Volatile organic compounds from Bacillus sp. PAR690 Promote plant growth by regulating photosynthesis and nitrogen metabolism

Abstract

Plant growth-promoting bacteria (PGPB) enhance plant growth through multiple mechanisms; however, the underlying plant molecular response pathways remain insufficiently understood, particularly those mediated by bacterial volatile organic compounds (VOCs). In this study, a Bacillus strain, designated PAR690, capable of producing growth-promoting VOCs, was isolated from the rhizosphere soil of Achnatherum inebrians. Split-plate assays demonstrated that PAR690 significantly increased chlorophyll a/b, carotenoids, and total chlorophyll contents in Arabidopsis, while simultaneously promoting the accumulation of reducing sugars, soluble sugars, and starch. GC/MS analysis revealed that growth-promoting VOCs produced by Bacillus sp. PAR690 include acetoin, 2,3-butanediol, and indole. Transcriptomic analyses revealed that PAR690 significantly upregulated multiple key genes involved in the “Photosynthesis-antenna proteins”, “Photosynthesis, light harvesting”, and “Chlorophyll catabolic process” pathways. In addition, PAR690 downregulated the expression of methyl esterase 16 while upregulating chlorophyllase 1, thereby maintaining chlorophyll homeostasis under conditions of elevated chlorophyll content. PAR690 also enhanced nitrogen uptake and accumulation by inducing the expression of nitrate transporter 2.1 and nitrate transporter 2.5. Moreover, PAR690 significantly increased anthocyanin accumulation in Arabidopsis, an effect closely associated with the upregulation of genes related to UDP-glucose: flavonoid 3-O-glucosyltransferase and production of anthocyanin pigment 1. Collectively, these findings elucidate the potential mechanisms by which Bacillus sp. PAR690 promotes plant growth via VOC-mediated regulation at both physiological and transcriptional levels, providing new insights into PGPB-plant interactions.

Supplementary Information

The online version contains supplementary material available at https://doi.org/10.1186/s12870-026-09489-4.

Introduction

The combined impacts of climate change and population have intensified the challenges faced by agricultural and forestry production, creating a pressing demand for sustainable production systems. Beneficial microorganisms, owing to their potential as biofertilizers and biocontrol agents, have attracted increasing attention and are regarded as effective alternatives to chemical inputs [1]. Plant growth-promoting bacteria (PGPB) represent a group of beneficial microbes that establish mutualistic associations with plants, primarily colonizing the rhizosphere or internal tissues, and enhance plant growth and development through multiple mechanisms [2, 3]. Although different types of PGPB vary in their taxonomic characteristics and ecological niches, they share common growth-promoting mechanisms, including nitrogen fixation, solubilization of inorganic phosphate, production of organic acids and siderophores to improve nutrient availability, as well as secretion of phytohormones or hormone analogs, induction of systemic resistance, and suppression of phytopathogens [4–6]. In recent years, PGPB have become a research hotspot, and advances in this field have led to the development and commercialization of numerous microbial products. Among them, genera such as Pseudomonas, Azospirillum, Burkholderia, and Bacillus are the most extensively studied, with representative strains including Azospirillum brasilense, Bacillus subtilis, and Pseudomonas fluorescens already commercialized as biofertilizers or biocontrol agents and widely applied in the sustainable production of cereals, vegetables, and fruit crops [7–9].

Among the various growth-promoting mechanisms, volatile organic compounds (VOCs) are low-molecular-weight compounds produced by microbial metabolism and act as short- or long-distance signaling molecules that regulate plant growth and development [10, 11]. Among them, 2,3-butanediol and acetoin, synthesized by Bacillus species, are the most extensively studied VOCs. These compounds were first identified in Bacillus subtilis strains GB03 and BSIP1171, and were subsequently demonstrated to promote plant growth [12, 13]. In recent years, an increasing number of VOCs, including alkanes, alcohols, esters, ketones, terpenes, and sulfur-containing compounds, have been reported [14–16]. Most of these compounds are intermediates or end products of microbial metabolic pathways. In soil, VOCs can adsorb onto clay particles and diffuse through soil pores or water films to reach the rhizosphere, where they mediate plant-microbe interactions [3, 17]. VOCs can enhance plant growth by regulating root development, improving nutrient acquisition, modulating hormone interactions, influencing photosynthesis, and alleviating both biotic and abiotic stresses [18–20].

Photosynthesis functions as a major pathway through which PGPB enhance plant performance, and its regulation by PGPB often involves multiple factors [21, 22]. Iron is an essential element for chlorophyll biosynthesis, electron transport, and photosystem activity [23]. Studies have demonstrated that VOCs released by PGPB can increase iron solubility and transport efficiency by stimulating proton extrusion in the rhizosphere, induce iron deficiency responses, and upregulate the expression of iron acquisition-related transcription factor MYB72 [24–26]. In addition, volatiles emitted by B. subtilis GB03 have been shown to inhibit HXK1-dependent glucose sensing, thereby alleviating hexose-mediated repression of photosynthesis [27]. Furthermore, B. subtilis GB03 can reduce endogenous glucose and abscisic acid levels, leading to increased chlorophyll accumulation in leaves and enhanced photosynthetic efficiency in Arabidopsis thaliana (Arabidopsis) [27]. Moreover, PGPB have been reported to alleviate chlorophyll degradation under stress conditions, thereby sustaining photosynthetic rates [28, 29]. Based on transcriptome analysis, Li et al. demonstrated that tobacco plants exposed to Cladosporium sphaerospermum TC09 exhibited a large number of differentially expressed genes involved in cell proliferation, cell cycle regulation, photosynthesis, hormone homeostasis, and defense responses [30]. Despite significant progress in elucidating PGPB mechanisms, systematic identification of novel strains and comprehensive mechanistic studies at the plant level remain essential to advance theoretical understanding and support their efficient and rational application in agriculture.

