Selective protein degradation: a rheostat to modulate cell-cycle phase transitions

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1 Journal of Experimental Botany, Vol. 65, No. 10, pp , 2014 doi: /jxb/ert426 Advance Access publication 18 December, 2013 Review paper Selective protein degradation: a rheostat to modulate cell-cycle phase transitions Pascal Genschik 1,2, *, Katia Marrocco 2, Lien Bach 2, Sandra Noir 1 and Marie-Claire Criqui 1 1 Institut de Biologie Moléculaire des Plantes, Centre National de la Recherche Scientifique, Unité Propre de Recherche 2357, Conventionné avec l Université de Strasbourg, Strasbourg, France 2 Laboratoire de Biochimie et Physiologie Moléculaire des Plantes, Institut de Biologie Intégrative des Plantes Claude Grignon, UMR CNRS/INRA/SupAgro/UM2, Place Viala, Montpellier Cedex, France * To whom correspondence should be addressed. pascal.genschik@ibmp-cnrs.unistra.fr Received 10 September 2013; Revised 12 November 2013; Accepted 17 November 2013 Abstract Plant growth control has become a major focus due to economic reasons and results from a balance of cell proliferation in meristems and cell elongation that occurs during differentiation. Research on plant cell proliferation over the last two decades has revealed that the basic cell-cycle machinery is conserved between human and plants, although specificities exist. While many regulatory circuits control each step of the cell cycle, the ubiquitin proteasome system (UPS) appears in fungi and metazoans as a major player. In particular, the UPS promotes irreversible proteolysis of a set of regulatory proteins absolutely required for cell-cycle phase transitions. Not unexpectedly, work over the last decade has brought the UPS to the forefront of plant cell-cycle research. In this review, we will summarize our knowledge of the function of the UPS in the mitotic cycle and in endoreduplication, and also in meiosis in higher plants. Keywords: APC/C, cell cycle, cullin, DNA replication, endoreduplication, meiosis, mitosis, SCF, ubiquitin. Introduction The typical eukaryotic cell cycle is divided into four phases, the M phase (mitosis) in which sister chromatids are separated and distributed to the newly forming daughter cells, the S phase in which the nuclear DNA becomes replicated, and two gap phases, G1 and G2, that separate the M and S phases. In particular, transition from G1 to S phase as well as progression and exit from mitosis are key steps that need multiple levels of control, one of which is assumed by the ubiquitin proteasome system. In this pathway, ubiquitin ligases (E3s) facilitate the transfer of ubiquitin moieties to a substrate protein, the preparative step for degradation via the 26S proteasome (Ciechanover et al., 2000). Similar to metazoans, based on specific commonly shared structural motifs, plant genomes also encode hundreds of different E3s (Vierstra, 2009). Two families of E3s, the SCF [Skp1, Cdc53 (cullin), and F-box] and the anaphase promoting complex/cyclosome (APC/C), dominate DNA duplication and cell division in all eukaryotes (Pesin and Orr-Weaver, 2008; Marrocco et al., 2010; Heyman and De Veylder, 2012; Mocciaro and Rape, 2012). SCF belongs to the Cullin-RING family of ubiquitin E3 ligases (CRLs), the most prevalent class of E3s (Petroski and Deshaies, 2005). SCF is a multimeric E3 in which the CULLIN1 (CUL1) protein serves as a molecular scaffold. This scaffold brings together a catalytic module composed of a RING-finger domain protein (RBX1), a ubiquitin-conjugating enzyme (E2), and a specific substrate-recognition module composed of the adaptor SKP1 (so-called ASK1/2 in Arabidopsis), and one protein of the F-box family that physically interacts with the target substrate(s) (Fig. 1B) (Hua and Viestra, 2011). The APC/C is composed of a larger Abbreviations: CDK, cyclin-dependent kinase; CKI, cyclin-dependent kinase inhibitor; MCC, mitotic checkpoint complex; QC, quiescent centre; SAC, spindle assembly checkpoint; UPS, ubiquitin proteasome system. The Author Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please journals.permissions@oup.com

2 2604 Genschik et al. Fig. 1. Different classes of ubiquitin E3 ligases involved in cell-cycle regulation. (A) Monomeric RING ubiquitin E3 ligases can interact directly with their substrates and the E2 ubiquitin-conjugating enzyme. (B D) The SCF/CRL1, CRL3, and CRL4 complexes are composed of the scaffold proteins CUL1, CUL3, and CUL4, respectively, and the RING-finger protein RBX1. These ubiquitin E3 ligases have a similar modular structure where cullins play the role of scaffold proteins bringing together the catalytic module (RBX1 and the E2 ubiquitin-conjugating enzyme) and the module in charge of substrate specificity, including F-box proteins, BTB domain proteins, and DCAFs as substrate recruiters of SCF/CRL1, CRL3, and CRL4, respectively. (E) APC/C is the largest multimeric ubiquitin E3 ligase, requiring WD40 activator proteins (CDC20/FZ and CDH1/FZR/CCS52) and most likely also the DOC1/APC10 subunit to recruit its substrates. The RING-H2 finger subunit APC11 (red) interacts with an E2 ubiquitin-conjugating enzyme to stimulate the ubiquitylation reaction. (This figure is available in colour at JXB online.) number of