Review Reports - Seed Nanopriming with Spirulina-Derived Carbon Dots Enhances Rice (Oryza sativa L.) Germination, Crop Establishment, and Seedling Metabolic Performance

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Reviewer Report (plants-4156051)

The manuscript plants-4156051, “Nanopriming with Spirulina-Derived Carbon Dots Enhances Rice Germination, Crop Establishment, and Seedling Metabolic Performance” investigate about the effects of Spirulina-derived carbon dots as nanopriming agents to enhance rice seed germination and early growth performance.

The authors investigated the use of Spirulina-derived carbon dots as a biogenic nanopriming agent for rice seeds with the aim to improve rice seed germination, breaking seed dormancy, and early growth traits.

The authors evaluated its effects on germination and early seedling vigor and explored the underlying mechanisms using biochemical and proteomic analyses. Their results demonstrated coordinated metabolic reprogramming, including enhanced carbohydrate reserve utilization, increased phenolic content and activation of growth-related pathways, alongside reduced constitutive defense activity.

The novelty is that this manuscript provides the first mechanistic insight into how Spirulina-based nanopriming promotes seedling establishment. The novelty of the manuscript is sufficiently emphasized.

The main contribution is that study introduces a sustainable, biologically derived nanopriming strategy with potential applications in crop improvement, stress-resilient agriculture, on rice seed germination, early growth, and seedling establishment.

However, there are few parts of the text which should be finalize in Conclusion:

Line: 584-596: Spirulina-derived CDs represent an effective biogenic nanopriming strategy for rice, promoting enhanced germination and early vigor. Beyond physiological improvements, Spirulina nanopriming elicited coordinated biochemical and proteomic reprogramming, including more efficient carbohydrate reserve utilization, increased phenolic pools, and enrichment of pathways related to translation, central energy metabolism, and ion/nutrient homeostasis. Simultaneously, it attenuated reserve-degrading and constitutive defense programs. These integrated shifts suggest a metabolic transition from stress maintenance to growth promotion, providing a mechanistic foundation for improved seedling establishment. These findings provide a basis for validating and optimizing Spirulina-based nanopriming under field conditions and for investigating its potential interactions with soil microbiota and plant microbiomes.

Delete last sentence: (A schematic model summarizing these findings is presented in Figure 6.)

Figure 6. (Schematic model summarizing the effect of seed nanopriming with Spirulina-derived CDs 598 for 12 h on rice seed germination, early seedling development, and associated proteomic and meta-599 bolic changes. The model highlights key physiological, biochemical, and molecular responses con-600 tributing to enhanced seedling vigor and growth.)

The figure should be moved to the Discussion section, as it does not belong in the Conclusion.

Line: 105-106: In 2.3. Section: The authiors provide a more detailed explanation of the application of X-ray fluorescence (XRF) analysis, as well as a clear description of how absolute and relative values were obtained and interpreted.

“Additionally, samples were analyzed by X-Ray Fluorescence spectrometer (XRF) (Shimadzu, EDX 7000) for elemental mapping.”

I believe that with the revisions suggested above, this manuscript will be significantly strengthened and will make a valuable contribution to the field of agronomy.

Author Response

However, there are few parts of the text which should be finalize in Conclusion:

Authors’ response: We thank the reviewer for careful reviewing the Conclusion section. Following the suggestions of reviewer 1 and 2, we modified this last section to: “In summary, nanopriming with Spirulina-derived CDs improved rice germination performance and early seedling vigor, accompanied by increased accumulation of starch, total carbohydrates, and phenolic compounds. Proteomic profiling revealed coordinated changes in proteins associated with translation, central energy metabolism, and nutrient homeostasis, alongside reduced abundance of reserve-degrading and constitutive defense-related proteins. These coordinated physiological and molecular adjustments provide mechanistic support for the observed improvements in germination dynamics and seedling vigor. The integrative framework proposed here (Figure 6) highlights how Spirulina-derived CDs may modulate early metabolic priorities, offering a promising strategy for improving crop establishment under controlled conditions. Although further functional validation will be necessary to confirm the specific roles of individual proteins, this study provides new insights into the biochemical and proteomic landscape associated with CD-mediated seed nanopriming”.

