Arabidopsis 14-3-3 epsilon members contribute to polarity of PIN auxin carrier and auxin transport-related development
- University of Tübingen, Germany
Figures
Figure 1 with 2 supplements
Phylogenetic tree of the Arabidopsis 14-3-3 isoforms and expression of genomic 14-3-3:GFP fusions under the control of the endogenous promoter.
(A) Unrooted phylogenetic tree of the Arabidopsis 14-3-3 protein family. Maximum parsimony analyses were performed using PAUP 4.0b10 (Altivec) with the bootstrap-algorithm and 1000 replica. (B, C) …
-
Figure 1—source data 1
Expression patterns of 14-3-3 isoforms in various tissues of six plant species based on publicly accessible RNA-seq or microarray data.
Normalized data for the expression of 14-3-3 isoforms (absolute signal intensities) were obtained from the eFP Browser of the Bio-Analytic Resource for Plant Biology (BAR) (http://bar.utoronto.ca) (microarray data: A. thaliana, M. truncatula, P. trichocarpa, O. sativa, P. patens) or the Tomato Functional Genomics Database (http://ted.bti.cornell.edu/cgi-bin/TFGD/digital/home.cgi) (RNA-seq data: S. lycopersicum). Expression of ubiquitin is depicted for comparison. Individual Excel sheets have been created for the different plant species. Each Excel sheet lists detailed information of 14-3-3s, such as gene ID and phylogenetic subgroup as well as the origin of expression data including references.
- https://doi.org/10.7554/eLife.24336.003
Figure 1—figure supplement 1
Phylogenetic relationship of 14-3-3 family members from six plant species.
Multiple alignment of 14-3-3 isoforms from A. thaliana (At), S. lycopersicum (TFT), M. truncatula (Mt), P. trichocarpa (Pt), O. sativa (Os), and P. patens (Pp) was performed using CLC Main Workbench …
Figure 1—figure supplement 2
Phenotype of the homozygous 14-3-3 mu T-DNA allele (SALK_004455) under continuous light.
The primers indicated in the schematic representation of the T-DNA insertion (lower panel) were used to identify homozygous plants. The control PCR reactions contain no input genomic DNA.
Figure 2 with 3 supplements
Ethanol-inducible emo-RNAi causes growth retardation phenotypes.
(A) to (F) Seedlings (wildtype and three independent emo-RNAi lines) grown either for 6 days in the light (A, B) or for 4 days in the dark (C–F) on noninductive (A, C, E) or inductive (0.1% (v/v) …
Figure 2—figure supplement 1
Ethanol-inducible amiRNA-(em)o causes growth retardation phenotypes.
(A) Schematic representation of the RNAi construct generated to reduce the expression of the 14-3-3 isoforms epsilon, mu and omicron. (B) Seedlings (two at a time, from left to right: wildtype, emo1-…
Figure 2—figure supplement 2
Auxin-induced gene expression is not compromised in emo-RNAi roots.
Semiquantitative RT-PCR analysis of the transcript level of selected auxin-induced genes in wildtype and emo1-RNAi seedlings grown for 7 days on MS medium followed by transfer to ethanol-containing …
Figure 2—figure supplement 3
Ethanol-inducible emo-RNAi causes defects in the gravitropic growth response and auxin transport in both roots and aerial tissues.
(A) to (D) Gravitropic root growth response of emo1-RNAi (B) as compared to wildtype (A). Seedlings grown for 3 days in the absence of ethanol were transferred to inductive medium for 2 days, …
Figure 3 with 1 supplement
Disorganized lateral root primordia and failure in the establishment of auxin response gradients caused by emo-RNAi induction.
Seedlings grown for 4 days in the absence of ethanol were transferred to inductive medium for 24 hr followed by treatment with exogenous auxin.(A) to (F) Lateral root primordia of wildtype (A–C) and …
Figure 3—figure supplement 1
Changes in auxin distribution caused by emo-RNAi induction.
(A, B, E–G) DR5rev::GFP activity in root tips of wild type (A, E) and emo1-RNAi (B, F) after transfer to inductive medium for 2 days (A, B) and gravistimulation (6 hr, 90°) (E, F). Arrowheads …
Figure 4 with 1 supplement
Dexamethasone-dependent expression of a constitutively active version of AHA2 does not rescue the emo-RNAi phenotype.
