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

TL;DR

Spirulina-derived carbon dots improve rice seed germination and seedling growth by enhancing metabolic activity and resource mobilization.

Contribution

Demonstrates that Spirulina-derived carbon dots act as effective nanobiostimulants for rice seed nanopriming.

Findings

Nanopriming with Spirulina CDs increased germination rate by 25% and root length by 37%.

Seedlings showed higher starch (24%) and phenolics (20%) accumulation with no oxidative stress.

Proteomic analysis revealed metabolic reprogramming favoring growth over defense mechanisms.

Abstract

Biogenic carbon dots (CDs) are emerging as promising plant biostimulants, yet their effects during early crop establishment remain underexplored. Here, we synthesized and characterized Spirulina-derived CDs and evaluated their efficacy as seed nanopriming agents in rice (Oryza sativa L.). CDs exhibited nanoscale size, abundant surface functionalities, and a highly negative ζ-potential, indicative of stable aqueous dispersions. Spectroscopic characterization (Raman and FTIR) confirmed a graphitic–amorphous carbon structure. Near-infrared spectroscopy coupled to principal component analysis revealed time-dependent metabolic changes during imbibition, identifying 8–12 h as the optimal priming window. Nanopriming with Spirulina CDs (0.2 mg mL−1 for 12 h) increased the seed germination rate (25%), the germination speed index (17%), vigor index I (22%), and root length (37%) compared to hydropriming. Biochemically, the nanoprimed seedlings accumulated higher levels of starch (24%), total carbohydrates (8%), and total phenolics (20%), without evidence of oxidative imbalance, based on antioxidant capacity measurements and proteomic profiling. Proteomic analysis revealed coordinated metabolic reprogramming, characterized by increased abundance of proteins involved in translation, energy metabolism, and ion/nutrient homeostasis, alongside reduced abundance of proteins associated with defense and catabolic processes. This shift from stress-preparation to growth-oriented metabolism supports improved seedling establishment. Overall, Spirulina-derived CDs function as effective nanobiostimulants that promote early metabolic activation and resource mobilization, offering a sustainable strategy to enhance rice seedling establishment.

Figures

Tables

Table 3. Differentially abundant proteins in rice seedlings 10 days after nanopriming (12 h) with Spirulina-derived CDs (0.2 mg mL−1) compared to hydroprimed control.

Spirulina × Control Conditions—Proteins Unique or More Abundant in Spirulina-Nanoprimed Seedlings
Functional Categories	Description	Uniprot	Reported Peptides	t-Test	Log2 Fold Change
Protein synthesis and translation machinery	Elongation factor 1 beta 2	Q40682	6	0.0036	1.2814
	Elongation factor 2	Q5JKU5	17	0.0147	0.8080
	DEAD-box ATP-dependent RNA helicase 37	Q75HJ0	8	0.0126	0.6206
	40S ribosomal protein S3-1	Q6YTY2	14	-	-
Energy metabolism and glycolysis/TCA cycle	Fructose-bisphosphate aldolase	Q6YPF1	6	0.0001	0.7853
	Methylthioribose kinase 1	Q7XR61	8	0.0403	1.0434
	Malate dehydrogenase	Q5QLS8	6	-	-
Nutrient mobilization and metal homeostasis	Nicotianamine synthase 2	Q0DSH9	13	0.0019	1.6612
	Nicotianamine synthase 2	Q10MI9	10	0.0010	1.9189
Protein quality control, folding, and degradation	26S proteasome regulatory particle triple-A ATPase subunit4	Q9FXT8	11	0.0012	1.9337
	Heat shock 70 kDa protein BIP5	Q6Z058	7	0.0390	1.4311
Transport and cellular homeostasis	Pyrophosphate-energized vacuolar membrane proton pump	Q8H616	5	0.0004	1.2782
Spirulina × Control conditions—Proteins unique or more abundant in control seedlings
Functional categories	Description	Uniprot	Reported peptides	t-Test	Log2 Fold Change
Amino acid metabolism	Cysteine synthase	Q7XS58	6	0.0047	−0.7234
	Glutamate decarboxylase	Q6YSB2	4	-	-
	S-methyl-5-thioribose kinase	A0A0P0WG91	5	-	-
	Phenylalanine ammonia-lyase	Q7X8V3	6	-	-
Defense and stress response	Pathogenesis-related protein	Q75T45	9	0.0094	−1.2663
	L-ascorbate peroxidase 1	Q10N21	10	0.0105	−1.0470
	Allene oxide cyclase	Q75KD7	6	0.0484	−0.7051
	Peroxidase	Q7XSU2	5	0.0052	−1.8970
	Heat shock 70 kDa protein, mitochondrial	Q10SR3	10	-	-
	Hypersensitive-induced response protein-like protein 2	Q6K550	3	-	-
	Probable aldo-keto reductase 3	Q7XQ45	2	-	-
	Putative 12-oxophytodienoate reductase 6	Q69TI2	3	-	-
Carbohydrate and energy metabolism	Malate dehydrogenase	Q0J5H3	7	0.0323	−1.2859
	Alpha-amylase	A0A0P0VPT7	14	-	-
	Isocitrate dehydrogenase [NADP]	Q0JBV4	4	-	-
	Soluble inorganic pyrophosphatase	Q0DYB1	4	-	-
	Adenosine kinase	Q0JBQ0	2	-	-
V-PPases	Pyrophosphate-energized vacuolar membrane proton pump 1	Q0JN26	4	0.0107	−1.3571
	Pyrophosphate-energized vacuolar membrane proton pump-like	A0A0P0VQV1	5	-	-
Cellular organization and protein turnover	26S proteasome regulatory subunit S10B homolog B	Q69Q32	9	-	-
	Coatomer subunit alpha-2	Q9AUR7	21	-	-
	40S ribosomal protein S4	Q0DIR7	4	-	-
Unknown	Os06g0549900 protein	Q0DBM6	5	0.0219	−1.2859