Delete last sentence: (A schematic model summarizing these findings is presented in Figure 6.)

Figure 6. (Schematic model summarizing the effect of seed nanopriming with Spirulina-derived CDs for 12 h on rice seed germination, early seedling development, and associated proteomic and metabolic changes. The model highlights key physiological, biochemical, and molecular responses contributing to enhanced seedling vigor and growth.)

The figure should be moved to the Discussion section, as it does not belong in the Conclusion.

Authors’ response: We thank the reviewer for this valuable suggestion. We agree that the conceptual framework illustrated in Figure 6 should be explicitly explained within the main text. In the revised manuscript, we have incorporated a dedicated paragraph at the end of Section 3.5.2 (Proteomic analysis) that provides a detailed discussion of the proposed integrative model. This section now synthesizes the physiological, biochemical, and proteomic findings, explicitly describing how increased abundance of proteins associated with translation, energy metabolism, and nutrient homeostasis, together with reduced abundance of reserve-degrading and constitutive defense-related proteins, are collectively interpreted within the framework presented in Figure 6. The model is now fully contextualized in the Discussion, rather than being introduced only in the Conclusion. The Conclusion has also been revised to avoid repetition and to refer to Figure 6 as a summarized integrative framework.

We believe this revision improves the clarity and coherence of the manuscript.

Line: 105-106: In 2.3. Section: The authors provide a more detailed explanation of the application of X-ray fluorescence (XRF) analysis, as well as a clear description of how absolute and relative values were obtained and interpreted.

“Additionally, samples were analyzed by X-Ray Fluorescence spectrometer (XRF) (Shimadzu, EDX 7000) for elemental mapping.”

Authors’ response: We thank the reviewer for this important observation. We agree that additional clarification regarding the XRF analysis and data interpretation strengthens the methodological transparency of the study.

In the revised manuscript, we have expanded Section 2.3 to: “The elemental composition was determined by energy-dispersive X-ray spectroscopy (EDS) coupled to SEM (Tescan VEGA 3, Czech Republic) using a Bruker Nano XFlash Detector 6-10, as well as by X-ray fluorescence (XRF) spectroscopy (Shimadzu, EDX 7000). Light elements (C and O) were quantified by EDS, as the XRF method employed does not allow their detection. To ensure representative values, ten independent EDS measurements were performed, and the average concentrations of these elements were subsequently fixed and applied to all samples analyzed by XRF. The remaining elements (Na to U) were quantified by XRF. This combined analytical approach enables reliable determination of both light and heavy elements in carbon-based materials.”

We believe this clarification improves methodological rigor and transparency.

I believe that with the revisions suggested above, this manuscript will be significantly strengthened and will make a valuable contribution to the field of agronomy.

Authors’ response: We really appreciate your comments to improve the quality of our manuscript.

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

Nano-biostimulants are finding increasing applications in regulating crop growth, development, and nutritional quality. This manuscript evaluated the effects of carbon dots (CDs) derived from Spirulina, used as a nanoprimer, on rice seed germination, seedling growth, and metabolism. The results confirmed that Spirulina-derived CDs function as effective nano-biostimulants, promoted seedling growth and development. This study provides a new paradigm for the development of bio-based nano-biostimulants.

