Volume 11, Issue 7, Pages 928-942 (July 2018) SEUSS and PIF4 Coordinately Regulate Light and Temperature Signaling Pathways to Control Plant Growth  Junling Huai, Xinyu Zhang, Jialong Li, Tingting Ma, Ping Zha, Yanjun Jing, Rongcheng Lin  Molecular Plant  Volume 11, Issue 7, Pages 928-942 (July 2018) DOI: 10.1016/j.molp.2018.04.005 Copyright © 2018 The Author Terms and Conditions

Figure 1 Phenotypic Characterization of the epp2 Mutant. (A) Phenotypes of the epp2 mutant grown under continuous darkness or various light conditions for 5 days. Scale bars, 2 mm. (B) Hypocotyl length of seedlings shown in (A). Data are means ± SD, n = 20. Asterisks indicate significant differences compared to the corresponding Col wild-type using Student’s t-test (∗∗P < 0.01). (C and D) Cell length (C) and cell number (D) of seedlings grown under red light for 5 days. Data are means ± SD, n = 30. Asterisks indicate significant differences compared to the corresponding Col wild-type using Student’s t-test (∗∗P < 0.01, ∗P < 0.05). (E) Cotyledon area of seedlings grown under red light for 5 days. Data are means ± SD, n = 20. Asterisks indicate significant differences compared to the corresponding Col wild-type using Student’s t-test (∗P < 0.05). (F) Cotyledon phenotype of a plant grown in darkness for 5 days. Scale bar, 200 μm. (G and H) Light-responsive expression of LHCA6 (G) and CHS (H). Col and epp2 seedlings were grown in darkness for 4 days and exposed to white light for various periods of time. Data are means ± SD, n = 3. Asterisks indicate significant differences compared to the corresponding Col wild-type using Student’s t-test (∗∗P < 0.01). (I) Phenotype of cop1 epp2 double mutant plants grown under long-day conditions for 6 weeks. Scale bar, 2 cm. Molecular Plant 2018 11, 928-942DOI: (10.1016/j.molp.2018.04.005) Copyright © 2018 The Author Terms and Conditions

Figure 2 Molecular Cloning and Confirmation of EPP2/SEU. (A) Genomic structure and cloning of SEU, and the location of the epp2 mutant locus. Black bars indicate exons. Fragments amplified by PCR are labeled (M0 to M6). (B) PCR genotyping of the epp2 mutant. (C) qRT–PCR of SEU in the epp2 and seu-4 mutants. Seedlings were grown in red light for 5 days. Data are means ± SD, n = 3. (D) Seedling phenotype of seu-4 grown under different light conditions for 5 days. Scale bars, 2 mm. (E) Hypocotyl length of seedlings shown in (D). Data are means ± SD, n = 20. Asterisks indicate significant differences based on Student's t-test (**P < 0.01). (F) Confirmation of SEU-GFP protein accumulation in the seu-6/SEUp:SEU-GFP transgenic plants by immunoblot analysis. The anti-SEU antibody recognized a specific endogenous protein of approximately 95 kDa in Col but not in seu-6. The upper band corresponds to the SEU-GFP fusion protein. Tubulin, detected by blotting with anti-tubulin antibody, served as a loading control. (G) Phenotypes of seu-6/SEUp:SEU-GFP seedlings grown under red light for 5 days. Scale bar, 2 mm. Molecular Plant 2018 11, 928-942DOI: (10.1016/j.molp.2018.04.005) Copyright © 2018 The Author Terms and Conditions

Figure 3 Transcriptional Activity Assay. (A) Diagram of full-length and truncated SEU proteins. LDB, LIM-domain binding. (B) Yeast one-hybrid assay. Sequences encoding full-length and truncated SEU proteins were fused with the LexA DNA-binding domain and co-expressed with the LexAop:LacZ reporter construct. (C) Diagram of various vectors used in (D). (D) Relative LUC activity in a transient expression assay using Arabidopsis protoplasts. Data are means ± SD, n = 6. Asterisks indicate significant differences based on Student's t-test (**P < 0.01). Molecular Plant 2018 11, 928-942DOI: (10.1016/j.molp.2018.04.005) Copyright © 2018 The Author Terms and Conditions

