Shade-induced nuclear localization of PIF7 is regulated by phosphorylation and 14-3-3 proteins in Arabidopsis
- Fudan University, China
Figures
Figure 1 with 1 supplement
Shade induces the nuclear localization of PIF7.
(a) Subcellular localization of GFP-PIF7 at the top of the hypocotyls of two independent transgenic seedlings grown under white light at different time points after transfer to shade. Transgenic Arab…
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Figure 1—source data 1
Source files for the ratios of the nuclear and cytoplasmic signal intensities in Figure 1b.
- https://doi.org/10.7554/eLife.31636.004
Figure 1—figure supplement 1
Shade induces the nuclear localization of PIF7 in the hypocotyl and cotyledon.
(a) Subcellular localization of GFP-PIF7 in the cotyledon, the top, middle and bottom portions of the hypocotyls, and the root of transgenic seedlings grown under white light or transferred to shade …
Figure 2 with 1 supplement
PIF7 interacts with 14-3-3 proteins.
(a) PIF7 interacts with 14-3-3 λ and 14-3-3 κ in a yeast two-hybrid assay. Each yeast clone containing pGADT7 (AD) or pGADT7-PIF7 (AD-PIF7), together with pGBKT7 (BD), pGBKT7-14-3-3 λ (BD-14-3-3 λ) …
Figure 2—figure supplement 1
PIF7 can interact with 14-3-3 χ, γ, μ and ε proteins.
(a) Phylogenic tree of 14-3-3 proteins in Arabidopsis. The tree was generated using predicted full-length amino acid sequences and neighbor-joining in MEGA5. The numbers on the nodes indicate the …
Figure 3 with 3 supplements
Interactions between PIF7 derivatives and 14-3-3 proteins.
(a) Interactions between PIF7, PIF7(2A) or PIF7(2D) and 14-3-3 λ or 14-3-3 κ in a yeast two-hybrid assay. Each yeast clone containing the pGADT7 (AD), or AD-PIF7 (2A), or AD-PIF7 or AD-PIF7 (2D) …
Figure 3—figure supplement 1
Potential phosphorylation sites on PIF7.
(a) Two known 14-3-3 binding sequences (mode I and mode II) and putative 14-3-3 binding sequences in PIF7, PHOT1 and CDC25C are listed. The consensus phosphorylation sites are labeled in red. S139 …
Figure 3—figure supplement 2
Expression of 14-3-3 proteins and PIF7, and quantification of YFP signals in Figure 3b.
(a) Anti-GFP antibody from Covance (MMS-118P-200G) was used to detect the nYFP-tagged protein. Anti-14-3-3 antibody from Agrisera (AS12 2119) was used to detect 14-3-3 λ-cYFP. The tobacco leaves …
Figure 3—figure supplement 3
Interactions between PIF7, PIF7(2A) or PIF7(2D) and 14-3-3 λ or 14-3-3 κ in BiFC and in a pull-down assay.
(a) Interaction between PIF7(2D) or PIF7(5D) and 14-3-3 λ as detected by BiFC. The C-terminal half of YFP was fused to 14-3-3 λ and the N-terminal half of YFP was fused to PIF7, PIF7(2D) or …
Figure 4 with 1 supplement
Phosphorylation sites of PIF7 are important for the localization and function of this protein.
(a) Subcellular localization of GFP-PIF7, GFP-PIF7(2A), GFP-PIF7 (2D), GFP-PIF7△, GFP-PIF7(5A) and GFP-PIF7 (5D) in tobacco cells. White scale bar represents 25 μm. (b) Subcellular localization of …
Figure 4—figure supplement 1
Subcellular localization of GFP-PIF7, GFP-PIF7(2A), GFP-PIF7(2D), GFP-PIF7△, GFP-PIF7(5A) and GFP-PIF7 (5D) in tobacco cells.
