Insights into Arabidopsis growth modulation by Microbacterium through volatile organic compounds and plant auxin biosynthesis and signaling

Insights into Arabidopsis growth modulation by Microbacterium through volatile organic compounds and plant auxin biosynthesis and signaling | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL This is a preprint and has not been peer reviewed. Data may be preliminary. 10 September 2025 V1 Latest version Share on Insights into Arabidopsis growth modulation by Microbacterium through volatile organic compounds and plant auxin biosynthesis and signaling Authors : Gonzalo Burgos Herrera 0000-0001-7744-8637 , Joaquín Inchaurrondo , Luciana Anabella Pagnussat , Mauro Do Nascimento , and Leonardo Curatti 0000-0002-8608-5791 [email protected] Authors Info & Affiliations https://doi.org/10.22541/au.175748638.86948481/v1 201 views 133 downloads Contents Abstract Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract Food security and environmental sustainability require innovative agricultural approaches, with plant growth-promoting microbes offering promising solutions. This study explores the growth modulation of plants by Microbacterium sp. MB15 through secreted compounds, distinguishing between the effects of total diffusible substances and volatile organic compounds (VOCs). Microbacterium sp. MB15 exhibited a dose-dependent influence on wheat and Arabidopsis germination, growth, and root architecture, shifting from stimulation at low exposure levels to inhibition at higher concentrations. Total diffusible compounds, including auxin produced by the bacterium, exerted its effect on Arabidopsis through the TIR1/AFB pathway and partially requires YUCCA genes for auxin biosynthesis by the plant. Microbacterium sp. MB15 VOCs, comprising ethanol, acetic acid, and dimethyl disulfide, also elicited strong modulation of seeds germination and seedlings growth through mechanisms only partially dependent on TIR1/AFB and YUCCA. A proteomic analysis of seedlings exposed to bacterial VOCs indicated stimulation of carbohydrate and lipid mobilization, overexpression of proteins associated with a non-canonical auxin pathway involving nitrilases and metabolism of indolic glucosinolates, defense responses, and sulfur/redox homeostasis. Additionally, high exposure to VOCs led to repression of genes for photosynthetic and chloroplast integrity. These findings contribute to unraveling a complex response of plant-microbe interactions at the molecular level. Insights into Arabidopsis growth modulation by Microbacterium through volatile organic compounds and plant auxin biosynthesis and signaling Gonzalo Burgos Herrera 1 , Joaquín Inchaurrondo 1 , Luciana Anabella Pagnussat 1,2 , Mauro Do Nascimento 1 , Leonardo Curatti 1 1 Instituto de Investigaciones en Biodiversidad y Biotecnología, INBIOTEC-CONICET, Mar del Plata, Buenos Aires, Argentina. Fundación para Investigaciones Biológicas Aplicadas 2 Laboratorio de Bioquímica Vegetal y Microbiana, Facultad de Ciencias Agrarias, Universidad Nacional de Mar del Plata, Mar del Plata, Buenos Aires, Argentina Correspondence: Leonardo Curatti ( [email protected] ) Funding: This research was financially supported by Agencia Nacional de Promoción Científica y Tecnológica, Argentina (Grant number PICT2018-3382) to LC. Keywords: plant-microbe interaction | PGPR | proteome | nitrilases | defense ABSTRACT Food security and environmental sustainability require innovative agricultural approaches, with plant growth-promoting microbes offering promising solutions. This study explores the growth modulation of plants by Microbacterium sp. MB15 through secreted compounds, distinguishing between the effects of total diffusible substances and volatile organic compounds (VOCs). Microbacterium sp. MB15 exhibited a dose-dependent influence on wheat and Arabidopsis germination, growth, and root architecture, shifting from stimulation at low exposure levels to inhibition at higher concentrations. Total diffusible compounds, including auxin produced by the bacterium, exerted its effect on Arabidopsis through the TIR1/AFB pathway and partially requires YUCCA genes for auxin biosynthesis by the plant. Microbacterium sp. MB15 VOCs, comprising ethanol, acetic acid, and dimethyl disulfide, also elicited strong modulation of seeds germination and seedlings growth through mechanisms only partially dependent on TIR1/AFB and YUCCA. A proteomic analysis of seedlings exposed to bacterial VOCs indicated stimulation of carbohydrate and lipid mobilization, overexpression of proteins associated with a non-canonical auxin pathway involving nitrilases and metabolism of indolic glucosinolates, defense responses, and sulfur/redox homeostasis. Additionally, high exposure to VOCs led to repression of genes for photosynthetic and chloroplast integrity. These findings contribute to unraveling a complex response of plant-microbe interactions at the molecular level. 1 | Introduction The rhizosphere, which includes plant roots, soil, and microorganisms, directly impacts crop growth and yield. Plant growth-promoting rhizobacteria (PGPR) improve plant health by converting nutrients into usable forms and producing phytohormones, enzymes, and stress-relieving compounds. They also enhance soil structure, fertility, and overall functioning, supporting plant growth in both normal and stressed conditions (Hakim et al. 2021; Bending et al. 2025). Rhizosphere engineering with PGPR is gaining attention as a “Nature-Based Solution” for sustainable agriculture. While some soil inoculants, like rhizobia, have shown consistent success, others perform well in the lab but yield mixed results in the field. To improve their effectiveness and ensure reliable applications for crop production, a deeper understanding of PGPR ecology and molecular interactions is crucial (Srivastava et al. 2025). The microbial production of volatile organic compounds (VOCs) is an important growth-promoting mechanism for plants. By using this mechanism, microbes can modulate plant growth without having direct contact. Microbial VOCs can enhance plant growth by modulating the synthesis and transport of plant hormones, as well as by promoting systemic resistance and stress tolerance (Bastías et al. 2022). Pioneering experiments demonstrated that VOCs produced by Bacillus subtilis promote the growth of Arabidopsis thaliana by modulating auxin homeostasis and the expression of auxin-related genes (Zhang et al. 2007). Furthermore, numerous prior research showed that bacterial VOCs might enhance plant resilience to abiotic challenges, including salinity, drought and nutrient scarcity (Liu and Zhang 2015), as well as biotic stresses such as herbivory, insect pests and microbial pathogens (Farag et al. 2013). Microbacterium spp . are rod-shaped, non-motile Gram-positive bacteria that can be found in soils. Although Microbacterium spp. were frequently isolated from different plant tissues for decades (Madhaiyan et al. 2010), demonstrations of its VOC-dependent PGPR properties are much more recent (Cordovez et al. 2018). Cordovez and colleagues (2018) demonstrated that VOCs from Microbacterium sp. can prime plant growth even after a short exposure to the roots, but not the shoots. They identified a wide array of sulfur-containing compounds, including dimethyl trisulfide, which has a concentration-dependent effect on plant growth, ranging from