subunits (Primorac and Musacchio, 2013), two of which (APC2 and APC11) are related to SCF and constitute the catalytic core of the complex (Fig. 1E). APC2 is a distant member of the CULLIN protein family, whereas APC11 is similar to the RING-H2 RBX1 protein. To be active, the APC/C requires crucial co-activators, called CELL DIVISION CYCLE (CDC20) and CDC20 HOMOLOG 1 (CDH1) in mammals, FIZZY and FIZZY-RELATED in Drosophila, and CDC20 and CELL CYCLE SWITCH52 (CCS52) in plants. These co-activators are WD40 repeat proteins that assume, with the APC10/DOC1 core subunit, most of the APC/C s selectivity. Indeed, throughout the cell cycle they recruit various substrates through short destruction motifs, predominantly the D-box and the KEN-box. For instance, in all eukaryotes, the D-box found in mitotic cyclins and other regulatory proteins is a small degenerated but conserved motif of 9 aa (RxxLxxIxN). Deletion or point mutations in this motif inhibit proteolysis (Brandeis and Hunt, 1996; Genschik et al., 1998; Yamano et al., 1998). Beside SCF and the APC/C, other classes of E3s have also been linked to cell-cycle regulation, such as monomeric RINGs and two other Cullin-RING ubiquitin ligases, CRL3 (formerly BTB CRLs) and CRL4 (formerly DCAF/DWD CRLs) (Fig. 1A, C and D) (Marrocco et al., 2010; Genschik et al., 2013). So far, the molecular mechanisms indicating a role of E3 ubiquitin ligases in the regulation of the cell cycle are far better described in yeast and animals than in plants. Thus, in the following sections, we will briefly introduce these proteolytic processes in more advanced systems and put them into perspective with plant research in this field. Progression from G1 to S phase When cells replicate their DNA, they are committed to divide, and therefore the transition from G1 to S phase is considered a key regulatory step in cell-cycle regulation (Nurse, 2000). Moreover, various mechanisms incorporating endogenous information, such as nutrient status and hormonal signals, with exogenous environmental conditions impact on the G1-to-S-phase transition. In both fungi and metazoans, this step requires the degradation of cyclin-dependent kinase inhibitors (CKIs) to release cyclin-dependent kinase (CDK) activity, which in turn allows the phosphorylation of regulatory proteins required to enter S phase. In budding yeast, one CKI, SIC1, is eliminated after its phosphorylation by the G1 cyclin CDK activity and ubiquitylation via SCF CDC4 (CDC4 being a WD40-type F-box protein) (Schwob

3 Selective protein degradation to modulate the cell cycle 2605 et al., 1994; Feldman et al., 1997). A similar mechanism exists in mammals, where the CKI protein p27 KIP1 becomes unstable when cells approach S phase and its degradation requires phosphorylation by the cyclin E CDK2 complex in order to be recognized by SCF SKP2 (SKP2 being a LRRcontaining F-box protein; Fig. 2A) (reviewed by Starostina and Kipreos, 2012). Furthermore, SCF SKP2 also targets other cell-cycle regulatory proteins such as p21 CIP1, another CKI, the transcription factor E2F1, and the chromatin licensing and DNA replication factor 1 (CDT1) among many others (Frescas and Pagano, 2008). Besides SCF SKP2, other E3 ligases are also required to fine-tune mammalian CKI protein levels during the cell cycle and development (Starostina and Kipreos, 2012). For instance, the RING-finger protein Fig. 2. Progression through G1 to S phase. The transition from G1 towards the S phase requires a decreased level of CKI proteins (green curve) in order to release CDK activity (red curve), resulting in interconnected regulation. (A) In mammals, two ubiquitin E3 ligases, at least, trigger the degradation of the p27 CKI protein: the RING-finger protein KPC targets non-phosphorylated p27 in the cytoplasm, whereas in the nucleus SCF SKP2 targets not only p27 once phosphorylated by the CyclinE/CDK2 complex but also another CKI, p21. The CRL4 CDT2 E3 complex also targets p21 when it interacts with proliferating cell nuclear antigen (PCNA) in order to prevent DNA overreplication. Additionally, CRL4 CDT2 is involved in the destruction of CDT1 to restrict its DNA replication activity. Fine-tuning of the S-phase progression further involves the SCF SKP2 complex in the regulation of many other cell-cycle regulators including CDT1 and the E2F1 transcription factor, which activates S-phase-promoting genes. (B) In plants, different CKI proteins, members of the KRP family, are also regulated by distinct ubiquitin E3 ligases and, as in mammals, their degradation might be conditioned by their phosphorylated status. In sporophytic tissues, the degradation of KRP1 is regulated by two distinct ubiquitin E3 ligases, the RING protein RKP and SCF SKP2B, while SCF SKP2A has been shown to mediate destruction of the E2Fc transcription factor, which negatively affects cell-cycle progression. Recent studies of Arabidopsis gametogenesis have demonstrated the implications of ubiquitin E3 ligases [the RING-H2 group F 1a (RHF1a) and RHF2a, and SCF FBL17 ] in post-meiotic cell-cycle regulation. Direct interaction and genetic evidence support a role of these ubiquitin E3 ligases in the proteasome-mediated degradation of KRP6 and KRP7. (This figure is available in colour at JXB online.)