Drunken horse grass (Achnatherum inebrians) is a native forage grass widely distributed across the arid and semi-arid grasslands of northwestern China [31, 32]. In natural ecosystems, this species frequently associates with Epichloë endophytic fungi, forming a symbiotic system characterized by enhanced environmental adaptability [33, 34]. Increasing evidence suggests that foliar endophytic fungi can reshape rhizosphere microbial communities by modulating soil physicochemical properties or altering root exudate composition. These changes promote nutrient uptake and utilization in A. inebrians and enhance its stress tolerance [35, 36]. For instance, Ju et al. reported that Epichloë alters the diversity of cultivable bacteria in the rhizosphere soil of A. inebrians [37]. They further showed that inoculation with three rhizosphere bacterial isolates (Arthrobacter sp., Bacillus sp., and Pseudomonas sp.) significantly enhanced seed germination of A. inebrians under NaCl stress [37]. These findings indicate that the rhizosphere of A. inebrians may harbor diverse and functionally important microbial resources [38, 39]. In-depth exploration of microbial resources in these ecological niches facilitates the discovery of novel plant growth-promoting strains and supports their efficient application.

In this study, we isolated a plant growth-promoting bacterium from the rhizosphere soil of healthy Epichloë-infected (E+) A. inebrians using King’s B (KB) medium. This bacterium was capable of releasing VOCs that significantly promoted plant growth. By integrating physiological assessments with transcriptomic analyses, we further elucidated the host’s responses, thereby uncovering the mechanisms underlying its growth-promoting effects. Collectively, this work expands the theoretical understanding of PGPB and offers new insights into the development and application of beneficial microbial resources.

Materials and methods

Isolation and identification of PGPB

Healthy, vigorously growing E+ A. inebrians plants were carefully uprooted from a multiplication plot at the Yuzhong Campus of Lanzhou University. Bulk soil adhering to the roots were gently removed by shaking, and rhizosphere soil was subsequently collected using a sterile brush into sterile sampling bags. 1 g of rhizosphere soil was suspended in 10 mL of sterile distilled water, vortexed thoroughly to ensure homogenization, and serially diluted in ten-fold increments up to 10⁻⁶. Aliquots of 200 µL from each dilution were evenly spread onto KB agar plates, which were then incubated at 28 °C for 72 h. During the initial screening stage, 200 single colonies were selected. After three rounds of successive purification, 32 representative isolates with distinct colony morphologies were selected. Subsequently, all isolates were simultaneously subjected to plant growth-promotion assays using Arabidopsis, along with 16S rRNA gene amplification and sequencing. The results showed that 9 strains exhibited clear differences in their 16S rRNA sequences and displayed significant growth-promoting effects. Based on these findings, the strain with the most pronounced growth-promoting activity was selected, designated as PAR690, and used for subsequent experiments. Next, PCR amplification was performed using universal primer sets targeting the 16S rRNA [40], recA, and rpoB genes [41]. The resulting amplicons were sequenced, and the obtained sequences were subjected to multi-locus sequence analysis (MLSA) for phylogenetic tree construction using the Neighbor-Joining method. In addition, bootstrap analysis with 1,000 resamplings was conducted using MEGA6 to evaluate the robustness of the inferred phylogeny.

Plant growth and treatments

The Arabidopsis seeds used in this study were of the Columbia-0 (Col-0) ecotype, provided by Professor Jianfeng Wang from Lanzhou University. The cultivated rapeseed cultivar “Qinyou 2” is a semi-winter-type hybrid of Brassica napus (Approval No.: GS070001-1992), purchased from a commercial seed company. Seeds of E+ A. inebrians were collected from the A. inebrians seed multiplication plot at the Yuzhong Campus of Lanzhou University. Seeds were surface-sterilized sequentially with 70% ethanol for 2 min and 1% available NaClO solution for 10 min, followed by five rinses with sterile distilled water, and then subjected to vernalization at 4 °C for 48 h. To evaluate the plant growth-promoting effects of PGPB on Arabidopsis, a divided Petri dish with 1/2 MS was employed: surface-sterilized seeds were sown in one compartment, while the other compartment received 10 µL of PGPB suspension (OD₆₀₀ = 0.8; 2.4 × 10⁸ CFU/mL); an equal volume of sterile water served as the control. The plates were incubated in a controlled-environment growth chamber under a photosynthetic photon flux density of 120 µmol·m⁻²·s⁻¹, a 16-h light/8-h dark photoperiod, 23 °C, and 60% relative humidity. Arabidopsis growth was assessed after 15 days of cultivation. Due to their larger size, rapeseed and A. inebrians seedlings were cultivated in culture bottles with a bottom diameter of 7.3 cm, a height of 10.9 cm, and a volume of 350 mL. To avoid indirect contact between bacteria and plants via the culture medium, a small Petri dish (35 mm diameter, containing 5 mL of 1/2 MS solid medium) with 10 µL of PAR690 suspension (OD₆₀₀ = 0.8; 2.4 × 10⁸ CFU/mL) was placed inside the culture bottle. After 18 days, rapeseed and A. inebrians seedlings were photographed, and growth parameters-including shoot height, fresh weight, and dry weight-were quantified.