promotion to inhibition. A genome-wide transcriptome analysis suggested that the plant’s response might involve the modulation of sulfur and nitrogen metabolism (Cordovez et al. 2018). A more recent transcriptome analysis conducted with M. aurantiacum VOCs, showed upregulation of Nicotiana benthamiana genes related to carbohydrate metabolic processes, and downregulation of genes involved in cell redox homeostasis, among other metabolic and regulatory processes. Analysis of A. thaliana mutants suggested growth modulation might comprise auxin, salicylic acid, and gibberellin signaling (Gao et al. 2022). This study aimed to elucidate the molecular mechanisms by which Microbacterium sp. MB15, isolated from a microalgal culture, modulates plant growth. We focused on its dose-dependent effects mediated by both volatile and non-volatile compounds. We hypothesized that Microbacterium sp. MB15 influences plant development by altering protein accumulation in key metabolic pathways. Our findings revealed a pronounced, dose-dependent impact on plant germination, growth, ranging from stimulation to strong inhibition and root architecture. These phenotypic changes were accompanied by significant shifts in protein profiles, comprising a non- pathway for auxin biosynthesis, as well as other developmental, defense and metabolic processes. Together, these results advance our understanding of the complex molecular interactions underlying microbial modulation of plant growth. 2 | Materials and Methods 2.1 | Bacterial material, isolation and culture conditions Microbacterium sp. MB15 (PQ206266) was isolated from microalgae cultures using AEX medium (Do Nascimento et al. 2013). Identification involved 16S rDNA sequencing with universal primers, followed by BLAST and Clustal W analysis. Phylogenetic trees were built using MEGA5 with Neighbor Joining and Maximum Likelihood methods. Bacterial cell density was determined by three different methods: direct cell counting in a Neubauer chamber under the microscope (50X); optical density (OD) (λ=600nm) in a spectrophotometer (Shimadzu, model RF-540); and number of colony forming units (CFU) onto solid medium containing Km and Gm. The determined relationships were: 1.0 ± 0.0 OD 600 = 3.9 ± 0.2 10 9 cells mL -1 = 5.4 ± 0.4 10 9 CFU mL -1 (Supporting Information: Annex S1). 2.2 | Plant material and plant-bacterium interaction assays Wheat seeds ( Triticum aestivum cv. Baguette 620) were disinfected and used to assess germination and seedling development under exposure to Microbacterium sp. MB15, either directly or via VOCs. VOC assays involved enclosing inoculated LB plates within seed-containing Petri dishes. Seedlings were grown in vermiculite with INTA13 medium and inoculated with bacterial suspensions. Arabidopsis thaliana Col-0, DR5::GUS, tir1-1afb2-3 , and yuc3,5,6,7,8 lines were surface sterilized and grown on MS-agar. Co-culture assays tested effects of TDSs and VOCs using modified plates. Bacterial density effects and seed inoculation via floral spraying were also evaluated, followed by bacterial recovery and growth analysis (Supporting Information: Annex S1 and Figure S1). 2.3 | Determination of IAA and VOCs IAA produced by Microbacterium sp. MB15 in spent LB medium was quantified via HPLC, following freeze-drying and methanol extraction. Analysis used a Zorbax Eclipse XDB C18 column with a methanol/acetic acid mobile phase (60:1, pH 4.0), at 1 mL·min⁻¹ and 23 °C, with fluorescence detection (282/360 nm) (De Marco et al ., 2024). For VOC analysis, LB agar cylinders colonized by MB15 were sealed in 13 mL vials, and headspace volatiles were sampled. VOCs were identified using GC-MS at Farestaie (Mar del Plata, Argentina). Control vials with non-inoculated LB showed no detectable VOCs, confirming microbial origin. 2.4 | Histochemical staining and determination of β-glucuronidase (GUS) activity DR5::GUS seedlings (Ulmasov et al. 1997) exposed to TDSs or VOCs were immersed in GUS staining buffer containing the chromogenic substrate 5-bromo-4-chloro-3-indolyl β-d-glucuronid acid, essentially as described (Jefferson et al. 1987). For quantitative determination of GUS activity, seedling exposed to both protein extracts were incubated in the presence of the chromogenic substrate p-nitrophenyl β-D-glucuronide and substrate-to-product conversion was determined spectrophotometrically at 415 nm using a standard curve made of 10 to 30 µg p-nitrophenol according to previous reports (Aich et al. 2001; Qamar et al. 2022) (Supporting Information: Annex S1). 2.5 | Proteomic analysis For proteomic analysis, protein samples (40 μg), excised from the SDS-polyacrylamide gels, were subjected to mass spectrometry and protein identification at the Center for Chemical and Biological Studies by Mass Spectrometry, University of Buenos Aires, Argentina (http://cequibiem.qb.fcen.uba.ar). Briefly, samples were analyzed by nano HPLC coupled to a mass spectrometer with Orbitrap technology and the spectra are analyzed with the Proteome Discoverer program, using the database UniProt for A. thaliana (https://www.uniprot.org/proteomes/UP000006548) (Supporting Information: Annex S1). The mass spectrometry proteomics data have been deposited at ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier XXXXXXX. 2.6 | Real-time quantitative PCR analysis (RT-qPCR) Total RNA was isolated from the plant material using TRIzol™ reagent (Thermo Fisher Scientific) according to the supplier’s instructions. cDNA was synthetized using Moloney murine leukaemia virus reverse transcriptase and random hexamer primers (Promega). For RT-qPCR, specific primers were designed for the target genes NIT1, NIT2, GSTF2 and the housekeeping genes Actine2 and TIP41-like (Urbancsok et al. 2018) (Supporting Information: Annex S1 and Table S1). 2.7 | Graphs and statistical analysis Graphs and statistical analysis of differences throughout the study were performed using the GraphPad Prism 8 software. For proteomics, the statistical analysis of the data was conducted using Perseus software version 1.5.8.5, Microsoft Excel, and GraphPad Prism version 8.4.3. For protein abundance variations, the raw data was filtered to retain proteins with at least two replicates per condition, a statistical significance determined by multiple t-tests of p ≤ 0.05 , and the specified threshold for protein up- or down-regulation. 