4 2606 Genschik et al. KPC1 (Kip1 ubiquitylation-promoting complex1) promotes the degradation of p27 KIP1 in the cytoplasm in a phosphorylation-independent manner during G1 phase (Nakayama and Nakayama, 2004; Fig. 2A), while the CRL4 CDT2 (CUL4- DDB1-CDT2) ubiquitin E3 ligase is in charge of p21 CIP1 turnover during S phase, and also after DNA damage (Abbas et al., 2008; Kim et al., 2008a). Notably, a physical interaction of p21 with proliferating cell nuclear antigen, mediated by the PIP box degron present in p21, is required for its efficient CRL4 CDT2 -dependent destruction. Another substrate of mammalian CRL4 CDT2 is the chromatin licensing factor CDT1, which also contains a PIP box and thus interacts with proliferating cell nuclear antigen, ensuring that its ubiquitylation occurs only on the active chromatin-bound pool of the substrate (Havens and Walter, 2011). Together, these findings illustrate that substrates sometimes need to be modified at the post-translational level and/or associate with specific factors to become ubiquitylated by multiple E3s that can act at different time points of the cell cycle and at different subcellular locations. In contrast to the complex, but well-documented situation occurring in mammalian cells, our understanding of proteolytic events at the G1-to-S-phase transition in plants is still limited. The Arabidopsis Cdk1 homologue CDKA;1, which is required for both S-phase entry and mitosis, is negatively regulated by CKIs (Verkest et al., 2005a). Two classes of CKIs have been identified so far in Arabidopsis: the INTERACTOR/INHIBITOR OF CDK, also called KIP-RELATED PROTEINS (with seven members in Arabidopsis, hereafter called KRP1 7) (Torres Acosta et al., 2011), and the SIAMESE-RELATED (SMRs), named after their founding member SIAMESE (Churchman et al., 2006; Peres et al., 2007). Both classes of plant CKIs show very restricted similarities with the mammalian Kip/Cip proteins. At the functional level, constitutive overexpression in transgenic Arabidopsis plants of all KRPs tested so far can block both M and S phases, leading not only to growth retardation, including a reduction in cell number and organ size, but also to different developmental abnormalities, such as leaf serration (Verkest et al., 2005a). Therefore, protein levels of plant CKIs must be tightly regulated. Indeed, it was shown that KRP2 proteasomal degradation is conditioned by its CDK-dependent phosphorylation (Verkest et al., 2005b), a situation reminiscent of the mammalian p27 KIP1 SCF SKP2 - dependent degradation. However, at present, the identity of the ubiquitin E3 ligases responsible for the ubiquitylation and degradation of plant CKIs at the sporophytic stage remains mysterious, although a picture is starting to emerge in gametogenesis (see below and Fig. 2B). Two distantly related Arabidopsis LRR-containing F-box proteins, called SKP2A and SKP2B, are proposed to be the metazoan homologues of SKP2; however, their function in the turnover of KRPs remains presently unclear. SKP2A is involved in cell-cycle regulation as it binds to the transcription factor E2Fc and its partner DPB to mediate their degradation (del Pozo et al., 2006). The E2Fc/DPB dimer acts as a negative regulator of cell division by counteracting the activation of E2F-responsive genes. Despite a high similarity in protein sequence between the two F-box proteins, SKP2B targets different substrates and KRP1 is likely to be one of them (Ren et al., 2008). Indeed, SKP2B overexpression in Arabidopsis was shown to suppress the leaf serration phenotype conferred by KRP1 overexpression. Nevertheless, a KRP1 GUS reporter protein did not accumulate to a higher level in a skp2a skp2b double mutant (Ren et al., 2008), nor did the endogenous KRP2 (Marrocco et al., 2010). This might suggest the implication of other ubiquitin E3 ligases. Based on protein similarities with the mammalian RING protein KPC1, the Arabidopsis RING protein named RKP (Related to KPC1) was identified and suspected to be also responsible for KRP1 proteasomal degradation (Ren et al., 2008). Nevertheless, no obvious developmental defects that would be expected for strong KRP protein accumulation could be observed in any of these mutant lines, not even in the triple skp2a skp2b rkp mutant, suggesting the existence of additional and/or even more critical ubiquitin E3 ligase(s) that await discovery. Possible candidates for such ubiquitin E3 ligases have emerged recently from studies of Arabidopsis mutants deficient in cell division during gametogenesis. For instance, RHF1a and RHF2a are two similar RING-finger proteins that, when mutated in the rhf1a rhf2a double mutant, impair normal cell divisions during pollen and embryo sac development (Liu et al., 2008). Interestingly, a reduction in KRP6 expression rescues in part the rhf1a rhf2a mutant phenotype, providing genetic evidence for a role of RHF E3s in KRP degradation during both male and female gametogenesis. Another key player in cell-cycle regulation during male gametogenesis is the F-box protein FBL17, as its corresponding loss-of-function mutants fail to undergo pollen mitosis II, which generates the two