Homeostasis of branched-chain amino acids is critical for the activity of TOR signaling in Arabidopsis
- Michigan State University, United States
- Great Lakes Bioenergy Research Center, Michigan State University, United States
Figures
Figure 1 with 2 supplements
Identification of a mutant with defects in vacuole morphogenesis.
(a – d) Confocal images of cotyledon epidermal cells expressing tonoplast marker GFP-δTIP in 10 day old (a and b) and 20 day old (c and d) wild type and eva1. The top panels present single images of …
Figure 1—figure supplement 1
eva1 mutants show vacuolar phenotypes of TVSs and presumably unfused vacuoles.
(a and b), Confocal images of cotyledon epidermal cells expressing GFP-δTIP in 10 day old wild type and eva1 plants. Arrows point to TVSs and arrowheads indicate small vacuoles, which are rarely …
Figure 1—figure supplement 2
The causal mutation of eva1 mutant is mapped to IPMS1.
(a) Mapping the causal mutation using bulk sergeant analysis. Horizontal and vertical axes are genomic position and observed allele frequency, respectively. Blue dots represent the SNPs identified …
Figure 2
Amino acid profiling of IPMS1 loss-of-function mutants.
(a) Schematic of the BCAA biosynthetic pathway in the chloroplast. Red lines show known feedback inhibitions of enzymes by end products. (b and c) Fold changes of each free amino acid in eva1, ipms1-…
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Figure 2—source data 1
The BCAA biosynthesis pathway is up-regulated in young seedlings of eva1 and IPMS1 loss-of-function mutants.
Amino acids were extracted from aerial tissues of 10 days old seedlings and rosette leaves of 20 days old plants. Each value represents the mean ± SEM. The asterisks indicate significant difference compared to the GFP-δTIP as wild type (WT) (*p≤0.05, **p≤0.01, ***p≤0.001, unpaired t test). aFW, fresh weight; bFC, fold change compared to GFP-δTIP; cFAA, total 19 free amino acids without cysteine.
- https://cdn.elifesciences.org/articles/50747/elife-50747-fig2-data1-v2.xlsx
Figure 3 with 3 supplements
Mutants of IPMS1 show defects in cotyledon architecture and chloroplast ultrastructure.
(a) Light microscopic images of cotyledon cross sections. Cotyledon thickness is denoted by red lines. Scale bar, 100 μm. (b) Measurement of cotyledon thickness. n = 9 for WT, eva1 and ipms1-4; n = 6…
Figure 3—figure supplement 1
Mutations of IPMS1 affect plant growth at early stage.
(a – d) Wild type (WT) and five mutants of IPMS1 were measured for fresh weight and primary root length at 10 day old stage (a and b) and 20 day old stage (c and d). tfl111 and tfl102 are two …
Figure 3—figure supplement 2
Root tip staining and analyses.
(a) Propidium iodide staining of the root tips of 10 day old wild type (WT), eva1, ipms1-4 and ipms1-5 seedlings. Meristem is the region between the quiescent center and the transition zone (TZ; …
Figure 3—figure supplement 3
Accumulation of anthocyanins in IPMS1 loss-of-function mutants.
(a) Visible accumulation of anthocyanins in 10 day old seedlings, particularly in cotyledon petioles and emerging true leaves. Scale bar, 1 cm. (b) Extracts of total anthocyanins from aerial tissues …
Figure 4 with 4 supplements
Mutation of IPMS1 affects the ER morphology and the F-actin organization.
(a and b) Confocal images of the wild type and eva1 cotyledon epidermal cells expressing ER marker ERYK. (a) Low-magnification Z-stack projection images show the ER morphology in eva1 is altered, …
Figure 4—figure supplement 1
The ER strands and trans-vacuolar strands are partially overlapping structures.
Confocal images shown in Figure 4a of ER marker (ERYK) are combined with a second channel of tonoplast marker (GFP-δTIP), showing ER strands and TVSs are only partially overlapping structures in …
Figure 4—figure supplement 2
The number and distribution of Golgi are altered in eva1 mutant.
(a) Confocal images of the wild type and eva1 mutant cotyledon epidermal cells expressing a Golgi marker GFP-CASP. Single images (top panel) show Golgi in the cortical focal plane. Maximal Z-stack …
Figure 4—figure supplement 3
The eva1 mutation does not affect secretion of a bulk flow marker to the apoplast.
Confocal images of the wild type (a) and eva1 mutant (b) cotyledon epidermal cells expressing SEC-RFP and GFP-δTIP. Scale bars, 10 μm.
Figure 4—figure supplement 4
Compared to wild type, IPMS1 loss-of-function mutants are less sensitive to Lat B.
(a and b), 10 day old wild type (Col-0), ipms1-4 and ipms1-5 seedlings germinated and grew on ½ LS and 1% sucrose medium was transplanted to ½ LS and 1% sucrose medium containing DMSO, 50 nM Lat B …
Figure 5 with 1 supplement
Chemical interventions can fully or partially rescue the vacuolar mutant phenotypes of eva1.
(a – h) Confocal images of cotyledon epidermal cells expressing GFP-δTIP from 10 day old eva1 plants. Images were acquired after 2 hr treatment of DMSO (a and b), wortmannin (Wm; c and d), …
Figure 5—figure supplement 1
Chemical treatment with PI3K/TOR dual inhibitors wortmannin and LY294002.
(a – h) Confocal images of cotyledon epidermal cells expressing tonoplast marker GFP-δTIP from 10 day old wild type (WT) and eva1 plants. Images were acquired after 3 hr treatment of DMSO (a and b), …
Figure 6 with 3 supplements
Vacuolar mutant phenotypes of eva1 are correlated with up-regulated TOR activity.