3 | Results 3.1 | Isolation and identification of Microbacterium sp. strain MB15 exhibiting PGPR activity on wheat and Arabidopsis seedlings Microbacterium sp. strain MB15 (PQ206266) was isolated from microalgae cultures enriched in AEX medium, which simulates typical microalgal exudates (Do Nascimento et al. 2013). Based on 16S rDNA sequencing, MB15 could not be definitively classified at the species level, showing 99.36% similarity to M. foliorum , M. phyllosphaerae , M. forte , M. testaceum , and M. oxydans , all associated with plant or algal environments. Phylogenetic clustering via Neighbor Joining (Supporting Information: Figure S2) and Maximum Likelihood (not shown) analyses confirmed this close relationship. Microbacterium sp. strain MB15 exhibited a cell density-dependent promotion of early wheat seedling development, with maximum activity observed at approximately 10 9 cells mL -1 in a bacterial suspension soaked under a filter paper that held the seeds. Higher bacterial densities resulted in a strong inhibition of early development (Supporting Information: Figure S3). Inoculation under hydroponic conditions (Supporting Information: Figure S4) also showed a significant increase in the length of the fully expanded third leaf (Figure 1a), shoot fresh weight (Figure 1b), and a remarkable increase up to about 80% in root fresh weight (Figure 1c). Bacterial VOCs also accelerated germination at low to moderate bacterial cell densities, particularly when using a single large bacterial spot (100 µL of cell suspension) for 24 h. Higher bacterial densities resulted in moderate to strong inhibition of germination (Figure 1d). Ethanol, acetic acid, methanethiol, and dimethyl disulfide were detected in the headspace of Microbacterium sp. MB15 cultured on solid medium within sealed vials, potentially acting as active compounds in the VOCs. When A. thaliana flowers were immersed in a suspension of Microbacterium sp. MB15 cells at 10 6 or 1.6 10 6 cells mg seed -1 (or an estimated 1.4 10 6 or 2.2 10 6 CFUs seed -1 ) (Supporting Information: Figure S1), 1.8 10 4 or 2.9 10 4 CFUs seed -1 were recovered in the seeds of self-pollinated plants, respectively. These seedlings germinated considerably faster than the non-inoculated seeds. Although some variability was observed in the germination rate of inoculated plants, the results suggested that most seeds carried enough bacteria to accelerate the germination process, regardless of the initial differences in bacterial density in the suspensions used to infiltrate the flowers (Figure 1e). However, no obvious difference in seedlings development and growth from these seeds was observed (Supporting Information: Figure S5). FIGURE 1 | Effect of inoculation or exposure to Microbacterium sp. strain MB15 on wheat and Arabidopsis germination and growth. (a-c) Wheat growth promotion by inoculation with Microbacterium sp . MB15. Plants on Zadoks phenological stage Z 1.4, Z 2.1 (Zadoks, 1974) were analyzed for (a) length of the third leaf; (b) shoot fresh weight; (c) fresh weight of roots. A total of 30 plants, distributed in 3 1-L pots were individually scored for (a) and (b), and the 10 plants from each pot were pooled for (c). The statistical analysis in (a-c) was performed by an unpaired t-test. (d) Effect of Microbacterium sp. MB15 VOCs on wheat seeds germination at two different doses and 3 different times. (e) Time course of germination of A. thaliana seeds from flowers immersed in a Microbacterium sp. MB15 cell suspensions. The statistical analysis was assessed using one-way ANOVA followed by Tukey’s multiple comparisons test for (d) and two-way ANOVA followed by Dunnett’s multiple comparisons test for (e). (a-e), *, p ≤ 0.05 ; **, p ≤ 0.01 ; ***, p ≤ 0.001 ; ****, p ≤ 0.0001 ; ns, p > 0.05 . 3.2 | Dissecting the mechanism of Microbacterium sp. MB15-dependent modulation of Arabidopsis growth Two alternative strategies were implemented to assess the impact of Microbacterium sp. MB15 exposure on A. thaliana growth: 1) TDSs assay plates containing agar-solidified MS medium with an LB medium cylinder, allowing free diffusion of substances (Figure 2a); and 2) VOCs assay plates, using I-plates with a central septum that isolates the bacteria and seedlings, enabling the analysis of effects produced solely by bacterial VOCs (Figure 2b). Figures 2a and 3a, show that TDSs produced by Microbacterium sp . MB15 modified seedlings growth and development, especially root architecture (branching and shortening of root elongation zone, and root hair density and length) according to its proximity to the source of bacterial diffusible substances, from strong inhibition to moderate growth promotion, respectively. Seedlings also exhibit sensitivity to the initial size of the bacterial inoculum in the TDSs plates. Therefore, we further analyzed the growth promotion and growth inhibition effects of TDSs using this proximity effect (I to IV) (Figure 2a). FIGURE 2 | Experimental approach to Microbacterium sp. MB15-mediated modulation of A. thaliana growth. (a) TDSs assay plates containing agar-solidified MS medium with an LB medium cylinder, allowing free diffusion of substances. Non-exposed plates contained the LB medium cylinder but no bacteria. From left to right: a cartoon, an image of a representative assay, and photomicrographs of root tips exposed to different doses TDSs according to its proximity to the bacteria. I to IV indicates the relative distance to the source of TDSs. Bars represent 100 µm for photomicrographs and 10 mm for Petri dishes. (b) VOCs assay plates containing a central septum that isolated the bacteria and seedlings, enabling the analysis of effects produced solely by bacterial VOCs. From left to right: cartoon and images of a representative assay using contrasting amounts of inoculated bacteria in the corresponding compartment. Seedlings also showed a pronounced sensitivity to the original size of the bacterial inoculum in the VOCs plates. Depending on the exposure level, the bacterium either enhanced growth or significantly impeded it (Figure 2b). However, as expected for volatile compounds, in contrast to TDSs plates, only marginal differences were observed according to the position of the seedlings relative to the source of VOCs. Thus, VOCs-influenced growth was evaluated independently for either growth promotion or inhibition using an appropriate size of the bacterial inoculum, referred as to low or high exposure (Figure 2b). FIGURE 3 | Arabidopsis thaliana DR5::GUS reactivity to Microbacterium sp. MB15 exposure. (a) Representative images of TDS-plates (left) and photomicrographs of histochemically stained roots for GUS activity. Bars represent 20 mm, or 1 mm, for plates or photomicrographs, respectively. (b) Photomicrographs of histochemically stained seedling roots showing GUS activity following exposure to bacterial VOCs decreasing from close proximity (I) to farther away (IV) from the VOC source. Bar represent 0.1 mm. (c) GUS activity of seedlings extracts. The statistical significance of the differences observed among treatments was assessed using one-way ANOVA followed by Dunnett’s multiple comparisons test. ** indicates statistically different, p ≤ 0.01 ; and ns, not statistically different, p > 0.05 . For each position (I-IV), 4 samples were used, one for each of four individual TDS-plates. Samples from all positions were pooled for non-exposed plants (n=16). For VOC-plates, both non-exposed (n=16) and exposed plant samples (n=16) corresponded of four plants from each one of four independent assays. Since both Microbacterium sp . MB15 TDSs and VOCs produce auxin-like effects in A. thaliana and wheat, and because IAA was identified in the spent medium of Microbacterium sp. MB15 at 0.07 ± 0.03 ng mL -1 , we conducted a more detailed analysis on this subject. When the A. thaliana auxin-reporter line DR5::GUS was challenged against bacterial TDSs, the seedlings exhibited not only enhanced root branching and reduced root length but also displayed auxin-responsive signals in the root maturation zone, particularly those nearer to the bacterial TDS source (Figure 3a). Seedlings not-exposed to bacterial TDSs showed a distinctive prominent signal in root tips, including the root cap, quiescent center, and xylem cells in the root apical meristem (Zhou et al. 2014). GUS activity showed very consistent results to that of the histochemical analyses for increased activity in the seedlings closer