The Anaphase-Promoting Complex/Cyclosome in Control of Plant Development

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1 Molecular Plant Volume 5 Number 6 Pages November 2012 REVIEW ARTICLE The Anaphase-Promoting Complex/Cyclosome in Control of Plant Development Jefri Heyman and Lieven De Veylder 1 Department of Plant Systems Biology, VIB and Department of Plant Biotechnology and Bioinformatics, Ghent University, Technologiepark 927, B-9052 Gent, Belgium ABSTRACT Temporal controlled degradation of key cell division proteins ensures a correct onset of the different cell cycle phases and exit from the cell division program. In light of the cell cycle, the Anaphase-Promoting Complex/Cyclosome (APC/C) is an important conserved multi-subunit ubiquitin ligase, marking targets for degradation by the 26S proteasome. However, whereas the APC/C has been studied extensively in yeast and mammals, only in the last decade has the plant APC/C started to unveil its secrets. Research results have shown the importance of the APC/C core complex and its activators during gametogenesis, growth, hormone signaling, symbiotic interactions, and endoreduplication onset. In addition, recently, the first plant APC/C inhibitors have been reported, allowing a fine-tuning of APC/C activity during the cell cycle. Together with the identification of the first APC/C targets, a picture emerges of APC/C activity being essential for many different developmental processes. Key words: cell cycle; CDC20; CCS52; endoreduplication; gametogenesis; ubiquitination. AN INTRODUCTION TO THE ANAPHASE-PROMOTING COMPLEX/ CYCLOSOME Selective degradation through ubiquitination of key proteins is pivotal to ensure unidirectional progression through the cell cycle. Attachment of ubiquitin moieties to substrate proteins occurs by a series of enzymatic reactions. First, an ATP-dependent activation of the ubiquitin molecule by an E1 activating enzyme takes place, followed by its transfer to an E2 conjugating enzyme, and finally attachment to the target protein by an E3 ubiquitin ligase (Hershko and Ciechanover, 1998; Fang and Weissman, 2004). The ubiquitin tag serves as a recognition motif for targeting proteins to the 26S proteasome that will subsequently degrade the ubiquitinated proteins using its ATP-dependent endopeptidase activity (Rechsteiner et al., 1993; Bedford et al., 2010; Gallastegui and Groll, 2010). One particular E3 ubiquitin ligase dedicated to cell division control is the highly conserved Anaphase-Promoting Complex/Cyclosome (APC/C), controlling progression through the cell cycle by the destruction of cell cycle proteins that contain the specific D- or KEN/GxEN-box destruction signals (Pfleger and Kirschner, 2000; Castro et al., 2003; De Veylder et al., 2007; Marrocco et al., 2010). The APC/C is activated from early mitosis onwards and remains active throughout mitosis and the G1 until early S phase (Peters, 2002; Capron et al., 2003). Its activity depends on the interaction of the core complex with one of two types of activating subunits: CELL DIVISION CYCLE 20 (CDC20) and CDC20 HOMOLOG 1 (CDH1), called FIZZY (FZY) and FIZZY-RELATED (FZR) in Drosophila, respectively. Activation of the APC/C by CDC20 occurs from late G2 phase on, whereas, from anaphase on, the CDC20 activator is exchanged for the CDH1 subunit until early S phase (Kevei et al., 2011) (reviewed in Pesin and Orr-Weaver, 2008). In plants, the CDH1 activators are known as the CELL CYCLE SWITCH 52 (CCS52) genes (Cebolla et al., 1999), and are classified into A- and B-type subgroups, namely CCS52A and CCS52B (Tarayre et al., 2004). COMPOSITION OF THE PLANT APC/C The APC/C is a large (1.7-MDa) multi-subunit complex, highly conserved within eukaryotes, as different APC/C subunits from distinct species are able to complement the corresponding yeast mutants (Capron et al., 2003; Eloy et al., 2011; Wang et al., 2012). Recently, the Arabidopsis thaliana APC/C was 1 To whom correspondence should be addressed. livey@psb.vib-ugent.be, tel. +32 (0) , fax +32 (0) The Author Published by the Molecular Plant Shanghai Editorial Office in association with Oxford University Press on behalf of CSPB and IPPE, SIBS, CAS. doi: /mp/sss094, Advance Access publication 3 October 2012 Received 14 June 2012; accepted 17 August 2012

