Reproducible growth ofBrachypodium distachyonin fabricated ecosystems (EcoFAB 2.0) reveals that nitrogen form and starvation modulate root exudation

Abstract

Understanding plant-microbe interactions requires examination of root exudation under nutrient stress using standardized and reproducible experimental systems. We grewBrachypodium distachyonhydroponically in novel fabricated ecosystem devices (EcoFAB 2.0) under three inorganic nitrogen forms (NO3-, NH4+, NH4NO3), followed by nitrogen starvation. Analyses of exudates with LC-MS/MS, 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 andp-coumaric acids) on the rhizobacteriumPseudomonas putidaorBrachypodiumseedlings revealed that shikimic acid promoted bacterial and root growth, while p-coumaric acid stunted seedlings. Our results suggest: (i)Brachypodiumalters exudation in response to nitrogen status, which can affect rhizobacterial growth; and (ii) EcoFAB 2.0 is a valuable standardized plant research tool.

Biophysical metabolic modeling of complex bacterial colony morphology

“…Here, we outlined an approach that combines genome-scale metabolic modeling with an appropriate physical model of biomass motility and demographic fluctuations. The ongoing efforts to construct metabolic networks for non-model organisms and to understand microbial motility in complex environments would enable application of our approach to practically relevant communities such as root-associated microbiota 22,20 or biomedically relevant (e.g. dental) biofilms.…”

“…[1][2][3][4][5][6][7] Microbial colonies on agar surfaces serve as a valuable model system to study ecological, evolutionary, and even developmental processes. 5,[7][8][9][10] Spatial and metabolic heterogeneity is ubiquitous across ecosystems, [11][12][13] from complex communities 14,15 of oceanic, [16][17][18] or soil 19 microbes to microbiota associated with plant roots, [20][21][22] or animal digestive tracts 23 to pathogenic communities growing on catheters 24 and other medical implants. The familiar and apparently simple experimental system -Escherichia coli growing on an agar-filled Petri dish, is no exception.…”

Assessing horizontal gene transfer in the rhizosphere ofBrachypodium distachyonusing fabricated ecosystems (EcoFABs)

“…Seedlings were germinated on 0.5x Murashige & Skoog (MS) plates (2.2 g l −1 MS medium, MSP01 (Caisson Labs, Smithfield, UT, USA) with 1650 mg l −1 ammonium nitrate, 6.2 mg l −1 boric acid, 332.2 mg l −1 calcium chloride, 0.025 mg l −1 cobalt chloride, 0.025 mg l −1 copper sulfate, 37.26 mg l −1 disodium EDTA, 27.8 mg l −1 ferrous sulfate heptahydrate, 180.7 mg l −1 magnesium sulfate, 16.9 mg l −1 manganese(II) sulfate monohydrate, 0.25 mg l −1 sodium molybdate dihydrate, 0.83 mg l −1 potassium iodide, 1900 mg l −1 potassium nitrate, 170 mg l −1 monopotassium phosphate, 8.6 mg l −1 zinc sulfate heptahydrate; 6% w/v phytoagar (Fisher Scientific, Waltham, MA, USA), pH adjusted to 5.8) in a 16 h : 8 h, light : dark regime at 24 °C with 150 µmol m −2 s −1 illumination in reach-in plant growth chambers (Controlled Environments Inc., Pembina, North Dakota, USA) [20]. EcoFAB 2.0 [6] were sterilized and seedlings transferred to EcoFAB chambers at 3 d after germination. The EcoFAB root chambers were filled with 10 g of sand (Sand, 50-70 mesh particle size, Sigma-Aldrich, St. Louis, MO) and 5 ml of 0.5 x MS before transferring to a humidity-controlled growth chamber.…”

“…Of these, fabricated ecosystems (EcoFABs) are open-source 3D printable chambers that are highly standardized reproducible tools enabling mechanistic studies of plant-microbe interactions under controlled laboratory conditions ( Fig. 1 ) [6,7]. Several complex processes occurring in the rhizosphere under natural environmental conditions have been simulated and studied in EcoFABs [6,8,9].…”

“…1 ) [6,7]. Several complex processes occurring in the rhizosphere under natural environmental conditions have been simulated and studied in EcoFABs [6,8,9]. In such studies, repeatability is critical.…”