(A) to (E) Wild-type and emo1-RNAi seedlings were grown for 4 days in the dark in the absence or presence of ethanol and Dex (10 μM). Semiquantitative RT-PCR analysis of the transcript level of the …
Figure 4—figure supplement 1
Yeast two-hybrid analysis and in planta interaction studies do not point to a direct interaction of 14-3-3 s with the PIN2 hydrophilic loop.
Upper panel: Yeast two-hybrid interaction analysis of the 14-3-3 isoforms epsilon or omega with the PIN2 hydrophilic loop region (top). As a positive control, the interaction of omega with the …
Figure 5
Ethanol-inducible emo-RNAi causes misexpression of PIN proteins in root tips.
(A) to (H) Expression of PIN1-GFP (A–D) and PIN2-GFP (G, H) in wildtype (A, B, G) and emo1-RNAi (C, D, H) seedlings grown for 4 days on inductive medium. The ratio of GFP intensity on the basal to …
Figure 6
Ethanol-inducible emo-RNAi alters membrane trafficking processes.
(A) to (F) PIN2-GFP in epidermal root cells of wild-type (A–C) and emo1-RNAi (D–F) seedlings grown for 4 days on inductive medium and treated with BFA for 10 min (A, D), 30 min (B, E) or 60 min (C, F…
Figure 7 with 1 supplement
Ethanol-inducible emo-RNAi causes defects in wortmannin-compartment formation and endocytic transport to the vacuole.
(A) to (G) PIN2-GFP (A, D) and mRFP-ARA7 (B, E) in epidermal root cells of induced (4d) wildtype (A–C) and emo1-RNAi (D–F) seedlings treated with cycloheximide (CHX, 50 μM) for 60 min followed by …
Figure 7—figure supplement 1
Ethanol-inducible emo RNAi delays endocytic trafficking from TGN/EE to the vacuole and alters endosome homeostasis.
(A) to (G) Root epidermal cells of induced (4d) wild-type (A–C) and emo1-RNAi (D–F) seedlings were stained with the endocytic tracer FM4-64 in the absence (A, B, D, E) or presence of 25 μM BFA (C, F)…
Tables
Table 1
Analysis of 14-3-3 epsilon-GFP immunoprecipitates via mass spectrometry (MS) based on two biological replicates. This table lists only proteins with a possible role in membrane trafficking. Proteins …
| AGI code | Gene name | Description |
|---|---|---|
| At1g08680 | AGD14 | ADP-ribosylation factor (ARF) GTPase-activating protein |
| At1g09630 | RAB-A2a | Member of the RAB-A subfamily of small Rab GTPases |
| At1g16920 | RAB-A1b/BEX5 | |
| At3g15060 | RAB-A1g | |
| At4g18800 | RAB-A1d | |
| At5g45750 | RAB-A1c | |
| At5g60860 | RAB-A1f | |
| At1g12360 | KEULE/SEC11 | SNARE-interacting protein Sec1 protein |
| At1g14670 | Endomembrane protein 70 protein family | |
| At2g01970 | Endomembrane protein 70 protein family | |
| At5g37310 | ||
| At2g20790 | AP5M | AP-5 complex subunit mu |
| At2g25430 | ENTH/ANTH/VHS superfamily protein | |
| At2g37550 | AGD7 | ARF GTPase-activating protein AGD7 |
| At2g43160 | EPSIN2 | ENTH/ANTH/VHS superfamily protein |
| At3g59290 | EPSIN3 | |
| At3g09900 | RAB-E1e | Member of the RAB-E subfamily of small Rab GTPases |
| At3g46060 | RAB-E1c/ARA3 | |
| At3g53610 | RAB-E1a | |
| At5g03520 | RAB-E1d | |
| At3g53710 | AGD6 | ARF GTPase-activating protein AGD6 |
| At4g12120 | SEC1B | Member of KEULE gene family |
| At4g32285 | ENTH/ANTH/VHS superfamily protein | |
| At4g35730 | IST1-LIKE 3 | Regulator of Vps4 activity in the MVB pathway protein |
| At5g52580 | RAB GTPase activator activity | |
| At5g54440 | ATTRS130 | TRAPII tethering factor, CLUB |
-
Table 1—source data 1
Complete list of 14-3-3 epsilon interactors based on two biological replicates.
Proteins listed in Table 1 are shown in bold face while well characterized 14-3-3 clients are highlighted in green.
- https://doi.org/10.7554/eLife.24336.019
