Integrative metabolomic and genetic analysis of in vitro regenerated Populus alba in response to nano hydroxyapatite
Asmaa Abdelsalam, Ehab Mahran, Arezue Boroujerdi, Ashraf B. Abdel Razik, Mohamed F. Ahmed, Eman Tawfik
TL;DR
This study shows that nano-hydroxyapatite boosts growth and changes metabolism and genetics in white poplar plants grown in the lab.
Contribution
The study reveals new insights into how nano-hydroxyapatite affects genetic and metabolomic profiles in woody plants.
Findings
nHAp improved vegetative growth and root formation in Populus alba.
nHAp caused genetic variation and altered metabolite levels, including increased phenylalanine and quinic acid.
Metabolic pathways like aminoacyl-tRNA biosynthesis were significantly enriched with nHAp treatment.
Abstract
Nano-elicitation has gained increasing attention as a strategy to enhance plant growth and metabolic performance in vitro. However, the genetic and metabolomic consequences of nano-hydroxyapatite (nHAp) application in woody plant species remain poorly understood. This study examined the influence of biosynthesized nHAp on growth performance, genetic variation, metabolomic remodelling, and metabolic pathway regulation in in vitro regenerated Populus alba. The biosynthesized nHAp exhibited a rod-like morphology with average dimensions of 60 nm × 213 nm and a negative surface charge (zeta potential − 22.1 mV). The effects of nHAp were examined at three concentration levels: 20, 40, and 60 mg/L. Supplementation with nHAp significantly improved vegetative growth parameters, including weight, shoot and root length, leaf number, and adventitious root formation, compared to the control. Random…
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Taxonomy
TopicsPlant tissue culture and regeneration · Plant Growth Enhancement Techniques · Polysaccharides and Plant Cell Walls
Introduction
Populus alba (white poplar) is a commercially important tree that belongs to the Salicaceae family. The plant is a promising bioenergy crop that is commonly used in forest management because of its abundant timber [1]. Populus alba (P. alba) plant extracts contain bioactive metabolites that exhibit antioxidant, anti-inflammatory, and antibacterial properties [2, 3]. It has been utilized in the treatment of rheumatism, arthritis, urinary ailments, and hepatic diseases [4]. Moreover, it is utilized as a topical paste for the management of hemorrhoids, chilblains, sprains, and infected wounds. It is also used therapeutically in the treatment of bone and dental cavities and has been reported to treat nasal congestion [4].
The long generation time of Populus spp., coupled with seasonal dormancy and environmental stress, substantially hampers traditional breeding efforts [5]. In vitro propagation techniques have been utilized to address these environmental difficulties, providing regulated conditions for accelerated plant growth and multiplication and enhancing the production of bioactive compounds [6]. The incorporation of nanoparticles into tissue culture systems is considered a novel technique to enhance plant growth, development, and nutrient availability [7]. Nano-hydroxyapatite (nHAp) has recently gained attention in plant science as a novel nanomaterial with potential applications in agriculture and plant biotechnology [8]. Owing to their high surface area, biocompatibility, and slow-release capacity for essential nutrients such as calcium and phosphorus, nHAp have been proposed as efficient nano fertilizers that can improve nutrient uptake and enhance plant growth, biomass accumulation, photosynthetic performance, and root development by improving phosphorus availability and cellular mineral balance [9, 10]. However, increasing evidence indicates that the biological effects of nHAp are highly concentration- and species-dependent. Elevated concentrations of nHAp showed adverse responses in Vigna radiata (mung bean). This phytotoxicity is characterized by a multifaceted assault culminating in organellar dysfunction, profound dysregulation of Ca²⁺ homeostasis, and the induction of programmed cell death via apoptotic signaling cascades [11, 12]. Whereas studies on Solanum lycopersicum (tomato) have shown no toxicity even at relatively high nHAp concentrations [13].
Despite the increasing interest in nHAp as phosphorus and calcium nano-fertilizers, their effects on plant in vitro regeneration and metabolic regulation remain poorly characterized. Most studies to date have focused on germination, seedling growth, or ex vitro applications, providing limited insight into how nHAp influences tissue culture regeneration and plant metabolic networks.
This study aims to investigate the effects of nHAp on in vitro regenerated Populus alba, using untargeted metabolomic profiling as the primary focus. It seeks to elucidate how nHAp-induced biochemical reprogramming, particularly changes in primary and secondary metabolite biosynthesis, correlates with enhanced plant growth and genetic polymorphism, thereby revealing the mechanistic basis of nHAp as a nano-elicitor under controlled in vitro conditions.