sperm cells in mature pollen grain (Kim et al., 2008b; Gusti et al., 2009). Genetic evidence here also supports a function of FBL17 in KRP degradation during gametogenesis, as different KRP loss-of-function mutations suppressed, at least partially, the pollen defect phenotype (Gusti et al., 2009; Zhao et al., 2012). However, whether FBL17 functions beyond gametogenesis is presently unknown. Progression through M phase APC/C: an evolutionarily conserved ubiquitin E3 regulating mitosis The key regulator for mitotic progression and exit is undoubtedly the APC/C (Thornton and Toczyski, 2006; Pesin and Orr-Weaver, 2008; van Leuken et al., 2008). This ubiquitin E3 ligase is composed of many different subunits and has an approximate size of 1.5 MDa. The APC/C is activated from early mitosis to late G1/early S phase. To be active and in order to select its substrates, the APC/C requires crucial co-activators, the CDC20/FIZZY and CDH1/FIZZY- RELATED proteins. Besides co-activators, the temporal regulation of APC/C activity during the cell cycle is also achieved by reversible phosphorylation and the action of

5 Selective protein degradation to modulate the cell cycle 2607 inhibitory proteins. The best-characterized APC/C inhibitory protein is the metazoan EARLY MITOTIC INHIBITOR 1 (EMI1). During the S and G2 phases, EMI1 restricts APC/C activity to enable the accumulation of cyclins A and B, allowing replication and promoting the transition from G2 phase to mitosis, respectively (reviewed by Peters, 2006; Fig. 3A). At the molecular level, EMI1 binds to the APC/C CDH1 using its D-box motif as a pseudosubstrate, thus blocking the accessibility of substrates to the D-box receptor site on the APC/C. Its zinc-binding region and RL tail domains also inhibit APC/C E3 ligase activity, mainly by interfering with ubiquitin chain elongation (Miller et al., 2006; Frye et al., 2013; Wang and Kirschner, 2013). Once EMI1 is degraded in prophase by the SCF β-trcp ubiquitin E3 ligase upon phosphorylation by Plk1 (Guardavaccaro et al., 2003; Margottin- Goguet et al., 2003), the APC/C activity is released and can thus orchestrate mitosis by inducing sequential ubiquitylation and finally proteolysis of different cell-cycle regulators (Pesin and Orr-Weaver, 2008). Expression of CDC20 precedes that of CDH1, and one of the first substrates of the APC/ C CDC20 in animals is cyclin A, which is degraded right after nuclear envelope breakdown, during prometaphase (Pines, 2006; Fig. 3A). Soon after, at metaphase, polyubiquitylation by the APC/C CDC20 and degradation of PDS1/SECURIN (a protease inhibitor) is required for sister chromatid separation (Peters, 2006). PDS1/SECURIN destruction leads to activation of the Separase protease, which cleaves the cohesin complex that physically attaches sister chromatids. Notably, this degradation step is part of a surveillance mechanism called the spindle assembly checkpoint (SAC), keeping the APC/ C CDC20 in check until all chromosomes are properly attached to the mitotic spindle and bioriented at the metaphase plane (Kim and Yu, 2011; Musacchio and Ciliberto, 2012). This mechanism involves the sequestration and, even more importantly, the degradation of CDC20 through the interaction with MAD2, BUB3, and BubR1, the mitotic checkpoint complex (MCC) proteins. Recent findings have revealed that the APC/C subunit APC15 (MND2 in yeast) actually targets CDC20 for proteolysis when bound to the MCC (Foster and Morgan, 2012; Uzunova et al., 2012; Fig. 3A). Moreover, P31 COMET, a MAD2-interacting protein identified only in higher eukaryotes, is operating in the same pathway as APC15, although their exact relationship remains unclear (Musacchio and Ciliberto, 2012). When the SAC is satisfied, the APC/C CDC20 sets off polyubiquitylation and thus the degradation of not only PDS1/SECURIN but also B-type cyclins (Fig. 3A). Consequently, the inhibition of Cdk1 activity induces different cellular processes such as disassembly of the mitotic spindle, chromosome decondensation, cytokinesis, and reformation of the nuclear envelope. In the last decade, substantial advances have been made in our understanding of plant APC/C composition and function, although we still know only a limited number of its substrates. While it has been recognized that most APC/C subunits are evolutionarily conserved in plants (Capron et al., 2003a), the complex was only recently biochemically isolated from Arabidopsis cell-suspension cultures (Van Leene et al., 2010). Most subunits were identified with the exception of APC9, APC13 (Bonsai), and CDC26, which, however, were classified as non-essential subunits of APC/C in yeast (Thornton and Toczyski, 2006). Likewise, the APC15 subunit was not identified by tandem affinity purification of the Arabidopsis APC/C (Van Leene et al., 2010), but is also conserved in plant genomes (Uzunova et al., 2012). Thus we can speculate that, as in mammals, APC15 is dispensable for APC/C activity directed against common APC/C targets (Mansfeld et al., 2011; Uzunova et al., 2012). As expected for such an important regulator of mitosis, knockout of different Arabidopsis APC/C subunits blocks female