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Multilayered Organization of Jasmonate Signalling in the Regulation of Root Growth.

Gasperini D, Chételat A, Acosta IF, Goossens J, Pauwels L, Goossens A, Dreos R, Alfonso E, Farmer EE - PLoS Genet. (2015)

Bottom Line: These effects, previously studied in leaves, require the activation of jasmonate (JA) signalling.The mutation weakly reduced root growth in undamaged plants but, when the upstream negative regulator NINJA was genetically removed, myc2-322B powerfully repressed root growth through its effects on cell division and cell elongation.In nature, growing roots are likely subjected to constant mechanical stress during soil penetration that could lead to JA production and subsequent detrimental effects on growth.

View Article: PubMed Central - PubMed

Affiliation: Department of Plant Molecular Biology, University of Lausanne, Lausanne, Switzerland.

ABSTRACT
Physical damage can strongly affect plant growth, reducing the biomass of developing organs situated at a distance from wounds. These effects, previously studied in leaves, require the activation of jasmonate (JA) signalling. Using a novel assay involving repetitive cotyledon wounding in Arabidopsis seedlings, we uncovered a function of JA in suppressing cell division and elongation in roots. Regulatory JA signalling components were then manipulated to delineate their relative impacts on root growth. The new transcription factor mutant myc2-322B was isolated. In vitro transcription assays and whole-plant approaches revealed that myc2-322B is a dosage-dependent gain-of-function mutant that can amplify JA growth responses. Moreover, myc2-322B displayed extreme hypersensitivity to JA that totally suppressed root elongation. The mutation weakly reduced root growth in undamaged plants but, when the upstream negative regulator NINJA was genetically removed, myc2-322B powerfully repressed root growth through its effects on cell division and cell elongation. Furthermore, in a JA-deficient mutant background, ninja1 myc2-322B still repressed root elongation, indicating that it is possible to generate JA-responses in the absence of JA. We show that NINJA forms a broadly expressed regulatory layer that is required to inhibit JA signalling in the apex of roots grown under basal conditions. By contrast, MYC2, MYC3 and MYC4 displayed cell layer-specific localisations and MYC3 and MYC4 were expressed in mutually exclusive regions. In nature, growing roots are likely subjected to constant mechanical stress during soil penetration that could lead to JA production and subsequent detrimental effects on growth. Our data reveal how distinct negative regulatory layers, including both NINJA-dependent and -independent mechanisms, restrain JA responses to allow normal root growth. Mechanistic insights from this work underline the importance of mapping JA signalling components to specific cell types in order to understand and potentially engineer the growth reduction that follows physical damage.

No MeSH data available.


