Magnesium Hydride Enhances Both Growth and Bioactive Compound Biosynthesis in Salvia miltiorrhiza: A Novel Eco-Friendly Approach for Medicinal Plant Cultivation

Abstract

Medicinal plants have garnered widespread attention owing to their broad range of biological activities and pharmaceutical value. However, achieving a balance between promoting plant growth and enhancing bioactive compound accumulation remains a major challenge in current research. In this study, Salvia miltiorrhiza was selected as the model organism to investigate the effects of MgH₂ on growth and the accumulation of bioactive compounds. Our results demonstrated that MgH₂ treatment at 10.0 mg kg⁻¹ soil increased the seed germination rate by 49.3%. At the optimal concentration of 12.5 mg kg⁻¹ soil, MgH₂ significantly promoted seedling growth, enhancing root biomass by 745.6% after 4 weeks of treatment. Furthermore, MgH₂ application dramatically boosted the accumulation of bioactive compounds, increasing the total content of rosmarinic acid and dihydrotanshinone by 1271.8% and 2407.7%, respectively. Transcriptome analysis revealed that these improvements were associated with the upregulation of genes involved in plant hormone signal transduction, energy metabolism, and the biosynthesis pathways of tanshinones and salvianolic acids. Our findings provide the first evidence that MgH₂ acts as a dual-functional regulator, simultaneously enhancing both plant growth and secondary metabolite accumulation in medicinal plants, offering a green and efficient cultivation strategy for the sustainable production of high-quality S. miltiorrhiza.

1. Introduction

Medicinal plants are widely acknowledged for their extensive bioactivities and pharmaceutical value [1] (Gao et al., 2024). Salvia miltiorrhiza Bunge is a perennial medicinal plant in the genus Salvia of the family Lamiaceae [2]. Salvia miltiorrhiza, commonly known as Danshen, typically 30–60 cm in height with erect stems, opposite leaves, and distinctive whorled purple-blue flowers, is extensively cultivated across provinces such as Shandong, Henan, Shanxi, and Anhui. Its primary medicinal part is the root, which contains various bioactive compounds with antioxidant [3], anti-inflammatory [4], antitumor [5], and cardiovascular protective properties [6], such as tanshinones and salvianolic acids [7]. Due to its wide range of pharmacological activities, S. miltiorrhiza has been extensively applied in traditional Chinese medicine for treating cardiovascular and cerebrovascular diseases, liver diseases, cancer, and other conditions [8]. Moreover, S. miltiorrhiza, owing to its relatively short life cycle, ease of propagation and cultivation, relatively small genome size (approximately 538 Mb) [9,10], and well-established genetic transformation [11,12,13] and gene editing systems [14], has emerged as one of the model systems in medicinal plant biology [15]. However, due to environmental factors, planting conditions, and cultivation techniques, the yield and quality of S. miltiorrhiza exhibit considerable variability [16,17,18,19], affecting its application in modern pharmaceutical industries. Various exogenous elicitors, including methyl jasmonate (MeJA), salicylic acid (SA), silver ions (Ag⁺), and yeast extract (YE), have been employed to enhance the accumulation of bioactive compounds in medicinal plants [20,21,22,23,24,25]. Nevertheless, achieving a balance between promoting plant growth and enhancing active compound biosynthesis remains a major challenge [26]. Therefore, there is an urgent need to develop eco-friendly methods capable of simultaneously promoting plant growth and increasing the accumulation of active compounds.

Magnesium hydride (MgH₂) is a metal hydride with a high hydrogen storage capacity. It reacts with water to produce hydrogen gas (H₂) and magnesium hydroxide (Mg(OH)₂) at room temperature [27]. This characteristic has led to its widespread use in energy storage and fuel cell technologies. Recently, with a deeper understanding of plant physiological regulation, the potential of MgH₂ in agriculture and horticulture has gradually gained attention. Studies have shown that H₂ released from MgH₂ can act as a signaling molecule, regulating various physiological processes in plants, such as seed germination, photosynthesis, and stress resistance [28,29,30,31]. Furthermore, Mg(OH)₂ can slowly release magnesium ions (Mg²⁺) in soil, participating in chlorophyll synthesis and improving photosynthetic efficiency [32,33]. Therefore, MgH₂, as a novel plant growth regulator, shows great promise for application. Previous studies have demonstrated that MgH₂ exhibits significant physiological regulatory effects across different plant species. For instance, MgH₂ can extend the vase life of cut flowers by regulating nitric oxide (NO) and hydrogen sulfide signaling [34,35] and alleviate copper toxicity in alfalfa seedlings [36]. Moreover, MgH₂ can mitigate cadmium-induced damage in rice seedlings by influencing RNA m6A methylation modifications [29] and reduce oxidative injury caused by osmotic stress through activation of the ascorbate-glutathione cycle [30]. These studies collectively indicate that MgH₂ plays a crucial role in plant stress tolerance and growth regulation. However, its application in medicinal plants, particularly its potential to concurrently enhance growth and bioactive compound accumulation, remains unexplored.

