Developmental patterning function of GNOM ARF-GEF mediated from the cell periphery
- Institute of Science and Technology Austria, Austria
- Plant Breeding and Acclimatization Institute – National Research Institute, Poland
Figures
Figure 1 with 11 supplements
GN localizes to the cell periphery at often stable punctate structures.
(A) CLSM images of GN-GFP and GNL1-GFP in the epidermis of seedling RAMs. In contrast with GNL1-GFP, GN-GFP localizes to the PM. Numbers in bottom right indicate ratios of RAMs with PM signals. …
Figure 1—figure supplement 1
gns mutant complementation data.
Seedlings (A) and adults (B) of gns complemented by GNpro:GN-GFP. No complementation of gns by GNpro:GNL1-GFP (C). (D) Root hair positioning in gns complemented by GNpro:GN-GFP, GNpro:GNfwr-GFP and G…
Figure 1—figure supplement 2
GN-GFP and GNL1-GFP subcellular localization data from two transgenic lines analyzed.
CLSM (A) and TIRF (B) images of GN-GFP and GNL1-GFP from two transgenic lines analyzed. Scale bars – (A) 10 µm, (B) 2 µm.
Figure 1—figure supplement 3
TIRF colocalization of GN-GFP and VAN3-mRFP following a BFA treatment.
TIRF colocalization of GN-GFP with VAN3-mRFP in etiolated hypocotyl epidermis, following a treatment with BFA at 50 µM for 1 hr. The BFA-induced, dense and dynamic punctate signals of GN-GFP do not …
Figure 1—figure supplement 4
Quantitative data on GN-positive peripheral structures.
(A) Lateral displacement of individual GN-GFP and GNfwr-GFP structures estimated from kymograph maximum projections of TIRF movies. GN-GFP: 0.555±0.280 µm (mean ± s.d.), n=103; GNfwr-GFP: …
Figure 1—video 1
TIRF time lapse of GN-GFP in seedling root epidermis.
Scale bar - 10 μm.
Figure 1—video 2
TIRF time lapse of GNL1-GFP in seedling root epidermis.
Scale bar - 10 μm.
Figure 1—video 3
TIRF time lapse of GN-GFP in hypocotyl epidermis following a mock treatment.
Scale bar - 10 μm.
Figure 1—video 4
TIRF time lapse of GN-GFP in hypocotyl epidermis following a treatment with BFA 50 µM for 1 hr.
Scale bar - 10 μm.
Figure 1—video 5
TIRF time lapse colocalization of GNOM-GFP and VAN3-mRFP in hypocotyl epidermis following a treatment with BFA 50 µM for 1 hr, green channel.
Scale bar - 10 μm.
Figure 1—video 6
TIRF time lapse colocalization of GNOM-GFP and VAN3-mRFP in hypocotyl epidermis following a treatment with BFA 50 µM for 1 hr, red channel.
Scale bar - 10 μm.
Figure 1—video 7
TIRF time lapse colocalization of GNOM-GFP and VAN3-mRFP in hypocotyl epidermis following a treatment with BFA 50 µM for 1 hr, merged channels.
Scale bar - 10 μm.
Figure 2 with 2 supplements
A functional GNfwr-GFP variant localizes to punctate structures at the cell periphery, but not to the GA.
(A) Eight-day-old seedlings of gns mutants complemented with GNpro:GN-GFP and GNpro:GNfwr-GFP transgenes. Two independent lines of each complemented mutant are shown. (B) Adult gns mutants …
Figure 2—figure supplement 1
Subcellular localization of GN-GFP and GNfwr-GFP at basal end of young trichoblasts.
TIRF images (A) and kymographs (B) of GN-GFP and GNfwr-GFP expressed in complemented mutant backgrounds, at basal ends of young trichoblasts. Scale bar – 2 µm.
Figure 2—video 1
TIRF time lapse of GNfwr-GFP in gns seedling root epidermis.
Scale bar - 10 μm.
Figure 3 with 8 supplements
GN-positive structures do not contain clathrin, and CME is normal in gns.
TIRF images (A) and kymographs (B) of GN-GFP and CLC-mKO in the epidermis of early elongation zone of seedling roots. The GN-positive structures at the PM are distinct from CCPs. Scale bar – 2 µm. …
Figure 3—figure supplement 1
Control TIRF co-localization of CME markers TPLATE-GFP and AP2A1-TagRFP.
TIRF images (A) and kymographs (B) of TPLATE-GFP and AP2A1-TagRFP in the epidermis of early elongation zone of seedling roots. Scale bar – 2 µm. Arrowheads in (A) indicate co-localizing punctae.
Figure 3—video 1
TIRF time lapse colocalization of GN-GFP and CLC-mKO in seedling root epidermis, green channel.
Scale bar - 2 μm.
Figure 3—video 2
TIRF time lapse colocalization of GN-GFP and CLC-mKO in seedling root epidermis, red channel.
Scale bar - 2 μm.
Figure 3—video 3
TIRF time lapse colocalization of GN-GFP and CLC-mKO in seedling root epidermis, merged channels.
Scale bar - 2 μm.
Figure 3—video 4
TIRF time lapse of CLC2-GFP in wild-type hypocotyl epidermis.
Scale bar - 10 μm.
Figure 3—video 5
TIRF time lapse of CLC2-GFP in gns hypocotyl epidermis.
Scale bar - 10 μm.
Figure 3—video 6
TIRF time lapse of TPLATE-GFP in wild-type hypocotyl epidermis.
Scale bar - 10 μm.