Lines 77-78: The effects of CDs on plant growth and development (e.g., mung bean sprouts, rice, lettuce, and tomato) have been previously reported.
Please include a particle size distribution histogram based on transmission electron microscopy (TEM) images to clearly define the actual size range. Additionally, stability data for the carbon dots should be provided.
Improve the readability of Figure 2.
Plant photographs are recommended.
Table 1 shows that the Arbolina treatment inhibits seed and seedling growth and development compared to the control. The authors should strengthen the discussion regarding this observation.
Although total phenolic content significantly increases in the Spirulina-derived CDs treatment group compared to the control, no significant difference is observed in antioxidant capacity (DPPH assay). The authors should include additional indicators of the antioxidant system or strengthen the discussion of these results.
Remove the text “10 more abundant…”from Figure 5.
Line 30: Regarding the statement “without inducing oxidative stress”, please provide measurements of MDA content or ROS levels.
In Table 2, please ensure correct units are indicated for each parameter (e.g., TPC and TFC should be expressed as gallic acid and quercetin equivalents, respectively).
Spirulina contains abundant bioactive substances such as amino acids, vitamins, and polysaccharides. It is possible that uncarbonized organic molecules remain after the synthesis of CDs. The authors are advised to include controls consisting of “nanoparticle-free Spirulina extract” and “nanoparticle carrier alone.”
Although the proteomics data reveal some changes, functional validation (e.g., gene knockout/overexpression) is lacking. The discussion should elaborate on the specific physiological significance of the differentially expressed proteins. For example, how exactly does the increase in NAS2 affect iron and zinc uptake? Furthermore, mechanistic inferences in the discussion should be weakened.
The proposed molecular mechanism (Figure 6) requires detailed explanation and discussion within the main text, rather than being merely mentioned in the conclusion (Line 596).

Author Response

Lines 77-78: The effects of CDs on plant growth and development (e.g., mung bean sprouts, rice, lettuce, and tomato) have been previously reported.

Authors’ response: We thank the reviewer for highlighting previous reports demonstrating the effects of CDs on plant growth and development. We agree that several studies have shown positive growth responses in species such as rice, lettuce, tomato, and mung bean. However, the novelty of the present study does not lie solely in demonstrating growth promotion by CDs, but rather in:

(i) the use of Spirulina-derived biogenic CDs as seed nanopriming agents;

(ii) the integration of NIR spectroscopy coupled with PCA to define the optimal priming window during seed imbibition;

(iii) the combined physiological, biochemical, and proteomic characterization revealing coordinated metabolic reprogramming associated with improved seedling establishment.

To better clarify this distinction, we have revised the Introduction to explicitly acknowledge prior studies while clearly defining the specific knowledge gap addressed here.

The revised text now reads: “Although several studies have demonstrated that different NPs can enhance seed germination and early plant development (Seifikalhor et al., 2020; Zhang et al., 2020; Liang et al., 2023; Abinaya et al., 2024), these investigations largely involve metal-based or inorganic nanomaterials. In contrast, studies specifically addressing the effects of CDs on seed nanopriming remain limited. While CDs have been reported to stimulate plant growth and development in crops such as rice (Li et al., 2018), mung bean (Abinaya et al., 2024), and lettuce and tomato (Kou et al., 2021), most reports emphasize phenotypic responses or general physiological changes rather than mechanistic insights. In particular, the biochemical and molecular processes underlying CD-mediated nanopriming during the critical window of seed imbibition and early seedling establishment remain poorly characterized. Integrative analyses linking reserve mobilization, metabolic activation, and proteomic reprogramming are still lacking. In this study, we evaluated the biochemical, physiological, and molecular effects of CDs synthesized from Spirulina biomass on rice seed germination, early growth, and seedling establishment. We hypothesized that Spirulina-derived CDs act as effective nanostructured biostimulants by enhancing germination, improving starch metabolism through interactions with key enzymes, and promoting early vigor through coordinated physiological and proteomic modulation.”

We believe this revision clarifies the contribution and novelty of our work.

Please include a particle size distribution histogram based on transmission electron microscopy (TEM) images to clearly define the actual size range. Additionally, stability data for the carbon dots should be provided.

Authors’ response: We thank the reviewer for this valuable suggestion. Regarding the particle size distribution, we acknowledge that statistical analysis based on multiple TEM micrographs can provide useful complementary information. In our study, TEM was primarily used for morphological characterization and visualization of particle shape and nanoscale dimensions. However, a statistically robust particle size distribution based exclusively on TEM images would require analysis of a substantially larger number of micrographs and individual particles than were acquired. To provide quantitative size distribution data, we employed Dynamic Light Scattering (DLS), which measures the hydrodynamic diameter of NPs in aqueous dispersion and is particularly suitable for assessing colloidal systems intended for biological applications. The DLS analysis showed an average particle size of 6.07 nm with a PDI of 0.62, indicating moderate polydispersity.

To clarify this point, we have revised the manuscript to explicitly state that DLS was used to determine particle size distribution in dispersion, while TEM provided morphological confirmation of nanoscale dimensions.