gametogenesis by arresting mitotic division after meiosis (Capron et al., 2003b; Kwee and Sundaresan, 2003; Pérez-Pérez et al., 2008; Wang et al., 2012, 2013). In these mutants, it was shown that mitotic cyclin reporter proteins carrying a D-box accumulated in the arrested embryo sacs, although it remains to be demonstrated that the cause of the arrest results from the non-degradation of one or more cellcycle proteins. Recently, it has been shown that the APC/C has also a function during male gametophyte development (Zheng et al., 2011). Interestingly, this work also highlighted that, in addition to its role in protein degradation, the APC/C seems also to play a role in transcriptional regulation of the CYCB1;1 gene. At the sporophytic stage, the APC/C is required for normal plant organ development, including roots, stems, leaves, and flowers (Blilou et al., 2002; Serralbo et al., 2006; Saze and Kakutani, 2007; Pérez-Pérez et al., 2008; Marrocco et al., 2009; Eloy et al., 2011; Zheng et al., 2011). In these organs, reduced APC/C activity alters both cell division and expansion rates, although the molecular details of this pleiotropic phenotype remain to be elucidated. Conversely, overexpression of certain APC/C subunits promotes plant growth, in part, by stimulating cell division (Rojas et al., 2009; Eloy et al., 2011). How this is achieved is also unknown at the mechanistic level. As well as APC/C core subunits, its co-activators have also been studied intensively in several plant species. Arabidopsis has five CDC20-like genes (CDC20-1 to CDC20-5) (Kevei et al., 2011) and three CCS52 genes (CCS52A1, CCS52A2, and CCS52B) (Tarayre et al., 2004). Expression of CDC20-1, CDC20-2, and also CCS52B peaks in M phase and organs with dividing cells (Fülöp et al., 2005; Kevei et al., 2011), suggesting mitotic functions. Indeed, knocking down both CDC20-1 and CDC20-2 genes reduces root meristem size and leads to smaller leaves with fewer cell numbers. While these observations strengthen the implication of the involvement of these proteins in cell-cycle regulation, the identity of the targets of the plant APC/C CDC20 is still mysterious. The function of CCS52 genes has been linked to cell-cycle exit and endoreduplication (see below). In particular, Arabidopsis CCS52A2 was shown to regulate mitosis exit and meristem maintenance in the root tip by controlling mitotic activity in quiescent centre (QC) cells (Vanstraelen et al., 2009). In order to identify potential targets of the APC/C CCS52A2 in the QC, Heyman et al. (2013) co-purified CCS52A2-associated proteins by tandem affinity purification. The identified candidates were further challenged for their ability to promote QC cell proliferation upon ectopic expression. This strategy led

6 2608 Genschik et al. Fig. 3. APC/C activity from entry towards exit of the M phase. (A) APC/C is activated from early mitosis and remains active throughout mitosis and the G1 phase until early S phase. In metazoans, two main inhibitors keep the APC/C complex in check during the cell cycle. EMI1 operates in the S and G2 phases, whereas the spindle assembly checkpoint (SAC) proteins target APC/C CDC20 in mitosis. The mitotic checkpoint complex (MCC), which contains the three SAC proteins (MAD2, BUBR1, and BUB3) together with CDC20, is regarded as the SAC effector. During SAC arrest, CDC20 turnover depends on its association with the SAC proteins and requires the APC/C subunit APC15. When proper alignment and attachment of the duplicated chromosomes to the mitotic spindle are achieved towards the end of metaphase, MCC and APC/C are dissociated from each other leaving the single MCC subunit protein, CDC20, with APC/C to polyubiquitylate SECURIN/PDS1 for 26S proteasomal degradation and thus successful chromosome segregation. APC/C CDC20 also polyubiquitylates mitotic CYCLIN B for proteasomal destruction to lower CDK activity and allows mitotic exit. (B) In plants, UVI4 and OSD1/ GIG1 are the functional homologues of EMI1. Similar to the EMI1 gene, E2Fa and E2Fb transcription factors regulate UVI4 transcription at the G1/S-phase transition in Arabidopsis. UVI4 mediates the inactivation of APC/C CCS52A during DNA replication and correspondingly allows the CYCA2;3 cyclin to accumulate. This A-type cyclin activates the CDKB1;1 kinase activity that is required for entering mitosis. A lossof-function uvi4 mutation causes a premature endocycle onset. Controlled plant endoreduplication in the dividing phase of Arabidopsis leaves is also orchestrated by the atypical E2Fe/DEL1 repressor that inhibits transcription of the CCS52A2 co-activator of the plant APC/C complex. The APC/C inhibitor OSD1/GIG1 interacts and inhibits the APC/C CDC20 complex during mitosis. Intriguingly, a loss-of-function gig1 mutation in cotyledon cells causes endomitosis. Therefore, UVI4 and OSD1/GIG1 operate sequentially during the mitotic cell cycle and inhibit the endocycle and endomitosis, respectively. As in fungi and metazoans, orthologous SAC proteins have been characterized in Arabidopsis, but their roles in this surveillance mechanism need to be clarified. (This figure is available in colour at JXB online.)