Full text

1. Introduction

Rice (Oryza sativa L.) is a major crop and a key cereal for global food security. It is a staple food for more than half of the world’s population, especially in Asia, Africa, and Latin America [1]. As a primary source of carbohydrates, minerals, and vitamins, its sustainable and resilient production is essential to meet growing food demand and address the challenges posed by climate change [2]. In this context, bio-inputs, particularly biostimulants, have gained attention for improving crop performance and reducing environmental impact [3]. Biostimulants are substances of natural origin that can significantly enhance yield and quality [4]. They are derived from various sources, including microorganisms, agro-industrial waste, macro- or microalgae, and cyanobacteria [5,6]. Recently, advances in nanoformulations have further improved the delivery and efficacy of biostimulants. When applied at low concentrations, nanoparticles (NPs) can induce beneficial physiological responses in plants, such as higher nutrient use efficiency and tolerance to abiotic and biotic stresses [7,8].

Among these, the class of small carbon-based NPs known as carbon-dots (CDs) has emerged as a promising tool in agriculture due to its biocompatibility, water solubility, stability, and low toxicity [9]. CDs can be synthesized from various carbon-rich sources, including the cyanobacteria Spirulina, which is a valuable source of biostimulants because it contains biologically active molecules, such as polysaccharides, vitamins, minerals, amino acids, phytohormones, phenolic compounds, and phycocyanin [10]. Using Spirulina to produce biostimulants reduces chemical dependence on fertilizers and environmental impacts, while supporting circular resource use in agriculture [11].

The application method (foliar spraying, soil incorporation, hydroponic media, or seed treatment) plays a key role in determining the effectiveness of NPs [12,13]. A promising approach is nanopriming, which involves pre-treating seeds with nanomaterials (NMs) before planting. This technique enhances water uptake, modulates enzymatic activity, and increases stress tolerance, resulting in more vigorous seedlings [14,15]. Nanopriming also induces a form of metabolic resilience by creating a “protective memory” that persists throughout the plant’s life cycle [13,16]. This memory is controlled by latent genetic mechanisms that can be reactivated under stress, even after seed desiccation [17].

Although several studies have demonstrated that different NPs can enhance seed germination and early plant development [18,19,20,21], 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 [22], mung bean [21], lettuce, and tomato [23], 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.

2. Materials and Methods

2.1. Materials

Powdered biomass of the microalga Arthrospira platensis was obtained from Olson Nutrição Ltd.a (Rio Grande do Sul, Brazil). Analytical-grade reagents and solvents were purchased from Sigma-Aldrich (Darmstadt, Germany) and used without further purification. Ultrapure water (≥18.25 MΩ.cm) was used in all experiments. Rice seeds (Oryza sativa ssp. indica cv. IRGA 424 RI) were purchased from a local farmer in Santa Vitória do Palmar, Rio Grande do Sul, Brazil.

2.2. Synthesis of Spirulina CDs Nanoparticles

Pyrolysis was carried out using 20 g of Arthrospira platensis biomass at 300 °C for 2 h, with a heating rate of 10 °C min^−1^ in a muffle furnace (Solidsteel, Piracicaba, Brazil). The resulting carbonized product was ground into a fine powder. For exhaustive extraction, approximately 10 g of the carbonized powder was dispersed in 100 mL of ultrapure water and then heated to 100 °C in a water bath for 30 min. The supernatant was filtered using a vacuum filtration system containing 25 μm filters, and then centrifuged at 13,000 rpm for 15 min. The final extract was stored at −80 °C in an ultra-freezer until lyophilization.

2.3. Morphological Characterization, Chemical Composition, and Size Distribution of the Spirulina CDs

The morphology of the CDs was observed by Scanning Electron Microscopy (SEM) (Tescan VEGA 3, Brno, Czech Republic) and Transmission Electron Microscopy (TEM) (Jeol, JEM-1400, Beijing, China). Samples were analyzed in high-vacuum mode at an accelerating voltage of 10 kV by SEM and at 120 keV by TEM. 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, Kyoto, Japan). 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. Functional groups were identified by Fourier Transform Infrared (FTIR) spectroscopy (Shimadzu, IRAffinity-1) using 60 scans in the spectral range of 500 to 4000 cm^−1^, with a resolution of 4 cm^−1^. Raman spectroscopy (Horiba, LadRAM HR Evolution, Kyoto, Japan) was performed in the range of 750 to 2000 cm^−1^ using a 633 nm excitation wavelength, a 20 s integration time, and three accumulations. The size distribution of the CDs was determined in terms of particle volumetric density by Dynamic Light Scattering (DLS) at 25 °C, and the zeta potential was measured using a Zetasizer Nano ZS (Malvern Panalytical, Malvern, UK).

