Photoactivated CRY1 and Phyb Interact Directly with AUX/IAA Proteins to Inhibit Auxin Signaling

Photoactivated CRY1 and phyB Interact Directly with AUX/IAA Proteins to Inhibit Auxin Signaling in Arabidopsis

Feng Xu1,3, Shengbo He2,3, Jingyi Zhang2, Zhilei Mao1, Wenxiu Wang1, Ting Li2, Jie Hua1, Shasha Du1, Pengbo Xu2, Ling Li2, Hongli Lian2, and Hong-Quan Yang1,*

1State Key laboratory of Genetic Engineering, Collaborative Innovation Center for Genetics and Development, School of Life Sciences, Fudan University, Shanghai 200438, China

2School of Agriculture and Biology/School of Life Sciences and Biotechnology, ShanghaiJiaotongUniversity, Shanghai 200240, China

3These authors contribute equally to this work

*Correspondence:

SUPPLEMENTAL INFORMATION

SUPPLEMENTAL FIGURE LEGENDS

Figure S1

Figure S1. Auxin Promotes Hypocotyl Elongation in Blue, Red, and Far-Red Light.

(A, C, E, G) Phenotypes of 5-day-old seedlings of different genotypes were grown under darkness (Dk), blue light (BL), red light (RL), and far-red light (FR), respectively. Scale bars, 2 mm.

(B, D, F, H) Statistical analyses of hypocotyl lengths as shown in (A). Data are mean ± SD (n=10 for Class II of tir1 afb123 in Dk, RL, and FR; n=20 for Class II of tir1 afb123 in BL; n=30 for others). *p < 0.05; **p < 0.001 (t test; compared to WT).

Figure S2

Figure S2. The Effects of TIR1 and Auxin Analog NAA on IAA12 Degradation in Tobacco Leaves.

(A) Schematic diagram depicting the vector used for Dual-LUC assay as shown in Figures 2A-2C. REN and LUC denote renilla and firefly luciferase, respectively. ter indicates transcriptional terminator.

(B) Schematic diagram depicting the constructs used for protein expression analyses in tobacco as shown in Figure 2F and Figure S4D.

(C) Western blot analysis of IAA12/iaa12 accumulation in tobacco. NAA denotes auxin analog 1-naphthalene acetic acid. Expression of IAA12 and iaa12 was determined by RT-PCR. ACT1 served as an internal control.

Figure S3

Figure S3. IAA7 and IAA17 Acts Genetically downstream of CRY1 to Regulate Hypocotyl Elongation under Blue Light.

(A) The schematic diagram depicting the dominant mutations of IAA7 (AXR2) and IAA17 (AXR3), respectively. The four conserved domains (I-IV) of IAA7 and IAA17 are also indicated.

(B) Genetic interaction analysis showing genetic epistasis of the gain-of-function mutants of IAA7 and IAA17 over CRY1 in the regulation of hypocotyl elongation under blue light. WT, cry1, axr2, cry1 axr2, axr3, and cry1 axr3 mutant seedlings were grown in blue light (30 μmol m-2 s-1) for 5 days, respectively. Scale bars, 2 mm.

(C) Statistical analyses of hypocotyl lengths as shown in (B). Data are mean ± SD (n=30).

Figure S4

Figure S4. CRY1 N Terminus Interacts with AUX/IAA Proteins and Inhibits the Degradation of AUX/IAA Proteins.

(A) GST pull-down assay showing that CCT1 fails to interact with AUX/IAA proteins. GST-tagged AUX/IAAs served as baits, MBP-CCT1 and MBP-CNT1 served as preys. GST-IAA7 pulled down MBP-CNT1, which served as a positive control.

(B) Co-IP assay showing that A462V and G283E mutations within CNT1 affect the interaction of CNT1 with IAA12. IAA12-Flag was co-expressed with YFP-NLS-GUS-CCT1 or YFP-tagged CNT1 or CNT1-A462V or CNT1-G283E in tobacco.

