Developmental patterning function of GNOM ARF-GEF mediated from the cell periphery

  1. Maciek Adamowski
  2. Ivana Matijević
  3. Jiří Friml  Is a corresponding author
  1. Institute of Science and Technology Austria, Austria
  2. Plant Breeding and Acclimatization Institute – National Research Institute, Poland
8 figures, 2 tables and 2 additional files

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-GEFOverall degree of GN functionAdult phenotypeSeedling growth rateRoot wavingRoot hair positioningApical patterningEtiolated seedling tropismApical hooks
GN (GGG)completenormalnormalnormalnormalnormalnormalnormal
LGGcompletenormalnormalnormalnormalnormalnormalnormal
GGLvery highnormalnormaldecreasednot analyzednormalnormalnormal
GLGmoderatedecreased growth and fertilitypartially decreaseddecreasednot analyzed~50% single or fused cotyledonsvariable growth directionspen apical hooks
LGLmoderate to lowvery small, infertilestrongly decreasedabsentnot analyzed~50% single or fused cotyledonsvariable growth directionspen apical hooks
GLLnone-identical to gn--identical to gn--
GNL1 (LLL)none-identical to gn--identical to gn--
LLGnot cloned
Key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
Biological sample (Arabidopsis thaliana) Col-0 (Columbia)Nottingham Arabidopsis Stock Centre (NASC) N1092
Gene (Arabidopsis thaliana) GNOMThe Arabidopsis Information Resource AT1G13980
Gene (Arabidopsis thaliana) GNOM-LIKE1The Arabidopsis Information Resource AT5G39500
Genetic reagent (Arabidopsis thaliana) gnsSALK T-DNA collection SALK_103014

Additional files

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
Transparent reporting form
https://cdn.elifesciences.org/articles/68993/elife-68993-transrepform1-v1.docx

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