**A Mass Spectrometry-Based Study Shows that Volatiles Emitted by** *Arthrobacter agilis* **UMCV2 Increase the Content of Brassinosteroids in** *Medicago truncatula* **in Response to Iron Deficiency Stress**

**Idolina Flores-Cortez 1, Robert Winkler 2, Arturo Ramírez-Ordorica 1, Ma. Isabel Cristina Elizarraraz-Anaya 2, María Teresa Carrillo-Rayas 2, Eduardo Valencia-Cantero <sup>1</sup> and Lourdes Macías-Rodríguez 1,\***


Received: 6 July 2019; Accepted: 10 August 2019; Published: 20 August 2019

**Abstract:** Iron is an essential plant micronutrient. It is a component of numerous proteins and participates in cell redox reactions; iron deficiency results in a reduction in nutritional quality and crop yields. Volatiles from the rhizobacterium *Arthrobacter agilis* UMCV2 induce iron acquisition mechanisms in plants. However, it is not known whether microbial volatiles modulate other metabolic plant stress responses to reduce the negative effect of iron deficiency. Mass spectrometry has great potential to analyze metabolite alterations in plants exposed to biotic and abiotic factors. Direct liquid introduction-electrospray-mass spectrometry was used to study the metabolite profile in *Medicago truncatula* due to iron deficiency, and in response to microbial volatiles. The putatively identified compounds belonged to different classes, including pigments, terpenes, flavonoids, and brassinosteroids, which have been associated with defense responses against abiotic stress. Notably, the levels of these compounds increased in the presence of the rhizobacterium. In particular, the analysis of brassinolide by gas chromatography in tandem with mass spectrometry showed that the phytohormone increased ten times in plants grown under iron-deficient growth conditions and exposed to microbial volatiles. In this mass spectrometry-based study, we provide new evidence on the role of *A. agilis* UMCV2 in the modulation of certain compounds involved in stress tolerance in *M. truncatula*.

**Keywords:** legumes; microbial volatiles; Fe deficiency; DLI-ESI-MS

#### **1. Introduction**

Mass spectrometry (MS) is gaining considerable popularity for profiling metabolites in complex biological samples. The increased applications have led to the improvement of MS technology in sample introduction, ionization source, mass analyzer, ion detection, and data acquisition and processing. Direct liquid introduction-electrospray ionization-mass spectrometry (DLI-ESI-MS, the acronym recommended by the Analytical Methods Committee [1]) is a rapid and high-throughput analytical tool that has been successfully applied in medicine and food and biological sciences [2–5]. DLI-ESI-MS does not require preliminary sample separation, and it can be applied to multiple biological matrices. The straightforward sample introduction allows for simultaneous fingerprinting of a vast

number of metabolites from different samples within a single period. In addition, different studies support the repeatability of DLI-ESI-MS data, and the quantification of the intensity of the ion signals (*m*/*z*) with a larger variance in plants because of environment, physiological state, or the genotype can also be performed [5–7]. High analytical performance (sensitivity, selectivity) allows it to be used for untargeted metabolomics screening approaches for different plant extracts; thus it offers an excellent cost-benefit ratio compared to other analytical platforms such as near-infrared reflectance spectroscopy (NIRS), ultra-performance liquid chromatography-mass spectrometry (UPLC-MS), gas chromatography with flame ionization detection (GC-FID), and GC-MS, which are slow and expensive to use for routine plant biochemistry studies [2]. Due to the various benefits reported for DLI-ESI-MS, we decided to conduct a study to determine its usefulness in microbial ecology research, as DLI-ESI-MS provides robust chemical information, is bioinformatically easy to handle, and could help us understand the chemical response of plants to abiotic or biotic factors.

Iron (Fe) is an essential micronutrient for plant growth and crop productivity. Plants acquire Fe mainly from the rhizosphere; therefore, the mechanisms that regulate Fe acquisition and homeostasis in the plant are of interest. The role of specific metabolites such as nitric oxide, ferritin, phenolic compounds, and brassinosteroids (BRs) have been highlighted in Fe-deficient growth conditions, indicating that plants undergo significant metabolic changes during Fe-adaptive processes [8–17].

In an attempt to make agriculture a viable component of a healthy and pleasant ecosystem, the application of plant growth-promoting rhizobacteria (PGPR) to enhance Fe uptake and transport in plants is an excellent biotechnology strategy. PGPR are soil bacteria that colonize the rhizosphere of plants, stimulating plant growth and health through different mechanisms, such as phosphorus solubilization or nitrogen-fixation, and the production of phytohormones or siderophores to capture Fe from the environment in biologically useful forms [18]. In 2003, Ryu et al. reported that some PGPR can modulate the growth and development of plants without physical contact with them. This mechanism involves the production of volatile compounds such as, acetoin and 2,3-butanediol, which modulate the mechanisms of phytohormone signaling and therefore stimulate morphogenesis programs in plants [19]. Six-years later, Zhang et al. (2009) noted, that the same microbial volatiles can modulate Fe uptake in *Arabidopsis* via deficiency-inducible mechanisms [20].