Materials and methods
Plant material
Nodal segment explants of P. alba were obtained from Vitro-lab, Alexandria Desert Road, Egypt.
Preparation of nHAp
Hydroxyapatite nanoparticles were synthesized utilizing chicken eggs as a calcium supply by the hydrothermal process reported by Wu et al., 2023 [14]. Waste chicken eggshells were initially rinsed thoroughly with distilled water to eliminate residual membranes and surface impurities. Cleaned shells were then oven-dried at 80 °C for 12 h to ensure complete dehydration, followed by grinding into a fine powder. Two grams of the powdered eggshells were mixed with 20 mL of a 25% HCl solution and stirred for 1 h, followed by 0.85 mL of 85% phosphoric acid slowly added to maintain a (Ca/P) molar ratio of 1.67. The pH of the solution was adjusted to 10 using aqueous ammonia. The resulting mixture was subjected to hydrothermal treatment at 150 °C for 24 h. After heating, the system was allowed to cool at room temperature, and then the product was washed thoroughly with distilled water to remove the residual ions. The final solid was recovered using vacuum filtration, dried at 80 °C for 24 h, and ground into a powder for further characterization of its properties.
Methods of characterization of nHAp
The biosynthesized nHAp crystals were analyzed using X-ray diffraction (XRD) on a Bruker D8 Advance X-ray Powder Diffractometer (Bruker AXS, Karlsruhe, Germany) equipped with Cu Kα radiation (λ₁ = 1.5406 Å, λ₂ = 1.5444 Å).
The morphological characteristics, particle size distribution, and structural features of the nHAp were analyzed using a JEOL JEM-2100 Transmission Electron Microscope (TEM) (JEOL Ltd., Tokyo, Japan).
High-resolution TEM (HR-TEM) micrographs were acquired using a LaB₆ filament and processed with Gatan Microscopy Suite^®^ software to determine particle size and shape. For crystallographic analysis, selected-area electron diffraction (SAED) patterns were recorded to confirm phase purity and crystallinity.
Dynamic Light Scattering (DLS) and zeta potential measurements were performed to determine the hydrodynamic size and surface charge of the nHAp in suspension using a Zetasizer Nano ZS (Malvern Panalytical, UK) equipped with a 633 nm laser. The zeta potential was used to evaluate the surface charge and colloidal stability of the synthesized nHAp.
In vitro culture of P. alba
The nodal explants of P. alba were surface sterilized as follows: The explants were defoliated and thoroughly washed with tap water. Surface sterilization was carried out using 1.05% (v/v) sodium hypochlorite for 20 min, followed by rinsing four times with sterile double-distilled water under aseptic conditions, 3 min for each rinse.
Surface-sterilized nodal cutting explants were initially cultured on hormone-free Murashige and Skoog (MS) basal medium [15] for four weeks. Morphologically uniform and contamination-free explants were transferred to MS medium supplemented with 0.075 mg/L 6-benzylaminopurine (BAP) to induce shoot proliferation for an additional four weeks. The regenerated shoots were subsequently dissected into single-node segments, defoliated, and aseptically transferred to MS basal medium supplemented with biosynthesized nHAp at concentrations of 0, 20, 40, and 60 mg/L. Each treatment comprised 5 replicate culture jars, each containing 4 nodal explants. Culture media was adjusted to pH 5.8 prior to autoclaving (121 °C, 20 min). The medium was solidified using 2 g/L Phytagel. Cultures were maintained under controlled environmental conditions: 25 ± 2 °C and a 16-h photoperiod under cool white fluorescent lamps (light intensity: 3000 lx). Following four weeks of culture on nHAp-supplemented medium, adventitious roots developed, and the regenerated plantlets were subsequently collected for morphological evaluation, genetic stability assessment, and metabolomic profiling. Morphological data were analyzed using Analysis of Variance (ANOVA), and the means of twenty independent replicas were compared using Tukey’s HSD post-hoc analysis (Honestly Significant Difference) (p ≤ 0.05) utilizing Minitab^®^ software.
Genetic polymorphism analysis
DNA isolation
Genomic DNA was isolated from both control (untreated) plants and nHAp-treated samples grown under identical culture and environmental conditions to ensure that any observed polymorphisms could be specifically attributed to nHAp exposure rather than environmental or cultural variation, using the cetyltrimethylammonium bromide (CTAB) extraction protocol, as standardized by [16]. DNA quality and quantity were evaluated using a NanoDrop™ 1000 spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, USA).
