CmRCD1 represses flowering by directly interacting with CmBBX8 in summer chrysanthemum

Abstract

The CmBBX8-CmFTL1 regulatory module is a key determinant in the transition from vegetative growth to reproductive development in summer-flowering chrysanthemum. However, the detailed regulatory mechanism of CmBBX8-mediated flowering remains elusive. In this study, we revealed that RADICAL-INDUCED CELL DEATH 1 (CmRCD1) physically associated with CmBBX8 through bimolecular fluorescence complementation (BiFC), pulldown and Coimmunoprecipitation (CoIP) assays. Furthermore, the RCD1-SRO1-TAF4 (RST) domain of CmRCD1 and the B-box of CmBBX8 mediated their interaction. In addition, Luciferase (LUC) assays and electrophoretic mobility shift assay (EMSAs) showed that CmRCD1 repressed the transcriptional activity of CmBBX8 and interfered with its binding to the CmFTL1 promoter, thereby leading to delayed flowering in the summer chrysanthemum ‘Yuuka’. These results provide insight into the molecular framework of CmRCD1-CmBBX8-mediated flowering in chrysanthemum.

References

[1] Wang, C., Guthrie, C., Sarmast, M. K. & Dehesh, K. BBX19 interacts with CONSTANS to repress FLOWERING LOCUS T transcription defining a flowering time checkpoint in Arabidopsis. Plant Cell 26, 3589-3602 (2014).

[2] Ordoñez-Herrera, N. et al. The transcription factor COL12 is a substrate of the COP1/SPA E3 ligase and regulates flowering time and plant architecture. Plant Physiol. 176, 1327-1340 (2018).

[3] Tripathi, P., Carvallo, M., Hamilton, E. E., Preuss, S. & Kay, S. A. Arabidopsis B-BOX32 interacts with CONSTANS-LIKE3 to regulate flowering. Proc. Natl Acad. Sci. USA 114, 172-177 (2017).

[4] Serrano-Bueno, G. et al. CONSTANS-FKBP12 interaction contributes to modulation of photoperiodic flowering in Arabidopsis. Plant J. 101, 1287-1302 (2020).

[5] Graeff, M. et al. Micro protein-mediated recruitment of CONSTANS into a TOPLESS trimeric complex represses flowering in Arabidopsis. PLoS Genet. 12, e1005959- e1005959 (2016).

[6] Wang, L. et al. CmBBX8 accelerates flowering by targeting CmFTL1 directly in summer chrysanthemum. Plant Biotechnol. J. 18, 1562-1572 (2020).

[7] Yang, Y. et al. A zinc finger protein regulates flowering time and abiotic stress tolerance in Chrysanthemum by modulating gibberellin biosynthesis. Plant Cell 26, 2038-2054 (2014).

[8] Ping, Q. et al. The heterologous expression in Arabidopsis thaliana of a chrysanthemum gene encoding the BBX family transcription factor CmBBX13 delays flowering. Plant Physiol. Biochem. 144, 480-487 (2019).

[9] Belles-Boix, E., Babiychuk, E., Van Montagu, M., Inzé, D. & Kushnir, S. CEO1 a new protein from Arabidopsis thaliana protects yeast against oxidative damage. FEBS Lett. 482, 19-24 (2000).

[10] D’Amours, D., Desnoyers, S., D’Silva, I. & Poirier, G. G. Poly (ADP-ribosyl) ation reactions in the regulation of nuclear functions. Biochem. J. 342, 249-268 (1999).

[11] Overmyer, K. et al. Ozone-sensitive arabidopsis rcd1 mutant reveals opposite roles for ethylene and jasmonate signaling pathways in regulating superoxide-dependent cell death. Plant Cell 12, 1849-1862 (2000).

[12] Ahlfors, R., Brosché, M., Kollist, H. & Kangasjärvi, J. Nitric oxide modulates ozone-induced cell death, hormone biosynthesis and gene expression in Arabidopsis thaliana. Plant J. 58, 1-12 (2009).

[13] Jaspers, P. et al. Unequally redundant RCD1 and SRO1 mediate stress and developmental responses and interact with transcription factors. Plant J. 60, 268-279 (2009).

[14] Teotia, S. & Lamb, R. S. The paralogous genes RADICAL-INDUCED CELL DEATH1 and SIMILAR TO RCD ONE1 have partially redundant functions during Arabidopsis development. Plant Physiol. 151, 180-198 (2009).

[15] Aravind, L. The WWE domain: a common interaction module in protein ubiquitination and ADP ribosylation. Trends Biochem. Sci. 26, 273-275 (2001).