(a) Images of GFP-PIF7, GFP-PIF7(2A), GFP-PIF7(2D), GFP-PIF7△, GFP-PIF7(5A) and GFP-PIF7(5D) in tobacco cells. White bar represents 25 μm. (b) Quantification of the ratios of the nuclear and …
Figure 5 with 2 supplements
14-3-3 proteins delay shade-induced nuclear translocation and dephosphorylation of PIF7.
(a) The effect of R18 on the shade-induced nuclear localization of GFP-PIF7. GFP-PIF7 #14 transgenic plants grown under white light were treated with R18 or R18(Lys) for 3 hr after 10 min of vacuum, …
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Figure 5—source data 1
Source files for the ratios of the nuclear and cytoplasmic signal intensities in Figure 5b and Figure 5e.
- https://doi.org/10.7554/eLife.31636.016
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Figure 5—source data 2
Source files for the ratios of phosphorylated PIF7(PIF7[p]) relative to total PIF7 proteins in Figure 5—figure supplement 1.
- https://doi.org/10.7554/eLife.31636.017
Figure 5—figure supplement 1
Quantification of phosphorylated PIF7(PIF7[p]) relative to total PIF7 proteins in Figure 5c and f.
(a) Quantification of R18 and R18(Lys) treatment in Figure 5c. (b) Quantification of PIF7-Flash/14-3-3λκ and PIF7-Flash/Col-0 background in Figure 5f. The intensities of phosphorylated PIF7 (top) …
Figure 5—figure supplement 2
R18(Lys) is a non-functional peptide control for R18.
(a) The effects of R18(Lys) and Mock on shade-induced nuclear localization of GFP-PIF7 do not differ. GFP-PIF7 #14 transgenic plants grown under white light were treated with R18 or R18(Lys) for 3 …
Figure 6 with 1 supplement
14-3-3 proteins negatively regulate shade-induced hypocotyl elongation and gene expression.
(a) Quantification of the hypocotyl length of Col-0 seedlings grown in plates containing 200 μg/ml R18 or R18(Lys) peptide under white light or shade conditions. Seedlings were grown under white …
Figure 6—figure supplement 1
Effects of shade on the mRNA level, protein level and localization of 14-3-3 λ.
(a) Relative gene expression of 14-3-3 λ in Col-0 grown under white light and shade. The seedlings were grown under white light for 5 days. Then, the seedlings were kept in white light or …
Figure 7
A molecular model illustrating the role of 14-3-3 proteins in PIF7-mediated SAS.
In response to shade light, de-phosphorylated PIF7 accumulates in the nucleus. 14-3-3 proteins retain phosphorylated PIF7 in the cytoplasm and hence regulate shade-induced hypocotyl elongation …
Author response image 1
(a) Quantification of ratios between nuclear and cytoplasmic intensities of YFP/GFP in tobacco leaf cells (n>10) which co-expressed 14-3-3λ-cYFP and nYFP-PIF7 or only expressed GFP-PIF7.
(b) Shade light doesn’t affect the localization of GFP-PIF7 expressed in tobacco leaf cells. (c) Co-expressed 14-3-3λ doesn’t affect the localization of PIF7 derivatives in tobacco leaf cells. …
Author response image 2
The effect of R18 on the localization of BES1-GFP.
BES1-GFP transgenic plants (a gift from Dr. Xuelu Wang’s lab) grown under white light were treated with R18 or R18(Lys) for 3 hr after 10 min of vacuum. ImageJ was used to quantify the fluorescence …
Author response image 3
The effect of 14-3-3λ and 14-3-3κ on PIF7-mediated activation of YUCCA8 promoter.
The effector constructs contain the CaMV 35S promoter fused to the transcription factor PIF7 or 14-3-3s. The reporter construct contains the 2.2 Kb upstream of the translation initiation site of YUCC…
Additional files
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Supplementary file 1
Primers used in this study.
- https://doi.org/10.7554/eLife.31636.021
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Transparent reporting form
- https://doi.org/10.7554/eLife.31636.022