RAPD and SCoT Polymorphism Assays
A preliminary screening of 10 RAPD markers was carried out for studying genetic polymorphism, and four primers, designated Deca-4, Deca-7, Deca-12, and Deca-13, were chosen for further investigation because of their capacity to generate unique, reproducible, and distinct bands. PCR reactions (50 µL) contained 25 µL Red Taq Master Mix (BioLene), 12.5 µL genomic DNA (50 ng/µL), 1 µL primers (10 µM; Biosearch Technologies), and 9.5 µL dd H₂O. Amplification included initial denaturation at 94 °C (3 min), followed by 35 cycles of denaturation (94 °C, 30 s), annealing (30 s), extension (72 °C, 1 min), and final extension (72 °C, 10 min).
For SCoT analysis, 6 SCoT primers were applied to focus on the short, conserved regions that surround the plant gene’s ATG translation start codon. Like ISSR and RAPD, SCoT is a gene-targeted marker with a stronger functional attachment to coding areas. Only 3 gave reproducible bands (SCoT-3, SCoT-5 and SCoT-11), and the thermal cycling conditions were as mentioned in the RAPD-PCR section. The data of both RAPD and SCoT primers are listed in Table 1.
Table 1RAPD analysis of Populus Alba plantlets regenerated in vitro and exposed to varying nHAp concentrationsNo.Primer namePrimer sequence (5’-3’)T (^o^C)GC%Polymorphic bandsTotal bandsPolymorphism %RAPDDeca-4CGTTGGCCCG44802540Deca-7CCGCCCGGAT45801616.67Deca-12CTTGCCCACG38.5704757.14Deca-13GTGGCAAGCC39703742.85Total102539.16%SCoTScoT-3CAACAATGGCTACCACCG53.9561333.33ScoT-6CAACAATGGCTACCACGC54.4561520ScoT-11AAGCAATGGCTACCACCA54.4501250Total31034.4%
PCR products were resolved on a 1.5% agarose gel stained with ethidium bromide (0.5 µg/mL) alongside a 1200 kb Plus DNA ladder, visualized via Gel Doc XR (Bio-Rad). Band patterns were analyzed using ImageLab™ software (Bio-Rad). From each treatment, 3 replications were carried out.
NMR-based metabolomic analysis
Metabolites extraction
The aerial parts of P. alba were collected in liquid nitrogen, frozen at -84 °C for 4 h, and lyophilized for 24 h. The dry samples were ground into a fine powder and thoroughly homogenized. Six replications were utilized for each nHAp concentration. The extraction of metabolites was performed as follows: To each dry sample (20 mg dry weight), 400 µL ice-cold methanol and 380 µL double-distilled water were added, followed by vortex mixing for 30 s, and maintained on ice for 10 min. The mixture was then transferred to a glass tube containing 400 µL ice-cold chloroform, vortexed for 30 s, and incubated on ice for an additional 10 min. The extract was centrifuged at 2000 x g for 10 min at 4 °C. The solvent volume for each sample was determined by calculating the water loss ratio [17]. The polar layer was subjected to vacuum drying with a Centrivap instrument (Labcono, Kansas City, MO, USA) until the solvents were completely removed.
NMR data collection
The dry polar fraction was reconstituted in 620 µL of NMR buffer (TMSP), which comprises the following components: 1 mM TMSP-d4 (deuterated trimethylsilylpropanoic acid), 100 mM sodium phosphate buffer, and 0.01% sodium azide dissolved in 99.9% D_2_O. The NMR data was acquired utilizing the 700 MHz Bruker Avance™ III spectrometer according to Abdelsalam et al., 2025 [18].
Metabolite identification, quantification, and metabolomic analysis
Metabolites were identified by comparing the collected 1D data to the Chenomx NMR Suite V12.0 (Chenomx Inc. of Edmonton, Alberta, Canada). The 2D data (^1^H^-13^ C HSQC) were verified using the Human Metabolome Database (HMDB; https://hmdb.ca/), the Biological Magnetic Resonance Bank (BMRB, https://bmrb.io/), and literature. Metabolite concentrations were determined by calibrating with the internal reference TMSP at a value of 1 mM in the sample spectrum.
MetaboAnalyst 6.0 was utilized to perform the statistical analyses on normalized NMR data. Principal Component Analysis (PCA) was adjusted to include 95% confidence intervals. The statistical significance of group patterns was determined using PERMANOVA, and distributions were estimated using the Euclidean distance. The hierarchical cluster analysis (HCA) used Euclidean distance measurement and Ward clustering linkag. Boxplots were constructed for the most significant metabolites discovered by one-way ANOVA, adjusted to p ≤ 0.05, and then Tukey’s HSD post-hoc analysis.
