Laser ablation electrospray ionization-mass spectrometry imaging (LAESI-MS) for spatially resolved plant metabolomics

There is increasing demand to bring the level of metabolomics analyses down to the tissue or cell level. Significant progress has been made involving the use of in situ metabolomics imaging techniques where no tissue collection or extraction is needed prior to analysis. In this chapter we describe a relatively new method which is simple and easy to use. No ectopic matrix or vacuum is required, and analyses are performed with living plant materials directly from (or even still attached to) the plant. Although relatively straightforward, there are still a few caveats as regards this method which are described at the end of the chapter

Root morphology and exudate availability are shaped by particle size and chemistry in Brachypodium distachyon

Root morphology and exudation define a plants' sphere of influence in soils. In turn, soil characteristics influence plant growth, morphology, root microbiome, and rhizosphere chemistry. Collectively, all these parameters have significant implications on the major biogeochemical cycles, crop yield, and ecosystem health. However, how plants are shaped by the physiochemistry of soil particles is still not well understood. We explored how particle size and chemistry of growth substrates affect root morphology and exudation of a model grass. We grew Brachypodium distachyon in glass beads with various sizes (0.5, 1, 2, 3 mm), as well as in sand (0.005, 0.25, 4 mm) and in clay (4 mm) particles and in particle-free hydroponic medium. Plant morphology, root weight, and shoot weight were measured. We found that particle size significantly influenced root fresh weight and root length, whereas root number and shoot weight remained constant. Next, plant exudation profiles were analyzed with mass spectrometry imaging and liquid chromatography-mass spectrometry. Mass spectrometry imaging suggested that both, root length and number shape root exudation. Exudate profiles were comparable for plants growing in glass beads or sand with various particles sizes, but distinct for plants growing in clay for in situ exudate collection. Clay particles were found to sorb 20% of compounds exuded by clay-grown plants, and 70% of compounds from a defined exudate medium. The sorbed compounds belonged to a range of chemical classes, among them nucleosides, organic acids, sugars, and amino acids. Some of the sorbed compounds could be desorbed by a rhizobacterium (Pseudomonas fluorescens WCS415), supporting its growth. This study demonstrates the effect of different characteristics of particles on root morphology, plant exudation and availability of nutrients to microorganisms. These findings further support the critical importance of the physiochemical properties of soils when investigating plant morphology, plant chemistry, and plant-microbe interactions

Impact of root-associated strains of three Paraburkholderia species on primary and secondary metabolism of Brassica oleracea

Abstract Several root-colonizing bacterial species can simultaneously promote plant growth and induce systemic resistance. How these rhizobacteria modulate plant metabolism to accommodate the carbon and energy demand from these two competing processes is largely unknown. Here, we show that strains of three Paraburkholderia species, P. graminis PHS1 (Pbg), P. hospita mHSR1 (Pbh), and P. terricola mHS1 (Pbt), upon colonization of the roots of two Broccoli cultivars led to cultivar-dependent increases in biomass, changes in primary and secondary metabolism and induced resistance against the bacterial leaf pathogen Xanthomonas campestris. Strains that promoted growth led to greater accumulation of soluble sugars in the shoot and particularly fructose levels showed an increase of up to 280-fold relative to the non-treated control plants. Similarly, a number of secondary metabolites constituting chemical and structural defense, including flavonoids, hydroxycinnamates, stilbenoids, coumarins and lignins, showed greater accumulation while other resource-competing metabolite pathways were depleted. High soluble sugar generation, efficient sugar utilization, and suppression or remobilization of resource-competing metabolites potentially contributed to curb the tradeoff between the carbon and energy demanding processes induced by Paraburkholderia-Broccoli interaction. Collectively, our results provide a comprehensive and integrated view of the temporal changes in plant metabolome associated with rhizobacteria-mediated plant growth promotion and induced resistance

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Disease-suppressive soils are ecosystems in which plants suffer less from root infections due to the activities of specific microbial consortia. The characteristics of soils suppressive to specific fungal root pathogens are comparable to those of adaptive immunity in animals, as reported by Raaijmakers and Mazzola (Science 352:1392-3, 2016), but the mechanisms and microbial species involved in the soil suppressiveness are largely unknown. Previous taxonomic and metatranscriptome analyses of a soil suppressive to the fungal root pathogen Rhizoctonia solani revealed that members of the Burkholderiaceae family were more abundant and more active in suppressive than in non-suppressive soils. Here, isolation, phylogeny, and soil bioassays revealed a significant disease-suppressive activity for representative isolates of Burkholderia pyrrocinia, Paraburkholderia caledonica, P. graminis, P. hospita, and P. terricola. In vitro antifungal activity was only observed for P. graminis. Comparative genomics and metabolite profiling further showed that the antifungal activity of P. graminis PHS1 was associated with the production of sulfurous volatile compounds encoded by genes not found in the other four genera. Site-directed mutagenesis of two of these genes, encoding a dimethyl sulfoxide reductase and a cysteine desulfurase, resulted in a loss of antifungal activity both in vitro and in situ. These results indicate that specific members of the Burkholderiaceae family contribute to soil suppressiveness via the production of sulfurous volatile compounds