1. Volume 11, Issue 4, Pages 523-541 (April 2018)
Photoactivated CRY1 and phyB Interact Directly with AUX/IAA Proteins to Inhibit Auxin Signaling in Arabidopsis 
Feng Xu, Shengbo He, Jingyi Zhang, Zhilei Mao, Wenxiu Wang, Ting Li, Jie Hua, Shasha Du, Pengbo Xu, Ling Li, Hongli Lian, Hong- Quan Yang 
Molecular Plant 
Volume 11, Issue 4, Pages (April 2018)
DOI: /j.molp
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2. Figure 1 CRY1 Mediates Blue Light Inhibition of Auxin Signaling.
(A and B) GUS activity analysis showing that DR5::GUS expression was repressed by blue light (A) and CRY1 (B). The relative GUS activity was normalized to that from mock-treated control arbitrarily set to 1. Data are mean ± SD (n = 3). In this and subsequent figures, Dk and BL denote darkness and blue light, respectively.
(C–F) qRT–PCR analyses showing CRY1 inhibition of auxin-induced auxin-responsive gene expression. Protoplasts were isolated from WT and cry1 seedlings and incubated in blue light (30 μmol m−2 s−1) with NAA treatment for 12 h, respectively, and used to perform qRT–PCR.
(G) Analysis of seedling responsiveness to auxin showing blue light inhibition of auxin-induced hypocotyl elongation. WT seedlings were grown on 1/2 MS plates supplemented with increasing amounts of picloram (0, 20, 40, 60, 80, and 100 nM) in the presence of both yucasin and NPA (see Methods) in the dark (0 μmol m−2 s−1) or increasing intensities of blue light (30–90 μmol m−2 s−1) for 7 days, then hypocotyl lengths were measured. Pic denotes picloram.
(H) Analysis of seedling responsiveness to auxin showing CRY1 mediation of blue light inhibition of auxin-induced hypocotyl elongation. WT, cry1, and CRY1-OX seedlings were grown on the same medium as in (G) in blue light (30 μmol m−2 s−1) for 7 days, then hypocotyl lengths were measured.
Data in (G) and (H) are means ± SD (n > 20); *p < 0.05; **p < 0.01; ***p < (two-way ANOVA). The curves shown above the columns depict the trends.
Molecular Plant  , DOI: ( /j.molp )
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3. Figure 2
CRY1 Mediates Blue Light Inhibition of Auxin-Induced Degradation of AUX/IAAs.
(A–C) Dual-LUC (LUC and REN, firefly luciferase, and renilla luciferase) assays showing blue light inhibition of auxin-induced degradation of AUX/IAAs. WT protoplasts were transfected with constructs expressing IAA12-LUC (A), IAA17-LUC (B), and iaa12-LUC (C), respectively, and incubated in the dark or blue light (30 μmol m−2 s−1) for 12–16 h with NAA treatments. Relative LUC/REN was normalized to that from mock-treated control arbitrarily set to 1. iaa12 denotes the gain-of-function mutant form of IAA12. Data are mean ± SD (n = 3). *p < 0.05; **p < 0.01; ***p < (two-way ANOVA).
(D) LUC assays showing blue light enhancement of IAA17 protein accumulation in Arabidopsis. Dark-adapted transgenic seedlings expressing IAA17-LUC in WT background treated with yucasin and NPA plus different concentrations of NAA (0, 0.05, 0.2, 2 μM) were kept in darkness for 2 h (Dk) or exposed to 30 μmol m−2 s−1 blue light (BL) for 2 h, and LUC activities were measured. **p < 0.01; ***p < (t-test, n = 3 biological replicates).
(E) LUC assays showing CRY1 enhancement of IAA17 protein accumulation in Arabidopsis. Dark-adapted transgenic seedlings expressing IAA17-LUC in WT and cry1 backgrounds with the same treatments as in (D) were exposed to 30 μmol m−2 s−1 blue light for 2 h, and LUC activities were measured. LUC activity/total protein was normalized to that from mock-treated control arbitrarily set to 1 (D and E). *p < 0.05 (t-test, n = 3 biological replicates).
(F) Western blot analysis showing CRY1 inhibition of TIR1- and auxin-triggered degradation of AUX/IAAs in tobacco. Nuclear localization signal (NLS)- and YFP-tagged GUS served as a control. GUS-NLS-YFP and CRY1-YFP were immunoblotted using anti-GFP antibody. TIR1-HA and YFP-HA were immunoblotted using anti-HA antibody. Expression of the indicated AUX/IAAs was determined by RT–PCR. ACT1 served as an internal control. See also Supplemental Figure 2B and 2C.