To investigate the role of MgH₂ in medicinal plants, we selected S. miltiorrhiza, a well-studied medicinal model plant, as the subject of our research. Seedlings were treated with varying concentrations of 10.0, 12.5, and 15 mg MgH₂ kg⁻¹ soil [30,35], and the results demonstrated that MgH₂ positively influenced seed germination and seedling growth and development, while significantly enhancing the accumulation of active compounds such as tanshinones and salvianolic acids. The mechanisms underlying this phenomenon were explored by integrating transcriptomic sequencing technology. This study provides the first evidence of these effects, highlighting the potential of MgH₂ to promote both plant growth and secondary metabolite biosynthesis in medicinal plants. Optimizing the concentration and timing of MgH₂ application has led to a green and efficient cultivation strategy for S. miltiorrhiza, which can be applied to other medicinal plants as well.

2. Materials and Methods

2.1. Plant Materials and Cultivation Conditions

The S. miltiorrhiza seeds used in this study were from a genetically stable line preserved in the germplasm repository of the College of Life Sciences and Medicine, Zhejiang Sci-Tech University. These seeds are maintained under standard conditions (4 °C, low humidity) to ensure viability and genetic integrity. After screening, plump and uniformly sized seeds were selected for experimentation. The experimental soil substrate consisted of a mixture of nutrient soil, vermiculite, and perlite at a volume ratio of 3:1:1, ensuring good aeration and water retention [36]. Soil moisture was maintained at approximately 60% water-holding capacity, ensuring no visible water discharge upon compression. The plants were cultivated in a growth chamber under the following conditions: a 16/8 h light/dark photoperiod with a photosynthetic photon flux density (PPFD) of 300 μmol m⁻² s⁻¹, day/night temperatures of 25/20 °C, and 65% relative humidity.

2.2. MgH₂ Treatments

The MgH₂ reagent was provided by the Center of Hydrogen Science, Shanghai Jiao Tong University. Based on literature reports and preliminary experimental results, the optimal application concentration range of MgH₂ was determined to be 10.0–15.0 mg kg⁻¹ soil. Three treatment concentrations and a control group were established: 10.0 mg MgH₂ kg⁻¹ soil (Group a), 12.5 mg MgH₂ kg⁻¹ soil (Group b), 15.0 mg MgH₂ kg⁻¹ soil (Group c), and an untreated control group (Group ck). Each treatment had at least 3 biological replicates to ensure data reliability. MgH₂ powder was uniformly applied into the soil at designated concentrations during soil preparation and thoroughly mixed.

The seeds of S. miltiorrhiza were selected and rinsed 3–5 times with distilled water. The treated seeds were then sown into soil mixtures containing different concentrations of MgH₂. Each culture pot was sown with 200 seeds, with 3 biological replicates per treatment group. The seed germination rate was determined after 14 days to assess the impact of MgH₂ on S. miltiorrhiza seed germination.

2.3. Growth and Photosynthetic Analysis of S. miltiorrhiza Seedlings

After germination and the emergence of two true leaves, S. miltiorrhiza seedlings were transplanted into pots. Each pot contained 1 kg of moist soil, with 8 seedlings planted per pot. Two weeks after transplantation, the seedlings, which had been growing for approximately 5 weeks, were treated with MgH₂. Samples were collected every fortnight, data were recorded, and fresh samples were stored for further analysis. From each treatment group, six seedlings were selected for measurement. Photosynthetic parameters were determined using the LCPRO+ photosynthesis system. The following growth parameters were recorded: plant height, root length, leaf length, leaf width, and leaf count. Additionally, the fresh and dry weights of the roots, stems, and leaves were measured. Note: Plant height was defined as the vertical distance from the highest growth point to the soil surface. Root length was measured from the soil surface to the tip of the longest primary root. Leaf length and width were measured at maximum expansion points. Leaf count: total number of leaves per plant. Fresh and dry weights: fresh weights of aerial parts (stems and leaves) and roots were weighed separately, followed by drying at 45 °C for 48 h to determine dry weights.