7 Selective protein degradation to modulate the cell cycle 2609 to identification of the transcription factor ERF115, a ratelimiting factor of QC cell division. Notably, ERF115 represents the first plant APC/C substrate that does not belong to the basic cell-cycle machinery. As in metazoans, APC/C inhibitory proteins have recently been identified and characterized in Arabidopsis (Fig. 3B). One of these is ULTRAVIOLET-B-INSENSITIVE 4 (UVI4), which co-purified with the APC/C by tandem affinity purification and was shown to interact physically with CCS52A1/ A2 (Van Leene et al., 2010; Heyman et al., 2011; Iwata et al., 2011). Although poorly conserved at the protein sequence level, UVI4 shares some structural features with the animal APC/C inhibitor EMI1 such as the D-box, which might interact with CCS52A1, whereas a C-terminal MR tail together with the GxEN motif might mediate the interaction with the APC/C holocomplex. Similarly to the EMI1 gene, UVI4 transcription at the G1-to-S-phase transition is regulated by E2F transcription factors (Heyman et al., 2011). UVI4 loss of function reduces cell division activity in roots and leaves. At the molecular level, good evidence has been provided that UVI4 restricts APC/C CCS52A1 activity in dividing cells, thus allowing the cyclin CYCA2;3 to accumulate at levels required for mitotic entry (Heyman et al., 2011; Fig. 3B). Arabidopsis UVI4 has a homologue called OMISSION OF SECOND DIVISION 1 (OSD1)/ GIGAS CELL 1 (GIG1) (Hase et al., 2006; d Erfurth et al., 2009; Heyman et al., 2011; Iwata et al., 2011). OSD1/GIG1 like UVI4 interacts with APC/C activators and associates in vivo with the APC/C (Van Leene et al., 2010; Iwata et al., 2011). OSD1/GIG1 seems to act during mitosis, most likely to repress APC/C CDC20 activity, which would result from the accumulation of CYCB (Iwata et al., 2011, 2012). Strikingly, an OSD1/GIG1 loss-of-function mutation triggers endomitosis in certain cotyledon cell types, a phenomenon even exacerbated when combined with CDC20 overexpression (Iwata et al., 2011). The exact mechanism of how OSD1/GIG1 represses endomitosis is unknown; however, UVI4 and OSD1/GIG1 may have temporally different activities during the cell cycle (UVI4 may function earlier than OSD1/GIG1 in the mitotic cell cycle) (Fig. 3B). Beside these conserved regulators, the APC/C also incorporates plant-specific components such as the SAMBA protein identified as a negative regulator of the APC/C (Eloy et al., 2012). However, the mode of action of SAMBA at the level of the APC/C or its substrate remains unclear. Inactivation of SAMBA leads to a higher accumulation of cyclin CYCA2;3 protein level during early development. This CYCA2;3 accumulation in samba mutant plants may explain the increased cell proliferation reported only at early developmental stages. Additionally, loss of function of SAMBA also leads to defective pollen mitosis I. Whether this defect in male gametogenesis relies on stabilized cyclins has not been addressed. A possible role of the APC/C CDC20 in the plant SAC While mitotic cyclins are probable substrates of the APC/ C CDC20, the targeting of plant securin-like proteins by this ubiquitin E3 ligase is still under debate. Moreover, these proteins are poorly conserved, which precludes their identification. However, several observations support a role for ubiquitin-dependent proteolysis in the plant SAC. Hence, when synchronized tobacco BY2 cells are treated with the proteasome inhibitor MG132, they arrest in metaphase (Genschik et al., 1998). Furthermore, the anti-microtubule drugs propyzamide and oryzalin inhibit the degradation of D-box containing reporter proteins and lead to the stabilization of cyclin CYCB1 (Genschik et al., 1998; Criqui et al., 2001), suggesting that the APC/C is inactivated when the SAC is on. Moreover, the APC15 subunit that triggers CDC20 proteolytic ubiquitylation when the SAC is activated is conserved in plants (Uzunova et al., 2012). Finally, orthologues of the SAC proteins BUBR1, BUB3, and MAD2 have recently been investigated in Arabidopsis and their function seems conserved in plants (Caillaud et al., 2009). Importantly, Arabidopsis CDC20-1 and CDC20-2 interact at least in yeast with MAD2 and BUBR1/MAD3, supporting a conserved function of CDC20 proteins in the formation of the MCC and SAC (Kevei et al., 2011). Although overall these data support a role of the plant APC/C CDC20 in SAC regulation, the observation that knockout of Arabidopsis APC/C does not block cells in metaphase (Capron et al., 2003b; Kwee and Sundaresan, 2003; Pérez-Pérez et al., 2008; Wang et al., 2012) points to the existence of other means of disassembly of the MCC, as reported in yeast and animals cells (Rieder and Maiato, 2004; Primorac and Musacchio, 2013). Endoreduplication The endocycle, also called endoreduplication or endoreplication, is an alternative cell cycle where cells duplicate their DNA without cell division, allowing them to increase their ploidy. Endoreduplication is widespread in plants, and often mitotically dividing cells switch into the endocycle as they differentiate (Breuer et al., 2010; De Veylder et al., 2011). Endoreduplication is, however, not restricted to plant cells and, although less common, this phenomenon is well described in some mammalian and insect cells (Edgar and Orr-Weaver, 2001; Lee et al., 2009). In particular, in Drosophila salivary gland cells, APC/C FZR activity is required in endocycling cells to mediate Geminin protein oscillation as well as other proteins (Narbonne-Reveau et al., 2008; Zielke et al., 2008). Geminin proteins are known to regulate DNA replication by inhibition of the DNA replication licensing factor CDT1, but, so far, orthologues of Geminin have not been identified in plants. A role of the APC/C in regulation of the plant endocycle was first demonstrated in leguminous Medicago plants where silencing of a CCS52 gene led to a reduction in DNA ploidy levels in leaves (Cebolla et al., 1999). The activity of CCS52A homologues is also required in developmental processes subjected to extensive endoreduplication such as tomato fruit development (Mathieu-Rivet et al., 2010) and endosperm development of rice kernels (Su udi et al., 2012). In Arabidopsis, downregulation of APC/C activity also leads to a significant reduction in endoreduplication in leaves and root cells (Serralbo et al., 2006; Marrocco et al., 2009).