to the source of bacterial TDSs (Figure 3c). The auxin-reporter seedlings positioned closer to the source of bacterial VOCs exhibited enhanced staining in the vascular bundle of the cell-division zone (Figure 3b). However, this localized increase was insufficient to elevate the total GUS activity across entire roots (Figure 3c), suggesting that the auxin signaling enhancement may be confined to a small subset of cells. It is also possible that this focal increase is offset by reduced expression in other root regions, resulting in no overall change in GUS activity. Notably, given the absence of a distance-dependent effect of VOCs on seedling growth, the physiological relevance of this spatially restricted signaling pattern remains uncertain. Figure 4a shows a distinctive profile of TDSs-dependent growth promotion and inhibition of A. thaliana Col-0. However, both responses were absent in an auxin-insensitive ( tir1-1afb2-3 ) seedlings. The mutant seedlings exhibit a negative response to proximity to one or more components of the LB medium, but mostly independently of the bacterium (Figure 4a and c). FIGURE 4 | Effect of the tir1-1afb2-3 mutation on Microbacterium sp. MB15 TDSs- and VOCs-dependent growth modulation of A. thaliana seedlings. Images of Col-0 or tir1-1afb2-3 seedlings exposed to Microbacterium TDSs at positions I-IV from left to right, respectively. (b) Images of plates containing Col-0 or tir1-1afb2-3 seedlings exposed to Microbacterium VOCs at a low or high dose. The bar in a and b represents 10 mm. (c) Dry weight of Col-0 or tir1-1afb2-3 seedlings exposed to Microbacterium TDSs at positions I (proximal) through IV (distal) relative to the TDS source. (d) Fresh weight of Col-0 or tir1-1afb2-3 seedlings exposed to low of high doses of Microbacterium VOCs. (e) Primary root length Col-0 or tir1-1afb2-3 seedlings exposed to a high dose of Microbacterium VOCs. (c–d) Ordinary one-way ANOVA followed by Dunnett’s multiple comparisons test (n = 15). Statistical significance is indicated as follows: **, p ≤ 0.01 ; ***, p ≤ 0.001 ; ****, p ≤ 0.0001 . On the other hand, tir1-1afb2-3 seedlings still exhibited either growth enhancement or inhibition in response to different doses of VOCs (Figure 4b). However, this response (expressed as a percentage of biomass relative to non-exposed seedlings) was attenuated in the mutant seedlings, which exhibited an increase in the mean residual growth after exposure to a high dose of VOCs about 3-fold higher than that of Col-0 plants (Figure 4d). This change, although modest, was also evident in the image of the seedlings (Figure 4b). Additionally, inhibition of elongation of the main root appeared less sensitive to high doses of bacterial VOCs in the tir1-1afb2-3 mutant to Col-0 seedlings (Figure 4e). FIGURE 5 | Effect of the simultaneous inactivation of the yucc3,5,7,8,9 genes on Microbacterium sp. MB15 TDSs- and VOCs-dependent growth modulation of A. thaliana seedlings. (a) Images of plates containing yucc3,5,7,8,9 ( yucQ ) seedlings exposed to Microbacterium VOCs at a low or high dose. (b) Images of plates containing Col-0 or yucQ seedlings exposed to Microbacterium TDSs. Bars in (a) and (b) represent 10 mm. (c) Photomicrographs of yucQ seedlings exposed to Microbacterium TDSs or VOCs. (d) Fresh weight of Col-0 or yucQ seedlings exposed to Microbacterium TDSs at positions I (proximal) through IV (distal) relative to the TDS source. (e) Fresh weight of Col-0 or yucQ seedlings exposed to low of high doses of Microbacterium VOCs. In (d-e), ns indicates not statistically different, p > 0.05 ; * statistically different at p ≤ 0.05 ; or *** p ≤ 0.001, according to Sidak’s multiple comparison test after two-way ANOVA, (n = 4) for each position (I-IV) in (d), or one-way ANOVA (n=16) in (e). A. thaliana seedlings with simultaneous inactivation of the yucc3,5,7,8,9 genes, ( yuc Q) displayed a characteristic growth-defect phenotype, which comprises the development of very short and agravitropic primary roots (Chen et al. 2014) (Figure 5a-c). Inoculation with bacteria onto TDS plates effectively rescued impaired root development and restored the gravitropic response. Although growth promotion and inhibition were still observed in seedlings placed farther or closer to the TDSs source, respectively, both responses were somewhat attenuated compared to Col-0 seedlings (Figure 5d). Bacterial VOCs moderately induced growth at low doses, similar to the response in Col-0 seedlings. However, yuc Q appeared to be significantly less sensitive to high doses of Microbacterium sp. MB15 VOCs than Col-0 or tir1-1afb2-3 seedlings (Figure 5e). 3.3 | Proteomic response of A. thaliana exposed to Microbacterium sp. MB15 VOCs To have enough biomass of VOCs-stimulated seedlings, especially in growth inhibition plates, seedlings were allowed to develop in the absence of bacteria for 11 days, and then Microbacterium sp. MB15 was inoculated at two different cell densities, regarded as low or high VOC doses. Under these experimental setting, growth inhibition was less severe than that shown in Figures 2-5. However, a distortion in root growth and architecture was still clearly noticeable ((Supporting Information: Figure S6). Overall, the most dramatic shift in the proteome was observed after stimulation with a high dose of bacterial VOCs (Supporting Information: Figure S7 and Table S2). Under these conditions, 0.8%, 2.3%, or 5.9% of the analyzed proteins were up-regulated by at least 16-, 8-, or 4-fold ( p≤0.05 ); and 0.2%. 0.4%, or 1.0% were down-regulated by at least 16-, 8-, or 4-fold ( p≤0.05 ), respectively. Exposure to a lower dose of bacterial VOCs resulted in a lower number of regulated genes/proteins (Supporting Information: Table S2). Supplementary Table S3 presents a list of A. thaliana proteins that exhibit strong responsiveness to high exposure to Microbacterium sp. MB15. The table includes protein names, gene names, and functions, retrieved from UniProt (https://www.uniprot.org/uniprotkb), along with their fold-change values and statistical analysis. A ShinyGO analysis of gene-set enrichment for proteins that accumulated more than 4-fold ( p≤0.05 ) following stimulation with a high dose of bacterial VOCs, using the Map.PlantGSAD database, primarily revealed enrichment in phytohormones/auxin metabolism; glucosinolate degradation; nitrilases; amino acid, lipids, sugars and glutathione metabolic pathways. A similar analysis using the GO Biological Processes, and other databases, showed similar pathway enrichment, being indole-containing compounds and IAA metabolism the top ranked pathways. On the other hand, 4-fold down-regulated proteins ( p≤0.05 ) by exposure to a high dose of VOCs were mainly grouped in pathways for photosynthesis and chloroplast functions, and glucosinolates metabolism (Supporting Information: Figure S8). FIGURE 6 | Proteomic changes in the A. thaliana glucosinolates/nitrilases-dependent biosynthesis of IAA pathway. Pathway was retrieved from (Piotrowski, 2008; Kumari et al. , 2015; Harun et al. , 2020). Specific values of fold-change and statistical significance by multiple t-test are stated in the main text and in Supplementary information: Table S3. The responses to low and high doses of VOCs showed minimal overlap. Only 15.6% of up-regulated proteins and 4.5% of down-regulated proteins were common between seedlings exposed to low or high VOC levels. Proteins that were similarly up-regulated primarily participated in lipid and amino acid metabolism. However, proteins exclusively up-regulated by low doses of VOCs were associated with protein biosynthesis, function, and photorespiration. In contrast, proteins up-regulated by higher doses of VOCs affected a wider range of metabolic processes, including IAA biosynthesis and signaling pathways (Supporting Information: Annex S2). On the other hand, we only observed down-regulation of glutaredoxin-C1, regardless of VOC dose. Low VOC exposure specifically led to the down-regulation of carbohydrate-related processes, such as cell wall and starch metabolism. Meanwhile, higher doses of VOCs resulted in the down-regulation of photosynthesis-related processes and abscisic acid metabolism (Supporting Information: Annex S2). 