VOCs identification by SPME-GC/MS

Solid-phase microextraction (SPME) GC/MS was employed to analyze VOCs produced by Bacillus sp. PAR690. Briefly, 10 µL of bacterial suspension (OD₆₀₀ = 0.8) was inoculated into a 250 mL Erlenmeyer flask containing 50 mL of 1/2 MS solid medium and incubated in a growth chamber (two independent biological replicates). The flasks were sealed with silicone stoppers to prevent VOCs loss. A 1/2 MS medium without Bacillus sp. PAR690 served as the control. The incubation conditions were consistent with those used for plant growth. After 8 days of cultivation, VOCs were collected using SPME methods and subsequently analyzed using the GC/MS system [42]. Briefly, a 0.2 g aliquot of the sample powder, ground with liquid nitrogen, was transferred to a headspace vial. Then, 0.2 g of NaCl powder was added to suppress enzyme activity, along with 20 µL of internal standard solution (10 µg/mL). The mixture was shaken at 60 °C for 5 min. A 120 μm DVB/CWR/PDMS extraction fiber (Agilent, CA, USA), preconditioned at 250 °C for 5 min prior to use, was inserted into the headspace of the vial for extraction at 60 °C for 15 min. Subsequently, the fiber was desorbed at 250 °C for 5 min, and the analytes were separated and identified by an Agilent Model 8890 gas chromatography and a 7000D mass spectrometer (Agilent, CA, USA). The chromatographic separation was performed on a DB-5MS capillary column (30 m × 0.25 mm × 0.25 μm, Agilent, CA, USA). High-purity helium was used as the carrier gas at a constant flow rate of 1.2 mL/min. The injector temperature was set at 250 °C, with a solvent delay of 3.5 min. The column temperature program was as follows: initial temperature of 40 °C held for 3.5 min, increased to 100 °C at 10 °C/min, then to 180 °C at 7 °C/min, and finally to 280 °C at 25 °C/min, held for 5 min. For mass spectrometry, the electron impact (EI) ion source is set at 230 °C, the quadrupole temperature at 150 °C, the transfer line at 280 °C, and the electron energy of 70 eV. Data acquisition was performed in selected ion monitoring (SIM) mode, with precise scanning of both qualitative and quantitative ions. The metabolome database was constructed by Metware Biotechnology Co., Ltd. and is primarily based on microbial species and literature references, partially authenticated standards, and retention indices. The potential differential metabolites were identified based on a threshold of |log₂(fold change)| ≥ 1.5 [43].

Determination of pigment, carbohydrate, amino acid, and anthocyanin contents

Photosynthetic pigments were extracted using 95% ethanol, and the concentrations of chlorophylls and carotenoids were calculated according to the following equations [44]: C_a=13.95×A₆₆₅–6.88×A₆₄₉, C_b=24.96×A₆₄₉–7.32×A₆₆₅, C_total=C_a+C_b, C_car=(1000×A₄₇₀–2.05×Ca–114.8×Cb)/245; and the final pigment contents were expressed on a fresh weight basis. The contents of reducing sugars, total soluble sugars, and starch content were quantified following the protocol described by Yang et al. [45]. Briefly, plant samples were extracted with 80% ethanol and incubated at 80 °C for 30 min to solubilize reducing and soluble sugars. Following removal of insoluble residues, the extract was concentrated in a boiling water bath, and the remaining carbohydrates were re-dissolved in water. Reducing sugars were determined using the 3,5-dinitrosalicylic acid (DNSA) method with absorbance measured at 540 nm, whereas total soluble sugars were quantified via the anthrone assay at 620 nm. The residue remaining after 80% ethanol extraction was used for starch determination; it was subjected to alkaline hydrolysis to convert starch into reducing sugars, followed by quantification. Anthocyanins were extracted using a solution of 45% methanol containing 5% acetic acid and subsequently quantified spectrophotometrically based on the formula [A₅₃₀−(0.25×A₆₅₇)]×5 [46].

Gas exchange measurements

Gas exchange parameters were measured using a LI-6800 Portable Photosynthesis System (LI-COR Biosciences, Lincoln, NE, USA). Measurements were performed on fully expanded rosette leaves of 15-day-old seedlings. Chamber conditions were set as follows: fan speed 7,000 rpm, flow rate 150 µmol·s⁻¹, relative humidity 50%, chamber temperature 22 °C, CO₂ concentration 400 µmol·mol⁻¹, and photosynthetically active radiation (PAR) at 500 µmol·m⁻²·s⁻¹. Net photosynthetic rate (A), transpiration rate (E), and stomatal conductance (gs) were calculated as described previously [47].

Quantification of amino acid, total N, P, and NH₄⁺ in Arabidopsis

Total amino acids were quantified by the ninhydrin-based colorimetric assay as described by Friedman [48]. Arabidopsis seedlings were blanched at 105 °C for 30 min and then dried to constant weight at 80 °C. The dried samples were ground and digested using H₂SO₄ digestion method with K₂SO₄ and CuSO₄ (10:1, w/w) as catalysts. The resulting digest was analyzed using a flow injection analyzer (FIAstar 5000, FOSS, Sweden) as described by Wang et al. [36]. The ammonium nitrogen content in plant samples was determined using a commercial assay kit (Cat. BC6040; Solarbio life sciences, Beijing, China), following the manufacturer’s instructions.