Pathway analysis
The metabolic responses of P. alba to three treatment levels (20, 40 and 60 mg/L) compared to the control were investigated. Differentially influenced compounds, identified via ANOVA, were retrieved from KEGG and mapped to pathways using the KEGGREST package in R. Affected pathways were identified, and enrichment analysis was performed with a p-value cutoff of 0.05 and BH adjustment. Results were visualized as a flow plot for compound-comparison-pathway flows.
Results
Nano-Hydroxyapatite characterization
TEM images (Fig. 1A & B) indicated that nHAp exhibits a rod-like particle shape, with an average width of 60 nm (Fig. 1C) and an average length of 213 nm (Fig. 1D), maintaining a consistent aspect ratio of 3.5.
Fig. 1TEM image of nHAp with magnification 200 nm (A) and 100 nm (B); (C, D) Histogram of width and length size distribution
Figure 2A displays the XRD pattern of nHAp, revealing distinct Bragg reflections at 2θ angles of 25.0°, 29.0°, 32.0°, 32.8°, 33.0°, 40.0°, 46.0°, 50.0°, and 54.0°, corresponding to the (002), (210), (211), (112), (300), (202), (222), (213), and (004) crystallographic planes.
Fig. 2A) XRD pattern; B) Zeta potential and C) Size distribution of nHAp
Dynamic light scattering analysis indicated that the biosynthesized nHAp had an average zeta potential of -22.1 mV (Fig. 2B) and a hydrodynamic diameter of 300.8 nm (Fig. 2C).
Effect of nHAp on P. alba in vitro culture
The application of biosynthesized nHAp at different concentrations (20, 40, and 60 mg/L) significantly improved the growth parameters of P. alba nodal cuttings (Fig. S1), including fresh weight, shoot length, leaf number, root length, and adventitious root number, compared to the control (Fig. 3). However, it did not show any effect on in vitro adventitious shoot regeneration, as all treatments as well as the control produced only one shoot. The addition of 20 mg/L nHAp into the culture medium resulted in a significant enhancement in total plant fresh weight (0.211 ± 0.002 g) relative to the control, which exhibited values of 0.14 ± 0.001 g. Leaf and root numbers increased significantly with nHAp, recording 9.33 ± 0.6 leaves (60 mg/L) and the highest root counts at 40–60 mg/L, both exceeding the control (5 ± 0.9 leaves). Shoot length was significantly greater at 20 and 40 mg/L (7.0 ± 0.3 cm and 6.8 ± 0.2 cm) than in the control (5.43 ± 0.2 cm), while root length increased across all nHAp treatments (4.1–4.33 cm) compared to control plantlets (3.03 ± 0.15 cm).
Fig. 3. Effects of biosynthesized nHAp on the in vitro growth of P. alba. Data are presented as mean (n = 20). Statistical analysis was performed using ANOVA followed by Tukey’s honestly significant difference (HSD) post hoc test (p ≤ 0.05). Different lowercase letters indicate statistically significant differences among treatments within the same parameter only. Letter groupings shown in different colors represent independent comparisons and indicate significant differences across treatments within each respective parameter (A) Fresh weight of individual plantlet; (B) Number of leaves and adventitious roots per explant; (C) Shoot and root lengths
Effect of nHAp on genetic polymorphism of P. alba
The RAPD analysis, performed with four primers (Deca-4, Deca-7, Deca-12, and Deca-13) on control and nHAp-treated samples, revealed distinct genetic polymorphism patterns (Fig. S2 and Table 1). A total number of 25 amplified fragments were generated with 10 polymorphic bands. Primer Deca-12 exhibited the highest polymorphism characteristics, generating 4 polymorphic bands out of 7 total bands (57.14% polymorphism). In contrast, Deca-7 showed the lowest polymorphism (16.67%), with only 1 polymorphic band detected among 6 total bands. For SCoT analysis, P. alba treated with different concentrations of nHAp shows moderate polymorphism. The primers (SCoT-3, SCoT-6 and SCoT-11) gave a total number of 10 bands, with only 3 polymorphic bands, resulting in 34.4% polymorphism (Fig. S3 and Table 1).