3.3.1 | Glucosinolates pathway and nitrilases-dependent biosynthesis of IAA Notably, some of the most responsive genes corresponded to the glucosinolates (GSL) pathway (Supporting Information: Table S3). Figure 6 depicts the A. thaliana glucosinolates pathway comprising nitrilases-dependent biosynthesis of IAA (Harun et al. 2020) and the observed changes in key protein abundance. In the aliphatic GSL biosynthesis branch, the most noticeable effect was down-regulation of the 3-isopropylmalate dehydratase small subunit 1 IPMI2/SSU2 (38-fold, p=0.058 ) and the cytochrome P450 83A1 CYP83A1 (25-fold, p=0.009 ). Other proteins of this metabolic branch, such as BCAT4, CYP79F1 were also down-regulated but to a lower extent and/or variation was less statistically supported (Supporting Information: Table S3). In the indolic GSL biosynthesis from chorismate, some proteins were strongly up-regulated by exposure to a high dose of VOCs. Most notably, the anthranilate synthase alpha subunit 1 ASA1 increased by 9-fold ( p=0.031 ); the UDP-glycosyltransferase 74B1 UGT74B1 (4-fold, p=0.024 ); the inactive GDSL esterase/lipase-like protein 23 GLL23 (10-fold, p=0.018) ; and the myrosinase-binding protein 1 MBP1 (8-fold, p=0.002 ). Other proteins of this pathway were also up-regulated but either to lower extent, and/or variation was less statistically supported (Supplementary Table S3). For the terminal branch indolic GSLs, comprising glucobrassicin degradation and production of IAA, the nitrile specifier protein 5, NSP5 and nitrilase 2, NIT2 were strongly up-regulated (both 19-fold, p=0.01 and p=0.002 , respectively). Nitrilase 1, NIT1, was also up-regulated (5-fold, p=0.02 ). Nitrilases NIT1 and NIT2 gene expression was further confirmed by RT-qPCR analysis (Figure 7). FIGURE 7 | RT-qPCR analysis of A. thaliana NIT1, NIT2 and GSTF2 in seedlings exposed to Microbacterium sp. MB15 VOCs. Each sample corresponded to two different seedlings from each biological replicate of non-exposed (n=4), or exposed to a low (n=4), or a high (n=5) dose of bacterial VOCs, which were pooled and processed together for RNA extraction. Asterisks indicate significant difference between samples, which were assessed by one-way ANOVA test together with Dunnett’s multiple comparisons test. *, p ≤ 0.05 ; **, p ≤ 0.01. 3.3.2 | Auxin metabolism/transport Stimulation with Microbacterium sp. MB15 VOCs significantly altered the abundance of proteins involved in auxin metabolism and transport. Notably, GSTF2 and GSTF6 were strongly up-regulated, suggesting enhanced IAA transport and camalexin biosynthesis via IAN conjugation. Other up-regulated proteins included GH3.15 (IAA conjugation), ILL4 (IAA release), IBR3 (IBA-to-IAA conversion), and UGT74E2 (auxin homeostasis and stress response), with expression levels increasing between 6- to 11-fold. In contrast, ROSY1 and MES9, linked to auxin transport and methylated hormone metabolism, were down-regulated. Additional functional information and relevant literature references is provided as Supporting Information: Annex S3. 3.3.3 | Response to pathogens/defense In addition to glucosinolate and IAA-related defense responses, several other defense-associated proteins were strongly up-regulated in A. thaliana upon exposure to Microbacterium sp. MB15 VOCs. These included BBE8, VLN3, and NHL3, all linked to stomatal immunity and pathogen resistance, with expression increases ranging from 19- to 29-fold. Proteins such as OGOX1, CORI3, SOT12, KTI1, NATA1, HIR3, LKR/SHD, PER71, and SAHH2—each involved in various defense mechanisms including cell wall reinforcement, systemic acquired resistance, and pest deterrence—also showed significant induction. In contrast, PER27 was notably down-regulated, suggesting selective modulation of peroxidase activity in VOC-triggered defense signaling. Additional functional information is provided as Supporting Information: Annex S4 3.3.4 | Other miscellaneous highly-responsive proteins Exposure to Microbacterium sp. MB15 VOCs triggered strong modulation of stress-related metabolic pathways in A. thaliana . Several proteins linked to osmotic stress, sugar metabolism, lipid mobilization, and nitrogen assimilation were significantly up-regulated, including CPK21, HPD, DIN10/RS6, RFS2/SIP2, SUS1, KJC1, LACS6/7, AEE15, and ASN1, with fold changes ranging from 5- to 64-fold. These changes suggest enhanced drought tolerance and metabolic reprogramming. Conversely, key regulators of senescence and redox balance, NEET, PER45, and ERD7, were markedly down-regulated. A more detailed description is provided as Supporting Information: Annex S5 4 | Discussion 4.1 | Microbacterium sp. MB15 modulates plants growth through IAA biosynthesis and signaling Production of IAA by microbacteria isolated from plants has been frequently reported (for example, see (Yadav et al. , 2022)) which, similarly to other plant-microbe interactions (Barazani and Friedman, 1999), tended to support the hypothesis that IAA mediates microbacteria plant-growth promotion or inhibition in a dose-dependent manner. However, other substances produced by the bacteria also participate in modulating the effect on plant growth (Barazani and Friedman, 1999). The direct determination of IAA in Microbacterium sp. MB15 exudates, the phenotype of seedlings exposed to TDSs (Figure 2), and the response of DR5::GUS (Figure 3), tir1-1afb2-3 (Figure 4) and yucQ (Figure 5) seedlings confirm that IAA mediates, at least in part, the response of A. thaliana to Microbacterium sp. MB15 TDSs. Failure of tir1-1afb2-3 plants to respond to TDSs indicates that IAA released by Microbacterium sp. MB15 accounts for a major part of both, growth promotion and inhibition, at low or high bacterial densities, respectively. TIR1/AFB is a key component of the canonical auxin signaling pathway, participating as an auxin receptor. Auxin binding to TIR1 increases its affinity for the Aux/IAA repressors, promoting the ubiquitination and subsequent degradation of Aux/IAAs, thus releasing their repression of ARF-mediated transcription (Qi et al. , 2022). IAA-dependent rapid inhibition of root growth comprises a TIR1/AFB receptors-dependent process that appears not to involve transcriptional reprogramming. However, it is later reinforced by the TIR1-mediated transcriptional regulation (Fendrych et al. , 2018). Additionally, the effective rescue of the gravitropic response in partially IAA-deficient yucQ seedlings, which are defective in root-specific yuc genes (Chen et al. , 2014), when exposed to Microbacterium sp. MB15 TDSs, further confirmed that the IAA produced