Figure 3—video 7
TIRF time lapse of TPLATE-GFP in gns hypocotyl epidermis.
Scale bar - 10 μm.
Figure 4 with 1 supplement
Bulk secretion is normal in gns.
(A) Maximum projections of z-stacks of CLSM images of secRFP in RAMs of wild-type seedlings and in basal ends of gns seedlings. Scale bars - 20 µm. (B) Maximum projection of a z-stack of CLSM images …
Figure 4—figure supplement 1
Additional controls for secRFP secretion.
(A) CLSM maximum projection of a z-stack of secRFP in RAMs of 2-day-old wild-type seedlings. (B) Maximum projections of z-stacks of CLSM images in gns basal ends with and without secRFP expression. …
Figure 5 with 1 supplement
A BFA-induced, GN-specific exocytic event.
(A) CLSM images of GN-GFP in seedling RAM epidermis following a treatment with BFA at 50 µM for 1 hr. After a treatment with BFA, GN-GFP locates to the core of BFA bodies and strongly localizes to …
Figure 5—figure supplement 1
A BFA-induced, GN-specific exocytic event.
(A) A CLSM image of GN-GFP in seedling RAM epidermis following a treatment with BFA at 50 µM for 1 hr, demonstrating strong localization of GN-GFP to the cell plates. Scale bar - 10 µm. (B) Control …
Figure 6 with 1 supplement
GNOM - GNOM-LIKE1 chimeric ARF-GEFs.
(A) Schematic representation of the domain composition of GN, GNL1, and chimeric GBF1-type ARF-GEFs. An LLG chimera was not cloned. (B) No complementation of gns by the expression of GLL-GFP. (C) …
Figure 6—figure supplement 1
Additional data related to GNOM - GNOM-LIKE1 chimeras.
(A) Terminal phenotypes of gns mutants complemented by the expression of LGL-GFP. (B) An additional picture of adult gns mutants complemented by the expression of GLG-GFP. (C) Control images showing …
Figure 7 with 2 supplements
Seedling phenotypes of gns mutants complemented with GGL-GFP, LGG-GFP, GLG-GFP, and LGL-GFP.
(A) Seedlings of gns mutants expressing LGG-GFP and GGL-GFP. Expression of LGG-GFP fully complements the mutant phenotype, while seedlings expressing GGL-GFP are characterized by roots growing more …
Figure 7—figure supplement 1
The effect of NPA on root waving.
Col-0 seedlings grown on media supplemented with 1 µM NPA on vertical and tilted plates. NPA causes loss of root waving as exhibited on plates tilted back from the vertical.
Figure 7—figure supplement 2
Additional data on seedling phenotypes of gns mutants complemented with GN-GNL1 chimeras.
Growth on tilted plates showing both general seedling morphology and root waving (A), single and fused cotyledons (B), dark-grown seedlings (C), and apical hooks (D) of indicated lines.
Figure 8 with 1 supplement
Subcellular localization and BFA-induced exocytosis of ARF-GEF chimeras.
(A) TIRF images of GN-GFP, LGG-GFP, GGL-GFP, GLG-GFP and LGL-GFP in hypocotyls of etiolated seedlings of complemented gns mutants. All complementing chimeras localize to the GN-specific structures …
Figure 8—figure supplement 1
Additional data on subcellular localization and BFA-induced exocytosis of ARF-GEF chimeras.
(A) TIRF images of LGG-GFP, GGL-GFP, GLG-GFP and LGL-GFP in hypocotyls of etiolated seedlings of complemented gns mutants from two transgenic lines tested. Scale bar - 2 µm. (B) CLSM images of …
Tables
Table 1
Summary of phenotypes of gns expressing ARF-GEF variants.
| ARF-GEF | Overall degree of GN function | Adult phenotype | Seedling growth rate | Root waving | Root hair positioning | Apical patterning | Etiolated seedling tropism | Apical hooks |
|---|---|---|---|---|---|---|---|---|
| GN (GGG) | complete | normal | normal | normal | normal | normal | normal | normal |
| LGG | complete | normal | normal | normal | normal | normal | normal | normal |
| GGL | very high | normal | normal | decreased | not analyzed | normal | normal | normal |
| GLG | moderate | decreased growth and fertility | partially decreased | decreased | not analyzed | ~50% single or fused cotyledons | variable growth directions | pen apical hooks |
| LGL | moderate to low | very small, infertile | strongly decreased | absent | not analyzed | ~50% single or fused cotyledons | variable growth directions | pen apical hooks |
| GLL | none | - | identical to gn | - | - | identical to gn | - | - |
| GNL1 (LLL) | none | - | identical to gn | - | - | identical to gn | - | - |
| LLG | not cloned | |||||||
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Biological sample (Arabidopsis thaliana) | Col-0 (Columbia) | Nottingham Arabidopsis Stock Centre (NASC) | N1092 | |
| Gene (Arabidopsis thaliana) | GNOM | The Arabidopsis Information Resource | AT1G13980 | |
| Gene (Arabidopsis thaliana) | GNOM-LIKE1 | The Arabidopsis Information Resource | AT5G39500 | |
| Genetic reagent (Arabidopsis thaliana) | gns | SALK T-DNA collection | SALK_103014 |
Additional files
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Supplementary file 1
Additional materials and methods.
(a) Lines generated as part of this study. (b) Primers used in this study. (c) Constructs generated in this study.
- https://cdn.elifesciences.org/articles/68993/elife-68993-supp1-v1.docx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/68993/elife-68993-transrepform1-v1.docx