(C) BiFC assay showing the interactions of CNT1 with AUX/IAA proteins in tobacco cells. CFP served as an internal control. The middle two panels show higher magnification. EV denotes empty vector. White scale bars, 50 μm; yellow scale bars, 5 μm.

(D) Western blot analysis showing CNT1 inhibition of TIR1- and auxin-triggered degradation of AUX/IAA proteins in tobacco. Expression of the indicated AUX/IAAs was determined by RT-PCR. ACT1 served as an internal control.

Figure S5

Figure S5. Deletion of the Conserved Core Amino Acids within DII or Dominant Point Mutation within DII of AUX/IAA Proteins Does Not Affect the Interactions of AUX/IAAs with CRY1.

(A and B) Pull-down assays showing that GST-IAA7-P87S and GST-IAA7Δ83-91 hardly interacted with TIR1 in the presence of 10 μmol IAA (A), but were able to interact with photoactivated CRY1 (B).

(C and D) Pull-down assays showing that GST-IAA17-P88L and GST-IAA17Δ83-91 barely interacted with TIR1 in the presence of 10 μmol IAA (C), but were able to interact with photoactivated CRY1 (D)

GST-tagged AUX/IAAs served as baits. TIR1-Flag-containing protein extracts from TIR1-Flag-OX seedlings (A and C), and Myc-CRY1-containing protein extracts from Myc-CRY1-OX seedlings that were exposed to blue (50 μmol m-2 s-1) for 3 hr (B and D) served as preys. GST did not interact with either TIR1 or CRY1, which served as a negative control.

Figure S6

Figure S6. phyB Mediates Red Light Inhibition of Auxin Signaling.

(A) Analysis of seedlings responsiveness to auxin showing red 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 red light (40, 80 μmol m-2 s-1) for 7 days, and then hypocotyl lengths were measured. The curves shown on the top of the columns (A and B) depict the trends.Pic denotes picloram.

(B) Analysis of seedlings responsiveness to auxin showing CRY1 mediation of red light inhibition of auxin-induced hypocotyl elongation. WT, phyAB, hy1 and phyB-OX seedlings were grown on the same medium in (A) in red light (80 μmol m-2 s-1) for 7 days, and then hypocotyl lengths were measured.

(C and D) GUS activity analysis showing DR5::GUS expression was repressed by red light (C) and phyB (D). The relative GUS activity was normalized to that from mock-treated control arbitrarily set to 1. Data are mean ± SD (n=3).

Data in (A) and (B) are means ± SD (n > 20);***p < 0.001 (two-way ANOVA).

Figure S7

Figure S7. PhyB Mediates Red Light Inhibition of Auxin-Induced Degradation of AUX/IAA Proteins.

(A-D) Dual-LUC assays showing red light inhibition of auxin-induced degradation of AUX/IAA proteins in Arabidopsis protoplasts. Protoplasts isolated from WT were transfected with constructs expressing IAA7-LUC (A), IAA12-LUC (B), IAA17-LUC (C), and iaa12-LUC (D), respectively, and incubated in the dark or red light (40 μmol m-2 s-1) for 12-16 hr with NAA treatments. Relative LUC/REN was normalized to that from mock-treated control arbitrarily set to 1. Data are mean ± SD (n=3).

(E and F) Dual-LUC assays showing phyB inhibition of auxin-induced degradation of IAA12 in red light. Protoplasts isolated from WT and phyB plants were transfected with constructs expressing IAA12-LUC (E) and iaa12-LUC (F), respectively, and incubated in red light (40 μmol m-2 s-1) for 12-16 hr with NAA treatments. Relative LUC/REN was normalized to that from mock-treated control arbitrarily set to 1. Data are mean ± SD (n=3).

*p < 0.05; **p < 0.01; ***p < 0.001 (two-way ANOVA).

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