2 Heyman & Veylder Control of Development by the APC/C 1183 purified using a Tandem Affinity Purification (TAP) approach, providing a biochemical framework for the composition of the APC/C in plants (Van Leene et al., 2010). The Arabidopsis APC/C consists of at least 11 core subunits (Capron et al., 2003; Van Leene et al., 2010) (Figure 1 and Table 1). Based on sequence similarity, the catalytic core of the plant APC/C is constituted of APC2 and APC11, the cullin and REALLY INTERESTING NEW GENE (RING-H2) domain subunits, respectively (Page and Hieter, 1999; Tang et al., 2001), which are essential for catalyzing the ubiquitin transfer reaction (Yu et al., 1998; Leverson et al., 2000; Tang et al., 2001). The APC10/Doc1 (Destruction of D-box) subunit is an important co-activator of the APC/C, recognizing and recruiting D-box containing proteins (da Fonseca et al., 2011), whereas the APC3, APC6, APC7, and APC8 subunits contain tetratricopeptide repeat (TPR) interaction domains and form together with APC1, APC4, and APC5 the APC/C backbone (Thornton and Toczyski, 2003; Schreiber et al., 2011). In Arabidopsis thaliana, all APC/C subunits are encoded by single genes, with the exception of APC3, for which two isoforms are present, namely APC3a/CDC27a and APC3b/HOBBIT (HBT) (Blilou et al., 2002; Capron et al., 2003; Lima Mde et al., 2010) (Table 1). The TPR repeat domains of the APC3 subunits allow interaction with the APC/C core complex and serve as a docking site for the CDC20 and CCS52 APC/C activator subunits, as well as for the APC10 co-activating subunit (Thornton and Toczyski, 2003; Schreiber et al., 2011). Correspondingly, also the CDC20 and CCS52 activator subunits co-purified with the core APC/C subunits (Van Leene et al., 2010), indicating that a complete APC/C holo-complex can be found in Arabidopsis cell cultures. In contrast to the majority of the core APC/C subunits, expression of the activator moieties appears to be strongly cell cycle phase-dependent, with transcript levels of the CDC20 and B-type CCS52 activators peaking from early G2 to M phase exit (Fülöp et al., 2005; Kevei et al., 2011). On the contrary, the A-type CCS52 activators show a late M to late S-to-G2 specific expression profile (Table 1) (Fülöp et al., 2005; Lammens et al., 2008). The observation that the expression of the CDC20 and CCS52 activator components is cell cycle phase-dependent indicates that these subunits are crucial factors controlling the temporal activation of the APC/C during the cell cycle. Based on sequence homology and their cell cycle phase-dependent expression, the E2 conjugating enzymes UBC19 and UBC20 were proposed to mediate the transfer of the activated ubiquitin specifically to the APC/C (Criqui et al., 2002). However, no co-purification of UBC19/20 could be seen with any of the APC/C subunits (Van Leene et al., 2010), which could be due to the transient interaction during the ubiquitination reaction, explaining the absence of UBC19/20 in the TAP purified complexes. APC/C ACTIVITY IS ESSENTIAL FOR GAMETOGENESIS APC/C-dependent protein degradation is essential for plant viability, since homozygous loss-of-function plants for each core subunit cannot be obtained, except for APC3, probably because Figure 1. Composition of the Arabidopsis thaliana APC/C. (A) Schematic