8 2610 Genschik et al. Consistent with a role of A-type CCS52 genes in regulation of the endocycle in different plant species, both Arabidopsis CCS52A1 and CCS52A2 are also required for endoreduplication in leaf (including trichomes) and root cells (Lammens et al., 2008; Vanstraelen et al., 2009; Kasili et al., 2010). Given that A-type CCS52 genes act as key factors for the switch from the mitotic to the endocycle, their expression must be tightly controlled. Hence, it was shown that the atypical E2F transcription regulators E2Fe/DEL1 repress CCS52A2 expression during the dividing phase of Arabidopsis leaf development to avoid premature entry into the endocycle (Hase et al., 2006; Lammens et al., 2008). It was shown as well that expression of CCS52A1 is repressed by the GT2-LIKE1 (GTL1) transcriptional regulator, in order to promote termination of ploidy-dependent cell growth such as in trichomes (Breuer et al., 2012). This raises the question of which are the critical substrates of the APC/C CCS52A in the regulation of endoreduplication. An interesting observation was that ectopic expression of a D-box-deficient and thus non-degradable CYCA2;3 significantly restrains endocycles in various plant organs (Imai et al., 2006), assigning this cyclin as a good candidate substrate for the APC/C in this process. Moreover, in UVI4- deficient plants, the presumed loss of APC CCS52A1 repression leads to increased degradation of CYCA2;3 and could thus explain the premature entry in endoreduplication (Heyman et al., 2011), showing that UVI4 plays an important function as an inhibitor of the endocycle onset. While we have slightly unravelled the function of the APC/C in plant endoreduplication, it is likely that other ubiquitin E3 ligases are also involved in this process. For instance, it was suggested recently that, while APC/C function is key for cell entry into endoreplication cycles by eliminating mitotic CDK activity, CULLIN4 (probably as part of a CRL4 complex) might be essential for progression through endoreplication cycles by degrading still unknown CDK inhibitor(s) to generate oscillating levels of S-phase CDK activity (Roodbarkelari et al., 2010). Another interesting ubiquitin E3 ligase is the HECT-type KAKTUS, which represses endoreduplication in trichomes (Downes et al., 2003; El Refy et al., 2003). Here again, the substrate(s) regulated by KAKTUS needs to be identified. Finally, as plant CKIs operate in a dosagedependent manner (e.g. at a low level these CKIs block only the mitotic cycle, whereas at high level both the endocycle and mitotic cycle are arrested; reviewed by De Veylder et al., 2011), it is likely that ubiquitin E3 ligases controlling CKI protein stability will also have an important impact on endoreduplication. Meiosis While mitosis produces genetically identical cells to the mother cell, meiosis is another type of cell division implicated in sexual reproduction to reduce the ploidy of the original cell and permit exchanges of genetic material by recombination. This process involves two rounds of chromosome segregation that follow a single round of chromosome duplication leading to the production of haploid gametes. The complexes of cyclins and CDKs are essential for progression through both mitotic and meiotic cell cycles. The transition from meiosis I to meiosis II requires a fine balance in cyclin CDK activity, which must be sufficiently low to exit meiosis I, but must nonetheless be maintained at a level sufficiently high to suppress DNA replication and promote entry into meiosis II. This fine-tuning of CDK activity has been shown to rely on the APC/C ubiquitin E3 ligase. Hence, in Xenopus oocytes, at the end of meiosis I, CyclinB is only partially degraded and the resulting low level of Cdc2/CyclinB activity is essential for entry into meiosis II (Iwabuchi et al., 2000). This partial degradation is controlled by inhibition of the APC/C by specific inhibitors, known as Emi2 in mouse and Xenopus (Madgwick et al., 2006; Ohe et al., 2007) and Mes1 in Schizosaccharomyces pombe (Kimata et al., 2008). In addition, budding yeast also encodes a meiosis-specific coactivator of the APC/C, called AMA1, which is more similar to Cdh1 than to Cdc20 (Cooper and Strich, 2011). The APC/C AMA1 targets several substrates including B-type cyclins (CLB in yeast), but not the anaphase inhibitor Pds1, and is required for meiosis I reductional division and spore formation. Recently, it has been shown that degradation by the APC/C AMA1 of NDD1, a subunit of a mitotic transcriptional activator complex inducing the expression of CLB1/2 and other regulators, suppresses mitotic cell-cycle control during prophase I (Okaz et al., 2012). Indeed, an extended duration of prophase I, controlled by the recombination checkpoint, is essential for homologous chromosome recombination to occur. In plants, recent progress has indicated that APC/C inhibition is also required to promote meiotic progression, and this is achieved by OSD1/GIG1. Mutations of OSD1 were shown initially to trigger failures to enter the second division during both male and female meioses producing diploid spores or gametes (d Erfurth et al., 2009). Moreover, the demonstration that OSD1 is structurally and functionally related to the animal Emi2 or yeast MES1 proteins was published recently (Cromer et al., 2012). OSD1/GIG1 contains three APC/C interaction domains (a D-box, a KEN-box, and an MR tail) and physically interacts with most Arabidopsis APC/C co-activators (Iwata et al., 2011; Cromer et al., 2012). Evidence that OSD1 inhibits