Role of NINJA in root jasmonate signaling

Abstract

Wound responses in plants have to be coordinated between organs so that locally reduced growth in a wounded tissue is balanced by appropriate growth elsewhere in the body. We used a JASMONATE ZIM DOMAIN 10 (JAZ10) reporter to screen for mutants affected in the organ-specific activation of jasmonate (JA) signaling in Arabidopsis thaliana seedlings. Wounding one cotyledon activated the reporter in both aerial and root tissues, and this was either disrupted or restricted to certain organs in mutant alleles of core components of the JA pathway including COI1, OPR3, and JAR1. In contrast, three other mutants showed constitutive activation of the reporter in the roots and hypocotyls of unwounded seedlings. All three lines harbored mutations in Novel Interactor of JAZ (NINJA), which encodes part of a repressor complex that negatively regulates JA signaling. These ninja mutants displayed shorter roots mimicking JA-mediated growth inhibition, and this was due to reduced cell elongation. Remarkably, this phenotype and the constitutive JAZ10 expression were still observed in backgrounds lacking the ability to synthesize JA or the key transcriptional activator MYC2. Therefore, JA-like responses can be recapitulated in specific tissues without changing a plant's ability to make or perceive JA, and MYC2 either has no role or is not the only derepressed transcription factor in ninja mutants. Our results show that the role of NINJA in the root is to repress JA signaling and allow normal cell elongation. Furthermore, the regulation of the JA pathway differs between roots and aerial tissues at all levels, from JA biosynthesis to transcriptional activation.

Keywords: herbivory; mapping by sequencing; plant fertility; root growth.

Fig. 1.

The JAZ10-GusPlus (JGP) reporter for JA-mediated wound signaling in 5-d.o. WT and aos mutant Arabidopsis seedlings. Detection of GUS activity was performed 2 h after wounding. Red arrows indicate cotyledon wounding sites. The reporter in the aos mutant background displays a constitutive faint blue staining in the aerial organs not observed in any other genotype and not attributable to a higher basal JAZ10 transcription (SI Appendix, Fig. S2). (Inset) Close-up of root. (Scale bars, 0.5 mm.)

Fig. 2.

jar1 mutations impair wound-induced JAZ10 expression mainly in roots. (A) JGP expression in 61E (jar1-13) and jar1-1 seedlings after cotyledon wounding. Black arrows indicate lack of staining in the root of wounded jar mutants. (Scale bars, 0.5 mm.) (B) Quantitative RT-PCR (qRT-PCR) of JAZ10 expression 1 h after wounding in aerial organs and roots of WT, jar1-1, and jar1-13 seedlings. JAZ10 transcript levels were normalized to those of UBC21 and displayed relative to the expression in the WT unwounded controls. Bars represent the means of three biological replicates (±SD), each containing a pool of organs from ∼60 seedlings.

Fig. 3.

ninja mutants display constitutive JAZ10 expression. (A) JGP expression in control and wounded seedlings of ninja-1 and ninja-1 aos (compare with WT and aos in Fig. 1). Arrows indicate constitutive reporter activity in hypocotyl and roots (black) and cotyledon wounding sites (red). (Inset) Close-up of root. The ninja-2 aos double mutant displays the same expression pattern as ninja-1 aos. (Scale bars, 0.5 mm.) (B and C) qRT-PCR of JAZ10 expression basally (B) and 1 h after wounding (C) in ninja-1 (n-1), ninja-2 (n-2), ninja-3 (n-3), ninja RNAi (n_i), jin1-7 (j1), ninja-1 jin1-7 (n-1 j1), and ninja-3 jin1-7 (n-3 j1). JAZ10 transcript levels were normalized to those of UBC21 and displayed relative to the expression of unwounded WT controls (B) or wounded WT samples (C); thus, WT levels are set to 1 in each plot and indicated with a dashed line. Bars represent the means of three biological replicates (±SD), except for n-2 and n_i (two biological replicates), each containing a pool of organs from ∼60 seedlings. Complete qRT-PCR data are in Dataset S1.

Fig. 4.

Reduced root growth and cell length in untreated ninja lines. (A) Representative 7-d.o. seedlings of WT and ninja mutants. (B and C) Quantification of root growth in WT and mutant lines grown in the absence or presence of 25 µM MeJA for 7 d. Data are the means (±SD) from at least 20 plants. Letters above bars indicate statistically significant differences between pairs of control samples as determined by Tukey’s honestly significant difference (HSD) test (P < 0.02). (D) Primary root meristem cell number in 5-d.o. seedlings of WT and several mutant lines. No statistically significant difference between genotypes was found by ANOVA (P = 0.475). (E) Representative cortex cells from WT and ninja-1 mutant roots. Yellow bars indicate cell length. (F) Box plot summary of cortex-cell length in 5-d.o. seedlings of WT and several mutant lines grown in the absence of MeJA. Medians and means are represented inside the boxes by solid and dashed lines, respectively. Circles depict outlier data points beyond ±1.5× the interquartile range defined by the whiskers. Letters indicate statistically significant differences between pairs as determined by Tukey’s HSD test (P < 2e-16).