2.4. Extraction and Quantification of Secondary Metabolites from S. miltiorrhiza Roots

Root samples of S. miltiorrhiza were thoroughly cleaned and weighed to 0.5 g. These samples were then dried in an oven at 45 °C for 48 h to ensure complete moisture evaporation. The dried samples were subsequently ground using a tissue homogenizer to obtain a fine powder. An accurate amount (0.02 g) of the powdered sample was weighed and transferred into a 2.0 mL centrifuge tube. To this, 1.5 mL of 70% methanol was added for extraction. The mixture was subjected to ultrasonic extraction for 1 h in an ultrasonic bath, with periodic shaking every 15 min to ensure even extraction. Following extraction, samples were centrifuged at 12,000 rpm for 10 min. The supernatant was carefully aspirated using a 2 mL syringe and filtered through a 0.22 μm membrane filter to remove any particulate impurities. Chromatographic analysis of metabolites was carried out using high-performance liquid chromatography (HPLC) under the following analytical conditions. Separation was performed on a reversed-phase C18 column (4.6 mm × 250 mm, 5 μm particle size), maintained at a constant temperature of 30 °C. The mobile phase consisted of two components: solvent A (acetonitrile) and solvent B (0.02% phosphoric acid in water). A gradient elution program was applied to optimize the separation of target compounds. The flow rate was set at 1.0 mL/min throughout the run. Detection was carried out using a photodiode array (PDA) detector. For fat-soluble diterpenes (e.g., tanshinones), the detection wavelength was set at 270 nm; for water-soluble phenolic acids (e.g., salvianolic acids), it was set at 288 nm. The identification of individual compounds was confirmed by comparing their retention times with those of authentic reference standards. All standard substances were purchased from the China National Institute for Food and Drug Control (Beijing, China). Data were analyzed using the Empower 3 software system (Waters Corporation, Milford, MA, USA). Metabolite concentrations were determined and calculated as described previously [37,38], ensuring accuracy and reproducibility across samples.

2.5. Transcriptome Sequencing of S. miltiorrhiza Seedling Roots

Root samples from Group b and Group ck at 4 weeks, which exhibited the most significant growth differences, were selected for transcriptomic sequencing. To further explore the molecular mechanisms by which MgH₂ promotes the growth and accumulation of active compounds in S. miltiorrhiza, root samples from the 4-week treatment group (12.5 mg MgH₂ kg⁻¹ soil, Group b) and the control group (ck) were subjected to transcriptomic sequencing. Sequencing was performed by Wuhan Benagen Tech Solutions Company Limited. RNA was extracted using the Trizol reagent (Invitrogen, Carlsbad, CA, USA), and RNA integrity was assessed by NanoDrop (Thermo Fisher, Waltham, MA, USA) and Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). High-quality RNA was then used for library construction, followed by paired-end sequencing (150 bp) on the Illumina NovaSeq 6000 platform. Rigorous quality control procedures were applied to filter out low-quality or contaminated sequences, ensuring data reliability and accuracy. The resulting data were analyzed through alignment to the reference genome, quantification of gene expression levels, identification of differentially expressed genes (DEGs), functional enrichment analysis, and investigation of novel genes and alternative splicing events. These analyses aimed to provide insights into the molecular mechanisms underlying the observed phenotypic differences between the experimental groups. The criteria for selecting DEGs were log₂FoldChange ≥1 or ≤−1 and FDR < 0.05. KEGG enrichment analysis was employed to identify key pathways associated with growth, development, and secondary metabolism. The quality of the transcriptome sequencing data was assessed by Principal Component Analysis (PCA). The raw RNA-seq data have been deposited in the NCBI SRA database. The accession number is PRJNA1369401.

2.6. RNA Extraction and Quantitative RT-PCR (qRT-PCR)

Total RNA was extracted using Transzol reagent (TransGen Biotech, Beijing, China) following the manufacturer’s protocol. First-strand cDNA was synthesized from 1 μg of total RNA using the M-MLV Reverse Transcriptase kit (Invitrogen, Grand Island, NE, USA). Quantitative real-time PCR (qRT-PCR) was performed on an ABI 7500 Real-Time PCR System (Applied Biosystems, Waltham, MA, USA) with FastStar Universal SYBR Green Master mix (Roche, Basel, Switzerland). The Salvia miltiorrhiza actin gene (XM_057912742.1) was used as the reference gene for normalization. Relative expression levels of target genes were calculated using the 2^−ΔΔCt method. All gene-specific primers used for qRT-PCR are listed in Table S2.

2.7. Data Statistics and Analysis

All experimental data were statistically analyzed using SPSS Statistics (v.27) and GraphPad Prism (v.10.1). One-way analysis of variance (ANOVA) was performed for statistical comparisons, followed by Tukey’s multiple comparison test (p < 0.05) or two-tailed Student’s t-test (p < 0.05) as appropriate. All experiments were conducted with at least 3 biological replicates to ensure data reliability and reproducibility. All figures were generated using GraphPad Prism (v.10.1), Adobe Photoshop (v.2024), and Adobe Illustrator (v.2024).

For the gene expression heatmap, the transcriptomic data were processed and visualized as follows to ensure clarity and accurate representation of the treatment effects. The analysis was based on Fragments Per Kilobase of transcript per Million mapped reads (FPKM) values obtained from root samples of the MgH₂-treated group (Group b) and the control group (Group ck) at 4 weeks.

