RcSRR1 interferes with the RcCSN5B-mediated deneddylation of RcCRL4 to modulate RcCO proteolysis and prevent rose flowering under red light

Abstract

Light is essential for rose (Rosa spp.) growth and development. Different light qualities play differing roles in the rose floral transition, but the molecular mechanisms underlying their effects are not fully understood. Here, we observed that red light suppresses rose flowering and increases the expression of sensitivity to red light reduced 1 (RcSRR1) compared with white light. Virus-induced gene silencing (VIGS) of RcSRR1 led to early flowering under white light and especially under red light, suggesting that this gene is a flowering repressor with a predominant function under red light. We determined that RcSRR1 interacts with the COP9 signalosome subunit 5B (RcCSN5B), while RcCSN5B, RcCOP1, and RcCO physically interact with each other. Furthermore, the RcCSN5B-induced deneddylation of Cullin4-RING E3 ubiquitin ligase (RcCRL4) in rose was reduced by the addition of RcSRR1, suggesting that the interaction between RcSRR1 and RcCSN5B relieves the deneddylation of the RcCRL4-COP1/SPA complex to enhance RcCO proteolysis, which subsequently suppresses the transcriptional activation of RcFT and ultimately flowering. Far-red light-related sequence like 1 (RcFRSL3) was shown to specifically bind to the G-box motif of the RcSRR1 promoter to repress its transcription, removing its inhibition of RcFT expression and inducing flowering. Red light inhibited RcFRSL3 expression, thereby promoting the expression of RcSRR1 to inhibit flowering. Taken together, these results provide a previously uncharacterized mechanism by which the RcFRSL3-RcSRR1-RcCSN5B module targets RcCO stability to regulate flowering under different light conditions in rose plants.

Acknowledgements

This work was supported by the Natural Science Foundation of Jiangsu Province (BK20221008), NSFC (32372744, 32302594, 32172615, 32102418), the Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions, and the ‘JBGS’ Project of Seed Industry Revitalization in Jiangsu Province (JBGS (2021)020), the China Postdoctoral Science Foundation (2023 T160325) and the Jiangsu Funding Program for Excellent Postdoctoral Talent (2022ZB344). This work was also supported by the high-performance computing platform of Bioinformatics Center, Nanjing Agricultural University. We also thank Dr. Yuehua Ma (Central laboratory of College of Horticulture, Nanjing Agricultural University) for assistance in using Stereo fluorescence microscope (M165FC, Leica, Germany), Fluorescence microscope (DM6B, Leica, Germany), and Quantitative real-time PCR (QuantStudio 6Flex, ABI, USA).

Author Contributions

Changquan Wang and Weinan Wang designed the research plan. Weinan Wang, Jingjing Sun, and Chunguo Fan performed the experiments. Weinan Wang and Jingjing Sun performed the data analyses and wrote the manuscript. Guozhen Yuan, Rui Zhou, Jun Lu, and Jinyi Liu involved in manuscript editing. All authors read and approved the final manuscript.

Data availability

The data supporting the findings of this study are available within the paper and its online supplementary data.

Conflict of interest statement:

The authors declare that they have no conflict of interest.

Supplementary Data

Supplementary data is available at Horticulture Research online.

References

[1] Cheng P, Bi D, Chen J. et al. Genome-wide identification and analysis of TCP transcription factor genes in Rosa chinensis in response to abiotic stress and fungal diseases. Ornam Plant Res. 2023;3:1-11

[2] Feng D, Zhang H, Qiu X. et al. Comparative transcriptomic and metabonomic analysis revealed the relationships between biosynthesis of volatiles and flavonoid metabolites in Rosa rugosa. Ornam Plant Res. 2021;1:1-10

[3] Harmon DD, Chen H, Byrne D. et al. Cytogenetics, ploidy, and genome sizes of rose (Rosa spp.) cultivars and breeding lines. Ornam Plant Res. 2023;3:10

[4] Iwata H, Gaston A, Remay A. et al. The TFL1 homologue KSN is a regulator of continuous flowering in rose and strawberry. Plant J. 2012;69:116-25

[5] Randoux M, Jeauffre J, Thouroude T. et al. Gibberellins regulate the transcription of the continuous flowering regulator, RoKSN, a rose TFL1 homologue. JExp Bot. 2012;63:6543-54

[6] Bendahmane M, Dubois A, Raymond O. et al. Genetics and genomics of flower initiation and development in roses. JExp Bot. 2013;64:847-57