by the bacterium was sensed by the seedlings, leading to altered root growth and development. The inhibitory effect of a higher dose of bacterial TDSs was also alleviated in yucQ seedlings, further confirming the participation of IAA in the observed response. In this study, we also analyzed separately the response of seedlings to bacterial VOCs in an attempt to delve deeper into a somehow less-investigated aspect of plant-microbe interaction. We showed that both wheat and A. thaliana respond strongly to Microbacterium sp. MB15 VOCs in a dose-dependent manner, exerting either beneficial or detrimental effects at low or high doses, respectively (Figures 1 and 2). The VOCs identified in the head space of sealed vials containing excised cylinders of LB culture medium containing Microbacterium sp. MB15 on the surface were ethanol, acetic acid, ethyl acetate, methanethiol and dimethyl disulfide. Although scarcely studied, the production of both ethanol (Hitchener et al. , 1979) and acetic acid (Fakhimi et al. , 2024) by Microbacterium spp. has been proposed. Determining whether the production of these typical fermentation products in other bacteria resulted from microaerobiosis/anaerobiosis in the sealed vials, or if it also occurred during co-culture with plants awaits further confirmation. Both ethanol (Bashir et al. , 2022) and acetic acid (Rahman et al. , 2024) have been shown to stimulate drought tolerance and other stress responses in plants, which may have promising implications for practical applications in agriculture. Methanethiol and dimethylpolysulfide were previously found in Microbacterium spp. VOCs, and were regarded as bioactive compounds modulating plants growth and funs (Cordovez et al. , 2018; Ballot et al. , 2023). While further analyses are required to determine whether these substances act individually, additively, or synergistically in modulating plant growth in response to Microbacterium sp. MB15 VOCs, it is also plausible that other, undetected compounds contribute to this effect. Nonetheless, the findings reported here provide a foundation for understanding this interaction. Sensitivity to Microbacterium sp. MB15 VOCs of the tir1-1afb2-3 seedlings suggests that the response would be only partially dependent on TIR1/AFBs. This contrasts with the marked insensitivity observed for tir1-1afb2-3 seedlings exposed to TDSs, as a source of exogenously supplied IAA. This is consistent with a previous report that showed that TIR1 and AFB2 are the dominant auxin receptors in the seedling root, and that tir1-1afb2-3 seedlings are insensitive to the exogenous IAA-dependent inhibition of root elongation (Parry et al. , 2009). Exposure of A. thaliana to M. oxydans EC8 VOCs up-regulated the expression of TIR1 in both shoot and root tissues (Cordovez et al., 2018), providing complementary evidence for IAA/TIR1/AFBs signal transduction of Microbacterium spp. VOCs. Although MB15 VOCs are present in TDSs, the TIR1/AFB-independent response seen with VOCs alone appears inactive when seedlings are exposed to TDSs. This suggests that dominant exogenous IAA/TIR1-AFB signaling may suppress alternative pathways, a hypothesis that warrants experimental validation. Also, Microbacterium sp. MB15 VOCs rescued the IAA-dependent gravitropic defect of yucQ seedlings. These plants remained noticeably less sensitive to both, growth promotion and inhibition, at low or high doses of VOCs, respectively. While this additionally suggests the effect of VOCs involves IAA production in roots, at least in a partially yucc3,5,7,8,9 -independent manner, it also suggests that the root yuc- pathway might additively participate in VOCs-dependent modulation of seedlings biomass production. Gao et al. (2022) found that M. aurantiacum GX14001VOC-induced growth promotion in A. thaliana was impaired in npr1 , gai1 or etr1 , implicating salicylic acid, gibberellin and ethylene signaling in the response. Although authors proposed a similar effect in arf1 mutants, the link to IAA signaling appears weaker, as ARF1 lacks critical roles in key auxin-regulated processes (Ellis et al., 2005). 4.2 | Insights from the proteomic analysis of A. thaliana proteins highly regulated by Microbacterium sp. MB15 VOCs Seelings showed complex shifts in the proteome after stimulation with VOCs, suggesting the participation of multiple signals and/or key regulatory factors, such as phytohormones. Indeed, one of the most noticeably effect was the up-regulation of key genes for indolic GSLs-nitrilases pathway for IAA biosynthesis, auxin metabolism and signaling, and fatty acids, amino acids and sugars metabolism, plant defense, and proteins related to other abiotic stresses (Supporting Information: Figure S8). Proteins for photosynthesis and chloroplast biogenesis and integrity were moderately (about 4-fold) down-regulated in seedlings exposed to a higher dose of VOCs, while exposure to a lower dose did not change the abundance of this set of proteins substantially. The proteomic shift observed after exposure to low or high doses of VOCs demonstrated limited overlap, with only modest quantitative changes detected in protein expression (Supporting Information: Annex S2). On one hand, this suggests that variations in stimulus threshold levels influence the activation of different responses. For instance, most defense proteins tend to be up-regulated in a dose-dependent manner in response to VOCs. Some IAA metabolism proteins were highly up-regulated mostly by a high dose of VOCs, and photosynthetic proteins were only down-regulated by a higher dose of VOCs. On the other hand, this would, at least in part, explain the final output of plants growth promotion or inhibition at the molecular level. We observed a strong down-regulation of ERD7 in A. thaliana following exposure to low doses of VOCs. Given that erd7 mutants show delayed senescence and overexpression accelerates leaf aging (Wu et al . , 2024), it would be interestingly to study whether VOC dose-dependent regulation of ERD7 may contribute to the distinct physiological responses triggered by the bacterium. As part of the defense response to pathogens, and due to its metabolic pathway involving IAA production in a yucca-independent manner, we conducted a more detailed analysis of the GLS pathway (Figure 7). GLSs are commonly found in cruciferous plants, including A. thaliana . Through the action of myrosinases and nitrile specifier proteins (NSPs), GLSs produce nitriles and other degradation products that contribute to plant defense against biotic stresses, including pathogens and herbivory (Malhotra et al . 