To prepare the data for visualization, a two-step transformation was applied. First, raw FPKM values were subjected to a log₂(FPKM + 1) transformation. The addition of 1 prevents undefined values when taking the logarithm of zero and helps mitigate the right-skewed distribution typical of expression data, making the distribution more suitable for downstream analysis. Subsequently, the log-transformed data were Z-score normalized across samples for each gene using the formula Z = (x−μ)/σ, where x is the log-transformed value, μ is the mean, and σ is the standard deviation. This standardization centers the data around a mean of 0 with a standard deviation of 1, allowing for an intuitive comparison of relative expression changes across different genes. The normalized Z-score matrix was used to generate the hierarchical clustering heatmap. The color scale was explicitly defined to reflect the direction and magnitude of change: a Z-score of 0 is represented as white, indicating expression at the average level. Positive Z-scores (up-regulation in the treated group) are represented in a gradient of red, while negative Z-scores (down-regulation) are represented in a gradient of blue.

Hierarchical clustering analysis was performed to visualize the global effects of MgH₂ treatment on phenotypic traits and metabolite accumulation. For phenotypic clustering (Figure S2), the dataset included all growth parameters (plant height, root length, leaf length, leaf width, leaf number) and physiological indices (aerial part/root fresh and dry weights, ΔC, Pn). For metabolic clustering (Figure S3), the contents of seven major bioactive compounds (caffeic acid, rosmarinic acid, salvianolic acid B, cryptotanshinone, tanshinone IIA, dihydrotanshinone, and tanshinone) were analyzed. All data were z-score normalized to give equal weight to each variable. Hierarchical clustering was performed in R (version 4.2.0) using the pheatmap package (version 1.0.12), employing the clustering distance rows parameter.

3. Results

3.1. MgH₂ Treatment Promotes Seed Germination in S. miltiorrhiza

To investigate the effects of MgH₂ on seed germination, plump and uniformly sized S. miltiorrhiza seeds were sown in pots with soil MgH₂ concentrations of 0 mg MgH₂ kg⁻¹ soil (Group ck), 10.0 mg MgH₂ kg⁻¹ soil (Group a), 12.5 mg MgH₂ kg⁻¹ soil (Group b), and 15.0 mg MgH₂ kg⁻¹ soil (Group c). After two weeks of treatment, germination rates were recorded. The results demonstrated that MgH₂ significantly enhanced seed germination. The germination rate was 33.5% in Group ck, 50.0% in Group a, 46.8% in Group b, and 42.0% in Group c (Figure 1). Compared to Group ck, the germination rates increased by 49.3% in Groups a, 39.7% in Groups b, and 25.4% in Groups c, with Group a displaying the most pronounced improvement. These findings indicate that MgH₂ effectively promotes S. miltiorrhiza seed germination within a specific concentration range, with 10.0 mg MgH₂ kg⁻¹ soil identified as the optimal concentration.

3.2. MgH₂ Enhances Seedling Shoot Growth and Development in S. miltiorrhiza

Five-week-old S. miltiorrhiza seedlings were treated with 0 mg (Group ck), 10.0 mg soil (Group a), 12.5 mg (Group b), or 15 mg (Group c) MgH₂ kg⁻¹ soil for eight weeks to systematically analyze its effects on seedling growth. Morphological and physiological parameters of the shoots were measured biweekly. From the second week onward, MgH₂-treated seedlings exhibited accelerated shoot growth, outperforming Group ck in plant height, leaf length, leaf width, leaf number, and aerial parts fresh/dry weights (Figure 2A–J and Figure S1). Notably, Group b displayed the most significant growth advantage after four weeks of treatment, with plant height increasing by 140.3% (Figure 2E), aerial parts fresh weight by 311.1% (Figure 2I), and aerial parts dry weight by 414.3% compared to Group ck (Figure 2J). These results suggest that MgH₂ markedly promotes shoot development in S. miltiorrhiza seedlings.

Given prior evidence that Mg²⁺ enhances photosynthesis, we evaluated the impact of MgH₂ on CO₂ assimilation (ΔC) and net photosynthetic rate (Pn) in seedlings. MgH₂ treatment, particularly in Group B at four weeks, significantly elevated ΔC and Pn by 125.9% (Figure 2N) and 169.0% (Figure 2O), respectively, indicating improved photosynthetic efficiency. As photosynthesis drives plant growth, these results imply that MgH₂ likely facilitates seedling development by augmenting energy production via enhanced photosynthesis.