[7] Dugo ML, Satovic Z, Millán T. et al. Genetic mapping of QTLs controlling horticultural traits in diploid roses. Theor Appl Genet. 2005;111:511-20

[8] Li S, Zhou N, Zhou Q. et al. Inheritance of perpetual blooming in Rosa chinensis ‘old blush’. Hortic Plant J. 2015;1:108-12

[9] Quail PH. Phytochrome photosensory signalling networks. Nat Rev Mol Cell Biol. 2002;3:85-93

[10] Wang H, Deng XW. Arabidopsis FHY3 defines a key phytochrome a signaling component directly interacting with its homologous partner FAR1. EMBO J. 2002;21:1339-49

[11] Wang H, Deng XW. Dissecting the phytochrome A-dependent signaling network in higher plants. Trends Plant Sci. 2003;8:172-8

[12] Zieslin N, Mor Y. Light on roses. A review. Sci Hortic. 1990;43:1-14

[13] Maas FM, Bakx EJ. Effects of light on growth and flowering of Rosa hybrids ‘Mercedes’. J Am Soc Hortic Sci. 1995;120:571-6

[14] Girault T, Bergougnoux V, Combes D. et al. Light controls shoot meristem organogenic activity and leaf primordia growth during budburstinRosa sp. Plant Cell Environ. 2008;31:1534-44

[15] Wubs AM, Heuvelink E, Marcelis LFM. et al. Axillary budbreak in a cut rose crop as influenced by light intensity and red: far-red ratio at bud level. J Am Soc Hortic Sci. 2014;139:131-8

[16] Valverde F, Mouradov A, Soppe W. et al. Photoreceptor regula-tion of CONSTANS protein in photoperiodic flowering. Science. 2004;303:1003-6

[17] Nishidate K, Kanayama Y, Nishiyama M. et al. Far-red light sup-plemented with weak red light promotes flowering of Gypsophila paniculata. J Jpn Soc Hortic Sci. 2012;81:198-203

[18] Yamada A, Tanigawa T, Suyama T. et al. Red:far-red light ratio and far-red light integral promote or retard growth and flow-ering in Eustoma grandiflorum(Raf.) shinn. Sci Hortic. 2009;120: 101-6

[19] Kristiansen K. Light quality regulates flower initiation, differ-entiation and development of Campanula carpatica Jacq.‘Karl Forster’. Sci Hortic. 1988;35:275-83

[20] Yoshimura T, Nishiyama M, Kanahama K. Effects of red or far-red light and red/far-red ratio on the shoot growth and flowering of Matthiola incana. J Jpn Soc Hortic Sci. 2002;71:575-82

[21] Mockler T, Yang H, Yu XH. et al. Regulation of photoperiodic flowering by Arabidopsis photoreceptors. Proc Natl Acad Sci USA. 2003;100:2140-5

[22] Andrés F, Coupland G. The genetic basis of flowering responses to seasonal cues. Nat Rev Genet. 2012;13:627-39

[23] Lazaro A, Mouriz A, Piñeiro M. et al. Red light-mediated degra-dation of CONSTANS by the E3 ubiquitin ligase HOS1 regu-lates photoperiodic flowering in Arabidopsis. Plant Cell. 2015;27: 2437-54

[24] Endo M, Tanigawa Y, Murakami T. et al. Phytochrome-dependent late-flowering accelerates flowering through physical interac-tions with phytochrome B and constans. Proc Natl Acad Sci USA. 2013;110:18017-22

[25] Iñigo S, Alvarez MJ, Strasser B. et al. PFT1, the MED25 subunit of the plant mediator complex, promotes flowering through CON-STANS dependent and independent mechanisms in Arabidopsis. Plant J. 2012;69:601-12

[26] Galv ¯ao VC, Fiorucci AS, Trevisan M. et al. PIF transcription fac-tors link a neighbor threat cue to accelerated reproduction in Arabidopsis. Nat Commun. 2019;10:4005

[27] Staiger D, Allenbach L, Salathia N. et al. The Arabidopsis SRR1 gene mediates phyB signaling and is required for normal circa-dian clock function. Genes Dev. 2003;17:256-68

[28] Brachi B, Faure N, Horton M. et al. Linkage and association mapping of Arabidopsis thaliana flowering time in nature. PLoS Genet. 2010;6:e1000940

