The karrikin signaling regulator SMAX1 controls Lotus japonicus root and root hair development by suppressing ethylene biosynthesis

L. japonicus smax1 mutants have a reduced primary root and increased root hair length. (A) Representative images of wild type, smax1-2, and smax1-3 grown on Petri dishes at 10 d postgermination. (Scale bars, 1 cm.) (B and C) Primary root length, postembryonic root number, and PER density of wild type, smax1-2, and smax1-3 (n ≥ 23) (B) and a population segregating for the smax1-2 LORE1 insertion (C) (n ≥ 13). (D) Representative images of the root tip. Red arrows indicate the position of the quiescent center (QC); green arrows indicate the nearest root hair. (Scale bars, 500 µm.) (E and F) Distance of the first RH from the QC (E) and RH length at 1.5 to 2 mm from the apex (F) in the wild type, smax1-2, and smax1-3 (n ≥ 6). (E) Asterisks indicate significant differences compared with the wild type (ANOVA, post hoc Dunnett test; N.S., not significant, P > 0.05; *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001). (B, C, E, and F) Letters indicate significant differences (ANOVA, post hoc Tukey test).

Differential gene expression in L. japonicus KAR/KL signaling mutants. (A) Hierarchical clustering of 3,748 unique DEGs (comparing each mutant vs. WT). Z scores were obtained by scaling the r–log-transformed count data. Red represents lower and green indicates higher transcript accumulation. The clusters are indicated by a dendrogram (Left) and illustrated by different colors and a number (Right). Gene ontology enrichment analysis was performed using AgriGO for each cluster separately. Negative log₁₀(FDR) represents the statistical significance (FDR, false discovery rate) of the enrichment, with higher values representing stronger enrichment (cutoff, −log₁₀FDR ≥ 1.3). (B) Transcript accumulation of D14like2 (DLK2) and ACS7 genes in roots of the indicated genotypes as determined by qRT-PCR. Expression values were normalized to the expression of ubiquitin. Letters indicate statistical differences between genotypes (ANOVA, post hoc Tukey test (n = 3 or 4). Numbers above the data points indicate, if significant, the log₂ fold change for the mutant vs. wild type comparison as determined by RNA-seq analysis.

Primary root and root hair phenotypes of smax1 mutants are caused by increased ethylene production. (A) Ethylene released by L. japonicus wild type, smax1-2, and smax1-3 seedlings and in response to treatment with 0.1 µM AVG or 1 µM ACC as determined by gas chromatography (n = 5). (B and C) Representative images (B) and quantification (C) of PR length, PER number, and PER density of 10-d-old wild-type and smax1-3 seedlings grown in the presence of 50 µM silver nitrate (AgNO₃) or 0.1 µM AVG (n ≥ 24). (Scale bars, 1 cm.) (D–F) Representative images of the root tip (D) and quantification of the distance between the first RH and the QC (E) and of the RH length (F) of wild-type and smax1-3 seedlings in the presence of 50 µM silver nitrate or 0.1 µM AVG (n ≥ 7). (Scale bars, 500 µm.) (A, C, E, and F) Letters indicate significant differences (ANOVA, post hoc Tukey test, P ≤ 0.001).