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. 2021 Oct 26;72(20):7107-7118.
doi: 10.1093/jxb/erab358.

Jasmonate inhibits adventitious root initiation through repression of CKX1 and activation of RAP2.6L transcription factor in Arabidopsis

Asma Dob  1 , Abdellah Lakehal  1   2 , Ondrej Novak  3   2 , Catherine Bellini  1   4
Affiliations

Jasmonate inhibits adventitious root initiation through repression of CKX1 and activation of RAP2.6L transcription factor in Arabidopsis

Asma Dob et al. J Exp Bot. .

Abstract

Adventitious rooting is a de novo organogenesis process that enables plants to propagate clonally and cope with environmental stresses. Adventitious root initiation (ARI) is controlled by interconnected transcriptional and hormonal networks, but there is little knowledge of the genetic and molecular programs orchestrating these networks. Thus, we have applied genome-wide transcriptome profiling to elucidate the transcriptional reprogramming events preceding ARI. These reprogramming events are associated with the down-regulation of cytokinin (CK) signaling and response genes, which could be triggers for ARI. Interestingly, we found that CK free base (iP, tZ, cZ, and DHZ) content declined during ARI, due to down-regulation of de novo CK biosynthesis and up-regulation of CK inactivation pathways. We also found that MYC2-dependent jasmonate (JA) signaling inhibits ARI by down-regulating the expression of the CYTOKININ OXIDASE/DEHYDROGENASE1 (CKX1) gene. We also demonstrated that JA and CK synergistically activate expression of the transcription factor RELATED to APETALA2.6 LIKE (RAP2.6L), and constitutive expression of this transcription factor strongly inhibits ARI. Collectively, our findings reveal that previously unknown genetic interactions between JA and CK play key roles in ARI.

Keywords: Adventitious roots; Arabidopsis; CKX1; MYC2; RAP2.6L; cytokinins; jasmonate; light; vegetative propagation.

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Figures

Fig. 1.
Fig. 1.
Dark–light transition affects CK pathways. (A) Representative scheme of the experimental set-up used to collect samples for RNA sequencing. Seedlings were grown in the dark until their hypocotyls were 6–7 mm long (T0) then shifted to the light for either 9 h (T9) or 24 h (T24). (B) Venn diagram showing numbers of differentially expressed genes during ARI in wild-type (Col-0) seedlings. (C) Illustrative model of CK pathways. (D–J) Heatmaps of the genes involved in the CK pathways: (D) response, (E and F) de novo biosynthesis, (G) inactivation, (H) irreversible cleavage, and (I and J) signaling. Heatmaps represent fold changes (log2) in transcript abundance in wild-type seedlings. Blue and red colors indicate down-regulated and up-regulated expression, respectively, at T9 or T24 (9 h and 24 h after transfer to light, at T0 respectively), relative to T0. Asterisks indicate significant differences.
Fig. 2.
Fig. 2.
Dark–light transition affects CK homeostasis. Contents in wild-type (Col-0) seedlings grown in the dark until their hypocotyls were 6–7 mm long (T0) then shifted to the light for either 9 h (T9) or 72 h (T72) of: (A) CK ribotides [isopentenyladenine riboside-5′-monophosphate (iPRMP), trans-zeatin riboside-5′-monophosphate (tZRMP), cis-zeatin riboside-5′-monophosphate (cZRMP)]; (B) CK ribosides [isopentenyladenine riboside (iPR), trans-zeatin riboside (tZR), cis-zeatin riboside (cZR)]; (C) CK free bases [isopentenyladenine (iP), trans-zeatin (tZ), cis-zeatin (cZ)]; (D and E) CK N-glucosides [isopentenyladenine-7-glucoside (iP7G), trans-zeatin-7-glucoside (tZ7G), cis-zeatin-7-glucoside (cZ7G), isopentenyladenine-9-glucoside (iP9G), trans-zeatin-9-glucoside (tZ9G), cis-zeatin-9-glucoside (cZ79G)]; and (F) CK O-glucosides [trans-zeatin-O-glucoside (tZOG), trans-zeatin riboside-O-glucoside (tZROG), cis-zeatin-O-glucoside (cZOG), cis-zeatin riboside-O-glucoside (cZROG)]. *, **, and *** indicate significant differences (0.05>P>0.01, 0.01>P>0.001, and P<0.001, respectively) at T9 and T72 relative to T0 according to ANOVA with t-tests. Means (bars) and SDs (whiskers). <LOD indicates under the limit of detection.
Fig. 3.
Fig. 3.
JA inhibits ARI by repressing CKX1 expression. (A–C) Relative amounts of CKX1 transcripts measured by qRT-PCR. Means and SEs (indicated by error bars) obtained from three technical replicates. All the qRT-PCR experiments were repeated with at least one other independent biological replicate and gave similar results. (A) Transcripts extracted from 6-day-old wild-type (Col-0) seedlings treated with JA (at the indicated concentrations) for 1 h relative to amounts in mock-treated controls. (B) Transcripts extracted from 6-day-old coi1-16 mutant (dark bars) or wild-type (gray bars) seedlings treated with 50 μM JA relative to amounts in mock-treated controls. (C) Transcripts extracted from dissected hypocotyls of ninja-1myc2-322B double mutant or wild-type seedlings etiolated in the dark until their hypocotyls were 6 mm long (T0) then moved to the light for 9 h (T9) or 24 h (T24) relative to amounts in wild-type (Col-0) seedlings. (D) Representative images showing emerged ARs. Red and white arrowheads indicate emerged ARs and root–hypocotyl junctions, respectively. Scale bars indicate 6 mm. (E) Average numbers of ARs. The non-parametric Kruskal–Wallis test combined with Dunn’s multiple comparison post-test indicates that the ninja-1myc2-322B35S:CKX1 triple mutant produced significantly more ARs than the ninja-1myc2-322B double mutant. Bars and whiskers indicate means and SEs, respectively (n>50; P<0.0007). (F) Illustrative scheme representing locations of G-box-like motifs in the CKX1 promoter.
Fig. 4.
Fig. 4.
CK and JA synergistically repress ARI through RAP2.6L induction. (A) Amounts of RAP2.6L transcripts in wild-type seedlings treated with the indicated concentrations of JA and/or tZ relative to amounts in mock-treated controls. Means and SEs obtained from three technical replicates. The qRT-PCR experiment was repeated with another independent biological replicate and gave similar results. (B) Average numbers of ARs. The t-test indicates that 35S:RAP2.6L produced significantly fewer ARs than wild-type seedlings (n>60; P<0.0001). (C) Average numbers of lateral roots (LRs). (D and E) Results of measurements respectively showing that 35S:RAP2.6L mutants produced longer primary roots than wild-type seedlings (t-test: n>24, P<0.0022), but their lateral root density did not differ (B–E) Bars and whiskers indicate means and SEs, respectively. (F) Representative images showing numbers of emerged ARs. Red and white arrowheads indicate emerged ARs and junction roots, respectively. Scale bars indicate 6 mm.
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