Related in: MedlinePlus

Spatial localization of NINJA, MYC2, MYC3 and MYC4 in the primary root meristem and contribution of the three TFs to the ninja mutant phenotype.Expression pattern overviews of (A) NINJApro-NLS3xVENUS, (B) MYC2pro-NLS3xVENUS, (C) MYC3pro-NLS3xVENUS and (D) MYC4pro-NLS3xVENUS fluorescent reporters in 5-do WT primary roots. Close-ups of MYC2pro-NLS3xVENUS (E and F), MYC3pro-NLS3xVENUS (G and H) and MYC4pro-NLS3xVENUS (I and J) expression in, respectively, the elongation and division zones of the primary root. Confocal microscopy images (A-J) represent merged overlays of the fluorescent (yellow) and propidium iodide (red) stained roots. Scale bars = 50 μm. (K-N) Distribution maps of NINJA, MYC2, MYC3 and MYC4 expression patterns (green) in the primary root meristem based on promoter and protein fusion reporters (S4 Fig). (O) qRT-PCR of basal JAZ10 expression in 5-do roots of ninja-1 (n-1), ninja-2 (n-2), myc2 (m2), myc3 (m3), myc4 (m4), ninja-1 myc2 (n-1 m2), ninja-1 myc3 (n-1 m3), ninja-1 myc4 (n-1 m4), myc2 myc3 (m23), myc2 myc4 (m24), myc3 myc4 (m34), ninja-1 myc2 myc3 (n-1 m23), ninja-1 myc2 myc4 (n-1 m24), ninja-1 myc3 myc4 (n-1 m34), myc2 myc3 myc4 (m234), ninja-1 myc2 myc3 myc4 (n-1 m234), and ninja-2 myc2 myc3 myc4 (n-2 m234). JAZ10 transcript levels were normalized to those of UBC21 and displayed relative to the expression of WT controls that are set to 1 and indicated with a dashed line. Bars represent the means of three biological replicates (±SD), each containing a pool of ~60 roots. Complete qRT-PCR data are in S1 File. (P) Root length of 7-do seedlings of the same genotype as indicated in (O). Data shown are means (± SD) from 22–48 plants; letters above bars indicate statistically significant differences between samples as determined by Tukey’s HSD test (P < 0.01).
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pgen.1005300.g003: Spatial localization of NINJA, MYC2, MYC3 and MYC4 in the primary root meristem and contribution of the three TFs to the ninja mutant phenotype.Expression pattern overviews of (A) NINJApro-NLS3xVENUS, (B) MYC2pro-NLS3xVENUS, (C) MYC3pro-NLS3xVENUS and (D) MYC4pro-NLS3xVENUS fluorescent reporters in 5-do WT primary roots. Close-ups of MYC2pro-NLS3xVENUS (E and F), MYC3pro-NLS3xVENUS (G and H) and MYC4pro-NLS3xVENUS (I and J) expression in, respectively, the elongation and division zones of the primary root. Confocal microscopy images (A-J) represent merged overlays of the fluorescent (yellow) and propidium iodide (red) stained roots. Scale bars = 50 μm. (K-N) Distribution maps of NINJA, MYC2, MYC3 and MYC4 expression patterns (green) in the primary root meristem based on promoter and protein fusion reporters (S4 Fig). (O) qRT-PCR of basal JAZ10 expression in 5-do roots of ninja-1 (n-1), ninja-2 (n-2), myc2 (m2), myc3 (m3), myc4 (m4), ninja-1 myc2 (n-1 m2), ninja-1 myc3 (n-1 m3), ninja-1 myc4 (n-1 m4), myc2 myc3 (m23), myc2 myc4 (m24), myc3 myc4 (m34), ninja-1 myc2 myc3 (n-1 m23), ninja-1 myc2 myc4 (n-1 m24), ninja-1 myc3 myc4 (n-1 m34), myc2 myc3 myc4 (m234), ninja-1 myc2 myc3 myc4 (n-1 m234), and ninja-2 myc2 myc3 myc4 (n-2 m234). JAZ10 transcript levels were normalized to those of UBC21 and displayed relative to the expression of WT controls that are set to 1 and indicated with a dashed line. Bars represent the means of three biological replicates (±SD), each containing a pool of ~60 roots. Complete qRT-PCR data are in S1 File. (P) Root length of 7-do seedlings of the same genotype as indicated in (O). Data shown are means (± SD) from 22–48 plants; letters above bars indicate statistically significant differences between samples as determined by Tukey’s HSD test (P < 0.01).

Mentions: Promoter activities of NINJA and the three TFs were further characterized at the cellular level in the primary root tip with a nuclear localized fluorescent VENUS reporter protein (Fig 3A–3J). NINJApro-NLS3xVENUS was strongly expressed in all cells of the primary root apex (Fig 3A). MYC2pro-NLS3xVENUS was expressed in elongating endodermal and epidermal cells of the elongation zone (Fig 3E) as well as epidermal, lateral root cap and columella cells of the root division zone (Fig 3F). A weaker MYC3pro-NLS3xVENUS signal was present in endodermal, cortex and epidermal cells of the elongation and differentiation zones, while it was not detected in cells of the division zone (Fig 3H). MYC4pro-NLS3xVENUS was absent from the elongation and early differentiation zone of the root (Fig 3I), and its expression was restricted to outer layers of the columella and lateral root cap (Fig 3J). The same expression patterns, although with much weaker fluorescent signals for the three MYC TFs, were observed with functional protein fusion reporters driven by endogenous promoters (S4 and S5 Figs). The localization of NINJApro-NLS3xVENUS, MYC2pro-NLS3xVENUS, MYC3pro-NLS3xVENUS and MYC4pro-NLS3xVENUS florescent reporters did not differ from WT when expressed in the aos background (S6 Fig), indicating that the basal expression of NINJA and the three TFs in the primary root tip is JA-independent.