3.3. MgH₂ Increases Root Biomass in S. miltiorrhiza

The medicinal value of S. miltiorrhiza primarily resides in its roots; thus, root biomass directly influences yield and quality. Previous studies have shown that H₂ promotes root elongation and lateral root formation [39]. Our data revealed that MgH₂ treatment significantly increased root length (Figure 2K), fresh weight (Figure 2L), and dry weight (Figure 2M), with the most pronounced effects observed at four weeks. Group b exhibited a 90.2% increase in root length, 745.6% higher fresh weight, and 584.5% greater dry weight relative to Group ck (Figure 2K–M). These findings demonstrate that MgH₂ not only stimulates shoot growth but also enhances root development, enabling more efficient water and nutrient uptake, thereby bolstering overall plant vigor. The dual mechanisms of MgH₂ action may involve (i) boosting carbon assimilation via photosynthesis and (ii) strengthening root systems to improve resource acquisition.

3.4. MgH₂ Elevates the Accumulation of Tanshinones and Salvianolic Acids in S. miltiorrhiza Roots

S. miltiorrhiza contains two major classes of bioactive constituents: the hydrophilic salvianolic acids (e.g., caffeic acid, rosmarinic acid, salvianolic acid B) and the lipophilic tanshinones (e.g., cryptotanshinone, tanshinone IIA, dihydrotanshinone, tanshinone). These compounds collectively exert potent antioxidant, anti-inflammatory, and cardioprotective activities. HPLC analysis revealed that MgH₂ treatment significantly increased the unit and total contents of these compounds in roots, and the differences were most obvious in group b after 2 and 4 weeks of MgH₂ treatment.

After two weeks of treatment, the unit content of caffeic acid (Figure 3A), rosmarinic acid (Figure 3B), salvianorin B (Figure 3C), cryptotanshinone (Figure 4A), tanshinone IIA (Figure 4B), dihydrotanshinone (Figure 4C), and tanshinone (Figure 4D) in the roots of group b rose by 53.2%, 704.1%, 376.5%, 568.0%, 9.6%, 222.3%, and 813.6%, respectively. By four weeks, the total root content of these compounds increased by 393.6%, 1271.8%, 1054.1%, 1529.4%, 634.1%, 2407.7%, and 2104.9%, respectively (Figure 3A–C and Figure 4A–D).

Strikingly, the magnitude of the increase in bioactive compounds varied significantly among individual metabolites. For instance, rosmarinic acid and dihydrotanshinone exhibited the most dramatic rises in both unit and total content, with rosmarinic acid showing increases of 704.1% in unit content and 1271.8% in total content after two and four weeks (Figure 3B), respectively, and dihydrotanshinone demonstrating 222.3% and 2407.7% increases (Figure 4C). In contrast, compounds such as caffeic acid and tanshinone IIA displayed relatively modest gains in unit content (rising by 53.2% and 9.6% at two weeks, respectively) (Figure 3A and Figure 4B). Crucially, however, the substantial enhancement in root biomass amplified the accumulation of these metabolites, driving their total contents to surge by 393.6% for caffeic acid and 634.1% for tanshinone IIA by four weeks (Figure 3A and Figure 4B). The unit content reflects the metabolic intensity within the root tissue, while the total content per plant represents the integrated yield. The remarkable increases in total content, particularly for metabolites with more moderate rises in unit concentration, underscore that the boost in root biomass is a major driver for enhancing the net production of bioactive compounds. These disparities likely reflect distinct regulatory mechanisms governing phenylpropanoid (e.g., involving PAL and 4CL) and terpenoid (e.g., involving HMGS and MVK) metabolic pathways, suggesting that MgH₂ differentially modulates secondary metabolite biosynthesis by both stimulating specific enzymatic pathways and leveraging root system expansion to boost overall resource acquisition and compound yield.

3.5. Multivariate Analysis Reveals Coordinated Enhancement by MgH₂

To gain a comprehensive understanding of MgH₂’s global effects, we performed hierarchical clustering analysis on both phenotypic and metabolic datasets. The phenotypic clustering (Figure S2) clearly separated MgH₂-treated samples from the control group, with Group b (12.5 mg MgH₂ kg⁻¹ soil) demonstrating the most consistent and substantial improvements across most key growth and photosynthetic parameters. Similarly, metabolic clustering (Figure S3) revealed that Group b formed a distinct cluster exhibiting the most pronounced and coordinated enhancement in the accumulation of both salvianolic acids and tanshinones. Based on these multivariate analyses demonstrating the superior and most consistent performance of the 12.5 mg kg⁻¹ treatment across the majority of measured traits, Group b was selected for subsequent transcriptome sequencing to elucidate the molecular mechanisms underlying MgH₂’s optimal efficacy.

