amino acids, marketed in >50 countries) and HICURE® (a highly concentrated 19‑amino‑acid formulation)—and cite recent field trials demonstrating yield improvements under drought and salinity in creeping bentgrass, rice, durum wheat, and soybean. We then explicitly identify key challenges (high production cost, lack of standardization, inconsistent field efficacy) and prioritize actionable goals: cost‑effective production, smart nano‑delivery systems, and crop‑specific, phenology‑adjusted formulations.

Comment 3:“Line 19-21: In short, amino acids are not just ……………..cope with stress. How, discuss briefly with future implications of the study.”

Response:Thank you for this point. The broader future implications are elaborated in the Conclusions (Section 3.3)(Lines 916–984), where we discuss translational potential for climate‑resilient crops, next‑generation biostimulants, and nano‑enabled delivery systems.

Comment 4:“Line 75-79: Through a critical examination……………nutritional quality. No information on how the literature review was carried out is mentioned anywhere in the text. The rationale and objective of the paper, the time period of the article, the literature selection criterion, the databases, the websites referred to, inclusion and exclusion basis of literature. It is necessary to briefly discuss how the study was performed and the factors considered for the study.”

Response:We apologize for this omission. In the revised manuscript, we have added a dedicated “Literature search strategy” section immediately after the Declarations and before the References (see Lines 1011–1019). It reads:
“A systematic literature search was conducted using PubMed, Web of Science, and Google Scholar for articles published up to March 2026. Search terms included combinations of ‘amino acids’, ‘plants’, ‘signaling’, ‘stress tolerance’, ‘nitrogen metabolism’, ‘biosynthesis’, and ‘transport’. Only peer‑reviewed articles, reviews, and book chapters written in English were considered. References cited in retrieved articles were also screened for relevance. No publication date restrictions were applied for landmark discoveries, but priority was given to studies published in the last decade.”

Comment 5:“Figure 3. Translating amino acid research into sustainable agricultural practices. Discuss the development of amino-acid-based biostimulants for the generation of stress-tolerant crops. What is the current progress/developments in this direction? Are there any commercially marketed formulations currently on the market? If yes, give examples.”

Response:As noted in our response to Comment 2, we have extensively expanded Section 3.3.2 (see Lines 938–947) to cover the current progress. We specifically list commercially available formulations, including Terra‑Sorb® and HICURE®, and summarize field evidence for their stress‑mitigating effects in multiple crop species. This information directly supports Figure 3 and its legend, which has also been updated to mention the example formulations. 

Comment 6:“Some minor corrections: Please be consistent with the use of scientific names for biological species. Line 225, Arabidopsis can be either in normal font or italicized, likewise in the entire manuscript.”

Response:We have carefully checked the entire manuscript and ensured that all scientific names (e.g., Arabidopsis thaliana, Oryza sativa) are now consistently italicized . This uniformity applies to both the main text and the reference list.

Comment 7:“Moderate English revisions are required for sentence clarity, spelling, and punctuation.”

Response:The manuscript has undergone thorough language editing. We have corrected typos, adjusted punctuation, and rephrased several long or ambiguous sentences to improve clarity. We believe the revised text now meets the required standard.

Comment 8:“Gene nomenclature: the complete name of the gene is usually written in normal font, while the abbreviation is written in italics.”

Response:We have revised gene nomenclature throughout the manuscript. At first mention, the full gene name is given in normal font, followed by the abbreviated symbol in italics (e.g., “Δ¹‑pyrroline‑5‑carboxylate synthetase 1 (P5CS1)”). Subsequent occurrences use only the italicized abbreviation. This applies to all genes discussed, including YUCCA, ACS, ADC, PAL, PAD3, *CYP79F1/F2*, GCS1, P5CS1, ProDH, BADH, GSH1, ATP‑PRT1, PCS1, etc. . The gene names in Tables 1 and 2 and in the figure legends have also been corrected to this format.

Comment 9:“The literature review includes a lot of information on the multiple structural and functional roles of different amino acids in plants. It is suggested to provide some information in a figure representation rather than an elaborate literature discussion.”

Response:We appreciate this constructive suggestion. We have strived to balance text and figures in the manuscript. In addition to the existing Figure 1 (metabolic network), Figure 2 (signaling pathways), and Figure 3 (agricultural translation), we have added Table 2, which concisely summarizes the functions of key amino acids in abiotic stress and the supporting genetic evidence in a tabular format, thereby reducing lengthy textual descriptions. Wherever possible, we have organized the multiple functions in a bullet-point style combined with illustrations to improve readability and reduce the reading burden.