[29] Johansson M, Staiger D. SRR1 is essential to repress flowering in non-inductive conditions in Arabidopsis thaliana. JExp Bot. 2014;65:5811-22

[30] Xiao D, Zhao JJ, Hou XL. et al. The Brassica rapa FLC homologue FLC2 is a key regulator of flowering time, identified through transcriptional co-expression networks. JExp Bo.. 2013;64: 4503-16

[31] Schiessl S, Williams N, Specht P. et al. Different copies of sensi-tivity to red light reduced 1 show strong subfunctionalization in Brassica napus. BMC Plant Biol. 2019;19:372

[32] Lyapina S, Cope G, Shevchenko A. et al. Promotion of NEDD-CUL1 conjugate cleavage by COP9 signalosome. Science. 2001;292: 1382-5

[33] Li L, Deng XW. The COP9 signalosome: an alternative lid for the 26S proteasome. Trends Cell Biol. 2003;13:507-9

[34] Wei N, Deng XW. Characterization and purification of the mammalian COP9 complex, a conserved nuclear regulator ini-tially identified as a repressor of photomorphogenesis in higher plants. Photochem Photobiol. 1998;68:237-41

[35] Wei N, Deng XW. The COP9 signalosome. Annu Rev Cell Dev Biol. 2003;19:261-86

[36] Schwechheimer C, Serino G, Callis J. et al. Interactions of the COP9 signalosome with the E3 ubiquitin ligase SCFTIRI in medi-ating auxin response. Science. 2001;292:1379-82

[37] Wang X, Feng S, Nakayama N. et al. The COP9 signalosome interacts with SCFUFO and participates in Arabidopsis flower development. Plant Cell. 2003;15:1071-82

[38] Hotton SK, Callis J. Regulation of Cullin RING ligases. Annu Rev Plant Biol. 2008;59:467-89

[39] Schwechheimer C, Isono E. The COP9 signalosome and its role in plant development. Eur J Cell Biol. 2010;89:157-62

[40] Genschik P, Marrocco K, Bach L. et al. Selective protein degrada-tion: a rheostat to modulate cell-cycle phase transitions. JExp Bot. 2014;65:2603-15

[41] Li G, Siddiqui H, Teng Y. et al. Coordinated transcriptional regu-lation underlying the circadian clock in Arabidopsis. Nat Cell Biol. 2011;13:616-22

[42] Lin R, Ding L, Casola C. et al. Transposase-derived transcription factors regulate light signaling in Arabidopsis. Science. 2007;318: 1302-5

[43] Liu Y, Wei H, Ma M. et al. Arabidopsis FHY3 and FAR1 regulate the balance between growth and defense responses under shade conditions. Plant Cell. 2019;31:2089-106

[44] Xie Y, Zhou Q, Zhao Y. et al. FHY3 and FAR1 integrate light signals with the miR156-SPL module-mediated aging pathway to regulate Arabidopsis flowering. Mol Plant. 2020;13:483-98

[45] Wang H, Wang HY. Multifaceted roles of FHY3 and FAR1 in light signaling and beyond. Trends Plant Sci. 2015;20:453-61

[46] Ritter A, Iñigo S, Fernández-Calvo P. et al. The transcriptional repressor complex FRS7-FRS12 regulates flowering time and growth in Arabidopsis. Nat Commun. 2017;8:15235

[47] Ma L, Li G. FAR1-related sequence (FRS) and FRS-related factor (FRF) family proteins in Arabidopsis growth and development. Front Plant Sci. 2018;9:692

[48] Zhong MC, Jiang XD, Cui WH. et al. Expansion and expression diversity of FAR1/FRS-like genes provides insights into flowering time regulation in roses. Plant Divers. 2020;43:173-9

[49] Lu J, Sun J, Jiang A. et al. Alternate expression of constans-like 4 in short days and constans in long days facilitates day-neutral response in Rosa chinensis. JExp Bot. 2020;71:4057-68

[50] SunJ LuJ, Bai M. et al. Phytochrome-interacting factors interact with transcription factor constans to suppress flowering in rose. Plant Physiol. 2021;186:1186-201

[51] Huq E, Tepperman JM, Quail PH. GIGANTEA is a nuclear protein involved in phytochrome signaling in Arabidopsis. Proc Natl Acad Sci USA. 2000;97:9789-94

[52] Gyula P, Schäfer E, Nagy F. Light perception and signalling in higher plants. Curr Opin Plant Biol. 2003;6:446-52