Regarding stability, we agree that this is an important parameter. Colloidal stability was assessed through zeta potential measurements, which yielded a value of −42.3 mV. NPs with absolute zeta potential values greater than ±30 mV are generally considered electrostatically stable due to strong interparticle repulsion. Therefore, the highly negative surface charge observed indicates good colloidal stability of Spirulina-derived CDs in aqueous solution.

To improve clarity, we have expanded the Results and Discussion section to explicitly interpret the zeta potential value in the context of colloidal stability.

The revised text now reads: “Particle size distribution in aqueous dispersion was determined by Dynamic Light Scattering (DLS), which showed an average hydrodynamic diameter of 6.07 nm with a polydispersity index (PDI) of 0.62. While this PDI value indicates moderate polydispersity, it likely reflects the hydrodynamic behavior of particles and minor agglomeration in suspension rather than structural heterogeneity. The measured pH (6.6), density (1.453 g cm^-3), and especially the highly negative zeta potential (−42.3 mV) indicate strong electrostatic repulsion between particles, supporting good colloidal stability and dispersion in aqueous medium. Homogeneity and nanoscale dimensions are critical parameters for efficient plant uptake (Wang et al., 2023). Moreover, the near-neutral pH and physicochemical properties of the dispersion suggest compatibility with biological systems and minimal interference with nutrient mobility or absorption (Santas-Miguel et al., 2023).”

We believe this clarification addresses the reviewer’s concern.

Improve the readability of Figure 2.

Authors’ response: Thanks for this comment. We have now improved the readability of Figures 2 and 3.

Plant photographs are recommended.

Authors’ response: We thank the reviewer for this suggestion. In the present study, plant growth responses were quantitatively assessed through objective morphophysiological measurements, including germination rate, vigor indices, seedling length, root length, fresh and dry biomass (Table 1), as well as biochemical and proteomic analyses. Because the observed differences were statistically supported and numerically reported, we prioritized quantitative data presentation over representative photographs. We agree that visual documentation can complement morphological evaluation. However, given that the study focused primarily on mechanistic and quantitative characterization rather than descriptive phenotyping, we believe that the existing data sufficiently support the conclusions drawn. We respectfully propose maintaining the current format, as the quantitative measurements provide a more robust and reproducible assessment of treatment effects than representative images alone.

Table 1 shows that the Arbolina treatment inhibits seed and seedling growth and development compared to the control. The authors should strengthen the discussion regarding this observation.

Authors’ response: We thank the reviewer for this insightful comment. We agree that the inhibitory effects observed for Arbolina treatments deserve further discussion. In the revised manuscript, we have expanded the Discussion section to address potential explanations for the reduced germination rate, vigor index, and seedling length observed under Arbolina exposure. Specifically, we now discuss that differences in NP composition, surface chemistry, concentration, and interaction with seed tissues may influence biological responses. Unlike the Spirulina-derived CDs synthesized in this study, commercial formulations may contain distinct surface functionalities or stabilizing agents that alter uptake dynamics, osmotic balance, or metabolic activation during seed imbibition. Additionally, seed nanopriming responses are known to be concentration-dependent, and supra-optimal NP concentrations can induce metabolic perturbations or transient stress responses that negatively impact early seedling growth.

The following paragraph has been added to the Discussion section: “In contrast to Spirulina-derived CDs, Arbolina treatments resulted in reduced germination rate, vigor index, and seedling length compared to the control. Such inhibitory effects may reflect differences in NP composition, surface chemistry, or formulation additives that influence seed-NP interactions during imbibition. Seed nanopriming responses are highly concentration-dependent, and excessive NP exposure may transiently disturb osmotic balance, metabolic activation, or reserve mobilization processes (Mahakham et al., 2017; Nile et al., 2022). These findings highlight the importance of material-specific characterization and dose optimization when evaluating nanobiostimulants for agricultural applications.”

We believe this addition clarifies the observed response while reinforcing the importance of careful formulation and dosage in nanopriming strategies.