Transcriptomic sequencing and bioinformatic analysis

The transcriptome analysis was performed following the methodology outlined by Wang et al. [32]. In brief, High-quality RNA samples from 10-day-old seedlings were employed to construct sequencing libraries using the Dual-mode mRNA Library Prep Kit (Cat:12301ES08; Yeasen Biotechnology; Shanghai). Library quality was assessed using the Agilent Bioanalyzer 2100 system, followed by high-throughput sequencing on the Illumina NovaSeq 6000 platform. Raw sequencing reads were processed to remove low-quality sequences and adapter sequences, generating high-quality clean reads. These clean reads were then aligned to the Arabidopsis thaliana reference genome (TAIR10) using HISAT2 (v2.0.4) via the BMKCloud online platform (www.biocloud.net). Differentially expressed genes (DEGs) were identified with DESeq2 (v1.30.1), applying a significance threshold of p-value < 0.05 and absolute fold change |FC| ≥ 2. Gene Ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were conducted using the clusterProfiler package (v4.4.4). Volcano plots and PCA were visualized using the ggplot2 package (v 3.1.1) in R. The heatmap was conducted using pheatmap package (v 1.0.12) based on Pearson correlation analysis.

qPCR analysis

For qPCR analysis, total RNA was isolated from 10-day-old seedlings using the RNeasy Plant Mini Kit (Qiagen, Hilden, Germany). Subsequently, 1 µg of RNA was reverse-transcribed into cDNA using the HiScript cDNA Synthesis Kit (Vazyme, Nanjing, China). qPCR was performed using a LightCycler^® 96 system (Roche, Indianapolis, USA) with SYBR Green dye (Cat. No. Q311; Vazyme; Nanjing). The qPCR reaction mixture (20 µL) consisted of 10 µL SYBR Green Mix, 0.4 µL each of forward and reverse primers (10 µM), 2 µL cDNA template (5 ng/µL), and 7.8 µL ddH₂O. The amplification protocol was as follows: an initial denaturation at 95 °C for 2 min, followed by 40 cycles of denaturation at 95 °C for 15 s and annealing/extension at 60 °C for 30 s, and concluded with a melting curve analysis. AtUBQ10 served as the internal reference, and the relative expression levels of the target genes were calculated using the 2^⁻ΔΔCt method [45]. The qPCR primers are provided in Table S1.

Determination of plant enzyme activities

Nitrate reductase (NR) and glutamine synthetase (GS) activities were determined spectrophotometrically according to the protocol of Wang et al. [49]. Briefly, plant tissues (0.3 g) were homogenized in 100 mM Hepes-KOH buffer (pH 7.5) containing 3% PVPP, 1 mM EDTA, and 7 mM cysteine, and centrifuged at 10,000 × g for 10 min at 4 °C. The supernatant was incubated in reaction buffer with 100 µM NADH and 5 mM KNO₃ at 25 °C in the dark, and nitrate reductase activity was determined by nitrite formation after color development at 540 nm. For GS activity, 0.3 g of plant tissue was homogenized in extraction buffer (50 mM Tris-HCl, pH 8.0; 2 mM MgSO₄; 2 mM DTT; 400 mM sucrose) and centrifuged at 13,000 × g for 20 min at 4 °C. The supernatant (0.7 mL) was incubated with reaction buffer and 40 mM ATP at 37 °C for 30 min, followed by addition of chromogenic reagent. After centrifugation, GS activity was determined spectrophotometrically at 540 nm. Glutamate dehydrogenase (GDH) activity was assayed by the amination and deamination reactions according to the method described by Turano et al. [50]. GOGAT activity was determined spectrophotometrically by monitoring NADH oxidation at 340 nm. The reaction mixture, containing 8 mM L-glutamine, 2 mM 2-oxoglutarate, 1 mM NADH, 0.6 mM EDTA, 10 mM KCl, and 0.5 mL enzyme extract in 25 mM phosphate buffer (pH 7.5), was initiated by adding L-glutamine, and the decline in absorbance was recorded over 5 min [51].

Statistical analysis

Statistical analyses were conducted using SPSS software (v. 17.0). Differences between the PAR690 and control groups for the measured parameters were assessed using a Student’s t-test, with “*”, “**”, and “***” indicating p < 0.05, p < 0.01, and p < 0.001, respectively. Data are presented as means ± standard deviations (SD).

Plant growth-promoting properties of Bacillus sp. PAR690

The bacteria were isolated from the rhizosphere soil of E+ A. inebrians during its growth period, and one of the strains was designated PAR690. Subsequently, the 16S rRNA, recA, and rpoB gene sequences were used to construct a phylogenetic tree based on MLSA among ten Bacillus species. The phylogenetic tree analysis indicated that the rhizosphere bacterium PAR690 exhibited a close evolutionary relationship with Bacillus velezensis and Bacillus amyloliquefaciens (Fig. 1A). The dual-compartment Petri dish was used to evaluate the regulatory effects of strain PAR690 on seedling growth of Arabidopsis. The results showed that PAR690 significantly promoted Arabidopsis seedling growth, with increasing fresh weight by 178.83% and dry weight by 291.67% compared to the control (Figs. 1B to 1D). Furthermore, a combination of culture bottles and small Petri dishes was employed to evaluate the plant growth-promoting traits of PAR690 on Brassica napus and E+ A. inebrians. The results showed that, compared to the control, PAR690 treatment significantly increased the fresh weight of B. napus seedlings by 54.79% (Figs. S1A and S1B), and the dry weight by 47.53% (Fig. S1C). Meanwhile, PAR690 increased the tiller number of E+ A. inebrians seedlings. Although plant height was not significantly enhanced (Fig. S2B), PAR690 treatment significantly increased seedling fresh weight by 11.91% (Figs. S2A and S2C) and dry weight by 17.53% (Fig. S2D) compared to the control (p < 0.05). These results indicate that Bacillus sp. PAR690 promotes the growth of multiple plant species through the production of VOCs.

