Changes in Amino Acid Distribution Across Populus trichocarpa Roots with and Without Microbes in a Rhizosphere-on-a-Chip Habitat

Authors:

John F. Cahill* (cahilljf@ornl.gov), Courtney Walton, Muneeba Khalid, Vilmos Kertesz, Scott T. Retterer, and Jennifer Morrell-Falvey

Institutions:

Oak Ridge National Laboratory

Goals

The goal of this project is to develop new technologies to image changes in chemistry occurring in the rhizosphere in living biosystems. The team created in situ Liquid Extraction Mass Spectrometry (in situ-LE-MS), a liquid microjuction-surface sampling probe mass spectrometry (LMJ-SSP-MS) imaging modality that enables non-destructive imaging of plant rhizospheres with broad chemical coverage and chemical specificity. In situ measure of amino acid distributions was achieved for Populus cuttings grown in a synthetic rhizosphere-on-a-chip system. Amino acid distributions varied across the Populus root structure. When co-cultured with and without rhizosphere bacteria, amino acid distributions were altered in a species-specific manner. These data shed unique insights into the high degree of spatial variance in root exudate occurring within the rhizosphere.

Abstract

The rhizosphere is an incredibly complex environment, containing thousands of unique exogenous chemical species oriented in a complex spatial network. Such compounds are known to affect plant-microbe organization, interactions, and, ultimately, growth and survivability. Due to its importance, the role of exogenous compounds in the rhizosphere is under much investigation, specifically the relation between plant physiology and the spatiotemporal distribution of molecular components. However, measure of the spatial distribution of exogenous compounds in the rhizosphere is challenging given the complex and dynamic nature of the environment. Compounds include, among others, organic acids, polysaccharides, proteins, and amino acids (AAs) which can exhibit a variety of roles in the rhizosphere including acting as a nutrient source for microbial colonization or a deterrent against pathogenic species. The exudation of AAs is one of the biggest components of plant carbon loss, which when released into carbon-deficient soil can lead to significantly enhanced hot-spots of microbial growth. In turn, microbes alter the relative distribution of exuded AAs, which can then be re-assimilated in the plant. Direct measurement of AA distribution within the rhizosphere is challenging due to the complex and dynamic nature of the environment and the limited accessibility of the rhizosphere for analysis. However, even for abundant molecules, like AAs, little is known of how they are spatially distributed along plant roots, how their distribution changes over time, and how their distribution affects microbial composition and, ultimately, plant health.

To understand the complex chemical dynamics of exudated compounds in the rhizosphere, the team developed in situ-LE-MS, an LMJ-SSP-MS imaging modality designed to measure exudate chemistry in situ from synthetic rhizosphere-on-a-chip environments. Uniquely, this technology extracts a very small volume of liquid from the rhizosphere and can measure exudates without additional sample preparation procedures enabling in situ, non-destructive MS imaging for the first time. Here, researchers chemically imaged AA distributions across Populus plants grown in rhizosphere-on-a-chip systems. Populus was cultured with rhizosphere bacteria CF313, PM419, and their co-culture relative to control (Populus without bacteria). Multivariate analyses were used to identify unique AA distributions measured between samples and related to root morphology annotated through brightfield imaging of root structure.

Funding Information

Oak Ridge National Laboratory is managed by UT-Battelle, LLC for the U.S. Department of Energy under contract no. DE-AC05-00OR22725. This project was sponsored by the U.S. Department of Energy, Office of Science, Biological and Environmental Research (BER) Bioimaging Program under FWP ERKP920.