3.6. Transcriptomic Analysis Reveals Potential Mechanisms

To elucidate the molecular mechanisms underlying MgH₂-induced growth promotion and bioactive compound accumulation, we performed transcriptome sequencing on root samples from Group b and Group ck at four weeks post-treatment, where both growth parameters and total metabolite content exhibited maximal differential responses (Figure 2K–M, Figure 3 and Figure 4). Comparative analysis identified 578 upregulated and 771 downregulated genes (Figures S4 and S5). GO biological processes and KEGG enrichment highlighted significant pathways, including “Metabolic pathways,” “Biosynthesis of secondary metabolites,” and “Plant hormone signal transduction” (Figure S6). Principal Component Analysis (PCA) of the transcriptome data revealed a clear separation between the MgH₂-treated and control groups along the principal component 1 (PC1), which accounted for 40.86% of the total variance (Figure S7). This distinct clustering of biological replicates within each group demonstrates the high reproducibility of our sequencing data and the pronounced effect of MgH₂ on the root transcriptome.

Within the “Plant hormone signal transduction” pathway, growth and energy metabolism related genes (e.g., GA2ox, YUCCA, CKX, GAPDH, NADPH, NADH) were upregulated (Figure 5). The upregulation of GA2ox and YUCCA (involved in gibberellin synthesis) and CKX (cytokinin degradation), alongside energy metabolism genes (GAPDH, NADPH, NADH), suggests that MgH₂ modulates hormonal balance and energy allocation to foster growth. In the “Biosynthesis of secondary metabolites” pathway, genes associated with tanshinone and salvianolic acid production (WRKY1, WRKY2, MYC2, HMGS, MVK, CYP76AH1, TAT, HCT, PAL, 4CL, CYP98A) were significantly upregulated (Figure 5). This transcriptional activation likely drives the observed accumulation of bioactive compounds.

To validate the reliability of our RNA-seq data, we selected 10 key DEGs (including WRKY1, WRKY2, MYC2, HMGS, MVK, CYP76AH1, TAT, PAL, 4CL, CYP98A75) for qRT-PCR analysis. The expression patterns of these genes determined by qRT-PCR were highly consistent with the RNA-seq results (Figure S8), confirming the accuracy of our transcriptomic profiling.

In summary, MgH₂ promotes S. miltiorrhiza growth and metabolite synthesis through physicochemical effects and transcriptional regulation of key pathways (Figure 6), providing a theoretical foundation for its application in medicinal plant cultivation.

4. Discussion

Medicinal plants hold immense value in healthcare due to their rich bioactive compound reservoir. However, a persistent challenge in this field lies in simultaneously enhancing plant growth and the accumulation of pharmacologically active secondary metabolites [26]. This study presents the first application of magnesium hydride (MgH₂) to the model medicinal plant S. miltiorrhiza, systematically investigating its effects on seed germination, seedling growth, photosynthesis, root biomass accumulation, and the content of key bioactive constituents, including tanshinones and salvianolic acids.

Our results demonstrate that MgH₂ treatment was associated with significantly enhanced seed germination efficiency (Figure 1) and accelerated seedling growth above ground, as evidenced by increased plant height, leaf area, leaf number, and biomass (Figure 2A–J). This growth promotion was accompanied by improved photosynthetic performance, indicated by elevated CO₂ assimilation (ΔC) and net photosynthetic rate (Pn) (Figure 2N–O). Crucially, MgH₂ treatment also substantially increased root biomass (Figure 2K–M). Moreover, it was linked to significantly boosting the accumulation of the core secondary metabolites, tanshinones and salvianolic acids (Figure 3 and Figure 4).

Notably, concentration-dependent effects were observed: 10 mg MgH₂ kg⁻¹ soil (Group a) yielded the highest germination rate (Figure 1), while 12.5 mg MgH₂ kg⁻¹ soil (Group b) provided superior growth promotion at the seedling stage (Figure 2). This suggests distinct physiological requirements during different developmental phases. The differential responses to MgH₂ concentrations at various growth stages may be attributed to complex interactions between internal hormonal regulation and external environmental factors. During seed germination, the optimal effect at 10 mg kg⁻¹ could be linked to H₂-mediated modulation of abscisic acid (ABA) and gibberellin (GA) metabolism [40,41], which are critical for breaking dormancy and initiating germination. The slightly higher concentration (12.5 mg kg⁻¹) required for optimal seedling growth might reflect the increased demand for magnesium as a cofactor in numerous enzymatic reactions involved in central metabolic pathways, including glycolysis, the tricarboxylic acid (TCA) cycle, and ATP synthesis. These metabolic processes provide essential energy and carbon skeletons necessary for vigorous vegetative growth. Furthermore, temperature fluctuations during different growth periods might influence MgH₂ hydrolysis rates and consequently affect H₂ and Mg²⁺ availability, adding another layer of regulation to the observed concentration-dependent effects [42]. Overall, the group b concentration demonstrated the most significant integrated effect at 4 weeks. This was characterized by enhanced photosynthetic efficiency, overall plant biomass, and root secondary metabolite content. It is noteworthy that this enhancement peaked at 4 weeks but subsequently diminished, consistent with the kinetics of H₂ release from MgH₂ [43]. This observation highlights a potential need for timed re-supply to sustain the effect in practical applications. Importantly, unlike conventional elicitors such as methyl jasmonate (MeJA) [21] or silver ions (Ag⁺) [25], which often enhance metabolite accumulation at the expense of growth, MgH₂ was observed to function as a bifunctional regulator, simultaneously promoting both plant growth and secondary metabolite biosynthesis. The coordinated enhancement of both growth and secondary metabolism suggests a synergistic action of MgH₂ hydrolysis products, primarily through H₂ signaling and Mg²⁺ nutrition, although direct mechanistic evidence for their specific signaling pathways is beyond the scope of this initial application study.

