CYCA3;4 Is a Post-Prophase Target of the APC/C^CCS52A2E3-Ligase Controlling Formative Cell Divisions in Arabidopsis

Abstract

32The Anaphase Promoting Complex/Cyclosome (APC/C) controls unidirectional progression 33 through the cell cycle by marking key cell cycle proteins for proteasomal turnover. Its 34 activity is temporally regulated by the docking of different activating subunits, known in 35 plants as CDC20 and CCS52. Despite the importance of the APC/C during cell 36 proliferation, the number of identified targets in the plant cell cycle is limited. Here, we 37 used the growth and meristem phenotypes of Arabidopsis CCS52A2-deficient plants in a 38 suppressor mutagenesis screen to identify APC/C CCS52A2 substrates or regulators, resulting 39 in the identification of a mutant cyclin CYCA3;4 allele. CYCA3;4 deficiency partially 40 rescues the early ccs52a2-1 phenotypes, whereas increased CYCA3;4 levels enhances them. 41 Furthermore, whereas CYCA3;4 proteins are promptly broken down after prophase in 42 wild-type plants, they remain present in later stages of mitosis in ccs52a2-1 mutant plants, 43 marking them as APC/C CCS52A2 substrates. Strikingly, CYCA3;4 overexpression results in 44 aberrant root meristem and stomatal divisions, mimicking phenotypes of plants with 45 reduced RBR1 activity. Correspondingly, RBR1 hyperphosphorylation was observed in 46 CYCA3;4-overproducing plants. Our data thus demonstrate that an inability to timely 47 destroy CYCA3;4 attributes to disorganized formative divisions, likely in part caused by 48 the inactivation of RBR1.49 4 109 ENHANCER (TE) / TILLERING AND DWARF (TAD1) was shown to mediate the 110 ubiquitination and subsequent degradation of MONOCULM 1 (MOC1), called LATERAL 111 6SUPPRESSOR (LAS) in Arabidopsis, a GRAS-family transcription factor that promotes shoot 112 branching and tillering (Lin et al., 2012; Xu et al., 2012). 113 Here, we have utilized an ethyl methanesulfonate (EMS) suppressor screen to identify 114 novel APC/C CCS52A2 targets, based on the growth inhibitory phenotype of ccs52a2-1 knockout 115 plants. We show that one of the identified revertants encodes a mutant allele of the CYCA3;4 116 gene and demonstrate this cyclin to be a specific target of APC/C CCS52A2 to ensure correct stem 117 cell organization. 118 119 RESULTS 120 Identification of pkn2 as a ccs52a2-1 Suppressor Mutant 121 Compared to wild type (WT, Col-0) plants, ccs52a2-1 mutant seedlings display a short root 122 phenotype (Figures 1A and 1B; Supplemental Figures 1A and 1B) (Vanstraelen et al., 2009; 123 Heyman et al., 2013). This phenotype was used to screen for putative targets or regulators of the 124 APC/C CCS52A2 ubiquitin ligase complex through a mutagenesis revertant screen. Therefore, ethyl 125 methanesulfonate (EMS)-mutagenized ccs52a2-1 plants were screened in the M 2 generation for a 126 recovered root growth. Out of a total of 260 initially identified revertants, 33 were confirmed in 127 the next generation. Among these, one revertant mutation, named pikmin 2 (pkn2), yielded a root 128 length in between that of WT and ccs52a2-1 mutant plants (Figures 1A to 1C; Supplemental 129 Figures 1A to 1C). 1...

Roles of plant retinoblastoma protein: cell cycle and beyond

“…Those experimentally demonstrated to be phosphorylated are shown (closed red circles). Data are taken from the UNIPROT database (Hansen et al , ; Farkas et al , ; Rubin, ; Willems et al , ).…”

“…It is interesting that phosphorylated RBR1 can be detected in the nucleus of interphase cells in the form of granules (Abrahám et al , ), although the functional relevance of such foci remains to be determined. Proteomic studies have demonstrated RBR1 phosphorylation in vivo at residues T406, S652, and S911 (Reiland et al , ; Willems et al , ). The identification of other phosphorylated residues, as well as their functions, remains as a future challenge.…”

“…RBR1 inactivation by phosphorylation likely occurs by a sequential activity of CDKB1;1 and CDKA;1, as revealed by the phenotypes of the corresponding mutants (Cruz‐Ramirez et al , ; Weimer et al , ). CYCA3;4 is another CDK partner controlling formative divisions in connection to RBR1 phosphorylation, as demonstrated by the abnormal root meristem and stomatal lineage pattern in the presence of high levels of this cyclin (Willems et al , ). Once ACD has been successfully completed, daughter cells acquire endodermal or cortex fate depending on the presence or absence of SCR, respectively.…”