2.4. Near-InfraRed (NIR) Spectroscopy

To determine the best time for pre-sowing treatment and metabolic activation, rice seeds were soaked in a suspension containing 0.2 mg mL^−1^ of Spirulina CDs (nanopriming) and collected at six time points: 0, 2, 4, 8, 12, and 24 h. As a control, seeds were soaked in distilled water (hydropriming) under the same conditions. After treatment, all seeds were dried in a forced-air oven at 45 °C for 24 h and ground into a fine powder using a knife mill. NIR spectra were recorded using an FT-NIR MPA spectrometer (Bruker Optic GmbH, Ettlingen, Germany) at a resolution of 8 cm^−1^ per data point. Aliquots (~5 g of powdered material) were transferred into the NIR glass vials and scanned in triplicate across the spectral range of 800–2500 nm, yielding 1154 spectral data points per sample. Pre-processing of the NIR spectra included baseline correction and Multiplicative Scatter Correction (MSC), both applied using algorithms available in Opus Lab software (v. 7.5, Bruker Optic GmbH, Ettlingen, Germany).

2.5. Germination Experiment (In Vitro Studies)

Rice seeds were separated, weighed, and surface-sterilized with a 2% sodium hypochlorite solution for 5 min, followed by five rinses with distilled water. The seeds were then soaked for 12 h in suspensions containing Spirulina CDs (nanopriming). The commercial biostimulant Arbolina (Krilltech, Brasília, Brazil) was used as a positive control at concentrations of 0.2 and 0.4 mg mL^−1^. Distilled water was used as the negative control (hydropriming). The treatments were divided into control, Spi (Spirulina CDs), and Arb (Arbolina CDs). After soaking, the seeds were drained and air-dried at room temperature for 48 h. Germination tests were conducted on Germitest paper (28 × 38 cm), previously autoclaved at 120 °C, using 100 seeds per replicate. The experiment was conducted in a BOD germination chamber under a 12 h photoperiod at 26 °C. Moisture was maintained by adding distilled water every three days. Germination was monitored daily for 10 days, starting three days after sowing. Seeds with a radicle length ≥ 4 mm were considered germinated. The test was performed in quintuplicate. At the end of the germination experiment, germination percentage, germination rate, germination speed index, and seedling vigor indices I and II were calculated according to the equations described elsewhere [14,24].

2.6. Morphophysiological Parameters

After 10 days of germination, 25 seedlings per treatment were collected and photographed for growth and biomass analyses, including fresh and dry weights and shoot and root lengths. Whole seedlings were weighed using an analytical balance, then dried in a forced-air oven at 60 °C for 72 h. The remaining seedlings were collected whole, immediately frozen in liquid nitrogen, and stored at −80 °C for subsequent biochemical and omics analyses.

2.7. Biochemical Analyses

All biochemical analyses were performed in triplicate. The seedlings were lyophilized and pulverized in liquid nitrogen.

2.7.1. Total Phenolic Content (TPC)

The TPC was determined using the method of Singleton et al. [25] with modifications. Phenolic compounds were extracted from 300 mg of whole seedlings using 3 mL of 80% methanol. For the assay, 100 μL of extract was added to 75 μL of Folin–Ciocalteu reagent and 825 μL of 2% sodium carbonate buffer. After 1 h incubation at room temperature, absorbance was measured at 750 nm using a microplate reader (ThermoPlate P-reader, Rayto, Dallas, TX, USA). The TPC was quantified using a gallic acid standard curve (7.81 to 500 μg mL^−1^; y = 0.006x, r^2^ = 0.9967).

2.7.2. Total Flavonoid Content (TFC)

The TFC was determined following the method of Woisky and Salatino [26] with modifications. Flavonoids were extracted from 300 mg of sample using 3 mL of 80% methanol. For the assay, 500 μL of extract was added to 2.5 mL of absolute ethanol and 500 μL of 2% aluminum chloride solution. After 1 h of incubation, absorbance was measured at 420 nm using a microplate reader (ThermoPlate P-reader, Rayto, Dallas, TX, USA). The TFC was quantified using a quercetin (Sigma-Aldrich, Germany) calibration curve (7.81 to 500 μg mL^−1^; y = 0.0056x, r^2^ = 0.9799).

2.7.3. Antioxidant Capacity (AC)

AC was assessed using the DPPH (2,2-diphenyl-1-picrylhydrazyl) assay [27]. A volume of 10 μL of extract (from the TPC supernatant) was added to 290 μL of the DPPH solution. After 30 min of agitation at room temperature in a microplate reader (ThermoPlate P-reader, Rayto, Dallas, TX, USA), absorbance was measured at 540 nm. The DPPH radical scavenging capacity was calculated using the following equation:

$$[eqn]$$

where the control absorbance is the absorbance of the DPPH solution, and the sample absorbance is the absorbance of the reaction mixture (DPPH + extract).

2.7.4. Total Carotenoids (TCN)

The TCN content was determined according to Aman et al. [28] with modifications. Carotenoids were extracted from 300 mg of sample using 3 mL of 80% methanol. Absorbance was measured at 450 nm using a microplate reader (ThermoPlate P-reader, Rayto, Dallas, TX, USA).

2.7.5. Total Soluble Sugars (TSS) and Total Starch (TS)

The TSS and TS contents were estimated following the methodology of Umbreit and Burris [29]. Briefly, 50 mg of sample was mixed with 2 mL of MCW solution (methanol:chloroform:distilled water, 12:5:3, v/v/v). For TSS quantification, extracts were centrifuged, and 1 mL of chloroform and 1.5 mL of distilled water were added to the supernatants. After phase separation, 1 mL of the upper aqueous phase was transferred to a new tube. Then, 2 mL of 0.2% anthrone solution in sulfuric acid was added, and the mixture was vortexed and heated in a water bath at 100 °C for 3 min. After cooling to room temperature, absorbance was measured at 630 nm using a microplate reader (ThermoPlate P-reader, Rayto, Dallas, TX, USA). TSS was quantified using a glucose (Sigma-Aldrich, Germany) calibration curve (62.50 to 2000 μg mL^−1^; y = 0.0018x, r^2^ = 0.9517).

