Effects of Sulfur Assimilation in Pseudomonas fluorescens SS101 on Growth, Defense, and Metabolome of Different Brassicaceae

Abstract

Genome-wide analysis of plant-growth-promoting Pseudomonas fluorescens strain SS101 (PfSS101) followed by site-directed mutagenesis previously suggested that sulfur assimilation may play an important role in growth promotion and induced systemic resistance in Arabidopsis. Here, we investigated the effects of sulfur metabolism in PfSS101 on growth, defense, and shoot metabolomes of Arabidopsis and the Brassica crop, Broccoli. Root tips of seedlings of Arabidopsis and two Broccoli cultivars were treated with PfSS101 or with a mutant disrupted in the adenylsulfate reductase cysH, a key gene in cysteine and methionine biosynthesis. Phenotyping of plants treated with wild-type PfSS101 or its cysH mutant revealed that sulfur assimilation in PfSS101 was associated with enhanced growth of Arabidopsis but with a reduction in shoot biomass of two Broccoli cultivars. Untargeted metabolomics revealed that cysH-mediated sulfur assimilation in PfSS101 had significant effects on shoot chemistry of Arabidopsis, in particular on chain elongation of aliphatic glucosinolates (GLSs) and on indole metabolites, including camalexin and the growth hormone indole-3-acetic acid. In Broccoli, PfSS101 sulfur assimilation significantly upregulated the relative abundance of several shoot metabolites, in particular, indolic GLSs and phenylpropanoids. These metabolome changes in Broccoli plants coincided with PfSS101-mediated suppression of leaf infections by Xanthomonas campestris. Our study showed the metabolic interconnectedness of plants and their root-associated microbiota.

Keywords: Pseudomonas fluorescens; flavonoids; glucosinolates (GLSs); induced systemic resistance; plant growth promotion; plant metabolomics.

Figure 1

Phenotypic changes in Arabidopsis and Broccoli seedlings upon root treatment by P. fluorescens SS101 or its cysH mutant 20H12. Photographs of MS agar plates with Arabidopsis, and two Broccoli cultivars (Coronado and Malibu) treated on the root tip with cell suspensions of PfSS101 (wild type) or its cysH mutant (20H12) (a). Percentage change in total fresh biomass (1), shoot (2), and root (3) of plants treated with rhizobacteria when compared to untreated plants (11dpi) (b). Means of percent changes in biomass with a different letter are significantly different among treatments according to two-way ANOVA (Tukey, p < 0.05). Asterisks denote statistically significant differences (two-tailed Student’s t test): * p < 0.05; ** p < 0.01 of rhizobacteria treated plants when compared to the controls. For each plant species, four independent biological replicates were used with 10 seedlings of Arabidopsis and five of Broccoli per biological replicate. PfSS101: Pseudomonas fluorescens SS101, 20H12: cysH mutant of PfSS101.

Figure 2

Rhizobacteria-mediated systemic resistance in two Broccoli cultivars, Coronado and Malibu, against the bacterial leaf pathogens. Disease severity was scored on an ordinal scale from 0 to 5, where 1 = no necrosis or migration, 2 = full infection of the treated leaf, 3 = migration of the infection to the leafstalk of the treated leaf, 4 = infection of the neighboring leaf, and 5 = infection of the entire seedling (a) (see Supplementary Materials Figure S1 for further details). Details on the conversion of the ordinal scales to disease severity index is provided in the Material and Methods section. Impact of priming roots of Broccoli cultivars with rhizobacteria on severity of leaf disease caused by Xanthomonas campestris pv. armoraciae (Xca) (b) and Xanthomonas campestris pv. campestris (Xcc) (c). Prior to pathogen inoculation on the leaves, roots of each Broccoli cultivar were treated with P. fluorescens SS101 or its cysH-mutant 20H12 and incubated for 11 days. For the disease severity caused by Xca or Xcc, Broccoli seedlings from four biological replicates were individually scored (n = 20). Different letters above bars indicate statistically significant differences based on beta regression analysis followed by Tukey test (p < 0.05).

Figure 3

Metabolome changes in Arabidopsis shoots upon root treatment by P. fluorescens SS101 or its cysH mutant 20H12. Shown are the results of the principal component analysis (PCA) (a) and hierarchical cluster analysis (HCA) (b) based on the differentially regulated semi-polar metabolites. In the HCA, various metabolite clusters are indicated by different colors and when none of the metabolites in a given cluster was annotated, the cluster number was omitted (clusters 3, 5, and 10 in panel b). * GLS, glucosinolate; ** d, derivative.

Figure 4

Metabolome changes in the shoots of two Broccoli cultivars, Coronado and Malibu upon root treatment by P. fluorescens SS101 or its cysH mutant 20H12. Shown are the results of the PCA (a) and HCA (b) based on differentially regulated semi-polar metabolites. In the HCA, various metabolite clusters are indicated by different colors; when none of the metabolites in a given cluster was annotated, the cluster number was omitted (clusters 3, 6, 8, 9, 12, 13, and 14 in panel b). * GLS, glucosinolate; ** d, derivative.