2023). In stimulated seedlings, the aliphatic GSL branch, was down-regulated, while the indolic branch was activated. These branches are reciprocally regulated by R2R3-MYB transcription factors, with indolic GSL regulators mainly expressed in vegetative tissues and aliphatic ones in reproductive organs (Gigolashvili et al., 2009). Notably, strong induction of nitrilases NIT1 and NIT2 supports activation of the YUCCA-independent IAA biosynthesis via the IAOx pathway (Cao et al., 2019). Beyond auxin production, NITs also contribute to plant defense by degrading nitriles from aliphatic GSLs. Mutants lacking NIT1, NIT2, and NIT3 showed an increased susceptibility to bacterial infection, with up to 20-fold higher pathogen loads (Janowitz et al., 2009; Yang et al., 2024), highlighting their dual role in hormone signaling and immunity. Another recent study by Chroston et al. (2024) revealed that Actinobacteria, mainly Rhodococcus spp. and to a lesser extent Microbacterium spp., dominate the rhizosphere of A. thaliana Col-0. In mutants impaired in GLS biosynthesis and/or NSP activity, the relative abundance of these bacteria varied but remained more stable than other microbial phyla. This resilience suggests that microbacteria may actively modulate GLS–IAA metabolic pathways to enhance their rhizosphere competence. Such interactions could reflect a selective advantage, allowing microbacteria to persist and thrive in chemically distinct root environments, though further experimental validation is needed to confirm this hypothesis. Exposure to VOCs uncovered the concerted over-expression of proteins that participate in stomatal closure upon challenge with pathogens, such as berberine bridge enzyme-like 8 (BBE8) (Rodrigues Oblessuc et al. , 2019) and 19 (OGOX1) (Benedetti et al. , 2018), and villin-3 (VLN3) (Zou et al. , 2021). In general, while plants overexpressing these proteins presented augmented resistance to pathogens, mutant plants lacking active copies of these genes are more susceptible. On one hand, this suggests that stomatal immunity could be a prominent aspect of the defense response triggered by a high dose of VOCs, preventing pathogen entry through open stomata. Additionally, this may confer drought tolerance, as observed in previous studies on Microbacterium -triggered drought resistance in plants (Siraj et al. , 2022). We showed promotion and/or anticipation of germination by VOCs (Fig. 1). The molecular mechanism behind that process may comprise the peroxisomal long chain acyl-CoA synthetases 6 and 7 (LACS6 and LACS7) which are key enzymes for the degradation of long-chain fatty acids via beta-oxidation during lipid mobilization for seed germination, postgerminative growth and seedling establishment. Also the long-chain-fatty-acid–[acyl-carrier-protein] ligase AAE15, which participate of triacylglycerols degradation was highly overexpressed. The soybean GmLACS2, which is apparently an orthologue to A. thaliana AAE15, participates in lipid degradation during seed germination (Yu et al. , 2014). Also the key proteins for raffinose metabolism DIN10/raffinose synthase 6 (RS6) and a probable galactinol–sucrose galactosyltransferase 2 RFS2/SIP2 were highly up-regulated upon challenging with bacterial VOCs. RS6, in conjunction with RS5, contributes to raffinose accumulation in drought-stressed plants. Mutant seeds deficient in RS4 and 5 showed a 5 days delayed germination phenotype in darkness (Gangl and Tenhaken, 2016). RFS2/SIP2 is an α-galactosidase which would participate in raffinose phloem unloading (Peters et al. , 2010) and galactose release from raffinose oligosaccharides (RFO) during seeds imbibition, as an integrator and signal multiplier of germination-promoting events through the PIF6 transcription factor. If no RFO storage is present, like in AtRS4,5 seeds, germination in the darkness is delayed (Gangl and Tenhaken, 2016). 4.3 | Comparison to previous transcriptomic studies Although A. thaliana shows similar responses to Microbacterium spp. VOCs, strain-level volatilome diversity and methodological differences complicate cross-study comparisons (Cordovez et al., 2018; Brilli et al., 2019). Cordovez et al. (2018), using Col-0 and related bacterial species, identified transcriptomic enrichment in cytokinin, ethylene, oxidative stress, sulfur metabolism, and purine pathways in shoots, and nitrate assimilation, jasmonic acid metabolism, actin regulation, and acetyl-CoA processes in roots. Down-regulated genes clustered around carbohydrate metabolism, plastid organization, and development in shoots, and anion transport, herbicide response, and syncytium formation in roots. Although broadly coincidental with our analysis, our research, especially stimulation with a higher dose of Microbacterium sp. MB15 VOCs, uncovered a more robust activation of defense genes/proteins and IAA metabolism. Both studies determined a strong regulation of sulfur metabolism/oxidative stress/redox signaling with significant change in the expression level of a variety of GST proteins. Metabolism of S-containing GSLs, including up-regulation of NIT2, was observed in both studies. However, this response, considering the number of gene/proteins and fold-change was much more prominent in our study. Contrary to Cordovez et al. (2018), we only detected a few VOCs-responsive gene/proteins for nitrogen assimilation. Notably, we observed a strong up-regulation (24-fold, p=0.0002 ) of asparagine synthetase [glutamine-hydrolyzing] 1 (ASN1). Seeds of ASN1 overexpressing lines presented higher soluble seed protein content and higher tolerance of young seedlings when grown on nitrogen-limiting media (Lam et al. , 2003). Gao et al. (2022) showed that VOCs from M. aurantiacum GX14001 modulate gene expression in Nicotiana benthamiana , affecting KEGG pathways related to hormone signaling, defense, and redox metabolism. These transcriptomic shifts suggest that Microbacterium spp. VOCs may trigger conserved responses across diverse plant species. 4.4 | Outlook and possible contributions towards sustainable agriculture Our study reveals a complex response of A. thaliana to VOCs emitted by Microbacterium sp. MB15, offering mechanistic insights into microbial modulation of plant physiology. While based on a model species, these findings contribute to growing evidence supporting VOC-mediated enhancement of stomatal immunity, stress tolerance, and nitrogen use efficiency, traits with clear relevance to crop systems. However, practical application of VOCs remains challenging due to their volatility and sensitivity to environmental factors like wind, humidity, and temperature, which complicate dose control and delivery (Zhao et al., 2002; Brilli et al., 2019; Ali et al., 2025). Advances in micro- and nano-encapsulation may help overcome these limitations. Additionally, MB15’s ability to colonize A. thaliana seeds via floral infection, persist in dry seeds, and stimulate germination highlights its potential for microbial seed inoculation. These findings support the development of nature-based microbial solutions for sustainable agriculture, though translational studies in crops and field conditions remain essential. Author contributions GBH and LC designed the research and analyzed the results. GBH performed all the major experiments. JI, LAP and MD performed or assisted in some experiments. GBH, MD and LC participated in data visualization tasks. LC wrote the draft manuscript, supervised the research and provided funding. All of the authors discussed the results and commented, and contributed to the edition of the manuscript. Acknowledgements We acknowledge the valuable technical assistance from Dr. Marisol Fassolari for determination of IAA. Data availability statement The data that support the findings of this study are available from the corresponding author upon reasonable request. ORCID Leonardo Curatti https://orcid.org/0000-0002-8608-5791 Declaration of competing interest The authors declare that they have no conflict of interest. References Aich, S., L. T. J. Delbaere, R. and Chen . 2001 . ”Continuous spectrophotometric assay for β-glucuronidase”. BioTechniques 30 : 846–850. Ali, Q., A. R. Khan, W. Raza, M. S. Bilal, S. Khalid, M. Ayaz, et al . 