Author Response File: Author Response.docx

Reviewer 3 Report

Comments and Suggestions for Authors

The core of a review lies in “Synthesis” (integration and critique), whereas the current manuscript reads more like a “Summary” (listing and summarizing) of a lot of papers.

(1) There is a lack of transitions and logical connectors between paragraphs. For example, at the end of Section 2.4.1, after discussing Proline, Section 2.4.2 directly begins with “Glutathione is one of the most important...” without any transitional sentence such as “Besides proline acting as an osmolyte, amino acids also contribute directly to redox homeostasis through...”. It is recommended that each paragraph switch should include a sentence bridging the sections, for instance, explaining to the reader why after reading about Proline, they are moving on to Glutathione.

(2) There is extensive use of “list-style” statements, lacking a critical perspective. For example, the description of hormone precursors on pages 7-8: “Tryptophan⟶⟶ Auxin ... Methionine⟶⟶ Ethylene ... Glutamate/Arginine ⟶⟶ Polyamines”. And pages 11: “Root development: AtGLR3.3…………Abiotic stress signaling:…………..”. A lot of these descriptions. A professional review should not merely list facts, but should compare the differences and similarities between pathways or point out current debates in research.

In addition, the current review mostly takes Arabidopsis thaliana as the research model, and it is suggested to supplement the functional differences of amino acids in monocotyledons (rice, corn) and dicotyledons (crops, horticultural plants), so as to improve the universality of the conclusion.

Author Response

We sincerely thank the reviewer for the insightful and rigorous assessment. The comments have helped us transform the manuscript from a descriptive summary into a truly integrative and critical review. Below, we provide a point-by-point response, indicating where and how each concern has been addressed in the revised manuscript.

Comment 1: “There is a lack of transitions and logical connectors between paragraphs. For example, at the end of Section 2.4.1, after discussing Proline, Section 2.4.2 directly begins with ‘Glutathione is one of the most important...’ without any transitional sentence… It is recommended that each paragraph switch should include a sentence bridging the sections…”

Response: We fully agree and have carefully revised the manuscript to insert logical transitions between all major sections and subsections. The specific example cited by the reviewer has been addressed: at the beginning of Section 2.4.2, we added the bridging sentence (Line 609-610):
“Beyond their osmotic functions, certain amino acids contribute directly to the antioxidant defense system.”
This sentence explicitly connects the osmolyte discussion (proline, glycine betaine) to the subsequent content on redox homeostasis. Similar transitional sentences have been added throughout the manuscript. Representative examples include:

(1) Between the introduction of GLRs, PII, and TOR in Section 2.3.3, we added: “While GLRs monitor extracellular amino acid concentrations, the PII protein functions as an intracellular metabolic sensor…”(Line 497-499) and later: “The Target of Rapamycin (TOR) kinase operates at a different timescale and organizational level from GLRs and PII.”(Line 520-521)

(2) Between the discussions of individual secondary metabolite pathways in Section 2.2.2, we included comparative segues such as: “Moving to methionine‑ and tryptophan‑derived glucosinolates, the regulatory logic shifts from transcriptional control of a gateway enzyme to substrate specificity at the entry level.” (Line 283-285) and “A third dimension comes from alkaloids…”(Line 298-299)

(3) At the end of the hormone precursor section (2.2.1), we summarized and transitioned: “Comparing these three pathways reveals not a simple list of precursor‑to‑product conversions, but a dynamic, interconnected network…”(Line 248-250)

These additions ensure that readers are continuously guided through the logical flow of the review.

Comment 2: “There is extensive use of ‘list-style’ statements, lacking a critical perspective. For example, the description of hormone precursors on pages 7-8… and pages 11… A professional review should not merely list facts, but should compare the differences and similarities between pathways or point out current debates in research."

Response: This is a crucial point, and we have substantially restructured the relevant sections to move beyond listing. Throughout the manuscript, we now explicitly compare pathways, contrast regulatory mechanisms, and highlight unresolved debates.

(1) Hormone precursors (Section 2.2.1): Instead of merely stating that tryptophan→auxin, methionine→ethylene, and arginine→polyamines, the revised text now compares the regulatory logic of each pathway. For example: “Moving from auxin to ethylene, the regulatory strategy shifts from localized biosynthesis to post‑translational control of enzyme stability.” It further contrasts polyamines with auxin and ethylene by noting their different concentration ranges and dual signaling/structural roles. We then identify open questions: “First, how do these pathways partition shared carbon and nitrogen resources under fluctuating environmental conditions? Second, what molecular mechanisms integrate auxin, ethylene, and polyamine signaling into coherent developmental outputs?” (See Section 2.2.1, paragraphs 2-4)(Line 222-260)

(2) Receptors and signaling (Section 2.3.3): Rather than listing GLR functions, we critically compare GLRs, PII, and TOR in terms of their subcellular location, temporal dynamics, and integrative hierarchy. For instance: “GLRs provide rapid, local Ca²⁺ signals. The PII protein continuously monitors intracellular glutamine levels, adjusting metabolism on a slower timescale. TOR integrates these local and metabolic signals into systemic decisions…” (Line 542-546)We also discuss the unresolved question of whether the diverse GLR functions arise from distinct subunits or from different regulatory partners, and the challenge of functional redundancy that complicates genetic analysis.