For the TS analysis, the pellet obtained from the TSS extraction was treated with 2 mL of 30% perchloric acid and centrifuged at 4000 rpm for 10 min. Supernatants were collected, and the extraction step was repeated. The final volume was adjusted to 4 mL with perchloric acid. The samples were centrifuged again, and 1 mL of supernatants was transferred to a new tube. Then, 2 mL of 0.2% anthrone solution in sulfuric acid was added, and the mixture was vortexed, heated in a water bath at 100 °C for 3 min, and cooled to room temperature. Absorbance was measured at 630 nm using a microplate reader (ThermoPlate P-reader, Rayto, Dallas, TX, USA). TS was quantified using a glucose (Sigma-Aldrich, Germany) calibration curve (62.50 to 2000 μg mL^−1^; y = 0.0016x, r^2^ = 0.9916).

2.7.6. Total Carbohydrates (TC)

The TC content was determined according to DuBois et al. [30] with modifications. Briefly, 50 mg of sample was extracted with 20 mL of distilled water. After centrifugation, 2 mL of the supernatant was transferred to a new tube, followed by the addition of 50 μL of 80% phenol and 5 mL of concentrated sulfuric acid. After standing for 10 min, the reaction mixture was stirred and incubated in a water bath at 25–30 °C for 15 min. After cooling at room temperature, samples were transferred to microplates, and absorbance was measured at 490 nm using a microplate reader (ThermoPlate P-reader, Rayto, Dallas, TX, USA). TC content was quantified using a galactose (Sigma-Aldrich, Germany) calibration curve (7.80 to 500 μg mL^−1^; y = 0.0025x, r^2^ = 0.9992).

2.7.7. Total Protein (TP)

The TP content was determined using the method of Bradford [31] with modifications. Proteins were extracted from 200 mg of sample using 10 mL of phosphate-buffered saline (PBS; pH 7.0), prepared by mixing 23 mL of monobasic sodium phosphate with 76 mL of dibasic sodium phosphate solution. The samples were shaken, allowed to stand for 15 min, and centrifuged. Then, 100 μL of the supernatant was transferred to a new tube, and 5 mL of Bradford reagent (diluted 1:5) was added. After 5 min of incubation at room temperature, aliquots were transferred to microplates. Absorbance was measured at 595 nm using a microplate reader (ThermoPlate P-reader, Rayto, Dallas, TX, USA). Bovine serum albumin (Sigma-Aldrich, Germany) was used as standard to construct a calibration curve (3.90 to 62.5 μg mL^−1^; y = 0.0032x, r^2^ = 0.9772).

2.7.8. Total Amino Acid (TAA)

The TAA content was determined according to Silveira and Furlong [32], with modifications. Amino acids were extracted from 100 mg of sample mixed with 2 mL of distilled water. After centrifugation, 1 mL of the supernatant was mixed with 3 mL of ninhydrin solution. The reaction mixture was heated in a water bath at 100 °C for 30 min and cooled to room temperature. Samples were transferred to microplates, and absorbance was measured at 570 nm using a microplate reader (ThermoPlate P-reader, Rayto, Dallas, TX, USA). TAA content was quantified using a proline (Sigma-Aldrich, Germany) calibration curve (1.95 to 499.20 μg mL^−1^; y = 0.0026x, r^2^ = 0.9962).

2.8. Differential Proteomic Analysis

2.8.1. Protein Extraction and Digestion

A differential proteomic analysis was performed to evaluate changes in the proteome of whole rice seedlings treated or not with Spirulina-derived CDs. Proteins were extracted from 250 mg of fresh seedlings using the Plant Total Protein Extraction kit (Sigma-Aldrich, St. Louis, MO, USA). Protein digestion was conducted using 100 μg of total protein per sample with trypsin as the proteolytic enzyme. To remove potential detergent residues, proteins were precipitated using a methanol/chloroform solution following the protocol of Nanjo et al. [33]. The resulting pellets were resuspended in a urea/thiourea buffer and desalted using Amicon Ultra-0.5 3 kDa filters (Merck Millipore, Darmstadt, Germany). Samples were then washed twice with 8 M urea and twice with 50 mM ammonium bicarbonate (pH 8.5). Finally, protein digestion was carried out according to Calderan-Rodrigues et al. [34].

2.8.2. Mass Spectrometry Analysis

Digested protein samples were analyzed by electrospray ionization/liquid chromatography tandem mass spectrometry (ESI-LC/MS-MS) using a Synapt G2-Si HMDS mass spectrometer (Waters, Cheshires, UK) coupled to a nanoACQUITY™ UPLC™ system, according to Xavier et al. [35]. Peptide separation was performed using a Waters Symmetry C18 trap column (180 μm × 20 mm) and a nanoAcquity UPLC HSS T3 analytical column (1.8 μm, 75 μm × 150 mm) maintained at 45 °C. An injection volume of 2 μg of digested peptides per sample was used. The mobile phase consisted of solution A (0.1% formic acid in water) and solution B (acetonitrile with 0.1% formic acid). Human [Glu1]-fibrinopeptide B (100 fmol μL^−1^) was used as an external calibration standard. The mass spectrometer was operated in positive mode with a resolution of V-35,000 full width at half maximum (FWHM). Data acquisition was performed using MassLynx V.4.0 (Waters, Cheshires, UK), and spectra were processed using Progenesis QI for Proteomics V.2.0 (Nonlinear Dynamics, Newcastle Upon Tyne, UK). For protein identification, the Oryza sativa proteome (Uniprot ID: UP000059680) available on UniProtKB (https://www.uniprot.org/ accessed on 14 March 2025) was used as the reference database.

