Reproducible Plant Growth in Fabricated Ecosystems (EcoFAB 2.0) Reveals that Nitrogen Supply Modulates Root Exudation

Authors:

Vlastimil Novak¹, Peter Andeer¹, Yi Zhai¹, John P. Vogel^1,2,3, Trent R. Northen^1,2* (trnorthen@lbl.gov), and Karsten Zengler⁴

Institutions:

¹Lawrence Berkeley National Laboratory; ²DOE Joint Genome Institute; ³University of California–Berkeley; and ⁴University of California–San Diego

Goals

This project couples novel lab and field studies to develop the first predictive model of grass-microbiomes based on new mechanistic insights into dynamic plant-microbe interactions in the grasses Sorghum bicolor and Brachypodium distachyon that improve plant N use efficiency (NUE). The results will be used to predict plant mutants and microbial amendments that improve low-input biomass production for validation in lab and field studies. To achieve this goal, the team will determine the mechanistic basis of dynamic exudate exchange in the grass rhizosphere, with a specific focus on the identification of plant transporters and proteins that regulate root exudate composition and how specific exudates select for beneficial microbes that increase plant biomass and NUE. Researchers will further develop a predictive plant-microbe model for advancing sustainable bioenergy crops and will predictively shift plant-microbe interactions to enhance plant biomass production and N acquisition from varied N forms.

Abstract

Understanding plant-microbe interactions requires examination of root exudation under nutrient stress using standardized and reproducible experimental systems. Researchers grew Brachypodium distachyon hydroponically in novel fabricated ecosystem devices (EcoFAB 2.0) under three inorganic nitrogen forms (NO₃−, NH₄+, or NH₄NO₃), followed by nitrogen starvation. In liquid chromatography with tandem mass spectrometry (LC-MS/MS) analyses of exudates, biomass, medium pH, and nitrogen uptake showed EcoFAB 2.0’s low intra-treatment data variability. Furthermore, the three inorganic nitrogen forms caused differential exudation, generalized by abundant amino acids/peptides and alkaloids. Comparatively, N-deficiency decreased N-containing compounds but increased shikimates/phenylpropanoids. Subsequent bioassays with two shikimates/phenylpropanoids (shikimic and p-coumaric acids) on the rhizobacterium Pseudomonas putida or Brachypodium seedlings revealed that shikimic acid promoted bacterial and root growth, while p-coumaric acid stunted seedlings. The next objective was to identify transport mechanisms for organic acids and inorganic nitrogen by creating plant mutants with knockout ABC or nitrogen transporters. These mutations caused significant phenotypic and exometabolic changes. In conclusion, results suggest: (1) Brachypodium alters exudation in response to nitrogen status, which can affect rhizobacterial growth; (2) EcoFAB 2.0 is a valuable standardized plant research tool; (3) the plant root exudation can be altered by membrane transport engineering.

Funding Information

The team gratefully acknowledges funding from the U.S. Department of Energy (DOE) Office of Science, Office of Biological and Environmental Research. The research described was funded under contract DE-AC02-05CH11231 to Lawrence Berkeley National Laboratory as part of a project lead by UC–San Diego (DE-SC0021234). The EcoFAB 2.0 was developed as part of the Trial Ecosystem Advancement for Microbiome Science (TEAMS) under contract DE-AC02-05CH11231 to Lawrence Berkeley National Laboratory project.