Molecular Plant  , DOI: ( /j.molp )
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4. Figure 3 CRY1 Physically Interacts with AUX/IAAs.
(A) Co-IP assay showing CRY1–AUX/IAAs interactions. The indicated FLAG-tagged AUX/IAAs or the control cLUC (C terminus of luciferase) were coexpressed with Myc-tagged CRY1 in tobacco, respectively.
(B) BiFC assay showing CRY1–AUX/IAAs interactions in tobacco cells. CFP served as internal control in each combination. The middle two rows, where only nuclei are shown, indicate higher magnification. EV denotes empty vector. White scale bars, 50 μm; yellow scale bars, 5 μm.
(C and D) Pull-down assay showing blue light-specific (C) and blue light intensity-dependent (D) interactions of CRY1 with AUX/IAAs. His-tagged AUX/IAAs served as bait. Myc-CRY1-containing protein extracts from Myc-CRY1-OX seedlings that were adapted to darkness or exposed to blue (50 μmol m−2 s−1), red (20 μmol m−2 s−1), or far-red light (10 μmol m−2 s−1) (C) for 10 h, or increasing fluence rates of blue light (0, 0.5, 5, 50 μmol m−2 s−1) for 3 h (D), served as prey. In this and other figures, RL and FR denote red and far-red light, respectively.
(E) Pull-down assay showing blue light-specific CRY2–AUX/IAAs interactions. His-AUX/IAAs served as bait. Myc-CRY2-containing protein extracts from Myc-CRY2-OX seedlings that were treated with MG132 for 4 h and then adapted to darkness or exposed to blue (50 μmol m−2 s−1), red (20 μmol m−2 s−1), or far-red light (10 μmol m−2 s−1) for 1 h served as prey.
(F) GST pull-down assay showing CNT1–AUX/IAAs interactions. CBB denotes Coomassie brilliant blue staining. See also Supplemental Figure 4A.
(G) Schematic depicting the domains of IAA12 and IAA17. N and C denote the N- and C-terminal domains, respectively.
(H and I) Co-IP assays showing that the N-terminal domains of IAA12 (H) and IAA17 (I) mediate the interactions of IAA12 and IAA17 with CRY1 in tobacco, respectively.
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5. Figure 4
Blue Light-Induced CRY1–IAA7/IAA17 Interactions Inhibit Auxin-Induced TIR1–IAA7/IAA17 Interactions.
(A and B) Co-IP assays showing auxin promotion of the interactions of TIR1 with IAA7 (A) and IAA17 (B) in a dosage-dependent manner in Arabidopsis protoplasts.
(C and D) Co-IP assays showing blue light-dependent interactions of CRY1 with IAA7 (C) and IAA17 (D) in Arabidopsis protoplasts. Dark-adapted TIR1-FLAG-OX protoplasts were transfected with constructs expressing Myc-CRY1 and IAA7-YFP, and Myc-CRY1 and IAA17-YFP, respectively, then kept in the dark or exposed to blue light (50 μmol m−2 s−1) for 1 h and subjected to Co-IP assays.
(E–H) Co-IP assays showing CRY1 inhibition of auxin-induced interactions of TIR1 with IAA7 (E) and IAA17 (F) in Arabidopsis protoplasts. Myc-GUS-CCT1 served as a control (G and H). Dark-adapted TIR1-FLAG-OX protoplasts were transfected with equal amounts of constructs expressing the indicated combinations of Myc- and YFP-tagged proteins and treated with 10 μM IAA, then kept in the dark or exposed to blue light (50 μmol m−2 s−1) for 1 h and subjected to Co-IP assays.
(I and J) Western blot analyses showing the input levels of Myc-CRY1 and Myc-GUS-CCT1 in (E) and (G), and (F) and (H), respectively. Tubulin protein served as an internal loading control.
Molecular Plant  , DOI: ( /j.molp )
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6. Figure 5 PhyB Interacts with and Inhibits the Degradation of AUX/IAAs.
(A) Co-IP assay showing interactions of phyB with AUX/IAAs. FLAG-tagged AUX/IAAs or the control cLUC were coexpressed with Myc-tagged phyB in tobacco, respectively.
(B) BiFC assay showing that phyB interacts with AUX/IAAs in tobacco cells. CFP served as an internal control in each combination. The middle two rows, where only single cells are shown, show higher magnification. EV denotes empty vector. White scale bars, 50 μm; yellow scale bars, 20 μm.