2.8.3. Proteomic Data Analysis

As a final quality control step, the data were refined by screening for proteins consistently detected across all three replicates of each treatment. Differentially accumulated proteins were categorized as “More abundant”, “Less abundant”, or “Unique”, based on a Student’s t-test (p < 0.05), and a Log_2_ Fold Change (FC) threshold of >0.585 or <−0.585. The mass spectrometry proteomics data have been deposited in the PRIDE repository [36] via the ProteomeXchange Consortium, under the dataset identifier PXD069738.

2.9. Statistical Analysis

Data were tested for normality and analyzed by a one-way analysis of variance (ANOVA), followed by Tukey’s post hoc test (p < 0.05), using SPSS software version 23.0. NIR spectral (800–2500 nm) data were processed using the Unscrambler^®^ X software (version 10.4). Principal component analysis (PCA) was performed using 1154 spectral data points per sample, computed via the non-iterative partial least squares (NIPALS) algorithm.

3. Results and Discussion

3.1. Characterization, Morphology, and Chemical Composition of Spirulina-Derived CDs

CDs are typically composed of sp^2^/sp^3^ hybridized carbon atoms with surface functional groups (amines, hydroxyls, and carboxylic acid), which contribute to their solubility and multifunctionality [37]. The SEM (Figure 1a,b) and TEM (Figure 1c,d) images revealed that the Spirulina CDs had a relatively uniform size, homogeneous morphology, and hexagonal shape, indicating successful and well-controlled synthesis.

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 [38]. Moreover, the near-neutral pH and physicochemical properties of the dispersion suggest compatibility with biological systems and minimal interference with nutrient mobility or absorption [39].

Zeta potential plays an important role in determining the mobility of NPs in plants [40]. The highly negative surface charge of the CDs suggests strong electrostatic repulsion between particles, which prevents aggregation and enhances long-term dispersion stability. Ristroph et al. [41] demonstrated that NPs with a high surface charge, whether negative or positive, exhibit greater mobility in the phloem, whereas near-neutral particles (≈±1 mV) move more slowly through the stem. Negatively charged NPs are easily translocated between plant organs, while positively charged NPs tend to accumulate at the application sites [42,43].

During pyrolysis, high temperatures (>300 °C), combined with long reaction times (>12 h), typically lead to the loss of surface functional groups in the raw material [5]. However, the degradation of polar functional groups is reduced in gradual and controlled carbonization, as observed in the FTIR spectrum (Figure 2a). Several characteristic bands were detected in the FTIR spectrum of the CDs: a broad peak at 3245 cm^−1^, attributed to O-H stretching vibrations; a medium, sharp peak at 1575 cm^−1^, associated with symmetric in-plane deformation of primary amines and amides (C=C or C=N bonds); a sharp peak at 1378 cm^−1^, indicating O-H bending or C-O stretching in carboxylic acids; and a large and sharp peak at 1113 cm^−1^, indicative of single C-O or C-N stretching vibrations, suggesting the presence of aromatic or aliphatic amines and alcohols.

The functional group composition of raw Spirulina biomass has been previously characterized [5]. Comparing the FTIR spectra of Spirulina-derived CDs and the original Spirulina biomass indicates that carbonization alters the surface chemistry by breaking chemical bonds and releasing volatile compounds. These changes are marked by reduced amino-acid-related signals, increased aromatic groups, and degradation of cellulose-derived components, typical outcomes of thermal decomposition during pyrolysis [44,45]. Similar spectral shifts have been reported in CDs synthesized from other algal species [46,47]. Carbonization also promotes the formation of aromatic carbon structures, which contribute to the elimination of volatile compounds [37]. 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.

Raman spectroscopy provides detailed information on molecular vibrations and structural features of carbon-based materials, including crystallinity and hybridization states [48]. The Raman spectrum of the CDs (Figure 2b) showed a peak at 1610 cm^−1^ (G or graphene band), indicating the presence of a sp^2^ hybridization pattern characteristic of the hexagonal arrangement of carbon atoms, serving as a distinct hallmark of materials with a regular crystalline structure. Another peak emerges at 1330 cm^−1^ (D or defect band), indicating the presence of disorder and defects within the carbon material, often associated with carbon atoms exhibiting a sp^3^ hybridization pattern. The intensity ratio of the D to G bands (I_D_/I_G_), commonly used to assess the degree of disorder in carbon-based materials, such as graphene, carbon nanotubes, and amorphous carbon [48], was 1.013. This ratio is commonly used to assess the degree of disorder in carbon-based materials, such as graphene, carbon nanotubes, and amorphous carbon [48]. This result suggests that Spirulina-derived CDs are partially ordered, with a balanced distribution of sp^2^ (graphitic) domains and sp^3^ (structural defects) domains. This degree of disorder is typical for well-synthesized CDs and correlates with favorable optical properties such as good fluorescence and colloidal stability [49].

EDS and XRF analyses of the CDs revealed a rich elemental composition, including C (43%), O (31%), and N (7%), and essential minerals such as Na (10.7%), K (3.6%), P (1.8%), Cl (1.37%), S (0.64%), Si (0.35%), Ca (0.33%), and Mg (0.27%). These elements are vital for plant growth and development, supporting processes such as osmotic regulation (Na, K), energy transfer (P), chlorophyll synthesis (Mg), and structural stability (Ca, Si). Previous studies have shown that different types of biochar can increase leaf biomass, shoot-to-root ratio, and K levels in the leaves of turmeric plants [50]. Spirulina-derived biochar also influenced rice productivity through similar nutrient contributions [51]. Since biochar is a precursor generated during CD synthesis, its nutrient profile directly influences the elemental composition of CDs. These embedded minerals likely contribute to the biostimulant properties of CDs by enhancing nutrient availability and physiological responses in plants.