The rhizobacterium *Arthrobacter agilis* UMCV2 used in this study, was isolated from the rhizosphere of maize (*Zea mays*) [21]. It emits a pool of volatiles that promotes the growth of leguminous and monocotyledonous plants with different levels of available Fe [22–24]. Notably, in *Medicago truncatula*, the UMCV2 strain increased the expression of genes involved in Fe uptake (*MtFRO1*, *MtFRO2*, *MtFRO3*, *MtFRO4*, and *MtFRO5*) under Fe-sufficient and -deficient conditions [25]. Nevertheless, those studies focused on elucidating the molecular mechanisms involved in the modulation of Fe acquisition responses. Thus, one question remaining is whether microbial volatiles modulate the production of other primary or secondary metabolites in plants to ameliorate Fe-deficiency stress.

Here, we used a DLI-ESI-MS method as an untargeted mass spectrometry strategy to study the metabolic profiles of *M. truncatula* seedlings grown under Fe-sufficient and -deficient conditions. We focused on the detection of significant differences among MS profiles to determine whether volatiles emitted by *A. agilis* UMCV2 alleviate plant stress and stimulate the accumulation of metabolites involved in abiotic stress tolerance; in addition, we used a complementary GC-MS method to confirm the identification of brassinolide, which is involved in Fe-adaptive processes in plants.

#### **2. Results**

#### *2.1. DLI-ESI-MS in the Analysis of Fe Deficiency in Medicago truncatula Seedlings and Response to Bacterial Volatiles*

Under conditions of Fe deficiency, plants adjust their metabolism to maintain cellular Fe homeostasis. Some visual symptoms of Fe deficiency, such as leaf yellowing (Figure 1d–f) and decreased plant size (Figure 1g) were observed in our experiments in comparison to plants grown under Fe sufficiency (Figure 1a–c). Additionally, we studied plants exposed to volatiles from *A. agilis* UMCV2, a rhizobacterium that induces Fe acquisition in plants (Figure 1c,f), and plants exposed to volatiles from *Bacillus* sp. L264, a commensal rhizobacterium (Figure 1b,e). As we expected, volatiles from the UMCV2 strain, had a stimulatory effect on plant growth under Fe-sufficient and -deficient growth conditions (Figure 1g).

**Figure 1.** Interaction between *Medicago truncatula* and rhizobacteria through the emission of volatile compounds. A 4 mL glass vial with 2 mL nutritive agar medium was inserted in each system; in the control system, 20 μL water was added into the vial instead of the bacterial inoculum. The interaction lasted for 10 days. Uninoculated 12-day-old plants grown under conditions of iron (Fe) sufficiency (**a**) and deficiency (**d**). (**b**) Plants were inoculated with the commensal strain *Bacillus* sp. L264 grown under Fe-sufficient and -deficient conditions (**e**). Inoculated plants exposed to volatiles from *A. agilis* UMCV2 and under Fe-sufficient (**c**) and -deficient conditions (**f**). (**g**) Dry weights of control plants and plants during interactions with bacterial volatile compounds. Data shown are means ± standard error (n = 15). White and black bars indicate Fe-sufficient and -deficient growth conditions, respectively. Different letters indicate significant differences (*p* ≤ 0.05) among treatments determined with two-way ANOVA and Tukey's test.

These plants were further analyzed by DLI-ESI-MS. The quadrupole analyzer allowed the collection of MS data with satisfactory spectral quality. Typical mass spectra of the broad range of molecular weights of compounds that are produced when plants undergo Fe stress, and microbial volatiles exposure are shown in Figure 2a,b, respectively. In total, 737 ions were obtained, mainly within the range 55.90–1592.52 *m*/*z*. All metabolite signals were extracted from a database with the MALDIquant package in the RStudio interface. Following purification, alignment, and normalization, a principal component analysis (PCA) was performed (Figure 3). The PCA (highly significant results *p* < 0.001, by permutational multivariate analysis of variance, PERMANOVA) showed that control plants grown under conditions of Fe sufficiency and those exposed to volatiles from L264 had similar mass spectra, since treatments were grouped together. Similarly, plants grown under Fe-deficiency stress and those exposed to volatiles from L264 had the same metabolic fingerprinting, and both treatments presented an overlap, indicating that only the absence of Fe affected the metabolic profile of the plants. The ion profiles of plants grown under conditions of Fe sufficiency and deficiency, and following exposure to UMCV2 volatiles were similar, indicating that UMCV2 promotes metabolic changes in plants under both growing conditions.

**Figure 2.** Non-targeted metabolomic profiling normalized from leaves of *Medicago truncatula* obtained by DLI-ESI-MS. (**a**) Control plants grown under Fe-sufficient (green) and -deficient conditions (orange). (**b**) Plants exposed to volatile compounds from *A. agilis* UMCV2 for 10 days and grown under iron-sufficient (green) and -deficient conditions (orange).

**Figure 3.** Principal component analysis (PCA) obtained from DLI-ESI mass spectra of *Medicago truncatula* leaves under Fe-sufficient and -deficient conditions and following exposure to microbial volatiles. Blue indicates control plants, orange represents plants exposed to volatiles emitted by L264 strain, and red shows the plants exposed to volatiles from *A. agilis* UMCV2. Circles (O) and triangles (Δ) indicate Fe sufficiency and deficiency, respectively. The ellipses represent 95% confidence intervals. Differences between groups were compared with a PERMANOVA test (*p* < 0.001).