Multilayered Organization of Jasmonate Signalling in the Regulation of Root Growth.

Gasperini D, Chételat A, Acosta IF, Goossens J, Pauwels L, Goossens A, Dreos R, Alfonso E, Farmer EE - PLoS Genet. (2015)

Spatial localization of NINJA, MYC2, MYC3 and MYC4 in the primary root meristem and contribution of the three TFs to the ninja mutant phenotype.Expression pattern overviews of (A) NINJApro-NLS3xVENUS, (B) MYC2pro-NLS3xVENUS, (C) MYC3pro-NLS3xVENUS and (D) MYC4pro-NLS3xVENUS fluorescent reporters in 5-do WT primary roots. Close-ups of MYC2pro-NLS3xVENUS (E and F), MYC3pro-NLS3xVENUS (G and H) and MYC4pro-NLS3xVENUS (I and J) expression in, respectively, the elongation and division zones of the primary root. Confocal microscopy images (A-J) represent merged overlays of the fluorescent (yellow) and propidium iodide (red) stained roots. Scale bars = 50 μm. (K-N) Distribution maps of NINJA, MYC2, MYC3 and MYC4 expression patterns (green) in the primary root meristem based on promoter and protein fusion reporters (S4 Fig). (O) qRT-PCR of basal JAZ10 expression in 5-do roots of ninja-1 (n-1), ninja-2 (n-2), myc2 (m2), myc3 (m3), myc4 (m4), ninja-1 myc2 (n-1 m2), ninja-1 myc3 (n-1 m3), ninja-1 myc4 (n-1 m4), myc2 myc3 (m23), myc2 myc4 (m24), myc3 myc4 (m34), ninja-1 myc2 myc3 (n-1 m23), ninja-1 myc2 myc4 (n-1 m24), ninja-1 myc3 myc4 (n-1 m34), myc2 myc3 myc4 (m234), ninja-1 myc2 myc3 myc4 (n-1 m234), and ninja-2 myc2 myc3 myc4 (n-2 m234). JAZ10 transcript levels were normalized to those of UBC21 and displayed relative to the expression of WT controls that are set to 1 and indicated with a dashed line. Bars represent the means of three biological replicates (±SD), each containing a pool of ~60 roots. Complete qRT-PCR data are in S1 File. (P) Root length of 7-do seedlings of the same genotype as indicated in (O). Data shown are means (± SD) from 22–48 plants; letters above bars indicate statistically significant differences between samples as determined by Tukey’s HSD test (P < 0.01).
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pgen.1005300.g003: Spatial localization of NINJA, MYC2, MYC3 and MYC4 in the primary root meristem and contribution of the three TFs to the ninja mutant phenotype.Expression pattern overviews of (A) NINJApro-NLS3xVENUS, (B) MYC2pro-NLS3xVENUS, (C) MYC3pro-NLS3xVENUS and (D) MYC4pro-NLS3xVENUS fluorescent reporters in 5-do WT primary roots. Close-ups of MYC2pro-NLS3xVENUS (E and F), MYC3pro-NLS3xVENUS (G and H) and MYC4pro-NLS3xVENUS (I and J) expression in, respectively, the elongation and division zones of the primary root. Confocal microscopy images (A-J) represent merged overlays of the fluorescent (yellow) and propidium iodide (red) stained roots. Scale bars = 50 μm. (K-N) Distribution maps of NINJA, MYC2, MYC3 and MYC4 expression patterns (green) in the primary root meristem based on promoter and protein fusion reporters (S4 Fig). (O) qRT-PCR of basal JAZ10 expression in 5-do roots of ninja-1 (n-1), ninja-2 (n-2), myc2 (m2), myc3 (m3), myc4 (m4), ninja-1 myc2 (n-1 m2), ninja-1 myc3 (n-1 m3), ninja-1 myc4 (n-1 m4), myc2 myc3 (m23), myc2 myc4 (m24), myc3 myc4 (m34), ninja-1 myc2 myc3 (n-1 m23), ninja-1 myc2 myc4 (n-1 m24), ninja-1 myc3 myc4 (n-1 m34), myc2 myc3 myc4 (m234), ninja-1 myc2 myc3 myc4 (n-1 m234), and ninja-2 myc2 myc3 myc4 (n-2 m234). JAZ10 transcript levels were normalized to those of UBC21 and displayed relative to the expression of WT controls that are set to 1 and indicated with a dashed line. Bars represent the means of three biological replicates (±SD), each containing a pool of ~60 roots. Complete qRT-PCR data are in S1 File. (P) Root length of 7-do seedlings of the same genotype as indicated in (O). Data shown are means (± SD) from 22–48 plants; letters above bars indicate statistically significant differences between samples as determined by Tukey’s HSD test (P < 0.01).
Mentions: Promoter activities of NINJA and the three TFs were further characterized at the cellular level in the primary root tip with a nuclear localized fluorescent VENUS reporter protein (Fig 3A–3J). NINJApro-NLS3xVENUS was strongly expressed in all cells of the primary root apex (Fig 3A). MYC2pro-NLS3xVENUS was expressed in elongating endodermal and epidermal cells of the elongation zone (Fig 3E) as well as epidermal, lateral root cap and columella cells of the root division zone (Fig 3F). A weaker MYC3pro-NLS3xVENUS signal was present in endodermal, cortex and epidermal cells of the elongation and differentiation zones, while it was not detected in cells of the division zone (Fig 3H). MYC4pro-NLS3xVENUS was absent from the elongation and early differentiation zone of the root (Fig 3I), and its expression was restricted to outer layers of the columella and lateral root cap (Fig 3J). The same expression patterns, although with much weaker fluorescent signals for the three MYC TFs, were observed with functional protein fusion reporters driven by endogenous promoters (S4 and S5 Figs). The localization of NINJApro-NLS3xVENUS, MYC2pro-NLS3xVENUS, MYC3pro-NLS3xVENUS and MYC4pro-NLS3xVENUS florescent reporters did not differ from WT when expressed in the aos background (S6 Fig), indicating that the basal expression of NINJA and the three TFs in the primary root tip is JA-independent.

