A DAO1-Mediated Circuit Controls Auxin and Jasmonate Crosstalk Robustness during Adventitious Root Initiation in Arabidopsis

Abstract

Adventitious rooting is a post-embryonic developmental program governed by a multitude of endogenous and environmental cues. Auxin, along with other phytohormones, integrates and translates these cues into precise molecular signatures to provide a coherent developmental output. Auxin signaling guides every step of adventitious root (AR) development from the early event of cell reprogramming and identity transitions until emergence. We have previously shown that auxin signaling controls the early events of AR initiation (ARI) by modulating the homeostasis of the negative regulator jasmonate (JA). Although considerable knowledge has been acquired about the role of auxin and JA in ARI, the genetic components acting downstream of JA signaling and the mechanistic basis controlling the interaction between these two hormones are not well understood. Here we provide evidence that COI1-dependent JA signaling controls the expression of DAO1 and its closely related paralog DAO2. In addition, we show that the dao1-1 loss of function mutant produces more ARs than the wild type, probably due to its deficiency in accumulating JA and its bioactive metabolite JA-Ile. Together, our data indicate that DAO1 controls a sensitive feedback circuit that stabilizes the auxin and JA crosstalk during ARI.

Keywords: adventitious roots; auxin; auxin oxidation; jasmonates; organogenesis.

Figure 1

Jasmonate (JA) controls the expression of DAO1 and DAO2. (A) Relative transcript amount of DAO1 and DAO2 quantified by qRT-PCR. mRNAs were extracted from six-day-old wild-type seedlings treated for 1 h either with different doses of JA or mock solution. The gene expression values are relative to the mock-treated control, for which the value was arbitrarily set to 1. The scale in the Y axis is indicated as a log2 unit. Error bars indicate ± SD obtained from three technical replicates. (B,C) Relative transcript amount of DAO1 and DAO2 quantified by qRT-PCR. mRNAs were extracted from six-day-old wild-type or coi1-16 mutant seedlings treated with 50 μM JA or mock solution at different time points. The gene expression values are relative to the mock-treated control, for which the value was arbitrarily set to 1. Error bars indicate ± SD obtained from three technical replicates. (D) Representative scheme of the location of G-box or G-box-like cis regulatory elements on the DAO1 and DAO2 promoters. (E,F) Relative transcript amount of DAO1 and DAO2 quantified by qRT-PCR. mRNAs were extracted from six-day-old wild-type or jin1-2 mutant seedlings treated for 1 h with different JA doses or mock solutions. The gene expression values are relative to the mock-treated control, for which the value was set to 1. Error bars indicate ± SD obtained from three technical replicates. A t-test indicates that the values indicated by an asterisk are significantly different from their mock counterpart (p < 0.01; n = 3). All wild-type and mutant seedlings were grown for five days under long-day conditions (16 h light/8 h dark), then they were acclimated overnight in liquid media before any treatment. All the experiments were repeated with another independent biological replicate and gave similar results.

Figure 2

JA controls the expression of DAO1 and DAO2 independently of TIR1/AFB-dependent auxin signaling. (A) Relative transcript amount of DAO1, DAO2, and GH3.3 quantified by qRT-PCR. mRNAs were extracted from six-day-old wild-type seedlings treated for 1 h with 10 μM indole-3-acetic acid (IAA) or mock solutions. The gene expression values are relative to the mock-treated control, for which the value was set to 1. (B) Relative transcript amount of DAO1 and DAO2 quantified by qRT-PCR. mRNAs were extracted from six-day-old wild-type seedlings pre-treated for 90 min with 10 μM auxinole, then they were co-treated with 50 μM JA and 10 μM auxinole for 1 h. The gene expression values are relative to the mock-treated control, for which the value was set to 1. The scale in the Y axis is indicated as a log2 unit. Error bars indicate ± SEM obtained from three technical replicates. Wild-type seedlings were grown for five days under long-day conditions (16 h light/8 h dark), then they were acclimated overnight in liquid media before any treatment. All the experiments were repeated at least twice and the biological replicates gave the same results.