3.2. Near-InfraRed (NIR) Spectra of Rice Seeds

The NIR spectra of rice seeds subjected to different imbibition times (0, 2, 4, 8, 12, and 24 h) in water or CDs (0.2 mg mL^−1^) showed broadly similar profiles. However, the seeds treated with CDs for 8 and 12 h had higher overall peak intensities, suggesting enhanced molecular activity or structural changes associated with nanopriming (Figure 3). We observed key absorption bands at 1000 nm (CH_2_ deformation, commonly found in lipids and carbohydrates such as starch), 1200 nm (C-O stretching in polysaccharides and glucose), 1950 nm (O-H deformation, primarily water, with potential contributions from proteins), and 2100 nm (C-H stretching associated with lipids or proteins) [52]. These spectral signatures reflect the major biochemical components of rice seeds and can vary depending on the processing methods used [53].

PCA was applied to the NIR spectral dataset to assess clustering patterns among treatments. The first two principal components accounted for 99% of the total variance, with PC1 and PC2 explaining 85% and 14%, respectively. The PCA revealed four distinct clusters: seeds subjected to nanopriming with CDs for 8 and 12 h, hydroprimed seeds at 0 and 2 h, hydroprimed seeds at 8 and 12 h together with nanoprimed seeds at 0 h, and hydroprimed and nanoprimed seeds treated for 24 h (Figure 4). Overall, the spectral and PCA results indicate that nanopriming for 0, 2, and 4 h produces molecular profiles similar to those of hydropriming for 8 and 12 h. This suggests that Spirulina-derived CDs accelerate early seed metabolic responses compared to water alone. In contrast, the rice seeds treated with CDs for 8 and 12 h showed the most distinct spectral signatures (Figure 3 and Figure 4), pointing to enhanced biochemical activity. These findings identify 8–12 h as the optimal immersion time for nanopriming with Spirulina-derived CDs. Longer exposure (24 h) did not produce further distinction, suggesting a plateau effect. Exposure time seems to play a key role in determining the effectiveness of nanopriming with Spirulina-derived CDs. To our knowledge, this is the first study to use NIR spectroscopy and PCA to determine the optimal duration for seed priming.

3.3. Morphophysiological Changes in Rice Seedlings Induced by Spirulina-Derived CDs

Nanopriming has proven successful in accelerating seed germination and increasing seedling vigor across different plant species [54]. Although we did not detect statistical differences in final germination percentages between hydroprimed and nanoprimed seeds, the germination rate increased by 25% in the seeds treated with Spirulina-derived CDs (0.2 mg mL^−1^) compared to hydropriming (Table 1). In contrast, nanopriming with Arbolina CDs (0.4 mg mL^−1^) reduced the germination rate by 17% (Table 1). The germination speed index also improved in the seeds treated with Spirulina-derived CDs at both concentrations (Table 1). A faster germination rate and shorter emergence times promote uniform field establishment, which is essential for producing vigorous, high-yielding plants [55].

Vigor index I (germination percentage × seedling length) increased by 22% in the seeds nanoprimed with Spirulina CDs (0.2 mg mL^−1^) compared to hydropriming, whereas a 23% decrease was observed with Arbolina CDs at the same concentration (Table 1). For vigor index II (germination percentage × seedling weight), no difference was detected with Spirulina CDs, but Arbolina CDs led to a 15% reduction (Table 1). Regarding seedling growth, no differences in fresh or dry weight were observed, except for a 13% reduction in fresh weight of the seedlings treated with Arbolina CDs (0.2 mg mL^−1^) (Table 1). In contrast, seedling length increased by 18% with Spirulina-derived CDs (0.2 mg mL^−1^), whereas the remaining three treatments showed approximately 20% reductions. These differences in seedling length were exclusively due to variations in root length, as shoot length remained unchanged across all treatments. Similar increases in root elongation have been reported in tomato, lettuce [23], and rice [56] seeds treated with CDs, likely associated with auxin synthesis and the upregulation of auxin-responsive genes. In rice, nanopriming with 50 mg L^−1^ of CDs modulated the expression of genes related to phytohormones and development in a time-dependent manner during germination, which may explain the phenotypic changes observed in roots [56]. Auxin plays a central role in cell division, elongation, and differentiation, contributing to faster germination and more vigorous seedling development [21].

In contrast to Spirulina-derived CDs, Arbolina treatments resulted in reduced germination rate, vigor index, and seedling length compared to the control (Table 1). 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 [14,57]. These findings highlight the importance of material-specific characterization and dose optimization when evaluating nanobiostimulants for agricultural applications.

Nanopriming improves seed germination and early seedling vigor by inducing reactive oxygen species (ROS), which trigger cellular signaling cascades that activate genes related to water uptake, starch metabolism, and embryo development [57]. Despite growing interest in this approach, the use of CDs in nanopriming remains underexplored. Liang et al. [20] reported that CDs derived from plastic wastes enhanced pea seed germination, increased seedling biomass and size, and stimulated enzymatic activity and root vigor in concentrations from 0.25 to 2 mg mL^−1^. Similarly, other studies demonstrated that seed treatment with CDs improved root elongation, seedling vigor, enzyme activity, and chlorophyll content in rice [22] and mung bean [58]. Biochar nanoparticles (BNPs) have also shown positive effects on germination and seedling growth in tomato and rice, particularly by promoting root development. However, high concentrations of BNPs reduced shoot length and biomass in reed seedlings, indicating potential phytotoxicity [19]. CDs synthesized by pyrolysis of peanut shells (200 ppm, 3 h) also improved seed imbibition, germination, and vigor index in mung bean (Vigna mungo) [21].