Based on the chemical properties of MgH₂, its hydrolysis in water to yield H₂ and Mg(OH)₂, and existing literature, we propose that the observed multifaceted promotive effects are likely mediated synergistically by the released hydrogen molecule (H₂) and magnesium ions (Mg²⁺). H₂ has been established as a novel gaseous signaling molecule in plants, regulating diverse processes including seed germination, root development, photosynthesis, and abiotic stress tolerance [28,29,30]. The significant improvements in seed germination rate and root length induced by MgH₂ align closely with previously reported physiological effects of H₂ [44,45]. Concurrently, the Mg(OH)₂ dissolves gradually in soil, releasing Mg²⁺. As an essential core component of chlorophyll, Mg²⁺ directly participates in the construction of the photosynthetic apparatus [46]. This mechanism may underpin the significant increases in Pn and ΔC observed in MgH₂-treated S. miltiorrhiza seedlings. Beyond magnesium’s fundamental role in chlorophyll structure, the hydrogen gas released from MgH₂ hydrolysis may further enhance photosynthetic performance through multiple mechanisms. Previous studies have demonstrated that H₂ can increase chlorophyll content by upregulating the expression of chlorophyll biosynthesis genes while suppressing chlorophyll degradation pathways [47,48]. Additionally, H₂ has been shown to modulate stomatal conductance through interactions with key signaling molecules such as nitric oxide (NO) and hydrogen sulfide (H₂S), thereby improving CO₂ uptake and utilization [49]. The combination of improved stomatal conductance from H₂ signaling and enhanced chlorophyll synthesis from Mg²⁺ nutrition may create a synergistic effect that significantly boosts photosynthetic efficiency. This enhanced photosynthetic capacity, in turn, provides increased photoassimilates and energy (ATP and NADPH) that support both growth-related processes and the biosynthetic demands of secondary metabolite production. The improved dry matter accumulation observed in MgH₂-treated plants, particularly in roots where bioactive compounds are stored, can be largely attributed to this coordinated enhancement of photosynthetic performance and carbon allocation. This synergistic action via multiple pathways appears crucial for the pronounced promotion of both shoot and root growth, especially the substantial enhancement in root development and biomass accumulation.

To elucidate the molecular basis of MgH₂’s regulation of root growth and secondary metabolism, we performed transcriptome sequencing on roots from the highly responsive Group b and Group ck seedlings. Analysis revealed significant alterations in the root gene expression profile following MgH₂ treatment. KEGG enrichment analysis highlighted “Metabolic pathways”, “Biosynthesis of secondary metabolites”, and “Plant hormone signal transduction” as major enriched pathways, suggesting potential integration of gene networks governing both growth regulation and metabolic responses by MgH₂. Numerous genes associated with growth regulation were upregulated, including GA2ox, YUCCA, CKX, GAPDH, NADPH, and NADH. Among these, GA2ox and CKX, involved in the degradation of gibberellins and cytokinins, respectively, are known to promote root development [50,51,52,53]. YUCCA encodes a key rate-limiting enzyme in auxin biosynthesis [54]. GAPDH, NADPH, and NADH are central to energy metabolism [55,56]. Furthermore, transcription factors and structural genes critically involved in the biosynthesis of phenolic acids (salvianolic acids) and diterpenoids (tanshinones) were significantly upregulated. These include WRKY1, WRKY2, MYC2, HMGS, MVK, CYP76AH1, TAT, HCT, PAL, 4CL, and CYP98A. Notably, WRKY transcription factors are pivotal regulators of tanshinone and salvianolic acid synthesis [57,58,59,60] while MYC2 participates in the jasmonate signaling pathway [61,62], governing secondary metabolite accumulation. Enzymes encoded by PAL, 4CL, and CYP98A are essential in the phenylpropanoid pathway, catalyzing the synthesis of phenolic compounds like Salvianolic acid [63,64]. HMGS and MVK function in the terpenoid backbone pathway, regulating tanshinone biosynthesis [65]. The coordinated upregulation of these genes provides molecular evidence consistent with the observed physiological and biochemical phenotypic alterations. Nevertheless, these transcriptional changes represent one layer of a potentially complex regulatory system, which may also involve post-transcriptional regulation and metabolite feedback mechanisms not captured in this study. For instance, the accumulation of salvianolic acids or tanshinones themselves could potentially exert feedback inhibition on their biosynthetic enzymes or upstream regulators, fine-tuning the metabolic flux. Furthermore, the stability and activity of key biosynthetic enzymes, such as those encoded by PAL, CYP76AH1, and others identified in our transcriptome data, could be subject to regulation through phosphorylation, ubiquitination, or other protein modifications downstream of MgH₂-derived signals. The precise regulatory mechanisms underlying the perception, transduction, and integration of H₂ and Mg²⁺ signals to orchestrate downstream gene networks necessitate further functional validation and pathway dissection.