Effect of nHAp on P. alba metabolomic and pathway analysis
A total of 35 metabolites were identified in the spectral region δ 0.5–10 ppm. ^1^H and HSQC chemical shifts of the identified metabolites are listed in Table S1 in the supplementary data. In the aliphatic region δ 0.3-3.0 ppm (Fig. 4A), a group of aliphatic amino acids, e.g. valine, leucine, and isoleucine has been identified along with organic acids. Five sugars were identified in the sugar abundant regions of the control sample (cellobiose, fucose, fructose, glucose and sucrose), with one sugar alcohol (myoinositol) (Fig. 4B). The aromatic region is characterized by aromatic amino acids, phenolic and alkaloid metabolites (Fig. 4C).
Fig. 4^1^H NMR of P. alba in vitro regenerated shoots polar extract. (A); Aliphatic regions between δH 0.8–3.3 ppm (B); Sugar abundant region δH 3.4–5.5 ppm and (C) Aromatic region δH 5.5–9.5 ppm
The addition of nHAp at different concentrations led to qualitative and quantitative changes in P. alba in vitro regenerated shoots (Table 2; Fig. 5). Sucrose was detected only in the control samples but not in the nHAp-treated samples. Also, π-methylhistidine was detected only in control and 20 mg/L nHAp-treated samples, while it was not detected at higher concentrations.
Table 2. Metabolites identified in the Polar extract of *P. alba *in vitro regenerated shoots treated with different nHAp concentrations. A check mark (√) indicates the presence of the metabolite in the respective treatmentCompoundCnHAp 20nHAp 40nHAp 6012-Hydroxyisobutyrate√√√√23-Hydroxyisobutyrate√√√√34-Aminobutyrate√√√√4Alanine√√√√5Arginine√√√√6Asparagine√√√√7Aspartate√√√√8Catechol√√√√9Cellobiose√√√√10Choline√√√√11Formate√√√√12Fructose√√√√13Fucose√√√√14Fumarate√√√√15Glucose√√√√16Glutamate√√√√17Glutamine√√√√18Guanine√√√√19Isoleucine√√√√20Lactate√√√√21Leucine√√√√22Lysine√√√√23Malate√√√√24Phenylalanine√√√√25Pyroglutamate√√√√26Quinic acid√√√√27Succinate√√√√28Sucrose√---29Threonine√√√√30Trigonelline√√√√31Uridine√√√√32Valine√√√√33myo-Inositol√√√√34π-Methylhistidine√√--35Xanthine√√√√
Fig. 51D NMR stacked spectra of polar extract of in vitro regenerated P. alba shoots exposed to different concentrations of nHAp
The 2D PCA score plot (Fig. 6A) showed a distinct clustering pattern among the treatment groups. The control and 20 mg/L nHAp-treated samples exhibited overlap. In contrast, samples treated with higher nHAp concentrations (40 mg/L and 60 mg/L) formed clearly separated clusters. The first two principal components (PC1 and PC2) account for 32.9% and 17.5% of the total variance, respectively. The 3D PCA plot (Fig. 6B) showed complete group separation across all treatment levels, with PC3 contributing 10.7% of explained variance.
Fig. 6. Multivariate statistical analysis of the polar metabolite extracts from in vitro regenerated P. alba in response to nHAp at different concentrations (20–60 mg/L), performed using MetaboAnalyst 6.0. The analysis was conducted on metabolite profiles obtained from 6 replicates per treatment. Prior to analysis, data were normalized, log-transformed, and autoscaled. (A,** B**) 2D and 3D PCA score plots illustrating metabolic separation among treatments; ellipses indicate 95% Hotelling’s T² confidence intervals. (C) PCA biplot highlighting metabolites contributing most strongly to group discrimination; the ten most statistically significant metabolites are labeled. (D) Hierarchical cluster analysis (HCA) dendrogram generated using Euclidean distance and Ward linkag, showing similarities among treatments based on polar metabolite profiles
Metabolites responsible for group separations are shown in the biplot (Fig. 6C). Metabolites such as cellobiose, isobutyrate and isoleucine are positively associated with the 60 mg/L nHAp group. Conversely, catechol was associated with 40 mg/L treated samples. Myo-inositol, uridine, malate, and aspartate are associated with the control and 20 mg/L nHAp groups.
The HCA dendrogram (Fig. 6D) supports the PCA findings. The dendrogram shows two main clusters, with control, 20 mg/L, and 40 mg/L nHAp grouped in one cluster, while 60 mg/L nHAp treatment is in the other cluster. Control and 20 mg/L nHAp samples form closely related clusters. The ANOVA analysis shows the metabolites that were significantly changed in the in vitro regenerated P. alba after addition of nHAp at p ≤ 0.05 (Fig. 7).