[53] Pham VN, Kathare PK, Huq E. Phytochromes and phytochrome interacting factors. Plant Physiol. 2018;176:1025-38

[54] Ronald J, Su C, Wang L. et al. Cellular localization of Arabidop-sis early flowering3 is responsive to light quality. Plant Physiol. 2022;190:1024-36

[55] Suárez-López P, Wheatley K, Robson F. et al. Constans mediates between the circadian clock and the control of flowering in Arabidopsis. Nature. 2001;410:1116-20

[56] Jang S, Marchal V, Panigrahi KC. et al. Arabidopsis COP1 shapes the temporal pattern of CO accumulation conferring a photope-riodic flowering response. EMBO J. 2008;27:1277-88

[57] Chen H, Shen Y, Tang X. et al. Arabidopsis CULLIN4 forms an E3 ubiquitin ligase with RBX1 and the CDD complex in mediating light control of development. Plant Cell. 2006;18:1991-2004

[58] Chen H, Huang X, Gusmaroli G. et al. Arabidopsis CULLIN4-damaged DNA binding protein 1 interacts with constitutively photomorphogenic1-suppressor of PHYA complexes to regulate photomorphogenesis and flowering time. Plant Cell. 2010;22: 108-23

[59] Lin H, Yan Y, Luo Y. et al. IP6-assisted CSN-COP1 competition reg-ulates a CRL4-ETV5 proteolytic checkpoint to safeguard glucose-induced insulin secretion against hyperinsulinemia. Nat Com-mun. 2021;12:2461

[60] Srikanth A, Schmid M. Regulation of flowering time: all roads lead to Rome. Cell Mol Life Sci. 2011;68:2013-37

[61] Fornara F, Montaigu AD, Coupland G. SnapShot: control of flow-ering in Arabidopsis. Cell. 2010;141:550-550.e2

[62] Jeong S, Clark SE. Photoperiod regulates flower meristem devel-opment in Arabidopsis thaliana. Genetics. 2005;169:907-15

[63] Hayama R, Yokoi S, Tamaki S. et al. Adaptation of photoperiodic control pathways produces short-day flowering in rice. Nature. 2003;422:719-22

[64] Chamovitz DA, Wei N, Osterlund MT. et al. The COP9 complex, a novel multisubunit nuclear regulator involved in light control of a plant developmental switch. Cell. 1996;86:115-21

[65] Saijo Y, Sullivan JA, Wang H. et al. The COP1-SPA1 interaction defines a critical step in phytochrome A-mediated regulation of HY5 activity. Genes Dev. 2003;17:2642-7

[66] Serino G, Deng XW. The COP 9 signalosome: regulating plant development through the control of proteolysis. Annu Rev Plant Biol. 2003;54:165-82

[67] Cañibano E, Bourbousse C, García-León M. et al. DET1-mediated COP1 regulation avoids HY5 activity over second-site gene targets to tune plant photomorphogenesis. Mol Plant. 2021;14: 963-82

[68] Yasui Y, Mukougawa K, Uemoto M. et al. The phytochrome-interacting vascular plant one-zinc finger1 and VOZ2 redun-dantly regulate flowering in Arabidopsis. Plant Cell. 2012;24: 3248-63

[69] Huang X, Ouyang X, Yang P. et al. Arabidopsis FHY3 and HY5 positively mediate induction of COP1 transcription in response to photomorphogenic UV-B light. Plant Cell. 2012;24: 4590-606

[70] Liu S, Yang L, Li J. et al. FHY 3 interacts with phytochrome B and regulates seed dormancy and germination. Plant Physiol. 2021;187:289-302

[71] Li D, Fu X, Guo L. et al. Far-red elongated hypocotyl3 activates sepallata2 but inhibits CLAVATA3 to regulate meristem deter-minacy and maintenance in Arabidopsis. Proc Natl Acad Sci USA. 2016;113:9375-80

[72] Tian J, Pei H, Zhang S. et al. TRV-GFP: a modified tobacco rattle virus vector for efficient and visualizable analysis of gene func-tion. JExp Bot. 2014;65:311-22

[73] Lu J, Bai M, Ren H. et al. An efficient transient expression system for gene function analysis in rose. Plant Methods. 2017;13:116

[74] Bai M, Liu J, Fan C. et al. KSN heterozygosity is associated with continuous flowering of Rosa rugosa purple branch. Hortic Res. 2021;8:26