Fig. 1.

Identification of the PAR690 strain and its regulatory role in Arabidopsis growth. A Phylogenetic tree of PAR690 within 10 species of the genus Bacillus. B The growth-regulating effect of PAR690 on Arabidopsis seedlings was evaluated using the dual-compartment Petri dish assay, with water serving as the control. Bar = 1 cm. Statistical analysis of fresh (C) and dry weights (D) of seedlings treated with PAR690 (n = 30)

Furthermore, the VOCs produced by strain PAR690 were identified using SPME–GC/MS. Between the control and treatment groups, a total of 171 potential differential metabolites were identified (Table S2). The identified differential metabolites mainly belong to “aldehydes and ketones, esters”, “alcohols and amines”, and “hydrocarbons” (Fig. S3A). Based on KEGG analysis, 11 VOCs were identified as significantly differential metabolites, and they were predominantly enriched in “metabolic pathways”, “microbial metabolism in diverse environments”, and “biosynthesis of secondary metabolites” (Fig. S3B). Among these 171 differential metabolites, several typical VOCs with plant growth-promoting traits were detected, including acetoin (5.22-fold increase relative to the control), 2,3-butanediol (5.14-fold increase), and indole (269.88-fold increase) (Fig. 2). Additionally, geraniol, a bioactive compound, was also identified as a potential differential metabolite (Fig. 2).

Fig. 2.

Chromatographic profiles of volatile compounds produced by Bacillus sp. PAR690. Compounds highlighted in red represent well-documented plant growth-promoting VOCs, whereas the one in black corresponds to compounds previously reported to possess biological activity

Bacillus sp. PAR690 enhances the content of photosynthetic pigments and carbohydrate biosynthesis in plants

Arabidopsis leaves co-cultivated with the PAR690 strain exhibited a darker green color. To investigate the underlying physiological basis, the contents of photosynthetic pigments in leaves under different treatments were determined. The results showed that PAR690 treatment significantly increased the levels of chlorophyll a (+66.35%; Fig. 3A), chlorophyll b (+54.31%; Fig. 3B), carotenoids (+41.43%; Fig. 3C), and total chlorophyll (+62.95%; Fig. 3D). Meanwhile, compared with the control, PAR690 treatment significantly increased the net photosynthetic rate (by 109.88%, Fig. S4A), stomatal conductance (by 124.13%, Fig. S4B), and transpiration rate (by 69.37%, Fig. S4C). Furthermore, the contents of photosynthetic carbohydrates were measured. The results showed that, compared with the control, PAR690 significantly increased the levels of reducing sugar by 20.89% (p < 0.05; Fig. 4A), soluble sugar by 91.09% (p < 0.01; Fig. 4B), and starch by 101.53% (p < 0.001; Fig. 4C).

Fig. 3.

Determination of photosynthetic pigment contents in Arabidopsis leaves. A Chlorophyll a; (B) Chlorophyll b; (C) Carotenoids; (D) Total chlorophyll. Data represent mean ± SD, n = 5

Fig. 4.

Carbohydrate contents in Arabidopsis leaves following PAR690 treatment. A Reducing sugar content. B Soluble sugar content. C Starch content. Data are presented as mean ± SD, n = 5

Transcriptome analysis and identification of DEGs

To investigate the transcriptional response of Arabidopsis seedlings to the PAR690 strain, we performed transcriptome analysis. The results showed that, compared with the control, PAR690 treatment significantly upregulated 151 genes and downregulated 77 genes (Fig. 5A). Principal component analysis (PCA) revealed a clear separation between the transcriptomic profiles of the PAR690-treated and control groups (Fig. 5B). The KEGG pathway enrichment analysis revealed that the DEGs were predominantly enriched in pathways including “Photosynthesis-antenna proteins”, “Monoterpenoid biosynthesis”, and “Anthocyanin biosynthesis” (Fig. 5C). In addition, GO enrichment analysis of the DEGs was performed across three categories: Biological Process, Molecular Function, and Cellular Component. Enrichment analysis revealed significant enrichment of multiple GO terms associated with photosynthesis. At the biological process level, these included “protein-chromophore linkage”, “photosynthesis, light harvesting”, and “chlorophyll catabolic process” (Fig. 5D). At the molecular function level, enriched GO terms included “chlorophyll binding” and “electron transfer activity” (Fig. 5E). At the cellular component level, significant enrichment was observed in “photosystem I”, “photosystem II”, “chloroplast thylakoid membrane”, “chloroplast”, and “photosystem II antenna complex” (Fig. S5).

Fig. 5.

Transcriptomic response of Arabidopsis to Bacillus sp. PAR690. A Volcano plot analysis of DEGs in Arabidopsis between the control and PAR690 treatment. B PCA of Arabidopsis transcriptomes from control and PAR690-treated groups. C KEGG pathway enrichment analysis of Arabidopsis DEGs induced by PAR690. Only pathways with p < 0.05 are shown. GO enrichment analysis of DEGs: Biological Process (D) and Molecular Function (E) categories (top 20, p < 0.05)

Bacillus sp. PAR690 regulated the expression of canonical genes associated with photosynthesis, nitrogen metabolism, and anthocyanin biosynthesis