Fig. 7. One-way analysis of variance (ANOVA) of the polar metabolic profiles of in vitro regenerated P. alba showing significant metabolite alterations in response to addition nHAp. Metabolomic data were obtained from 6 biological replicates per treatment and processed using MetaboAnalyst 6.0
The relative concentrations of the significantly changing metabolites (due to the addition of nHAp) are represented with box plots based on ANOVA (Table S2). Treatment of P. alba with nHAp induced a dose-dependent reprogramming of metabolite profiles, as revealed by box plot analysis (Fig. 8). Different concentrations of nHAp upregulated phenylalanine. On the other hand, higher concentrations of nHAp enhanced the accumulation of amino acids γ aminobutyrate, asparagine, isoleucine, and valine. Sugars had different responses to nHAp: higher concentrations (60 mg/L), increased cellobiose, while lower concentration (20 mg/L) increased fucose. Xanthine levels consistently declined following the addition of nHAp. Catechol concentrations increased significantly under higher nHAp treatments (40 and 60 mg/L), while trigonelline content was markedly enhanced at 20 and 40 mg/L.
Fig. 8. Boxplots illustrating the relative concentrations of the twenty most significant metabolites, identified based on one-way ANOVA, in in vitro regenerated P. alba treated with different concentrations of nHAp (C; 20, 40, and 60 correspond to 0, 20, 40, and 60 mg L⁻¹ nHAp, respectively). Black dots represent individual biological replicates, and yellow diamonds indicate the mean metabolite concentration for each treatment. The Y-axis shows relative metabolite abundance, and the X-axis indicates the treatment groups. Statistically significant differences (p ≤ 0.05) are marked by different lowercase letters above the boxes
Pathway enrichment analysis (adjusted p ≤ 0.05) revealed distinct, concentration-dependent metabolic responses with partial overlap driven by shared metabolites such as γ-aminobutyrate, phenylalanine, fucose, and xanthine (Fig. 9). Compared with the control, the low nHAp treatment (20 mg/L) significantly enriched twelve pathways; the most significant pathways were aminoacyl-tRNA biosynthesis, ABC transporters, and D-amino acid metabolism. The medium concentration (40 mg/L) altered ten pathways, primarily aminoacyl-tRNA biosynthesis, alanine, aspartate and glutamate metabolism, and ABC transporters. The high concentration (60 mg/L) influenced eight pathways, with the strongest effects observed on aminoacyl-tRNA biosynthesis, ABC transporters, and cyanoamino acid metabolism.
Fig. 9. Pathway-metabolite relationships under different nHAPs concentrations. ABTR: ABC transporters, ALAS: Alanine, aspartate and glutamate metabolism, AMBI: Aminoacyl-tRNA biosynthesis, ARAN: Arginine and proline metabolism, BUME: Butanoate metabolism, CICY: Citrate cycle (TCA cycle), D-AC: D-Amino acid metabolism, GLBI: Glucosinolate biosynthesis, TAAN: Taurine and hypotaurine metabolism, TRPI: Tropane, piperidine and pyridine alkaloid biosynthesis, VALE1: Valine, leucine and isoleucine biosynthesis, VALE2: Valine, leucine, and isoleucine degradation
Discussion
This study aimed to comprehensively evaluate the morphological, genetic, and metabolomic responses of in vitro regenerated P. alba plants to biosynthesized nHAp. The results revealed that nHAp acts not merely as a source of macronutrients but as a dynamic nano-elicitor, inducing a concentration-dependent, systemic adaptive response. The nanoparticles promote robust morphological growth while simultaneously triggering a stress acclimation program at the metabolic and genomic levels.
The successful biosynthesis of nHAp was confirmed using physicochemical characterization. TEM analysis revealed that the biosynthesized nHAp are rod-like in morphology with average widths of 60 nm and lengths of 213 nm. Previous studies have reported the rod-like shape of nHAp but with smaller dimensions: a width range of 20–50 nm and a length up to ~ 139 nm [15]. The larger dimensions observed in our system may reflect a slight difference in biosynthetic conditions, such as variations in precursor concentration, reaction kinetics, or ionic interactions, all of which are well known to shape size and morphology in nHAp synthesis [19]. The X-ray diffraction displayed clear Bragg reflections characteristic of highly crystalline, hexagonal-phase hydroxyapatite, confirming successful phase formation [15, 19]. Hydrodynamic diameter measured by DLS was ~ 300 nm – substantially larger than the TEM-determined physical sizes, suggesting significant agglomeration and the presence of hydration layers. These differences between physical and hydrodynamic dimensions are common in nHAp characterization and have been documented in previous studies [20].
