A Versatile Glass Jar System for Semihydroponic Root Exudate Profiling

Our intensive agricultural systems are based on high inputs of fertilizers and pesticides. This is harmful to the environment and also not sustainable. Those key questions in research are how to lower this input while maintaining yield of our crops.

One promising avenue to do that is to supply plants with beneficial microbes in the field. However, to be able to do that successfully, we need to understand the complex chemical crosstalk between the partners. We studied this interaction by looking at root exudates in our lab.

We found that root exudation is very dynamic. It differs between plant species developmental stages and also diurnal time points. Plants also react to the presence of different kind of microbes by changing their metabolic profile.

Because root exudates are nutrients and signals to the microbial community, studying exudation profiles is crucial to understand how plants interact with microbes in their environment. Developments in metabolomics, but also in next-generation sequencing technologies really pushed the field of plant microbiome interaction forward. Obviously, you need metabolomics workflow to detect the compound or the compounds of interest in a given system.

And next-generation sequencing technologies are really essential in understanding the structure, but also the function of plant microbiomes. Also, it is very helpful to use specialized growth systems or simplified growth systems like the one we present here to understand the molecular mechanisms of plant microbe interactions. Our system can be kind of sterile, but we can also inoculate it with target microorganisms.

One challenge we have is to detect metabolites in low concentration in exudates. If the background is low then it's easy to do it. If we have a much more complex environment such as soil, then it's more difficult.

Another challenge that is unresolved is when we add microbes to the picture, then it's not possible to distinguish between the compounds produce by plants or by the microbes. We developed a system that is low cost and reasonably easy. Its sterile design allows for consecutive experiments.

First, plant inert metabolites are discarded, then plants are inoculated with microbes, and here the changes in the metabolite profile are evaluated. It also allows for the growth of different plant species for an extended period of time. So in summary, this system is well-suited for many applications.

To begin, add 150 milliliters of clean five millimeter glass beads to the clean jar and close the lid. Cover the closed jar with enough aluminum foil to conceal the lid to jar junction, and autoclave at 121 degrees Celsius for 20 minutes. When the seedlings are ready to transfer into the jar, on the sterile bench, remove the aluminum foil and lid of the jar.

Add 35 milliliters of half strength Murashige and Skoog medium of pH 5.7 to 5.9 to the jar, and stir the beads to coat evenly with the medium. Introduce three to five seedlings into the jar, ensuring the root systems are placed between the glass beads. Adjust the beads with sterile forceps or spoons to cover the roots and lift the leaves out of the medium.

Secure two strips of 1.25 centimeter micropore tape across the top of the jar, and gently place the lid on top. Seal the gap between the lid and jar with 2.5 centimeter micropore tape to maintain sterility while permitting air exchange, and place the jar into a growth chamber. When the plants reach the desired age, remove the micropore tape band lid from the jar.

Aliquot 20 microliters of growth medium onto LB agar plate for sterility testing. Using a 25 milliliter volumetric pipette, remove as much growth medium as possible without damaging the roots. Add 50 milliliters of collection medium along the jar wall to prevent wetting the leaves.

Close the jar with a lid and incubate in sterile conditions for two hours. After incubation, remove the desired amount of collection medium into tubes. Measure the weight of the root and shoot from the plant in one jar to normalize the root exudates by plant weight.

Arabidopsis thaliana plants looked healthy across two different laboratories. The glass jar system proved adaptable for various plant species and developmental stages. Wheat had the highest shoot weights, followed by sorghum and tomato, while sorghum had the highest root weights, followed by wheat and Medicago truncatula.

Inoculation of Arabidopsis thaliana with commensal bacteria did not alter the plant phenotype, and inoculated bacteria persisted in the system for at least 12 days, with a slight increase in colony forming units.