bioRxiv preprint doi: https://doi.org/10.1101/2020.03.17.995506; this version posted March 19, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1 CYCA3;4 Is a Post-Prophase Target of the APC/CCCS52A2 E3-Ligase Controlling Formative Cell 2 Divisions in Arabidopsis [W] 3 AUTHORS: 4 Alex Willems,a,b Jefri Heyman,a,b Thomas Eekhout,a,b Ignacio Achon,a,b Jose Antonio Pedroza- 5 Garcia,a,b Tingting Zhu,a,b Lei Li,a,b Ilse Vercauteren,a,b Hilde Van den Daele,a,b Brigitte van de 6 Cotte,a,b Ive De Smet,a,b and Lieven De Veyldera,b,1 7 AFFILIATIONS: 8 aDepartment of Plant Biotechnology and Bioinformatics, Ghent University, Ghent, B-9052, 9 Belgium 10 bCenter for Plant Systems Biology, VIB, Ghent, B-9052, Belgium 11 ORCID IDs: 12 0000-0002-7211-8388 (A.W.); 0000-0003-3266-4189 (J.H.); 0000-0002-2878-1553 (T.E.); 0000- 13 0002-4553-1257 (I.A.); 0000-0001-8258-0157 (J.A.P.G); 0000-0002-0904-7636 (T.Z.); 0000- 14 0002-1820-9852 (L.L.); 0000-0001-9561-8468 (I.V.); 0000-0002-4271-6694 (H.V.d.D.); 0000- 15 0001-7236-9243 (B.v.d.C.); 0000-0003-4607-8893 (I.D.S.); 0000-0003-1150-4426 (L.D.V.) 16 RUNNING TITLE: CYCA3;4 control by APC/CCCS52A2 17 ONE-SENTENCE SUMMARY: 18 Timely post-prophase breakdown of the Arabidopsis cyclin CYCA3;4 by the Anaphase Promoting 19 Complex/Cyclosome is essential for meristem organization and development. 20 CORRESPONDING AUTHOR: 21 Lieven De Veylder 22 Department of Plant Systems Biology 23 VIB-Ghent University 24 Technologiepark 71, B-9052 Gent (Belgium). 25 Tel.: +32 9 3313800; Fax: +32 9 3313809; E-mail: [email protected] 1 bioRxiv preprint doi: https://doi.org/10.1101/2020.03.17.995506; this version posted March 19, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 26 FOOTNOTES: 27 1Address correspondence to [email protected]. 28 The author responsible for distribution of materials integral to the findings presented in this 29 article in accordance with the policy described in the Instructions for Authors 30 (www.plantcell.org) is: Lieven De Veylder ([email protected]). 31 [W]Online version contains Web-only data. 2 bioRxiv preprint doi: https://doi.org/10.1101/2020.03.17.995506; this version posted March 19, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 32 ABSTRACT 33 The Anaphase Promoting Complex/Cyclosome (APC/C) controls unidirectional progression 34 through the cell cycle by marking key cell cycle proteins for proteasomal turnover. Its 35 activity is temporally regulated by the docking of different activating subunits, known in 36 plants as CDC20 and CCS52. Despite the importance of the APC/C during cell 37 proliferation, the number of identified targets in the plant cell cycle is limited. Here, we 38 used the growth and meristem phenotypes of Arabidopsis CCS52A2-deficient plants in a 39 suppressor mutagenesis screen to identify APC/CCCS52A2 substrates or regulators, resulting 40 in the identification of a mutant cyclin CYCA3;4 allele. CYCA3;4 deficiency partially 41 rescues the early ccs52a2-1 phenotypes, whereas increased CYCA3;4 levels enhances them. 42 Furthermore, whereas CYCA3;4 proteins are promptly broken down after prophase in 43 wild-type plants, they remain present in later stages of mitosis in ccs52a2-1 mutant plants, 44 marking them as APC/CCCS52A2 substrates. Strikingly, CYCA3;4 overexpression results in 45 aberrant root meristem and stomatal divisions, mimicking phenotypes of plants with 46 reduced RBR1 activity. Correspondingly, RBR1 hyperphosphorylation was observed in 47 CYCA3;4-overproducing plants. Our data thus demonstrate that an inability to timely 48 destroy CYCA3;4 attributes to disorganized formative divisions, likely in part caused by 49 the inactivation of RBR1. 3 bioRxiv preprint doi: https://doi.org/10.1101/2020.03.17.995506; this version posted March 19, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 50 INTRODUCTION 51 Cell division represents an essential biological process, not only allowing the transfer of genetic 52 information from one generation to the next, but also permitting multicellular organisms to grow 53 and develop. The latter implies the control of cell proliferation in such a way that a building plan 54 can be carried out. When a new cell arises through cell proliferation from the stem cells, it 55 frequently undergoes a number of cell divisions that are eventually followed by the execution of a 56 cell cycle exit program. Both the proliferative activity of the stem cells and the timing of cell 57 cycle exit need to be strictly regulated, as perturbations in either impair growth (De Veylder et 58 al., 2007; Polyn et al., 2015; Shimotohno and Scheres, 2019). One of the key players that controls 59 both events is the Anaphase Promoting Complex/Cyclosome (APC/C) (see Heyman and De 60 Veylder (2012) for an extensive review on the plant APC/C). The APC/C is a conserved E3 61 ubiquitin ligase that provides unidirectional transit through the cell cycle by targeting key cell 62 cycle proteins for degradation by the 26S proteasome (Marrocco et al., 2010). The plant APC/C 63 consists of at least 11 core subunits, of which most are coded for by single-copy genes that are 64 essential for plant viability (Page and Hieter, 1999; Capron et al., 2003; Van Leene et al., 2010; 65 Heyman and De Veylder, 2012). Its structural backbone consists of the tetratricopeptide repeat 66 (TPR) interaction domain-containing proteins APC6, APC7, APC8 and APC3 (the latter being 67 present in two copies in Arabidopsis: APC3a/CDC27 and APC3b/HOBBIT) and is completed by 68 APC1, APC4 and APC5. Together, they correctly position the catalytic subunits APC2 and 69 APC11, which perform the ubiquitin transfer reaction, the co-activator APC10, and one activator 70 subunit belonging to one of two classes, respectively called CELL DIVISION CYCLE 20 71 (CDC20) or CDC20 HOMOLOG 1 (CDH1), the latter also known in plants as CELL CYCLE 72 SWITCH 52 (CCS52) (Tarayre et al., 2004; Kevei et al., 2011; Heyman and De Veylder, 2012). 73 The activator proteins recruit the APC/C ubiquitination targets through recognition of conserved 74 amino acid motifs such as the Destruction box (D-box) (Pfleger and Kirschner, 2000; De Veylder 75 et al., 2007; da Fonseca et al., 2011). 76 The plant CCS52 gene was first identified in Medicago, where it plays an important role 77 in the establishment of the polyploid tissues of the root nodules (Cebolla et al., 1999). The 78 described link between CCS52 expression, initiation of differentiation, and the onset of the 79 endocycle was later confirmed in other plant species. For example, in tomato, decreased CCS52A 80 levels were found to cause a reduction in endoreplication and fruit size, whereas in rice, mutation 4 bioRxiv preprint doi: https://doi.org/10.1101/2020.03.17.995506; this version posted March 19, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 81 of CCS52A resulted in dwarf growth and problems with kernel development due to a reduction of 82 endoreplication in the endosperm (Mathieu-Rivet et al., 2010; Su'udi et al., 2012; Xu et al., 83 2012). 84 In Arabidopsis, three isoforms of CCS52 are present, two A-types (CCS52A1 and 85 CCS52A2) and one plant specific B-type (CCS52B) (Tarayre et al., 2004; Kevei et al., 2011). 86 Prophase-confined expression of CCS52B indicates that it might play a role in the degradation of 87 M-phase proteins necessary for the progression of mitosis (Yang et al., 2017). Contrary, the 88 CCS52A1 and CCS52A2 genes are thought to repress cell division in a tissue-specific manner that 89 is determined by their expression pattern. Within the root, CCS52A1 is predominantly expressed 90 at the root elongation zone where it controls cell cycle exit, illustrated by an increased root 91 meristem size in ccs52a1 knockout plants (Vanstraelen et al., 2009). Additionally, CCS52A1 92 expression can be found in leaves and trichomes, where it controls the number of endocycles 93 (Lammens et al., 2008; Boudolf et al., 2009; Larson-Rabin et al., 2009; Baloban et al., 2013; 94 Heyman et al., 2017). Next to controlling endocycle progression in the leaf, CCS52A2 appears to 95 be important for maintaining the low proliferation status of the quiescent center (QC) and the 96 organizing center (OC) of respectively the root and the shoot, seeing that ccs52a2-1 mutant plants 97 show a severe disruption of meristem organization, leading to a short root, dwarf growth and a 98 strong reduction in the development of reproductive organs (Vanstraelen et al., 2009; Liu et al., 99 2012). 100 Currently, only a relatively limited set of proteins have been thoroughly characterized as 101 targets of the CCS52-activated APC/C. In Arabidopsis, protein stability of the A-type cyclin 102 CYCA2;3 was found to be reduced by APC/CCCS52A1 to control the onset of endoreduplication 103 (Boudolf et al., 2009). The ETHYLENE RESPONSE FACTOR 115 (ERF115) transcription 104 factor was initially identified as an interactor of CCS52A2 in a tandem affinity purification 105 experiment and was shown to be an important rate-limiting factor of QC cell division (Heyman et 106 al., 2013).