Bacterial volatile organic compounds (VOCs) promote growth and induce metabolic changes in rice

Abstract

Plant growth-promoting bacteria (PGPB) represent an eco-friendly alternative to reduce the use of chemical products while increasing the productivity of economically important crops. The emission of small gaseous signaling molecules from PGPB named volatile organic compounds (VOCs) has emerged as a promising biotechnological tool to promote biomass accumulation in model plants (especially Arabidopsis thaliana) and a few crops, such as tomato, lettuce, and cucumber. Rice (Oryza sativa) is the most essential food crop for more than half of the world's population. However, the use of VOCs to improve this crop performance has not yet been investigated. Here, we evaluated the composition and effects of bacterial VOCs on the growth and metabolism of rice. First, we selected bacterial isolates (IAT P4F9 and E.1b) that increased rice dry shoot biomass by up to 83% in co-cultivation assays performed with different durations of time (7 and 12 days). Metabolic profiles of the plants co-cultivated with these isolates and controls (without bacteria and non-promoter bacteria-1003-S-C1) were investigated via ¹H nuclear magnetic resonance. The analysis identified metabolites (e.g., amino acids, sugars, and others) with differential abundance between treatments that might play a role in metabolic pathways, such as protein synthesis, signaling, photosynthesis, energy metabolism, and nitrogen assimilation, involved in rice growth promotion. Interestingly, VOCs from IAT P4F9 displayed a more consistent promotion activity and were also able to increase rice dry shoot biomass in vivo. Molecular identification by sequencing the 16S rRNA gene of the isolates IAT P4F9 and E.1b showed a higher identity with Serratia and Achromobacter species, respectively. Lastly, volatilomes of these and two other non-promoter bacteria (1003-S-C1 and Escherichia coli DH5α) were evaluated through headspace solid-phase microextraction coupled with gas chromatography-mass spectrometry. Compounds belonging to different chemical classes, such as benzenoids, ketones, alcohols, sulfide, alkanes, and pyrazines, were identified. One of these VOCs, nonan-2-one, was validated in vitro as a bioactive compound capable of promoting rice growth. Although further analyses are necessary to properly elucidate the molecular mechanisms, our results suggest that these two bacterial isolates are potential candidates as sources for bioproducts, contributing to a more sustainable agriculture.

Keywords: Oryza sativa; bioactive compounds; metabolomics; microbial volatile organic compounds; plant growth promotion.

Figure 1

Effects of bacterial VOCs on (A) dry shoot biomass of rice plants, after 7 days of co-cultivation, and on (B) dry shoot and (C) root biomass, after 12 days of co-cultivation. Plants were co-cultivated with the bacterial isolates (full colored), the negative control E. coli DH5α (white striped), and grown without bacteria (control, gray). Significant differences (ANOVA followed by Tukey’s test, p < 0.05) among treatments are indicated by letters (n = 7).

Figure 2

Heatmap of the metabolome of rice plants co-cultivated with the bacterial isolates (E.1b, IAT P4F9, and 1003-S-C1) and control plants (cultivated without bacteria). Columns represent each treatment and rows represent the different metabolites identified. The color code indicates the abundance of each metabolite (blue = low abundance; red = high abundance) based on the average of the Log₁₀-transformed values (concentrations determined based on the reference signal of 0.5 mM TMSP-d₄) (n = 4). The darkest blue indicates that the metabolite was not identified in plants subjected to the respective treatment.

Figure 3

Heatmap of VOCs produced by the bacterial isolates (E.1b, IAT P4F9, and 1003-S-C1) and E. coli DH5α grown in LB medium. Columns represent each bacterium and rows represent the different VOCs detected. The color code indicates the abundance of each compound (blue = low abundance; red = high abundance), based on the average of the Log₁₀-transformed values (peak areas were normalized by cis-3-Hexenyl acetate) (n = 5). The darkest blue indicates that the VOCs were not identified in the volatilome of the respective bacterium.