Given that five out of ten variables (seed germination rate, germination speed index, vigor index I, seedlings length, and root length) showed higher values with Spirulina-derived CDs at 0.2 mg mL^−1^, subsequent analyses were focused on this concentration.

3.4. Effects of CDs on the Metabolism and Establishment of Seedlings

Biochemical analyses showed no significant differences in total flavonoid or carotenoid contents, nor in antioxidant capacity (DPPH) between rice seedlings submitted to nanopriming or hydropriming (Table 2). However, total phenolic content increased by 20% in seedlings treated with Spirulina-derived CDs compared to the control. 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 [59]. Moderate increases in phenolic compounds may contribute to signaling modulation or metabolic priming rather than substantially altering bulk antioxidant capacity [60]. Moreover, the reduced abundance of stress-associated proteins (Table 3), 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.

NPs often affect plants by inducing ROS, Ca^2+^, and mitogen-activated protein kinases (MAPKs). Together with other messengers, they play an important role in plant signaling and the activation of secondary metabolic pathways [57]. For instance, the addition of 6 μg mL^−1^ of CDs to the culture medium stimulated the production of phenolic compounds, flavonoids, and triterpenoids in Evolvulus alsinoides without affecting plant development [61]. Similarly, Li et al. [62] demonstrated that CDs alleviated the inhibitory effects of 2,4-dichlorophenoxyacetate sodium (2,4-D-Na) and increased rice cell biomass under stress conditions such as 2,4-D, NaCl, and high light intensity. In addition, CDs enhanced antioxidant activity (peroxidase, phenolics, and flavonoids) and nutrient uptake, contributing to greater abiotic stress tolerance.

Plant secondary metabolites are key molecules known for their protective function against biotic and abiotic stresses [63]. Carbon-based NMs, such as CDs, can interact with signaling pathways and stimulate the biosynthesis of phenolic compounds (particularly polyphenols) and flavonoids that help scavenge ROS [64]. Such metabolic responses enhance plant tolerance to various environmental stresses, including salinity, heavy metals, drought, heat, cold, and UV radiation. By mitigating oxidative stress, these compounds also contribute to improved crop quality and productivity [59].

No significant differences were observed in the total contents of soluble sugars, proteins, and amino acids following treatment with Spirulina-derived CDs. However, total starch and carbohydrate levels increased by 23% and 8%, respectively (Table 2). The higher starch content suggests enhanced photosynthetic activity, as Spirulina extracts promote stress tolerance in plants and act as biostimulants [65]. When applied as a seed treatment (priming), Spirulina can stimulate photosynthesis and increase carbohydrate accumulation [66]. These higher energy reserves may help support seedling growth and recovery. Nanopriming corn seeds using a 3% Spirulina suspension accelerated germination, improved growth and photosynthetic performance, and protected seedlings against Cd-induced stress [18].

In seedlings treated with Arbolina CDs, protein content increased by 10%, while amino acid levels decreased by 33% (Table 2). No significant differences were observed in the other evaluated parameters compared to the control. These results suggest that free amino acids may have been redirected toward protein synthesis, leading to lower amino acid concentrations and higher total protein contents. Since no additional changes in primary metabolism were detected, this modulation might be specific to protein-related pathways.

Several studies showed that seed nanopriming can act by remodeling plant metabolism through the synthesis of carbohydrates, lipids, and proteins [67]. For example, nanopriming using gold NPs (10 ppm) increased the vigor index, biomass, chlorophyll, and total soluble sugar contents in corn seedlings [68]. Rai-Kalal et al. [69] showed that nanopriming using silicon oxide NPs in wheat seeds (15 mg L^−1^) improved photosynthetic parameters, maintained biochemical balance, and increased biomass in the treated seedlings, even under drought conditions. An increase in biomass in mung bean plants treated with peanut shell CDs (200 ppm) was linked to the rise in carbohydrate content and the expression of the genes ZmSUT1 and ZmSUT4, involved in carbohydrate transport [21].

Carbon-based NMs have been shown to enhance the activity of key photosynthetic enzymes such as RuBisCO and phosphoenolpyruvate carboxylase (PEPC), thereby improving CO_2_ assimilation and promoting biomass accumulation in plants [70]. For instance, nanopriming rice seeds with CDs synthesized from Syzygium cumini juice functionalized with betaine (0.2 to 1 g mL^−1^) activated photophysical parameters such as carbohydrates, chlorophyll, and carotenoids, in addition to increasing the activity of antioxidant enzymes, promoting seedling growth [71]. These findings underscore the potential of CDs as next-generation bio-stimulants, offering a more sustainable approach to improve crop performance and resilience under diverse environmental conditions.

3.5. Effects of Spirulina-Derived CDs on the Protein Abundance in Rice Seedlings

A total of 995 proteins were identified in rice seedlings when comparing the control and Spirulina CD-treated samples. Among these, 35 proteins (3.5%) were either unique or differentially abundant among treatments. Ten proteins were more abundant (and two unique) in Spirulina CD-treated seedlings, while eight were more abundant (and 15 unique) in the control (Figure 5). The identified proteins were classified into functional categories based on their predicted molecular functions and existing literature (Table 3). The potential roles of selected proteins are discussed below in relation to seedling growth, development, and establishment.

