Raman-Deuterium Isotope Probing and Metagenomics Reveal the Drought Tolerance of the Soil Microbiome and Its Promotion of Plant Growth

Abstract

Drought has become a major agricultural threat leading crop yield loss. Although a few species of rhizobacteria have the ability to promote plant growth under drought, the drought tolerance of the soil microbiome and its relationship with the promotion of plant growth under drought are scarcely studied. This study aimed to develop a novel approach for assessing drought tolerance in agricultural land by quantitatively measuring microbial phenotypes using stable isotopes and Raman spectroscopy. Raman spectroscopy with deuterium isotope probing was used to identify the Raman signatures of drought effects from drought-tolerant bacteria. Counting drought-tolerant cells by applying these phenotypic properties to agricultural samples revealed that 0% to 52.2% of all measured single cells had drought-tolerant properties, depending on the soil sample. The proportions of drought-tolerant cells in each soil type showed similar tendencies to the numbers of revived pea plants cultivated under drought. The phenotype of the soil microbiome and plant behavior under drought conditions therefore appeared to be highly related. Studying metagenomics suggested that there was a reliable link between the phenotype and genotype of the soil microbiome that could explain mechanisms that promote plant growth in drought. In particular, the proportion of drought-tolerant cells was highly correlated with genes encoding phytohormone production, including tryptophan synthase and isopentenyl-diphosphate delta-isomerase; these enzymes are known to alleviate drought stress. Raman spectroscopy with deuterium isotope probing shows high potential as an alternative technology for quantitatively assessing drought tolerance through phenotypic analysis of the soil microbiome. IMPORTANCE Soil microbiome has played a critical role in the plant survival during drought. However, the drought tolerance of soil microbiome and its ability to promote plant growth under drought is still scarcely studied. In this study, we identified the Raman signature (i.e., phenotype) of drought effects from drought-tolerant bacteria in agricultural soil samples using Raman-deuterium isotope probing (Raman-DIP). Moreover, the number of drought-tolerant cells measured by Raman-DIP was highly related to the survival rate of plant cultivation under drought and the abundance of genes encoding phytohormone production alleviating drought stress in plant. These results suggest Raman-DIP is a promising technology for measuring drought tolerance of soil microbiome. This result give us important insight into further studies of a reliable link between phenotype and genotype of soil microbiome for future plant-bacteria interaction research.

Keywords: Raman-DIP; drought; drought tolerance; metagenomics; phenotype; soil microbiome.

FIG 1

SCRS of tolerant and sensitive bacteria after incubation. SCRS are for media with either with 0% (black line) or 25% PEG (red line), including 40% deuterium water. SCRS of 20 cells were averaged (solid line) with SD shown in gray shading. (A) DTB: A. piechaudii, A. halopraeferens, and A. chlorophenolicus. (B) DSB: R. soli, D. gummosa, and A. liporerum.

FIG 2

DAPC plot of SCRS. SCRS of fingerprint region for (A) DTB and (B) DSB. Distances are averages and SD between each centroid of the control group and each dot of the PEG-treated group (C and D). The six colors in the plots represent the six species. The solid circles and triangles represent the control and PEG treatments, respectively.

FIG 3

Statistical significance of Raman shifts, comparing spectra of control and PEG treatments. SCRS were measured for 20 individual cells of (A) DSB and (B) DTB under each condition. The average of SCRS is shown for control (black line) and PEG (red line) treatments. The red and blue boxes represent the Raman shifts that were significantly different between two conditions (p <0.01).

FIG 4

Ratio of Raman intensity for Protein/Lipid (1,209 cm⁻¹/1,267 cm⁻¹) with statistical analysis via t test. Control group was cultured in non-PEG treated media; PEG group was cultured in 25% PEG-treated media (*, P < 0.05; **, P < 0.005; and ***, P < 0.0005).

FIG 5

Evaluation of drought-tolerant capabilities of soil samples using C-D ratio. The C-D ratios are plotted as box plots. The mean of the C-D ratios of DTB in the control treatment is shown by the dashed blue line; that of DTB in the PEG treatment is shown by the dashed red line. DTB comprise the SCRS of three different model bacteria (A. piechaudii, A. halopraeferens, and A. chlorophenolicus).

FIG 6

Functional classification and correlation with proportion of drought-tolerant cells. (A) Functional classification based on COG for the microbiome in each soil; upper plot shows a similarity tree. (B) Correlations between abundances of functional products involved in important metabolic pathways for drought tolerance and proportions of drought-tolerant cells in soil samples. Correlations between relative abundances of the bacteria producing (C) tryptophan synthase or (D) isopentenyl-diphosphate delta-isomerase, shown alongside proportions of drought-tolerant cells in soil samples (*, P < 0.05).