(a – f) TOR inhibitor AZD-8088 treatment rescues vacuolar mutant phenotypes of eva1. Confocal images were acquired before (a and b), after 2 hr (c and d), and after 4 hr (e and f) 5 μM AZD-8055 …
Figure 6—figure supplement 1
Another TOR inhibitor Torin2 exerts more potent effects on the vacuolar phenotypes of eva1.
Confocal images of cotyledon epidermal cells expressing tonoplast marker (GFP-δTIP) from 10 day old wild type (WT) or eva1 mutant. The images were acquired before (a and b) and after (c and d) 2 hr …
Figure 6—figure supplement 2
The effect of TOR inhibitor AZD-8055 on ipms1 primary root elongation is dose-dependent.
(a and b) ipms1 primary root elongation is promoted by lower concentrations (0.1 and 0.2 μM) of AZD-8055 (a), but inhibited by higher concentrations (0.4, 0.6 and 1.0 μM) of AZD-8055 (b). …
Figure 6—figure supplement 3
Effects of PI3K/TOR dual inhibitor wortmannin and F-actin depolymerizer Lat B on ipms1 primary root elongation.
(a) Wortmannin confers minimal impacts on ipms1 primary root elongation. Photographs of 10 day old wild type (WT, Col-0), eva1, ipms1-4 and ipms1-5 seedlings germinated and grew on medium containing …
Figure 7 with 2 supplements
Feeding of exogenous BCAAs and over-accumulation of endogenous BCAAs induce actin bundling, which is dependent on functional TOR but not RAPTOR.
Organization of actin cytoskeleton presented by confocal images of cotyledon epidermal cells expressing F-actin marker YFP-ABD2. Higher fluorescence intensity of the actin marker suggests more …
Figure 7—figure supplement 1
Exogeneous feeding of BCAAs or over-accumulation of BCAAs due to mutations of the BCAA biosynthetic pathway alters morphology of the ER network to form enhanced ER strands.
Architecture of the ER network presented by confocal images of cotyledon epidermal cells expressing ER marker ERYK in 10 day old wild type (Col-0) and mutants with distinct BCAA profiles, which were …
Figure 7—figure supplement 2
Exogeneous feeding of BCAAs affects morphology of the lytic vacuole.
Confocal images of cotyledon epidermal cells expressing tonoplast marker GFP-δTIP in 10 day old wild type that were growing on Arabidopsis growth medium without (a) or with (b) 1 mM BCAA …
Figure 8
Working model of TOR-regulated subcellular processes.
Over-accumulation of BCAA Val, Leu and Ile stimulates TOR signaling. Except for the established downstream processes such as protein synthesis and cell proliferation, vacuole fusion and actin …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Gene (Arabidopsis thaliana) | AtIPMS1 | TAIR: AT1G18500 | ||
| Gene (Arabidopsis thaliana) | AtOMR1 | TAIR: AT3G10050 | ||
| Gene (Arabidopsis thaliana) | AtAHASS1 | TAIR: AT2G31810 | ||
| Gene (Arabidopsis thaliana) | AtAHASS2 | TAIR: AT5G16290 | ||
| Gene (Arabidopsis thaliana) | AtTOR | TAIR: AT1G50030 | ||
| Gene (Arabidopsis thaliana) | AtRaptor1B | TAIR: AT3G08850 | ||
| Genetic reagent (Arabidopsis thaliana) | eva1 | this paper | EMS line with a mutation of IPMS1 | |
| Genetic reagent (Arabidopsis thaliana) | ipms1-4 | Xing and Last, 2017 | SALK_101771 | |
| Genetic reagent (Arabidopsis thaliana) | ipms1-5 | Xing and Last, 2017 | WiscDsLoxHs221_05F | |
| Genetic reagent (Arabidopsis thaliana) | tfl111 (ipms1-1D) | Xing and Last, 2017 | TAIR: CS69734 | |
| Genetic reagent (Arabidopsis thaliana) | tfl102 (ipms1-1D) | Xing and Last, 2017 | TAIR: CS69733 | |
| Genetic reagent (Arabidopsis thaliana) | ahass1-1 | Xing and Last, 2017 | SALK_096207 | |
| Genetic reagent (Arabidopsis thaliana) | ahass2-7 | Xing and Last, 2017 | WiscDsLoxHs009_02G | |
| Genetic reagent (Arabidopsis thaliana) | ahass2-1D | Xing and Last, 2017 | TAIR: CS69724 | |
| Genetic reagent (Arabidopsis thaliana) | omr1-11D | Xing and Last, 2017 | TAIR: CS69720 | |
| Genetic reagent (Arabidopsis thaliana) | tor-es | Xiong and Sheen, 2012 | TAIR: CS69829 | |
| Genetic reagent (Arabidopsis thaliana) | raptor1b | Salem et al., 2017 | SALK_022096 | |
| Antibody | Anti-S6K (Rabbit polyclonal) | Agrisera | AS12 1855 | Western blotting (1:1000 dilution) |
| Antibody | Anti-S6K-phosphorylated (Rabbit polyclonal) | Abcam | ab207399 | Western blotting (1:1000 dilution) |
| Antibody | HRP conjugated anti-rabbit (Goat polyclonal) | Sigma-Aldrich | A0545 | Western blotting (1:10000 dilution) |
| Commercial assay or kit | Click-iT EdU Alexa Fluor 488 Imaging Kit | Invitrogen | C10337 |
Additional files
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Supplementary file 1
LC gradient used for amino acid separation.
- https://cdn.elifesciences.org/articles/50747/elife-50747-supp1-v2.xlsx
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Supplementary file 2
Primers used in this study.
- https://cdn.elifesciences.org/articles/50747/elife-50747-supp2-v2.xlsx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/50747/elife-50747-transrepform-v2.pdf