3.5.1. Proteins Stimulated by Nanopriming with Spirulina-Derived CDs

Spirulina CD-nanoprimed seedlings showed higher abundance of proteins associated with growth, nutrient mobilization, and stress responses (Table 3). Among the most responsive proteins was nicotianamine synthase 2 (NAS2), a key enzyme responsible for the biosynthesis of nicotianamine, a non-proteinogenic amino acid that chelates divalent metal ions such as Fe^2+^ and Zn^2+^ and facilitates their intracellular trafficking and long-distance transport [72,73]. Nicotianamine plays a central role in maintaining metal homeostasis, particularly during periods of rapid growth when micronutrient demand is elevated. Therefore, the increased abundance of NAS2 in Spirulina-CD-treated seedlings is consistent with a potential enhancement of metal chelation capacity and micronutrient redistribution during early seedling establishment. However, while these findings suggest a potential contribution of NAS2 to improved nutrient homeostasis, functional validation would be required to directly confirm its role in modulating Fe and Zn uptake dynamics under nanopriming conditions. Vacuolar H^+^-pyrophosphatase (V-PPase), an enzyme that energizes ion transport across vacuolar membranes [74], was also more abundant in nanoprimed seedlings. This is consistent with a potential enhancement of vacuolar function and ion compartmentalization; processes often associated with enhanced tolerance to abiotic stress [75].

Elongation factors (EF-1β2, EF-2), ribosomal protein S3-1, and RNA helicase 37 were more abundant, suggesting a potential increase in translational activity and RNA metabolism, processes associated with cell division and elongation during seedling establishment [76,77,78]. This aligned with increased root length, vigor index, and germination rate (Table 1). Increased abundance of fructose-bisphosphate aldolase, malate dehydrogenase, and methylthioribose kinase are consistent with a shift in central carbon and amino acid metabolism, reflecting higher energy demands and biosynthetic activity during early growth [79,80,81]. These changes are consistent with higher starch and carbohydrate availability detected in Spirulina CD-nanoprimed seedlings (Table 2).

In parallel, the enrichment of heat shock protein BiP5 and a 26S proteasome ATPase subunit is consistent with modulation of protein folding and degradation pathways, supporting proteome stability under stress or developmental transitions [82,83,84]. The higher total phenolics (Table 2) in these plants reinforce the hypothesis that Spirulina-derived CDs stimulate antioxidant and proteostasis networks involved in stress mitigation [85]. Together, these changes are consistent with a multifaceted molecular response associated with nanopriming, enhancing metabolism, nutrient utilization, and protein quality control, thereby promoting more vigorous seedling establishment in rice.

3.5.2. Proteins Inhibited by Nanopriming with Spirulina-Derived CDs

Spirulina-CD nanopriming not only increased the abundance of proteins linked to growth and metabolism but also led to a marked reduction in several proteins (more abundant in hydroprimed controls—Table 3). These included enzymes involved in amino acid and secondary metabolism, such as cysteine synthase, glutamate decarboxylase, and phenylalanine ammonia-lyase, as well as stress-related proteins (e.g., pathogenesis-related proteins, peroxidases, and ascorbate peroxidase). The reduced contents of these defense-associated proteins suggest that nanoprimed seedlings may exhibit a reduced abundance of constitutive defense-associated proteins, and therefore invest less in constitutive defense mechanisms. Instead, they appear to rely on enhanced protein quality control systems (e.g., BiP5, proteasome ATPase) and higher phenolic accumulation (Table 2) to maintain cellular homeostasis. This interpretation is supported by the reduced abundance of an aldo-keto reductase isoform, an enzyme that detoxifies reactive carbonyl species [86], which may reflect differences in oxidative or carbonyl-related metabolism in Spirulina-nanoprimed seedlings, aligning with the higher phenolic pool and the reduced levels of peroxidases and APX (Table 3). Similarly, the reduced abundance of α-amylase is consistent with the greater starch content observed in Spirulina-nanoprimed seedlings (Table 2), indicating a slower mobilization of carbohydrate reserves and a potential strategy to sustain energy supply during early development [87,88]. The presence of isoforms with higher and reduced abundances (e.g., malate dehydrogenase and vacuolar proton pumps) further suggests that nanopriming with Spirulina-derived CDs induces a selective metabolic remodeling rather than a broad, uniform activation. Together, these findings indicate that nanopriming is associated with a selective remodeling of the rice seedling proteome, enhancing translational and core metabolic processes while reducing proteins related to defense and catabolic pathways. This fine-tuned molecular balance likely underpins the improved seedling vigor and reserve accumulation at the physiological level.

The integrated physiological, biochemical, and proteomic data allow the proposal of a conceptual framework summarizing the effects of Spirulina-derived CDs on early seedling development (Figure 6). Nanopriming was associated with increased accumulation of starch, total carbohydrates, and phenolic compounds, alongside enhanced abundance of proteins involved in translation, energy metabolism, and nutrient homeostasis. Concurrently, the proteins related to reserve breakdown and constitutive defense responses were less abundant. Collectively, these coordinated changes are consistent with a metabolic profile favoring growth-associated processes over constitutive stress-related pathways during early seedling establishment. Rather than indicating a direct causal reprogramming event, this model represents an integrative interpretation of the observed molecular and physiological adjustments associated with Spirulina-CD nanopriming.

4. Conclusions

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.