Figure 4 SEU Interacts with PIF4. (A) Yeast two-hybrid assay examining the interaction between SEU and PIF proteins. SEU was fused with the LexA DNA-binding domain. (B) Yeast two-hybrid assays examining the interaction between different domains/fragments of SEU and PIF4. (C) Semi-in vitro pull-down assay. The 35S:GFP and 35S:PIF4-GFP constructs were transiently expressed in N. benthamiana for 2 days. The proteins were extracted and incubated with MBP-FLAG-SEU fusion proteins. The mixtures were incubated and precipitated with anti-GFPmAb-agarose, and proteins were analyzed by immunoblotting with anti-GFP and anti-FLAG antibodies. Due to the relatively weak signal of PIF4-GFP compared with GFP alone, a longer exposure of the same blot is shown in the upper panel. (D) Co-immunoprecipitation assay. The 35S:GFP or 35S:PIF4-GFP constructs were co-transformed with 35S:SEU-nLUC and transiently expressed in N. benthamiana. Total proteins were extracted and precipitated with anti-GFPmAb-agarose, and the proteins were analyzed by immunoblotting with anti-GFP and anti-SEU antibodies. The star indicates a non-specific band. For (C) and (D), the experiments were repeated three times with similar results. IP, immunoprecipitation. Molecular Plant 2018 11, 928-942DOI: (10.1016/j.molp.2018.04.005) Copyright © 2018 The Author Terms and Conditions

Figure 5 Transcriptome Analyses of SEU- and PIF4-Regulated Genes. (A) IAA content. Seedlings were grown in red light for 5 days. Data are means ± SD, n = 4. DW, dry weight. Asterisks indicate significant differences based on Student's t-test (**P < 0.01). (B) GUS staining of Col and seu-6 plants expressing the DR5:GUS reporter. Scale bar, 1 mm. (C) Venn diagram of genes differentially regulated by SEU and PIF4 and targeted by PIF4. (D) Venn diagram of genes differentially induced or repressed by SEU and PIF4. (E) Hierarchical clustering of transcripts co-expressed in seu-6 and pif4. (F) Hierarchical clustering of auxin biosynthetic and responsive genes co-expressed in seu-6 and pif4. Molecular Plant 2018 11, 928-942DOI: (10.1016/j.molp.2018.04.005) Copyright © 2018 The Author Terms and Conditions

Figure 6 SEU and PIF4 Co-regulate Auxin-Responsive Gene Expression. (A) Phenotype of the seu pif4 double mutant grown in red light for 5 days. Scale bar, 2 mm. (B) Hypocotyl length of seedlings shown in (A). Data are means ± SD, n = 20. (C) qRT–PCR of representative genes in Col, seu, pif4, and seu pif4. (D) Diagram of the promoter structure of IAA19 and YUC8 and the fragments used for ChIP–PCR. The positions of the fragments are shown. (E) ChIP–PCR assay using anti-GFP antibody. (F) ChIP–PCR assay using anti-H3K4me3 antibody. (G) ChIP–PCR assay using anti-GFP antibody. For (C) and (E–G), seedlings were grown in red light for 5 days. Data are means ± SD, n = 3. Asterisks indicate significant differences based on Student's t-test (**P < 0.01, *P < 0.05). Molecular Plant 2018 11, 928-942DOI: (10.1016/j.molp.2018.04.005) Copyright © 2018 The Author Terms and Conditions

Figure 7 SEU Is Involved in High-Temperature-Mediated Responses. (A) Phenotypes of seedlings grown in white light at 22°C or 28°C for 5 days. Scale bars, 2 mm. (B) Hypocotyl length of seedlings shown in (A). Data are means ± SD, n = 20. Asterisks indicate significant differences from Col based on Student's t-test (**P < 0.01). (C) Phenotypes of seedlings grown at 22°C for 4 days, followed by 22°C or 28°C for an additional 3 days. Scale bars, 2 mm. (D) Hypocotyl length of seedlings shown in (C). Data are means ± SD, n = 20. Asterisks indicate significant differences from Col based on Student's t-test (**P < 0.01). (E) qRT–PCR of IAA19, IAA29, and YUC8 after the 28°C treatment. Data are means ± SD, n = 3. Asterisks indicate significant differences from Col based on Student's t-test (**P < 0.01). (F) A proposed working model for the coordinated regulation of light and temperature signaling pathways by SEU and PIF4. The SEU transcriptional co-activator physically interacts with PIF4 and promotes its binding to the promoter of auxin biosynthetic and signaling genes (such as IAA19 and YUC8), leading to subsequent gene activation and hypocotyl elongation. Red light triggers while warm temperature represses the active form (Pfr) of the phyB photoreceptor, which regulates SEU and PIF4. Arrow, activate; bar, repress. Dashed bar indicates unknown mechanism. Molecular Plant 2018 11, 928-942DOI: (10.1016/j.molp.2018.04.005) Copyright © 2018 The Author Terms and Conditions