the APC/C is also supported by the fact that its expression in mouse oocytes leads to a metaphase I arrest. Interestingly, mutation of TARDYASYNCHRONOUS MEIOSIS (TAM) encoding the A-type cyclin CYCA1;2 leads to a similar phenotype as osd1 (i.e. blocking of the transition from meiosis I to meiosis II; d Erfurth et al., 2010). Combining tam and osd1 mutations leads to a failure of the transition from prophase to the first meiotic division (d Erfurth et al., 2010). As the level of CDK activity is important for regulation of these transitions, a moderate decrease in CDK activity in osd1 and cyca1;2/tam single mutants may cause failure to enter meiosis II without impairing the prophase-to-meiosis-i transition. In contrast, the coincident depletion of OSD1 and CYCA1;2/TAM may further decrease CDK activity, impairing entry into meiosis I. To date, CYCA1;2/TAM is the only known cyclin affecting meiotic progression, but its exact function will need further investigation. As CDKA;1 associated

9 Selective protein degradation to modulate the cell cycle 2611 with CYCA1;2/TAM is able to phosphorylate OSD1/GIG1, at least in vitro, this meiotic cyclin may control OSD1/GIG1 activity and/or stability. Conclusion and some perspectives Although we are still far from understanding the mechanistic details of how ubiquitin E3 ligases control the plant cell cycle, it is clear that, in the last decade, a number of important contributions from several laboratories have provided a first picture of these regulations. In particular, new results have highlighted the importance of the plant APC/C core complex and its activators during different type of processes such as gametogenesis, plant growth, and endoreduplication onset. Conversely, less understood is the role of SCF-type E3s in cell-cycle regulation and especially in the transition from G1 to S phase. While the SCF FBL17 and the RINGfinger proteins RHF1a/RHF2a are key cell-cycle regulators during gametogenesis, the situation is far less clear at the sporophytic stage where essential E3s still wait to be discovered. It is also unclear whether other CRL families such as CRL3 and CRL4 contribute to the regulation of the plant cell cycle as they do in metazoans. Another important challenge for the future will be to identify substrates for all these plant E3s, and it is likely, based on animal models, that each will have more than one substrate. Identifying these substrates will be complicated further by the fact that some need to be modified at the post-translational level in order to bind their ubiquitin E3 ligases. This is of peculiar importance for SCFs where phosphorylation of the substrate is often a prerequisite for their recognition by these E3s. At present, we know very little about post-translational modifications of plant regulatory cell-cycle proteins. After binding of the substrate by its dedicated E3 ubiquitin ligase, the ubiquitylation reaction can proceed, and it is well established that the formation of polyubiquitin chains through lysine 48 (K48) triggers degradation of the substrate by the proteasome. Interestingly, some substrates are also modified by atypical polyubiquitin chains and/or by monoubiquitylation. For instance, in mammals it was found that the APC/C assembles polyubiquitin chains linked to lysine 11 (K11) (Jin et al., 2008) that nevertheless also lead to proteasomal degradation. Whether this is true for the degradation of mitotic substrates by the plant APC/C remains to be demonstrated. In mammals, CRL3 E3 ligases have been shown to play important functions during mitosis (reviewed by Genschik et al., 2013). In particular, some of these E3s mono-ubiquitylate cell-cycle kinases such as Aurora B and Polo-like kinase 1 (PLK1) (Maerki et al., 2009; Beck et al., 2013), controlling their subcellular localization but not their stability. Whether such mechanisms exist in plants remains presently also at the level of pure speculation. Besides unravelling conserved cell-cycle functions of plant E3s, another interesting research avenue is to elucidate how these enzymes regulate and are regulated by endogenous and exogenous plant signals. For instance, it was proposed recently that an abiotic stress such as drought mediates mitotic exit and earlier onset of endoreplication by modulating the activity of the APC/C (Claeys et al., 2012). Drought, but also other stresses, acts by decreasing the amount in bioactive gibberellins, resulting in an increase in the accumulation of the nuclear-localized growth repressing DELLA proteins (reviewed by Achard and Genschik, 2009). How DELLAs are linked to APC/C activity will require further investigations. A link between the APC/C and auxin has also been highlighted in several reports (Blilou et al., 2002; Lindsay et al., 2011; Wang et al., 2012) but remains poorly understood at the mechanistic levels. Auxin has also been shown to positively affect the stability of the cell-cycle transcription factor E2Fb that promotes cell division (Magyar et al., 2005). However, the mechanism by which auxin avoids E2Fb proteolysis is still not known. Moreover, a recent striking finding is that auxin binds directly to the Arabidopsis F-box protein SKP2A to promote E2Fc/DPB degradation, directly connecting auxin with cell-cycle control (Jurado et al., 2010). Auxin also induces the degradation of SKP2A, thus adding another level of control. Thus, the above-mentioned examples represent only a few challenges among others that await our research community in the near future. 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