Figure 3

DAO1, but not DAO2, plays a major role in IAA oxidation during ARI. (A,B) Average number of ARs in dao mutants. Seedlings were grown in AR phenotyping conditions. (A) A t-test indicates that the dao1-1 mutant exhibits significantly more ARs than the wild type (inidicated by an asterisk). Error bars indicate ± SEM (n > 30; p < 0.001). (B) One-way ANOVA combined with Tukey’s multiple comparison post-hoc test indicates that the dao1-1 single and dao1–1dao2C double mutants exhibit significantly more ARs compared to the wild type. The values indicated by different letters are significantly different from each others. Error bars indicate ± SEM (n > 20; p < 0.001). (C) Density of lateral roots (LRs) (i.e., the number of LRs per cm of primary root) in dao mutants grown in AR phenotyping conditions. One-way ANOVA combined with Dunnett’s multiple comparison post-hoc test indicated that the LR density was significantly affected in dao1-1 single and dao1–1dao2C double mutants (inidicated by an asterisk). Error bars indicate ± SEM (n > 20; p < 0.001). (D) Wild-type and dao mutant seedlings were grown in the dark until their hypocotyls reached 6 mm long, when they were transferred to fresh medium containing either mock solution or 1 μM IAA. The seedlings were kept for seven more days under long-day conditions to induce ARs. Arrow heads indicate hypocotyl–root junction. (E) Average number of ARs in the wild type and dao mutants in response to IAA grown as in (D). One-way ANOVA combined with Tukey’s multiple comparison post-hoc test indicates that the dao1-1 single and dao1–1dao2C double mutant produce significantly more ARs than the wild type. The values indicated by different letters are significantly different from each others. Error bars indicate ± SEM (n > 30; p < 0.001 (F) Ratio of AR number from IAA-treated/mock-treated seedlings. One-way ANOVA combined with Tukey’s multiple comparison post-hoc test indicates that the dao1-1 single and dao1–1dao2C double mutant produce significantly more ARs than the wild type. Error bars indicate ± SEM (n > 30; p < 0.001). (G) Spatiotemporal activity and dynamics of DAO1 promoter. Seedlings expressing the pDAO1:GUS construct were grown in the dark until their hypocotyls were 6 mm long (T0), 9 h (T9L), and 24 h (T24L) after their transfer to the light and their respective controls, which were kept in the dark for 9 h (T9D) and 24 h (T24D). The seedlings were stained for 2 h. (D–G) All scale bars represent 6 mm.

Figure 4

The AR phenotype of the dao1-1 mutant is probably linked to its deficiency in JA and JA-Ile. (A–D) Endogenous hormone contents. (A) Free IAA, (B) free JA, (C) JA-Ile, and (D) cis-OPDA were quantified in the hypocotyls of wild-type and dao1-1 mutant seedlings grown in the dark until the hypocotyl reached 6 mm long (T0) and after their transfer to the light for 9 h (T9), 24 h (T24) and 72 h (T72). Asterisks indicate a statistically significant difference in the mutant lines versus the wild type in an ANOVA analysis (* and **correspond to p-values of 0.05 > p > 0.01, 0.01 > p > 0.001). Error bars indicate ± SD of six biological replicates. (E) Relative transcript amount of two key genes in the JA biosynthesis, AOC2 and OPR3, as well as (F) GH3.3, GH3.5, and GH3.6 quantified by qRT-PCR. mRNA was extracted from hypocotyls of wild-type and dao1-1 mutant seedlings grown in AR phenotyping conditions as indicated above. The gene expression values are relative to the wild type, for which the value was set to 1. The scale in the Y axis is indicated as a log2 unit Error bars indicate ± SEM obtained from three technical replicates.

Figure 5

DAO1 controls a feedback circuit to stabilize IAA–JA crosstalk during ARI. Auxin promotes ARI by modulating the homeostasis of the negative regulator JA. COI1-dependent JA signaling induces the expression of DAO1, which in turn controls the thresholds of IAA by irreversible degradation. Arrows indicate positive regulation, whereas dashes indicate negative regulation.