(3) Secondary metabolite pathways (Section 2.2.2): We systematically compare phenylpropanoids, glucosinolates, and alkaloids, identifying shared challenges (how to partition common precursors) but divergent regulatory solutions (transcriptional control of a single entry enzyme vs. substrate‑specific CYP79 isoforms vs. dual‑route putrescine biosynthesis). This comparative framework allows us to highlight current debates, such as whether ADC or ODC is the primary route for nicotine production, and whether findings in Arabidopsis apply to crops.

Throughout the review, we now regularly pose “Unresolved questions” and “Key open questions”, ensuring that a critical perspective is maintained rather than a passive summary.

Comment 3: “The current review mostly takes Arabidopsis thaliana as the research model, and it is suggested to supplement the functional differences of amino acids in monocotyledons (rice, corn) and dicotyledons (crops, horticultural plants), so as to improve the universality of the conclusion.”

Response: We greatly appreciate this recommendation. The revised manuscript now systematically incorporates evidence from monocot and dicot crop species and, equally importantly, highlights where Arabidopsis-based knowledge may not be generalizable. Specific additions include:

(1) Secondary metabolism: We explicitly contrast the tryptophan-derived phytoalexin camalexin in Arabidopsis with the diterpenoid phytoalexins (momilactones) in rice, noting the lineage-specific chemical arsenals (Section 2.2.2)(Line 285-287). We also discuss glucosinolate pathway evolution across Brassicales and compare regulatory responses in cabbage versus Arabidopsis(Line 292-296).

(2) Long‑distance signaling and receptors: In Section 2.3.1, we state that most long‑distance amino acid signaling work has been conducted in Arabidopsis and call for comparative studies in rice, maize, and tomato(Line 417-419). In Section 2.3.3, we note that TOR signaling has been studied almost exclusively in Arabidopsis and tobacco cell cultures, and that natural variation in crop TOR components could be a rich source of breeding targets(Line 538-540). We also point out that the canonical glutamine‑binding motif of PII is not conserved in Brassicaceae (including Arabidopsis), underscoring the risk of overgeneralizing from a single model(Line 511-513).

(3) Stress responses: We include data from Triticeae species (barley vs. rye) on cold‑induced amino acid accumulation (Section 2.4.4)(Line 662-666), from nickel hyperaccumulator Alyssum  species for histidine‑mediated metal tolerance (Section 2.4.3)(Line 633-635), and from rice and pea for amino acid transporter engineering to improve seed protein and NUE (Sections 2.6.3 and 3.3.3)(Line 858-861 962-964).

(4) The Conclusions and Perspectives section (Section 3.2) now explicitly identifies the Arabidopsis-centric bias as a major challenge and dedicates Section 3.3.3 to the transition “From Arabidopsis to the Field and Beyond,” outlining concrete strategies for translating findings into cereals, legumes, and horticultural crops using CRISPR, synthetic biology, and multi‑environment field validation.

We believe these additions substantially broaden the phylogenetic scope of the review and strengthen the universality of the conclusions.

We hope that the above revisions fully address the reviewer’s concerns. The manuscript has been transformed, in accordance with these suggestions, into a more integrative, critical, and broadly applicable synthesis. We thank the reviewer again for the time and expertise that have significantly improved our work.

Author Response File: Author Response.docx

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript can be considered for publication in the present form.

Comments on the Quality of English Language

Moderate English revisions are required.

Author Response

Comment 1:The manuscript can be considered for publication in the present form. Moderate English revisions are required.

Response：We sincerely thank the reviewer for taking the time to evaluate our manuscript and for the encouraging recommendation that it can be considered for publication in its present form. We greatly appreciate this positive assessment and the recognition of our work.We have also further polished the language of the manuscript.

Author Response File: Author Response.docx

Reviewer 3 Report

Comments and Suggestions for Authors

1. The manuscript is excessively long and text-heavy. Many paragraphs are dense with mechanistic details. Considering reducing text.
2. The manuscript lacks sufficient visual aids to guide readers through complex parallel pathways. While Figures 1–3 are useful, they are not referenced frequently enough in the text.
3. Add a concluding sentence or bullet-point summary at the end of each major subsection.