[16] Ruf, A., Mennissier De Murcia, J., de Murcia, G. & Schulz, G. E. Structure of the catalytic fragment of poly(AD-ribose) polymerase from chicken. Proc. Natl Acad. Sci. USA 93, 7481-7485 (1996).

[17] Oliver, A. et al. Crystal structure of the catalytic fragment of murine poly (ADP-ribose) polymerase-2. Nucleic Acids Res. 32, 456-464 (2004).

[18] Sousa, F. et al. PARPs and the DNA damage response. Carcinogenesis 33, 1433-1440 (2012).

[19] Marr, M. T. N. TAF4 takes flight. Proc. Natl Acad. Sci. USA 106, 1295-1296 (2009).

[20] Wang, M. et al. ERF109 of trifoliate orange (Poncirus trifoliata (L.) Raf.) contributes to cold tolerance by directly regulating expression of Prx1 involved in antioxidative process. Plant Biotechnol. J. 17, 1316-1332 (2019).

[21] Wang, L. et al. Overexpression of CmSOS1 confers waterlogging tolerance in Chrysanthemum. J. Integr. Plant Biol. 62, 1059-1064 (2020).

[22] An, H. CONSTANS acts in the phloem to regulate a systemic signal that induces photoperiodic flowering of Arabidopsis. Development 131, 3615-3626 (2014).

[23] Liu, S. et al. A wheat SIMILAR TO RCD-ONE gene enhances seedling growth and abiotic stress resistance by modulating redox homeostasis and maintaining genomic integrity. Plant Cell 26, 164-180 (2014).

[24] Shapiguzov, A. et al. Arabidopsis RCD1 coordinates chloroplast and mitochondrial functions through interaction with ANAC transcription factors. Elife 8, e43284 (2019).

[25] Vainonen, J. P. et al. RCD1-DREB2A interaction in leaf senescence and stress responses in Arabidopsis thaliana. Biochem. J. 442, 573-581 (2012).

[26] Hiltscher, H. et al. The radical induced cell death protein 1 (RCD1) supports transcriptional activation of genes for chloroplast antioxidant enzymes. Front. Plant Sci. 5, 475- 475 (2014).

[27] Böhlenius, H. et al. CO/FT regulatory module controls timing of flowering and seasonal growth cessation in trees. Science 312, 1040-1043 (2006).

[28] Fan, C. et al. Conserved CO-FT regulons contribute to the photoperiod flowering control in soybean. BMC Plant Biol. 14, 9- 9 (2014).

[29] Shim, J. S., Kubota, A. & Imaizumi, T. Circadian clock and photoperiodic flowering in Arabidopsis: CONSTANS is a hub for signal integration. Plant Physiol. 173, 5-15 (2017).

[30] Zhang, B., Wang, L., Zeng, L., Zhang, C. & Ma, H. Arabidopsis TOE proteins convey a photoperiodic signal to antagonize CONSTANS and regulate flowering time. Genes Dev. 29, 975-987 (2015).

[31] Tamura, K., Dudley, J., Nei, M. & Kumar, S. MEGA4: molecular evolutionary genetics analysis (MEGA) software version 4.0. Mol. Biol. Evol. 24, 1596-1599 (2007).

[32] Schütze, K., Harter, K. & Chaban, C. Bimolecular fluorescence complementation (BiFC) to study protein-protein interactions in living plant cells. Methods Mol. Biol. 479, 189-202 (2009).

[33] Lai, H. & Chiang, C. Bimolecular fluorescence complementation (BiFC) assay for direct visualization of protein-protein interaction in vivo. Bio-Protocol 3, e935 (2013).

[34] Kost, B., Schnorf, M., Potrykus, I. & Neuhaus, G. Non-destructive detection of firefly luciferase (LUC) activity in single plant cells using a cooled, slow-scan CCD camera and an optimized assay. Plant J. 8, 155-166 (1995).

[35] Francisco-Velilla, R., Fernandez-Chamorro, J., Ramajo, J. & Martinez-Salas, E. The RNA-binding protein Gemin5 binds directly to the ribosome and regulates global translation. FEBS Lett. 44, 8335-8351 (2016).

[36] Cheng, Z. et al. Pathogen-secreted proteases activate a novel plant immune pathway. Nature 521, 213-216 (2015).

[37] Yoo, S. D., Cho, Y. H. & Sheen, J. Arabidopsis mesophyll protoplasts: a versatile cell system for transient gene expression analysis. Nat. Protoc. 2, 1565-1572 (2007).

[38] Livak, K. J. & Schmittgen, T. D. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25, 402-408 (2001).

[39] Blanchard, M. G. & Runkle, E. S. Use of a cyclic high-pressure sodium lamp to inhibit flowering of chrysanthemum and velvet sage. Sci. Hortic. 122, 448-454 (2009).