Bottom Line: These effects, previously studied in leaves, require the activation of jasmonate (JA) signalling.The mutation weakly reduced root growth in undamaged plants but, when the upstream negative regulator NINJA was genetically removed, myc2-322B powerfully repressed root growth through its effects on cell division and cell elongation.In nature, growing roots are likely subjected to constant mechanical stress during soil penetration that could lead to JA production and subsequent detrimental effects on growth.

View Article: PubMed Central - PubMed

Affiliation: Department of Plant Molecular Biology, University of Lausanne, Lausanne, Switzerland.

ABSTRACT
Physical damage can strongly affect plant growth, reducing the biomass of developing organs situated at a distance from wounds. These effects, previously studied in leaves, require the activation of jasmonate (JA) signalling. Using a novel assay involving repetitive cotyledon wounding in Arabidopsis seedlings, we uncovered a function of JA in suppressing cell division and elongation in roots. Regulatory JA signalling components were then manipulated to delineate their relative impacts on root growth. The new transcription factor mutant myc2-322B was isolated. In vitro transcription assays and whole-plant approaches revealed that myc2-322B is a dosage-dependent gain-of-function mutant that can amplify JA growth responses. Moreover, myc2-322B displayed extreme hypersensitivity to JA that totally suppressed root elongation. The mutation weakly reduced root growth in undamaged plants but, when the upstream negative regulator NINJA was genetically removed, myc2-322B powerfully repressed root growth through its effects on cell division and cell elongation. Furthermore, in a JA-deficient mutant background, ninja1 myc2-322B still repressed root elongation, indicating that it is possible to generate JA-responses in the absence of JA. We show that NINJA forms a broadly expressed regulatory layer that is required to inhibit JA signalling in the apex of roots grown under basal conditions. By contrast, MYC2, MYC3 and MYC4 displayed cell layer-specific localisations and MYC3 and MYC4 were expressed in mutually exclusive regions. In nature, growing roots are likely subjected to constant mechanical stress during soil penetration that could lead to JA production and subsequent detrimental effects on growth. Our data reveal how distinct negative regulatory layers, including both NINJA-dependent and -independent mechanisms, restrain JA responses to allow normal root growth. Mechanistic insights from this work underline the importance of mapping JA signalling components to specific cell types in order to understand and potentially engineer the growth reduction that follows physical damage.

No MeSH data available.


Related in: MedlinePlus