FIG 4.

(A) Schematic of controlled atmosphere plant-microbe co-culture labeling study. A. brasilense wild-type (WT) and ΔglnE strains with chromosomally integrated constitutive and inducible uAT10 circuits (Fig. S16 and S21) were introduced at an OD₆₀₀ of 0.1 to 14-day-old S. viridis plants growing gnotobiotically in modified 1/5-strength NFbHP. At the time of inoculation, the on-state replicates were induced with 200 ng ml⁻¹ anhydrotetracycline while off-state replicates had no anhydrotetracycline added. The atmosphere of replicates in the label group was modified by replacing 50% of the volume with ¹⁵N₂ gas immediately after inoculation (resulting in a final mixture of 90/10 N₂/O₂). UI, uninoculated controls. After an additional 14 days, samples for determination of shoot dry weight (B) and total chlorophyll content (C) were collected. Chlorophyll was converted to pheophytin for isotopic analysis (D) to determine fractional enrichment of the ¹⁵N isotope (see Fig. S18 for calculation and S19 for fertilized controls). (E) Representative pheophytin isotopic envelopes showing mass shifts caused by ¹⁵N incorporation. All error bars are standard deviations for 6 biological replicates; stars denote P values determined to be of statistical significance compared to WT using a two-sided homoscedastic t test.