Although total phenolic content significantly increases in the Spirulina-derived CDs treatment group compared to the control, no significant difference is observed in antioxidant capacity (DPPH assay). The authors should include additional indicators of the antioxidant system or strengthen the discussion of these results.

Authors’ response: We thank the reviewer for this important observation. We acknowledge that although total phenolic content increased significantly in Spirulina-CD-treated seedlings, no corresponding increase was detected in DPPH radical scavenging activity. While additional antioxidant markers (e.g., enzymatic assays) could provide further insights, the present study focused primarily on early metabolic reprogramming rather than a comprehensive oxidative stress profiling. To address this point, we have expanded the Discussion to clarify the apparent discrepancy between phenolic accumulation and DPPH results.

Specifically, we now emphasize that:

(i) DPPH measures in vitro radical scavenging capacity and may not fully reflect in vivo antioxidant dynamics;

(ii) moderate increases in phenolic compounds may contribute to signaling or priming functions rather than bulk antioxidant capacity;

(iii) the reduced abundance of stress-related proteins (e.g., peroxidases, APX) in nanoprimed seedlings suggests that these plants were not under elevated oxidative stress, which may explain the absence of increased DPPH activity.

The following paragraph has been added to the Discussion: “Although total phenolic content increased in Spirulina-CD-treated seedlings, no significant difference was observed in DPPH radical scavenging activity. The DPPH assay reflects in vitro free radical neutralization capacity and may not fully capture dynamic in vivo antioxidant processes (Rao and Zheng, 2025). Moderate increases in phenolic compounds may contribute to signaling modulation or metabolic priming rather than substantially altering bulk antioxidant capacity (Guo et al., 2023). Moreover, the reduced abundance of stress-associated proteins, including peroxidases and ascorbate peroxidase, suggests that nanoprimed seedlings were not experiencing elevated oxidative stress. Therefore, the enhanced phenolic pool may reflect metabolic reprogramming associated with growth promotion rather than a stress-induced antioxidant response.”

We believe this clarification strengthens the interpretation of the data.

Remove the text “10 more abundant…”from Figure 5.

Authors’ response: We thank the reviewer for this suggestion. The text indicating “10 more abundant in Spirulina-nanoprimed seedlings” and “8 more abundant in Control seedlings” was included to clarify the directionality of differential protein accumulation among the 18 shared proteins identified between treatments. While the Venn diagram effectively illustrates overlap, it does not explicitly convey how these shared proteins are distributed in terms of relative abundance. The annotation was therefore intended to facilitate rapid interpretation of the number of proteins more/less abundant in Spirulina-nanoprimed seedlings compared to control seedlings. Therefore, we respectfully propose maintaining this information in Figure 5, as it enhances interpretability without introducing redundancy.

Line 30: Regarding the statement “without inducing oxidative stress”, please provide measurements of MDA content or ROS levels.

Authors’ response: We thank the reviewer for this important comment. We agree that direct assessment of oxidative stress markers such as malondialdehyde (MDA) or reactive oxygen species (ROS) levels would provide more direct evidence regarding oxidative status. In the present study, oxidative stress was not evaluated through lipid peroxidation or ROS quantification assays. To avoid overinterpretation, we have revised the statement accordingly. Rather than asserting the absence of oxidative stress, we now indicate that no indirect evidence of oxidative imbalance was observed based on the available biochemical and proteomic data. Specifically, antioxidant capacity (DPPH assay) did not differ between treatments, and stress-associated proteins such as peroxidases and ascorbate peroxidase were not increased in Spirulina-CD-treated seedlings.

The sentence has been revised to: “without evidence of oxidative imbalance based on antioxidant capacity measurements and proteomic profiling”.

We believe this modification more accurately reflects the scope of the data presented.

In Table 2, please ensure correct units are indicated for each parameter (e.g., TPC and TFC should be expressed as gallic acid and quercetin equivalents, respectively).