Further analysis revealed 14 DEGs involved in photosynthesis-related pathways. Among them, 12 genes were significantly upregulated under PAR690 treatment, while the NAD(P)-binding Rossmann-fold superfamily protein gene and the methyl esterase gene were significantly downregulated (Fig. 6A). The upregulated genes were primarily enriched in the light-harvesting complex II (LHCB) family, as well as in processes related to photosynthetic electron transport and ATP synthesis (Fig. 6A). qPCR validation results showed that the expression trends of all tested genes were consistent with the RNA-seq data (Figs. S6A to S6N), except for ATP synthase CF1 epsilon subunit, which exhibited a discrepancy in expression direction (Fig. S6L). This discrepancy may arise from the presence of multiple transcript isoforms, which may not be fully captured by the qPCR primers used in this study. This overall concordance indicates the high reliability of the transcriptomic data. Notably, two genes involved in the nitrogen metabolism pathway-nitrate transporter 2.5 (NRT2.5) and nitrate transporter 2.1 (NRT2.1)-were significantly upregulated under PAR690 treatment (Fig. 6B). Additionally, genes associated with anthocyanin biosynthesis, including production of anthocyanin pigment 1 (PAP1) and UDP-glucose: flavonoid 3-O-glucosyltransferase (UF3GT)-were upregulated (Fig. 6B). Furthermore, a GATA zinc finger protein gene implicated in the transcriptional regulation of photosynthesis-related genes also showed increased expression following PAR690 treatment (Figs. 6B and S6O).

Fig. 6.

Heatmaps showing the expression patterns of DEGs identified between control and PAR690 treatment. A Hierarchical clustering heatmap of DEGs associated with photosynthesis (based on transcriptomic data). B Heatmap showing the expression patterns of DEGs involved in nitrogen metabolism and anthocyanin biosynthesis pathways. “C” represents the control, and “T” represents PAR690 treatment group

Effect of Bacillus sp. PAR690 on anthocyanin biosynthesis in Arabidopsis

Compared with the control, at 10 days after germination (DAGs), Arabidopsis seedlings treated with PAR690 developed distinct purplish-brown spots on the abaxial surface of the cotyledons, suggesting potential anthocyanin accumulation (Fig. 7A). Consistent with the transcriptomic data, qPCR analysis confirmed that PAR690 treatment significantly upregulated the expression of key anthocyanin biosynthesis genes PAP1 (Fig. 7B) and UF3GT (Fig. 7C). Anthocyanin quantification revealed an overall decline in anthocyanin levels during plant growth. Notably, at 10 and 15 days after PAR690 incubation, anthocyanin contents in the PAR690-treated group were 636.9% (p < 0.001) and 415.4% (p < 0.05) higher than those in the control group, respectively (Fig. 7D).

Fig. 7.

Response of anthocyanin biosynthesis in Arabidopsis to Bacillus sp. PAR690. A Purplish-brown pigmentation on the abaxial surface of cotyledons in Arabidopsis seedlings at 10 DAGs. Expression levels of key anthocyanin biosynthesis genes-production of anthocyanin pigment 1 (PAP1; B) and UDP-glucose: flavonoid 3-o-glucosyltransferase (UF3GT; C)-in response to PAR690 treatment, as determined by qPCR. D Quantification of anthocyanin content in Arabidopsis seedlings at 10 and 15 DAGs (n = 5)

Regulation of nitrogen metabolism in Arabidopsis by Bacillus sp. PAR690

Consistent with the transcriptomic results, qPCR analysis revealed that strain PAR690 significantly upregulated the expression of NRT2.1 (Fig. 8A) and NRT2.5 (Fig. 8B). Correspondingly, PAR690 treatment significantly increased nitrogen content in Arabidopsis plants from 39.87 mg·g⁻¹ F.W. in the control group to 53.91 mg·g⁻¹ F.W. (Fig. 8C). However, no significant effect was observed on plant phosphorus content (p > 0.05; Fig. 8D). Furthermore, compared with the control, PAR690 significantly elevated amino acid levels in fresh plant tissues, from 1.54 to 3.06 mg·g⁻¹ F.W. (Fig. 8E), and significantly enhanced nitrate reductase activity by 100.88% (Fig. 8F). Meanwhile, PAR690 treatment significantly reduced NH₄⁺ content (Fig. S7A), as well as the activities of glutamine synthetase (GS; Fig. S7B), glutamate synthase (GOGAT; Fig. S7C), and glutamate dehydrogenase (GDH; Fig. S7D).

Fig. 8.

Bacillus sp. PAR690 regulates nitrogen metabolism in Arabidopsis. qPCR analysis of nitrate transporter 2;1 (NRT2.1, A) and nitrate transporter 2;5 (NRT2.5, B) gene expression following PAR690 treatment. Nitrogen (C), phosphorus (D), and amino acid content (E) in Arabidopsis plants under control and PAR690-treated conditions. F Determination of nitrate reductase activity. n = 5

Discussion

Microbial VOCs play a crucial role in regulating plant growth and triggering induced systemic resistance. In this study, we clearly demonstrated that VOCs produced by a novel strain of Bacillus sp. PAR690 exhibit plant growth-promoting properties via the coordinated optimization of carbon assimilation (photosynthesis), nitrogen acquisition, and anthocyanin accumulation.

VOCs identified from Bacillus sp. PAR690

To date, more than 1300 species and isolates have been documented to produce approximately 3500 distinct VOCs [52]. Meanwhile, various Bacillus species from different genera, including Bacillus subtilis, Bacillus amyloliquefaciens, and Bacillus megaterium, have been shown to promote plant growth by producing VOCs such as acetoin, 2,3-butanediol, and 2-pentylfuran [13, 53–55]. It has been reported that VOCs application can increase plant biomass up to nine-fold compared to the control [56]. As known plant growth-promoting volatiles, 2,3-butanediol and acetoin have been shown to significantly enhance plant growth by inducing systemic resistance and regulating auxin homeostasis in plants [54, 57].

