Natural Arabidopsis brx Loss-of-Function Alleles Confer Root Adaptation to Acidic Soil  Bojan Gujas, Carlos Alonso-Blanco, Christian S. Hardtke  Current Biology  Volume 22, Issue 20, Pages 1962-1968 (October 2012) DOI: 10.1016/j.cub.2012.08.026 Copyright © 2012 Elsevier Ltd Terms and Conditions

Figure 1 Effect of Media pH and Brassinosteroid Treatment on Arabidopsis Root Growth (A) Primary root length at 8 days after germination (dag) in Arabidopsis wild-type (Col-0 background line) or brx mutants (brx-2) grown on media with different pH. (B) Root length at 10 dag in Col-0 and brx-2 grown on standard (5.7) or neutral (7.0) pH in the presence of increasing amounts of brassinolide, the most active brassinosteroid in Arabidopsis. (C) Corresponding meristem sizes at 5 dag. (D) Length to width (l/w) ratio progression of cortex cell development in a Col-0 root meristem, with developing stem cell daughters in red (l/w ≈ 1.0), intensive division zone in green (l/w ≈ 0.5), and transition-elongation zone in purple (l/w ≥ 1.0). (E and F) Size of the different meristem zones in Col-0 and brx-2 at 5 dag when grown on mock or 100 pM brassinolide (E), and on standard or neutral pH (F). All error bars represent standard error. n.s., not significant; ∗∗p < 0.01; ∗∗∗p < 0.001. Scale bars represent 50 μm. Current Biology 2012 22, 1962-1968DOI: (10.1016/j.cub.2012.08.026) Copyright © 2012 Elsevier Ltd Terms and Conditions

Figure 2 Override of Impaired Auxin Signaling in the Root Meristem by Neutral Media and Shifted pH Optimum of brx Roots (A) Confocal microscopy analysis of the inverse auxin activity marker DII-VENUS in Col-0 and brx-2 root meristems when transferred to or grown on media with different pH at 5 dag. (B) Expression levels of indicated reporter genes after growth on pH 5.7 at 6 dag or 24 hr after transfer from pH 5.7 to 8.0 at 5 dag. (C) Root growth of the aux1 auxin uptake carrier mutant grown in the presence of auxins in different pH media at 10 dag. (D) Dissociation curves of 1-NAA and IAA. (E) Root growth of Col-0 and brx-2 on media of different pH or standard pH (5.7) media supplemented with 1-NAA or its inactive analog 2-NAA. (F) Root meristem size as indicated by meristematic cortex cell number in different genotypes when grown on different pH. (G) Comparison of axr3-1 root meristems when grown on standard or neutral pH at 5 dag. See Figure 1D for cell color codes. (H) DII-VENUS fluorescence in Col-0 and axr3-1 root meristems at 5 dag when grown on different pH. All error bars represent standard error. n.s., not significant; ∗∗p < 0.01; ∗∗∗p < 0.001. Scale bars in (A), (B), and (H) represent 100 μm; scale bar in (C) represents 1 cm; scale bar in (G) represents 50 μm. Current Biology 2012 22, 1962-1968DOI: (10.1016/j.cub.2012.08.026) Copyright © 2012 Elsevier Ltd Terms and Conditions

Figure 3 PM-H+-ATPase Hyperactivity in brx Mutants (A) Root growth of Col-0 and brx-2 on increasing amounts of the PM-H+-ATPase activator drug fusicoccin. (B) Standard media acidification by transferred 6-day-old Col-0 and brx-2 seedlings. (C) Standard media acidification by roots of transferred 3-day-old Col-0 and brx-2 seedlings. (D and E) Time course of media acidification by roots of 3-day-old Col-0 and brx-2 seedlings transferred to near neutral (D) or standard (E) media. (F) Relative root length of 10-day-old Col-0 and brx-2 seedlings grown on increasing amounts of the PM-H+-ATPase inhibitor dicyclohexylcarbodiimide. All error bars represent standard error. n.s., not significant; ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001. Current Biology 2012 22, 1962-1968DOI: (10.1016/j.cub.2012.08.026) Copyright © 2012 Elsevier Ltd Terms and Conditions

Figure 4 Adaptive Value of brx Loss of Function in Acidic and Salt Stress Conditions (A) Representative rosettes of Col-0 and brx-2 plants transferred from standard (pH 5.7) to standard or acidic media at 3 dag. Images were taken 21 days after transfer. (B) Representative rosettes of Col-0 and brx-2 plants germinated and grown on standard or acidic media at 17 dag. (C) Proportion of bleached and/or growth-arrested Col-0 and brx-2 plants germinated and grown on acidic media at 17 dag (proportion on standard media was 0% for both genotypes). (D) Leaf formation in Col-0 and brx-2 plants germinated and grown on acidic media at 17 dag. Values for median (m), first quartile (q1), and third quartile (q3) for the two genotypes and media are indicated. The difference is statistically significant (p < 0.01) for Col-0 grown on pH 4.5 versus the three other samples, which are not significantly different from each other. (E) Representative rosettes of Col-0 and brx-2 plants transferred from standard media to standard media containing 100 mM NaCl at 3 dag. Images were taken 21 days after transfer. (F) Biomass of Col-0 and brx-2 plants germinated and grown on standard media containing 110 mM NaCl at 51 dag. Wide horizontal bars indicate average; boxes and vertical bars indicate quartiles. (G) Representative Col-0 and brx-2 plants at the end of their life cycle, grown on soil of different pH. (H) Average mature silique number and average number of seeds in five mature siliques at the end of the life cycle for Col-0 and brx-2 plants grown on soil of different pH. a: statistical significance compared to the other genotype in the same condition; b: statistical significance compared to the same genotype in the other condition. (I) Root growth vigor of different Arabidopsis accessions from alkaline or acidic collection sites (see Table S1) on different pH. (J) Location of the novel brx loss-of-function mutation identified in the Gue-0 and Gue-2 accessions. All error bars represent standard error. n.s., not significant; ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001. Scale bars represent 1 cm. Current Biology 2012 22, 1962-1968DOI: (10.1016/j.cub.2012.08.026) Copyright © 2012 Elsevier Ltd Terms and Conditions