Author Response

We sincerely thank the reviewer for the insightful and rigorous assessment. We have carefully considered all your suggestions and have revised the manuscript accordingly. Below we provide a point-by-point response to each of your comments. All changes are highlighted in the revised manuscript.

Comment 1. “The manuscript is excessively long and text-heavy. Many paragraphs are dense with mechanistic details. Considering reducing text.”

Response：We appreciate this concern. The original manuscript was indeed lengthy due to the broad scope of amino acid multifunctionality. In response, we have carefully streamlined the text by removing redundant descriptions, condensing several mechanistic explanations, and merging overlapping discussions without losing essential scientific content. Specifically for Section 2.4, we have retained the more representative subsections on amino acid responses to drought and heavy metal stresses (original Sections 2.4.1 and 2.4.3), while removing the original subsections on oxidative stress and cold stress (original Sections 2.4.2 and 2.4.4). Furthermore, we recognized that the original Section 2.6 (homeostatic regulation of amino acids) focused primarily on agricultural applications rather than on the intrinsic functions of amino acids in plants. We therefore deleted this section entirely and integrated its relevant content into other sections of the manuscript where appropriate. As a result of these revisions, the overall word count of the manuscript has been reduced by more than 1,200 words.We believe the manuscript is now more concise and reader-friendly while preserving the depth required for a comprehensive review.

Comment 2. “The manuscript lacks sufficient visual aids to guide readers through complex parallel pathways. While Figures 1–3 are useful, they are not referenced frequently enough in the text.”

Response：Thank you very much for your valuable comments. To further enhance the visual presentation of the manuscript, we have removed the original Table 1 and Table 2. Correspondingly, we have enriched the content of Figure 1 and have added a new Figure 3 entitled “Metabolic and transcriptional regulatory networks underlying plant responses to abiotic and biotic stresses.” We agree that frequent cross‑referencing to figures can greatly improve readability. Therefore, we have added additional citations to Figures 1, 2, 3 and 4throughout the text where the corresponding pathways or concepts are discussed. For example: (1) In Section 2.1, we now explicitly refer to Fig. 1 when describing the GS/GOGAT cycle and amino acid branching pathways (Line 120, 123 and 127).

(2) In Section 2.2.1, additional references to Fig. 1 have been inserted for auxin, ethylene, polyamine, and SAM pathways (Line 210, 228, 240 and 127).

(3) In Section 2.3.3, we have added calls to Fig. 2 when discussing GLR‑mediated Ca²⁺ signaling, PII‑NAGK interaction, and TOR‑autophagy regulation (Line 475, 483, 516 and 537).

(4) In Sections 2.4 and 2.5, we have increased citations to Fig. 3 for proline metabolism, histidine chelation, phytochelatin synthesis, glutamine signaling, and L‑canavanine action (Line 583, 588, 596, 630, 642, 660, 705 and 761).

(5) In Section 3.3.2 and 3.3.3, we refer to Fig. 4 for biostimulant applications, nano‑delivery, and breeding strategies (Line 859, 867, 872 and 875).

These additional citations guide the reader more effectively between the text and the visual summaries.

Comment 3. “Add a concluding sentence or bullet-point summary at the end of each major subsection.”

Response：We have followed this recommendation precisely. At the end of each major subsection (2.1, 2.2, 2.3, 2.4, 2.5), a concise concluding sentence (or short paragraph) has been added to summarize the key take‑home message. These concluding sentences are clearly marked in the revised manuscript (in italics or as separate paragraphs). For clarity, they are as follows: (1) Section 2.1: “In summary, amino acids are not merely the building blocks of proteins but are also central integrators of plant carbon and nitrogen metabolism…”(Line 172-175)

(2) Section 2.2: “In summary, the capacity of amino acids to serve as precursors for hormones, secondary metabolites, and small redox‑active molecules illustrates a complex, interconnected network…”(Line 394-398)

(3) Section 2.3: “Thus, amino acid signaling in plants operates as a multi‑temporal, multi‑compartmental network…”(Line 561-565)

(4) Section 2.4: “Thus, under abiotic stress, plants rapidly repurpose amino acids as osmoprotectants, antioxidants, and metal chelators…”(Line 682-686)

(5) Section 2.5: “In conclusion, amino acids contribute to biotic stress responses through diverse mechanisms… a trade‑off that sits at the heart of the growth‑defense balance.”(Line 784-788)

These additions significantly improve the logical flow and allow readers to quickly grasp the essential points of each subsection.

We thank you again for your thorough review and believe that the revised manuscript has been substantially strengthened by your suggestions. 

Author Response File: Author Response.docx

Round 3

Reviewer 3 Report

Comments and Suggestions for Authors

The authors' response have addressed all my concerns.