Indole is an important microbial metabolite that plays key roles in diverse physiological processes, including biofilm formation, drug resistance regulation, and polysaccharide biosynthesis [58]. Meanwhile, indole also serves as a bacterial VOC with significant plant growth-promoting potential. Bailly et al. demonstrated that volatile indole emitted by Escherichia coli significantly enhanced plant biomass accumulation and increased lateral root formation [59]. Similarly, Yu and Lee reported that indole produced by Proteus vulgaris effectively stimulated cabbage growth [60]. Notably, through mutant-based analyses, Bhattacharyya et al. further demonstrated that the growth-promoting effects of indole released by rhizosphere-associated P. vulgaris are mediated by the coordinated regulation of auxin, cytokinin, and brassinosteroid signaling pathways [61]. In our results, indole was upregulated by nearly 270-fold compared with the control, suggesting that it may play a more prominent role in the growth-promoting process mediated by PAR690. Furthermore, VOC of geraniol is considered to possess antimicrobial activity [62], indicating that PAR690 may also exert an effect in shape the rhizosphere microbial community.

VOCs from Bacillus sp. PAR690 regulate photosynthetic processes

The leaf photosynthetic rate is highly associated with plant biomass. Enhanced photosynthesis in plants is widely regarded as one of the key mechanisms by which VOCs promote plant growth. Chloroplasts play a pivotal role in plant photosynthesis by harvesting light energy and converting it into chemical energy [63]. Previous studies have demonstrated that VOCs enhance photosynthetic efficiency, as evidenced by increased maximum quantum yield of photosystem II (Fv/Fm) and elevated chlorophyll content, likely through the promotion of iron assimilation and reduced ABA levels in plants [25, 64]. Li et al. demonstrated that the VOCs emitted by Bacillus velezensis CanL-30 significantly enriched GO terms related to photosynthetic processes, particularly “Photosynthesis, light harvesting in photosystem I”, “Photosynthesis, light harvesting in photosystem II”, which is consistent with our findings [65]. Furthermore, the VOCs from CanL-30 were found to upregulate chlorophyll and carotenoid biosynthetic genes, potentially mediated by enhanced expression of GOLDEN2-LIKE (GLK) transcription factors, which are pivotal for plastid development and pigment biosynthesis [65]. In the present study, our transcriptomic detected the GATA zinc finger protein gene, a transcription factor involved in the regulation of chloroplast development [66, 67] and chlorophyll biosynthetic pathway [68]. This finding suggests that: (1) GATA and GLK play distinct, stage-specific roles in regulating chlorophyll biosynthesis; (2) plant growth-promoting bacteria may employ distinct molecular mechanisms to enhance plant growth, potentially attributable to differences in their profiles of VOCs.

In plants, GATA transcription factors are evolutionarily conserved and play crucial roles in developmental regulation, responses to phytohormones, external stresses, and nitrogen metabolism [69, 70]. For instance, overexpression of Glycine max GATA58 in the Arabidopsis gnc (AtGATA21) loss-of-function mutant rescued the chlorophyll-deficient phenotype, while overexpression in wild-type Arabidopsis significantly enhanced leaf chlorophyll accumulation by upregulating multiple genes involved in the chlorophyll biosynthetic pathway [68]. Meanwhile, transcriptome profiling by An et al. indicated that GATA transcription factors in Populus trichocarpa regulate photosynthetic activity in leaves, growth and carbohydrate metabolism in stems, and nitrogen uptake in roots [71]. Moreover, transcriptomic analysis revealed that, in this study, Bacillus strain PAR690 significantly modulated the transcriptional expression of two genes involved in chlorophyll metabolism: methyl esterase 16 (MES16) and chlorophyllase 1 (CLH1). Chlorophyll degradation represents an efficient strategy for detoxifying the potentially phototoxic pigment. In Arabidopsis, the cytosolic enzyme MES16 plays a pivotal role in chlorophyll catabolism by specifically demethylating chlorophyll-derived intermediates of fluorescent chlorophyll catabolites [72]. The Arabidopsis chlorophyllase 1 localizes to developing chloroplasts and contributes to the protection of young leaves against prolonged photodamage by facilitating chlorophyll turnover during the PSII repair cycle [73]. Chlorophyll content in leaves of clh1 null mutant seedlings was approximately 25% lower than that in wild-type plants, whereas seedlings overexpressing CLH1 and CLH2 exhibited a ~30% increase in chlorophyll levels [74]. Furthermore, Streptomyces cellulase RFS-23 promotes the expression of the CLH1 gene, thereby inhibiting the decrease in chlorophyll content under plant viral infections [75].

VOCs-mediated regulation of nitrogen metabolism and anthocyanin biosynthesis

Nitrogen is a pivotal nutrient for plant growth and development, serving as an essential component in the biosynthesis of fundamental biomolecules-including amino acids, chlorophyll, nucleic acids, protein, and ATP [76]. PGPB have been demonstrated to enhance nitrate uptake through the upregulation of nitrate transporter gene expression, consequently improving nitrogen use efficiency and fostering plant growth [77]. The Arabidopsis NRT2 transporter family comprises seven members (NRT2.1-NRT2.7) that function as high-affinity, nitrate-specific transporters, among which NRT2.1 is the principal mediator of root high-affinity nitrate uptake under most physiological conditions [78]. Zhao et al. demonstrated that VOCs from Lysinibacillus macroides Xi9 up-regulated the expression of NRT2.1, NRT2.3, and NRT2.6, increased the nitrate and amino acid contents, and remodeled the root architecture of Arabidopsis seedlings [79]. Meanwhile, Lee et al. reported that Arabidopsis seedlings treated with B. subtilis strain L1 exhibited elevated NRT2.1 expression and increased nitrate reductase activity [77]. Similarly, our study revealed that PAR690 treatment significantly upregulated the expression of NRT2.1 and NRT2.5 in Arabidopsis, concomitantly enhancing amino acid content, total nitrogen content, and NR activity. GATA transcription factor may act as a key regulator, as GATA-binding motifs are present in the promoter regions of numerous genes associated with nitrogen assimilation, including those encoding nitrate reductase and nitrite reductase [80].