(C and D) Pull-down assay showing red light-induced and red light intensity-dependent interactions of phyB with AUX/IAAs. His-tagged AUX/IAAs served as bait. Myc-phyB-containing protein extracts from Myc-PHYB-OX seedlings that were adapted to darkness or exposed to red (50 μmol m−2 s−1) or far-red light (10 μmol m−2 s−1) (C) or increasing fluence rates of red light (D) served as prey.
(E) LUC assays showing red light enhancement of IAA17 protein accumulation in Arabidopsis. Dark-adapted transgenic seedlings expressing IAA17-LUC in WT background treated with yucasin and NPA plus different concentrations of NAA (0, 0.05, 0.2, 2 μM) were kept in darkness for 2 h (Dk) or exposed to 60 μmol m−2 s−1 red light for 2 h, and LUC activities were measured. *p < 0.05; **p < 0.01; ***p < (t-test, n = 3 biological replicates).
(F) LUC assays showing phyB enhancement of IAA17 protein accumulation in Arabidopsis. Dark-adapted transgenic seedlings expressing IAA17-LUC in WT and phyB backgrounds with the same treatments in (E) were exposed to 60 μmol m−2 s−1 red light for 2 h, and LUC activities were measured. LUC activity/total protein was normalized to that from mock-treated control arbitrarily set to 1 (E and F). **p < 0.01 (t-test, n = 3 biological replicates).
Molecular Plant  , DOI: ( /j.molp )
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7. Figure 6
Antagonistic Regulation of Global Auxin-Responsive Gene Expression by Photoreceptors and Auxin Receptors.
(A) Venn diagrams showing the overlapping genes regulated by blue light, CRYs, and TIR1/AFBs. BL, CRYs, and TIR1/AFBs denotes the genes regulated by blue light, CRYs, and TIR1/AFBs, respectively, which were obtained by analyses of differential expression from samples of WT grown in blue light and darkness, blue light-grown WT and cry1 cry2 mutant, and dark-grown WT and tir1 afb123 mutant, respectively.
(B) Hierarchical clustering analyses of the overlapping genes shown in (A). Scale bar denotes the log2 value of fold change.
(C–E) Venn diagrams showing the overlapping genes regulated by blue light, CRYs, iaa7 (C), iaa12 (D), and iaa17 (E). iaa7, iaa12, and iaa17 denote genes regulated by iaa7, iaa12, and iaa17, respectively, which were obtained with the gain-of-function mutants of IAA7/IAA12/IAA17, axr2/bdl/axr3, respectively.
(F–H) Hierarchical clustering analyses of the overlapping genes shown in (C) to (E). Scale bar denotes the log2 value of fold change.
(I) Gene Ontology (GO) analysis showing antagonistic regulation of the 923 overlapping genes in (A) by CRYs and TIR1/AFBs. The numbers on each column denote the percentage of genes in each GO category. Total denotes all Arabidopsis genes.
(J) Venn diagrams showing the overlapping genes regulated by phyA and TIR1/AFBs. phyA denotes phyA-regulated genes.
(K) Hierarchical clustering analysis of the overlapping genes shown in (J). Scale bar denotes the log2 value of fold change.
(L) Venn diagrams showing the overlapping genes regulated by red light, phyB, and TIR1/AFBs. RL and phyB denote the genes regulated by red light and phyB, respectively, which were obtained by analyses of differential expression from samples of WT grown in red light (60 μmol m−2 s−1) and darkness, and red light (60 μmol m−2 s1)-grown WT and phyB mutant, respectively.
(M) Hierarchical clustering analysis of the overlapping genes shown in (J). Scale bar denotes the log2 value of fold change.
Molecular Plant  , DOI: ( /j.molp )
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8. Figure 7
A Proposed Model Illustrating Light Inhibition of Auxin Signaling by CRY1 and phyB.
In darkness CRY1 is inactive, and phyB is at its Pr form and localized in the cytoplasm. They are not able to regulate auxin signaling, since they are unable to interact with AUX/IAAs (A and C); in the light CRY1 is activated, and phyB undergoes Pr to Pfr conformational change and enters the nucleus, and they may compete with TIR1 to interact with AUX/IAAs to inhibit their degradation, thereby repressing ARF activity and auxin signaling (B and D). N and C denote the N- and C-terminal domains of CRY1 and AUX/IAAs, respectively. Thicker and thinner red arrows denote higher and lower gene expression, respectively. +auxin denotes the availability of auxin in plants.
Molecular Plant  , DOI: ( /j.molp )
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