The coordinated responses observed across different organizational levels, spanning from morphological traits to metabolic accumulation, were further confirmed by multivariate clustering analysis (Figures S2 and S3). The clear separation of MgH₂-treated groups in both phenotypic and metabolic clustering demonstrates the consistency and system-wide nature of MgH₂’s effects, supporting our hypothesis of its dual regulatory function.

In summary, this study reveals the unique potential of MgH₂ application in S. miltiorrhiza. Its key advantage over conventional growth regulators or elicitors lies in its association with concurrent enhancement of both growth performance and medicinal quality. This represents a significant advance and an innovative approach towards resolving the persistent “growth-quality trade-off” dilemma in medicinal plant cultivation. It extends the application of MgH₂, which was previously studied for preservation or stress resistance in non-medicinal plants, into the realm of medicinal plant cultivation for quality improvement.

As a novel, environmentally friendly additive, the practical application of MgH₂ necessitates further research to optimize dosage, timing, and application frequency (considering the temporal limitation of the H₂ release), elucidate its long-term stability and transformation in soil, assess potential residue risks, evaluate its impact on soil microbial communities for long-term ecological safety, and investigate its compatibility with other agronomic practices. Furthermore, species-specific responses to MgH₂ require evaluation to optimize protocols for different medicinal plants.

5. Conclusions

In conclusion, this study demonstrates that MgH₂ application is an effective strategy for synchronously enhancing both the growth and the medicinal quality of Salvia miltiorrhiza. We achieved our objective of investigating MgH₂’s effects by showing that it significantly improves seed germination, promotes photosynthetic efficiency and biomass accumulation in seedlings, and substantially increases the root content of pharmaceutically valuable tanshinones and salvianolic acids. Transcriptomic analysis revealed that these promotive effects were accompanied by the upregulation of genes involved in hormone signaling, energy metabolism, and the biosynthesis pathways of these secondary metabolites. Our findings confirm the dual regulatory role of MgH₂ and provide a foundational, eco-friendly cultivation technique for the sustainable production of high-quality S. miltiorrhiza.

6. Study Limitations and Future Perspectives

While this study demonstrates the promising potential of MgH₂ as a novel plant growth regulator for medicinal plants, several limitations should be acknowledged to properly contextualize the findings and guide future research.

First, the potential environmental effects of MgH₂ soil application, such as soil pH alteration due to Mg(OH)₂ formation and the persistence of residues, were not directly monitored in this pot-based study. Although the hydrolysis product Mg(OH)₂ is a mild base and our soil mixture likely provided sufficient buffering capacity, future field-scale studies should systematically measure soil pH, electrical conductivity, and other physicochemical parameters throughout the growth period to fully assess its environmental impact and long-term stability. Second, our observations were conducted over a relatively short-term period (8 weeks) under controlled laboratory conditions. This timeframe, while sufficient to capture significant physiological and transcriptional changes, may not fully represent the long-term effects of MgH₂ application on the yield and quality of S. miltiorrhiza across its entire growth cycle, especially under field conditions with fluctuating environmental factors. Therefore, multi-season field trials are essential to validate the efficacy and economic viability of this approach. Third, the present work focused exclusively on the species-specific response of S. miltiorrhiza. The transferability of these findings to other medicinal plants remains an open question. Given that the proposed mechanism involves fundamental processes like H₂ signaling and Mg²⁺ nutrition, which are conserved across plant species, it is plausible that MgH₂ could benefit a wider range of medicinal species. However, the optimal concentration and timing of application are likely to vary. Future research should therefore explore the effects of MgH₂ on other high-value medicinal plants with different metabolic profiles and growth habits to determine the broader applicability of this strategy.

In conclusion, addressing these limitations in future work will be crucial for transitioning MgH₂ from a promising laboratory discovery to a reliable, eco-friendly tool for the sustainable cultivation of medicinal plants.