2025 . ”Mechanisms of microbial VOC‐mediated communication in plant ecosystems and agricultural applications”. Journal of Sustainable Agriculture and Environment 4 : e70044. Ballot, A., J. Dore, M. Rey, G. Meiffren, T. Langin, P. Joly, et al. 2023 . ”Dimethylpolysulfides production as the major mechanism behind wheat fungal pathogen biocontrol, by Arthrobacter and Microbacterium actinomycetes” (KL Hockett, Ed.). Microbiology Spectrum 11 : e05292-22. Barazani, O. and J. 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Plants on Zadoks phenological stage Z 1.4, Z 2.1 (Zadoks, 1974) were analyzed for (a) length of the third leaf; (b) shoot fresh weight; (c) fresh weight of roots. A total of 30 plants, distributed in 3 1-L pots were individually scored for (a) and (b), and the 10 plants from each pot were pooled for (c). The statistical analysis in (a-c) was performed by an unpaired t-test. (d) Effect of Microbacterium sp. MB15 VOCs on wheat seeds germination at two different doses and 3 different times. (e) Time course of germination of A. thaliana seeds from flowers immersed in a Microbacterium sp. MB15 cell suspensions. The statistical analysis was assessed using one-way ANOVA followed by Tukey’s multiple comparisons test for (d) and two-way ANOVA followed by Dunnett’s multiple comparisons test for (e). (a-e), *, p ≤ 0.05 ; **, p ≤ 0.01 ; ***, p ≤ 0.001 ; ****, p ≤ 0.0001 ; ns, p > 0.05 . FIGURE 2 | Experimental approach to Microbacterium sp. MB15-mediated modulation of A. thaliana growth. (a) TDSs assay plates containing agar-solidified MS medium with an LB medium cylinder, allowing free diffusion of substances. Non-exposed plates contained the LB medium cylinder but no bacteria. From left to right: a cartoon, an image of a representative assay, and photomicrographs of root tips exposed to different doses TDSs according to its proximity to the bacteria. I to IV indicates the relative distance to the source of TDSs. Bars represent 100 µm. (b) VOCs assay plates containing a central septum that isolated the bacteria and seedlings, enabling the analysis of effects produced solely by bacterial VOCs. From left to right: cartoon and images of a representative assay using contrasting amounts of inoculated bacteria in the corresponding compartment. FIGURE 3 | Arabidopsis thaliana DR5::GUS reactivity to Microbacterium sp. MB15 exposure. (a) Representative images of TDS-plates (left) and photomicrographs of histochemically stained roots for GUS activity. Bars represent 20 mm, or 0.1 mm, for plates or photomicrographs, respectively. (b) Photomicrographs of histochemically stained seedling roots showing GUS activity following exposure to bacterial VOCs decreasing from close proximity (I) to farther away (IV) from the VOC source. Bar represent 0.1 mm. (c) GUS activity of seedlings extracts. The statistical significance of the differences observed among treatments was assessed using one-way ANOVA followed by Dunnett’s multiple comparisons test. ** indicates statistically different, p ≤ 0.01 ; and ns, not statistically different, p > 0.05 . For each position (I-IV), 4 samples were used, one for each of four individual TDS-plates. Samples from all positions were pooled for non-exposed plants (n=16). For VOC-plates, both non-exposed (n=16) and exposed plant samples (n=16) corresponded of four plants from each one of four independent assays. FIGURE 4 | Effect of the tir1-1afb2-3 mutation on Microbacterium sp. MB15 TDSs- and VOCs-dependent growth modulation of A. thaliana seedlings. Images of Col-0 or tir1-1afb2-3 seedlings exposed to Microbacterium TDSs at positions I-IV from left to right, respectively. (b) Images of plates containing Col-0 or tir1-1afb2-3 seedlings exposed to Microbacterium VOCs at a low or high dose. The bar represents 10 mm and applies to (a) as well. (c) Dry weight of Col-0 or tir1-1afb2-3 seedlings exposed to Microbacterium TDSs at positions I (proximal) through IV (distal) relative to the TDS source. (d) Fresh weight of Col-0 or tir1-1afb2-3 seedlings exposed to low of high doses of Microbacterium VOCs. (e) Primary root length Col-0 or tir1-1afb2-3 seedlings exposed to a high dose of Microbacterium VOCs. (c–d) Ordinary one-way ANOVA followed by Dunnett’s multiple comparisons test (n = 15). Statistical significance is indicated as follows: **, p ≤ 0.01 ; ***, p ≤ 0.001 ; ****, p ≤ 0.0001 . FIGURE 5 | Effect of the simultaneous inactivation of the yucc3,5,7,8,9 genes on Microbacterium sp. MB15 TDSs- and VOCs-dependent growth modulation of A. thaliana seedlings. (a) Images of plates containing yucc3,5,7,8,9 ( yucQ ) seedlings exposed to Microbacterium VOCs at a low or high dose. (b) Images of plates containing Col-0 or yucQ seedlings exposed to Microbacterium TDSs. Bars in (a) and (b) represent 10 mm. (c) Photomicrographs of yucQ seedlings exposed to Microbacterium TDSs or VOCs. (d) Fresh weight of Col-0 or yucQ seedlings exposed to Microbacterium TDSs at positions I (proximal) through IV (distal) relative to the TDS source. (e) Fresh weight of Col-0 or yucQ seedlings exposed to low of high doses of Microbacterium VOCs. In (d-e), ns indicates not statistically different, p > 0.05 ; * statistically different at p ≤ 0.05 ; or *** p ≤ 0.001, according to Sidak’s multiple comparison test after two-way ANOVA, (n = 4) for each position (I-IV) in (d), or one-way ANOVA (n=16) in (e). FIGURE 6 | Proteomic changes in the A. thaliana glucosinolates/nitrilases-dependent biosynthesis of IAA pathway. Pathway was retrieved from (Piotrowski, 2008; Kumari et al. , 2015; Harun et al. , 2020). Specific values of fold-change and statistical significance by multiple t-test are stated in the main text and in Supplementary information: Table S3. FIGURE 7 | RT-qPCR analysis of A. thaliana NIT1, NIT2 and GSTF2 in seedlings exposed to Microbacterium sp. MB15 VOCs. Each sample corresponded to two different seedlings from each biological replicate of non-exposed (n=4), or exposed to a low (n=4), or a high (n=5) dose of bacterial VOCs, which were pooled and processed together for RNA extraction. Asterisks indicate significant difference between samples, which were assessed by one-way ANOVA test together with Dunnett’s multiple comparisons test. *, p ≤ 0.05 ; **, p ≤ 0.01. Information & Authors Information Version history V1 Version 1 10 September 2025 Copyright This work is licensed under a Non Exclusive No Reuse License. Keywords defense hormones nitrilases plant-microbe interaction proteome volatile emissions Authors Affiliations Gonzalo Burgos Herrera 0000-0001-7744-8637 Instituto de Investigaciones en Biodiversidad y Biotecnologia View all articles by this author Joaquín Inchaurrondo Instituto de Investigaciones en Biodiversidad y Biotecnologia View all articles by this author Luciana Anabella Pagnussat Instituto de Investigaciones en Biodiversidad y Biotecnologia View all articles by this author Mauro Do Nascimento Instituto de Investigaciones en Biodiversidad y Biotecnologia View all articles by this author Leonardo Curatti 0000-0002-8608-5791 [email protected] Instituto de Investigaciones en Biodiversidad y Biotecnologia View all articles by this author Metrics & Citations Metrics Article Usage 201 views 133 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Gonzalo Burgos Herrera, Joaquín Inchaurrondo, Luciana Anabella Pagnussat, et al. 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