Authors’ response: We thank the reviewer for pointing out the need for clarification regarding units in Table 2. We have carefully revised Table 2 to ensure that all parameters are presented with appropriate and clearly defined units. Specifically, total phenolic content is now expressed as mg gallic acid equivalents (GAE) per g dry weight, and total flavonoid content is expressed as mg quercetin equivalents (QE) per g dry weight. The corresponding units have been added directly to the table headers and clarified in the table legend. We have also verified that all other biochemical parameters (Total soluble sugars content, Total starch content, Total carbohydrates content, Total proteins content, and Total amino acids content) are consistently reported with their correct units: GluE: glucose equivalent; GalE: galactose equivalent; BSAE: bovine albumin serum equivalent; and PE: proline equivalent.

Spirulina contains abundant bioactive substances such as amino acids, vitamins, and polysaccharides. It is possible that uncarbonized organic molecules remain after the synthesis of CDs. The authors are advised to include controls consisting of “nanoparticle-free Spirulina extract” and “nanoparticle carrier alone.”

Authors’ response: We thank the reviewer for raising this important point regarding the possibility of residual bioactive compounds from Spirulina biomass. In the present study, CDs were synthesized through pyrolysis of Spirulina biomass at 300°C for 2 h, followed by aqueous extraction, filtration, centrifugation, and lyophilization. This carbonization process promotes extensive thermal decomposition of native biomolecules such as amino acids, vitamins, and polysaccharides, leading to the formation of carbonized nanostructures with distinct physicochemical properties. FTIR and Raman analyses confirmed the presence of graphitic–amorphous carbon structures and characteristic functional groups typical of CDs, indicating substantial structural transformation of the original biomass. While we acknowledge that inclusion of a NP-free Spirulina extract control could further dissect potential contributions of residual soluble compounds, the synthesis protocol employed here is designed to minimize the presence of uncarbonized organic molecules. Moreover, the physicochemical characterization (Raman ID/IG ratio, FTIR functional group shifts, nanoscale dimensions by TEM/DLS, and negative zeta potential) supports the formation of carbonized nanomaterials rather than a crude extract. We agree that inclusion of additional controls could provide further insight into the relative contributions of precursor-derived compounds versus nanostructured carbon materials. This represents an interesting direction for future investigation and has been acknowledged in the revised Discussion section: “Although the pyrolysis and purification procedures are expected to minimize the presence of uncarbonized Spirulina biomolecules, future studies including NP-free precursor extracts would help further distinguish the specific contributions of carbon nanostructures from potential residual soluble compounds”.

Although the proteomics data reveal some changes, functional validation (e.g., gene knockout/overexpression) is lacking. The discussion should elaborate on the specific physiological significance of the differentially expressed proteins. For example, how exactly does the increase in NAS2 affect iron and zinc uptake? Furthermore, mechanistic inferences in the discussion should be weakened.

Authors’ response: We thank the reviewer for this thoughtful and constructive comment. We agree that functional validation experiments (e.g., gene knockout or overexpression approaches) would provide direct evidence of causality. The present study was designed as an integrative physiological and proteomic analysis aimed at identifying candidate proteins and metabolic pathways associated with Spirulina-CD-mediated nanopriming, rather than performing targeted genetic validation.

To address the reviewer’s concern, we have revised the Discussion section to:

(i) Elaborate on the physiological roles of key differentially abundant proteins, including NAS2;

(ii) Clarify the known function of NAS2 in nicotianamine biosynthesis and metal chelation, and its potential implications for iron and zinc homeostasis during early seedling establishment;

(iii) Moderate mechanistic language to avoid implying direct causality.

Specifically, we now state that increased NAS2 abundance is consistent with enhanced nicotianamine production, which may facilitate intracellular chelation and mobilization of Fe²⁺ and Zn²⁺, thereby supporting nutrient homeostasis during rapid early growth. However, we explicitly indicate that these interpretations are correlative and based on established functional annotations.

We have also revised the manuscript to replace definitive mechanistic statements (e.g., “indicates” or “demonstrates”) with more cautious phrasing such as “is consistent with”, “suggests”, or “may contribute to”.

We believe these revisions strengthen the physiological interpretation while appropriately acknowledging the exploratory nature of the proteomic analysis.

The proposed molecular mechanism (Figure 6) requires detailed explanation and discussion within the main text, rather than being merely mentioned in the conclusion (Line 596).

We believe this revision improves the clarity and coherence of the manuscript.

Author Response File: Author Response.docx