nHAp significantly enhanced vegetative growth of P. alba nodal cuttings, increased plant weight, length and adventitious root number. This response pattern is consistent with the nature of nHAp, which acts primarily as a slow-release source of phosphorus and calcium that supports biomass accumulation, cell elongation, chlorophyll content and root system development [21], rather than functioning as a substitute for the cytokinin, which are necessary to induce shoot formation. In woody species, including Populus, shoot regeneration and multiplication are complex processes tightly governed by several factors including phytohormones, especially cytokinin [22]. Enhanced rooting with higher nHAp concentrations aligns with the role of Ca²⁺ as a messenger in root development as it interacts with auxin transport/signaling during root initiation and growth [23]. In Populus spp., the adventitious root formation has been reported to depend mainly on auxin type and concentration [24].
This robust enhancement in vegetative growth parameters correlates directly with the metabolic reprogramming revealed by NMR analysis. This growth phenotype appears to be driven by a coordinated metabolic flux where energy reserves are reallocated toward structural and developmental pathways. Specifically, the depletion of sucrose in nHAp-treated plants, coupled with the accumulation of cell wall precursors (fucose and cellobiose), suggests a shift from carbon storage to structural carbohydrate synthesis necessary for supporting increased biomass. In plants, sucrose is the primary form of transported carbon and a central metabolite in energy storage, signaling, and a precursor in the biosynthesis of key metabolites like cellulose and starch [25]. Fucose is involved in cell wall biosynthesis, which is a critical process for growth and development [26]. Fucose plays a multifaceted role in plant immunity by supporting stomatal and apoplastic defenses, mediating pattern- and effector-triggered immune responses, and facilitating protein fucosylation [27].
Phenylalanine and quinic acid were significantly elevated in nHAp-treated samples, while catechol levels increased noticeably at higher concentrations (40 and 60 mg/L). Phenylalanine, a central precursor in the phenylpropanoid pathway, supplies essential building blocks for lignin and flavonoids, which are critical for cell wall reinforcement, vascular differentiation, and biomass formation [28–30]. This pathway also provides precursors for bioactive secondary metabolites, supporting plant development and adaptive metabolism [30, 31]. The phenylalanine metabolic pathway has been documented in many Populus spp., governed by numerous trans-regulatory gene networks, with the differential expression of these genes being a critical determinant of growth rate and wood quality [32]. Quinic acid accumulation is particularly noteworthy; beyond its role as a potential calcium carrier [33], it functions as a metabolic modulator, promoting secondary metabolite biosynthesis and influencing phytohormone networks related to growth and development [34], consistent with its identification as a biomarker for enhanced biomass in species such as Brassica napus [35]. Catechol, a basic phenolic compound, serves as a precursor for complex lignin and flavonoid polymers and contributes to antioxidant capacity, supporting plant growth and cellular integrity [36–38].
Furthermore, the significant enrichment of ABC transporters provides the molecular machinery required to sustain the high protein synthesis rates demanded by this accelerated growth. ABC transporters significantly changed in all nHAp treated samples. ABC transporters form one of the largest membrane-protein families in plants, where they drive the ATP-dependent translocation of diverse substrates including phytohormones, lipids, and xenobiotic conjugates across cellular membranes. By mediating nutrient distribution, hormone signaling, and detoxification, these transporters are crucial for plant growth, organ development, and adaptation to both biotic and abiotic stress conditions [39–42]. Also, the aminoacyl-tRNA biosynthesis pathway significantly changed across all nHAp concentrations. This pathway plays a pivotal role in plant development. By catalyzing the covalent attachment of specific amino acids to their cognate transfer RNAs (tRNAs), this pathway generates aminoacyl-tRNAs the essential substrates for ribosomal protein synthesis [43]. Numerous investigations have shown that chloroplast aspartyl-tRNA synthetase is essential for chloroplast formation and enhancement of photosynthetic efficiency [44].