PAR690 treatment also significantly reduced the content of NH₄⁺, and the enzymatic activities of GS, GOGAT, and GDH. NH₄⁺ generated by nitrite reductase (NiR) is first incorporated into the amide group of GS. Subsequently, glutamate is synthesized primarily through the action of GOGAT. In higher plants, over 95% of assimilated NH₄⁺ is channeled through this GS/GOGAT cycle [81]. Previous studies have also reported that B. cereus G2 leads to a reduction in GS activity [82]. From the perspective of whole-plant energy metabolism, once photosynthetic activity nears saturation, the capacity for additional energy gain becomes limited, which may constrain energy allocation and lead to trade-offs with other energy-demanding processes, such as nitrogen assimilation [83]. The reduction in GS and GOGAT activities induced by PAR690 may be attributed to three potential mechanisms: (i) the accumulation of amino acids within plant tissues may exert feedback inhibition on these enzymes [84]; (ii) PAR690 enhances nitrogen assimilation by accelerating NH₄⁺ incorporation into amino acids, reducing intracellular NH₄⁺ and inducing adaptive decreases in GS and GOGAT activities [85]; and (iii) the enhanced plant growth stimulated by the bacterial strain could deplete nitrogen reserves in the growth medium, thereby triggering nitrogen starvation and consequently downregulating the activities of both GS and GOGAT [86]. PAR690 incubation reduced GDH activity in Arabidopsis, indicating that the plants adjusted their C-N metabolism under this condition to sustain a more efficient growth state.

Anthocyanins represent a major subgroup of plant flavonoids and play pivotal roles in mitigating oxidative damage triggered by various abiotic and biotic stresses [87]. Consequently, enhancing anthocyanin accumulation has become a key research focus, and the application of PGPB is considered an effective strategy. Paenibacillus pabuli P7S, a novel PGPB strain, has been shown to significantly increase both anthocyanin content and biomass in Arabidopsis seedlings by upregulating key genes involved in anthocyanin biosynthesis, including F3′H, DFR, and PAP1 [88]. Similarly, PAR690 significantly elevated anthocyanin levels by inducing PAP1 and UF3GT. UF3GT catalyzes flavonoid glycosylation, a crucial step that generates the first stable intermediate in the anthocyanin pathway [89]. The concurrent increase in total anthocyanin content suggests that PAR690 treatment may enhance the plant’s capacity to cope with stress conditions.

Conclusion

Elucidating the mechanisms by which PGPB enhance plant performance, together with the associated plant response pathways, is essential for both the effective application of beneficial microbes and the rational design of targeted plant improvement strategies. Our findings suggest that the Bacillus sp. PAR690, isolated from the rhizosphere of A. inebrians, promotes plant growth primarily through VOCs-mediated regulation of key transcriptional and metabolic processes. GC/MS analysis indicated that the potential VOCs produced by PAR690 with plant growth‑promoting activity include acetoin, 2,3‑butanediol, and indole. Transcriptomic analyses revealed that PAR690 significantly upregulated the expression of multiple genes involved in photosynthesis, which was physiologically manifested as enhanced chlorophyll accumulation and increased carbohydrate biosynthesis. In addition, the induction of the high-affinity nitrate transporter genes NRT2.1 and NRT2.5 implies that PAR690 may improve nitrogen uptake and accumulation, a mechanism frequently associated with improved plant biomass. Notably, the upregulation of GATA zinc finger transcription factor suggests a potential regulatory node linking microbial VOCs perception to photosynthetic control and nitrogen metabolism. Nevertheless, future studies should integrate multiple lines of genetic evidence to validate the roles of GATA transcription factors in the response to PAR690. It will also be important to clarify how specific VOCs produced by PAR690 regulate GATA expression and contribute to plant stress tolerance.

Authors’ contributions

Y.Y. Writing-original draft, Methodology, Investigation. J.J. Methodology, Investigation and Data curation. L.R., W.X., M.B., W.B. Investigation, Data curation. W.C., W.J. Writing-review & editing, Supervision, Project administration, Funding acquisition.

Funding

Yili Normal University Research Start-up Fund for High-Level Talents (2025RCYJ14); Xinjiang Leading Talent Introduction Program-Key University Talent Recruitment Project (XJRC-2025-JY-YJ-GX-QNQZ-046); Natural Science Foundation of Gansu Province (26JRRA176); Karst Rocky Desertification Water-Fertilizer Coupling and Biodiversity Restoration (Guizhou Education Technology [2023]004).

Data availability

The raw sequence data reported in this paper have been deposited in the Genome Sequence Archive in National Genomics Data Center, China National Center for Bioinformation/Beijing Institute of Genomics, Chinese Academy of Sciences (GSA: CRA038581) that are publicly accessible at https://ngdc.cncb.ac.cn/gsa/browse/CRA038581.

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.