These metabolic adjustments occur in parallel with genomic changes, as revealed by RAPD and SCoT analyses. RAPD profiling was performed to assess genetic alterations induced by the addition of nHAp at different concentrations. The RAPD assay indicated a polymorphism percentage of 39.16%. Such variations may result from multiple nanoparticle-induced mechanisms, including oxidative stress that generates DNA strand breaks, base modifications, or altered methylation patterns. Indeed, nanoparticles at certain concentrations are known to interact with nuclear DNA directly or indirectly via the production of reactive oxygen species, which may lead to mutation [45, 46]. According to SCoT markers, the current study shows that P. alba exposed to varying nHAp concentrations developed moderate genetic variability. SCoT markers are highly repeatable and beneficial in identifying genetic changes brought on by stress because they target the small, conserved area that surrounds the ATG start codon. The bioactive nHAp may function as an abiotic elicitor, possibly inducing genomic reactions and genetic diversity in P. alba, based on the reported average polymorphism rate of 34.4%. According to related research, nanoparticles can cause genomic changes and DNA polymorphisms in plants that represent stress or adaptive responses [47]. The highest polymorphism was notably created by SCoT-11, suggesting that primer selection has a significant impact on variability detection. These findings are in line with earlier publications from other plant species where genomic alterations under stress or treatment were successfully identified using SCoT analysis. Therefore, in addition to being a growth-promoting factor in tissue culture, nHAp may also help P. alba undergo modest genetic changes that might be used for biotechnology and tree enhancement initiatives [48]. This genomic variability may represent adaptive responses, providing a substrate for stress acclimation and potential biotechnological applications. The elicited metabolic response likely serves as protective acclimation, buffering against genomic instability. For example, high nHAp concentrations (60 mg/L) induced the accumulation of GABA, asparagine, isoleucine, and valine metabolites. GABA functions as stress signaling molecule, which regulates cytosolic pH, carbon–nitrogen balance, and reactive oxygen species scavenging under abiotic stress conditions [49, 50]. Asparagine is a pivotal nitrogen carrier that plays a crucial function in the plant development process, encompassing seed germination, vegetative growth, and seed maturity, especially under stress conditions [51]. Isoleucine and valine, as branched-chain amino acids, play multifaceted roles in plant metabolism. They serve as essential building blocks for protein synthesis and act as precursors for the production of different secondary metabolites. Different studies indicate that branched-chain amino acids contribute to stress tolerance by facilitating osmoprotection, scavenging reactive oxygen species, and participating in defense pathways against abiotic stresses [52].
Furthermore, significant changes in the aminoacyl-tRNA biosynthesis pathway are crucial for maintaining translational fidelity under stress. By ensuring the accurate aminoacylation of tRNAs, aminoacyl-tRNA synthetases prevent the incorporation of incorrect amino acids and the formation of misfolded or toxic proteins, a risk that is particularly heightened during stress conditions [53]. Numerous metabolomic studies have identified this pathway as one of the most responsive pathways to stress conditions. For example, significant alterations in this pathway have been reported in wheat plants exposed to heat stress [54] and in Vicia faba subjected to lead stress [55]. This pathway also influences plant immunity, promoting the production of defensive metabolites and regulating hormone signaling networks [56].
The metabolic response also extended to cyanoamino acid metabolism, which was significantly altered at 60 mg/L nHAp. This shift suggests a targeted activation of cyanide detoxification pathways, contributing to cellular protection and enhanced tolerance to the abiotic stress [57, 58]. Furthermore, this pathway is closely linked to the biosynthesis of cyanogenic glycosides. These compounds serve a dual role: as defensive metabolites against herbivores and pathogens and as contributors to oxidative stress mitigation by scavenging reactive oxygen species [59].
Conclusion
This study provides a comprehensive evaluation of the interactions between biosynthesized nHAp and in vitro regenerated P. alba. nHAp application elicited a concentration-dependent and multifaceted response, acting as an effective growth-promoting agent. The integration of metabolomic analyses reveals that this growth promotion is driven by a coordinated metabolic reprogramming. Specifically, the depletion of sucrose coupled with the accumulation of fucose, cellobiose, and amino acids indicates a shift in carbon partitioning toward cell wall biosynthesis and nitrogen assimilation, supported by the enrichment of aminoacyl-tRNA biosynthesis and ABC transporter pathways. Furthermore, the moderate genetic polymorphism observed via RAPD and SCoT markers suggests that nHAp triggers an adaptive genomic response that facilitates stress acclimation rather than causing genomic instability. Overall, the study demonstrates the potential of nHAp as a promising nanobiotechnological tool in plant biotechnology, particularly for enhancing in vitro growth and promoting secondary metabolite production. The observed growth-promoting effects and associated metabolic/genetic responses were assessed under controlled in vitro conditions, and their long-term stability during ex vitro acclimatization and under field environments remains to be verified in future experiments.
Supplementary Information
Supplementary Material 1.
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