representation of the APC/C, based on the direct interaction studies and resolved APC/C structure from Saccharomyces cerevisiae by Schreiber et al. (2011) and da Fonseca et al. (2011). The APC/C core complex subunits are indicated in red, catalytic core subunits in orange, activator subunits in green, and positive/negative regulators (UVI4/PYM, OSD1/UVI4-Like/GIGAS, and SAMBA) in blue. (B) Interactome of the plant APC/C, composed from literature described yeast two-hybrid interactions. The APC/C core complex subunits are colored as in (A). it is encoded by two isoforms. Accordingly, cdc27a hbt double mutants are not viable (Pérez-Pérez et al., 2008). In the case of APC2, self-pollination of hemizygous knockout mutants results in 50% aborted ovules due to defects in female gametogenesis (Capron et al., 2003). Similarly, apc4 (Figure 2A) and nomega/ apc6 and cdc27a hbt double mutant plants show defects during megagametogenesis (Kwee and Sundaresan, 2003; Wang et al., 2012). Considering the importance of the APC/C in the control

3 1184 Heyman & Veylder Control of Development by the APC/C Table 1. The Arabidopsis thaliana APC/C Components. AGI Nomenclature Type subunit Cell cycle phase expression Corresponding mutant phenotype At5g05560 APC1 Core Constitutive n.d. At2g04660 APC2 Catalytic Constitutive Defected female gametogenesis At3g16320 APC3a/CDC27a Core S-G2 Increased proliferation upon overexpression At2g20000 APC3b/HOBBIT Core S/G2-M Dwarf phenotype At4g21530 APC4 Core Constitutive Defected female gametogenesis At1g06590 APC5 Core Constitutive n.d. At1g78770 APC6/Nomega Core Constitutive Defected female gametogenesis At2g39090 APC7 Core Constitutive n.d. At3g48150 APC8 Core Constitutive Defected male gametogenesis At2g18290 APC10 Core Constitutive Increased proliferation upon overexpression At3g05870 APC11 Catalytic Constitutive n.d. At1g73177 APC13 Non-core n.d. Defected male gametogenesis At4g33270 CDC20-1 Activator Early G2 M phase exit Reduced meristem size At4g33260 CDC20-2 Activator Early G2 M phase exit Reduced meristem size At5g27080 CDC20-3 Activator At5g26900 CDC20-4 Activator At5g27570 CDC20-5 Activator At4g22910 CCS52A1 Activator Late M late S-G2 Delayed endocycle onset At4g11920 CCS52A2 Activator Late M late S-G2 Delayed endocycle onset At5g13840 CCS52B Activator Early G2 M phase exit n.d. At2g42260 UVI4/PYM Inhibitor G1-S Premature endocycle onset At3g57860 OSD1/UVI4-Like/GIGAS Inhibitor G2-M Endomitosis At1g32310 SAMBA Activator n.d. Increased organ size n.d., not determined. of mitosis exit through the targeted proteolysis of mitotic cyclins (reviewed in Kim and Yu, 2011), these observations are not surprising. Accordingly, APC/C mutants have been observed to accumulate cyclin proteins in the embryo sacs (Table 2). However, gametophytic phenotypes are not always directly linked to the inability to degrade cell cycle proteins. Mutation of either APC8 or APC13 results in a decreased biogenesis of the micro- RNA mir159, which targets the DUO POLLEN 1 (DUO1) gene, a transcriptional activator of CYCB1;1 cyclin (Palatnik et al., 2007; Brownfield et al., 2009; Zheng et al., 2011). As a consequence, CYCB1;1 transcription is stimulated in the apc8 and apc13 mutant pollen, likely contributing to the male gametophytic defect of these APC/C loss-of-function plants (Zheng et al., 2011). EFFECTS OF HYPOMORPHIC CORE APC/C MUTATIONS ON PLANT DEVELOPMENT Sporophytic effects of reduced APC/C activity have been studied mainly using silencing lines (Table 2), revealing its necessity for correct growth and development. For example, Arabidopsis thaliana apc6 mutants show a reduced leaf size that is accompanied by an altered vein patterning (Marrocco et al., 2009). Similarly, rice apc6 mutants display a dwarfed phenotype that originates from a reduced vascular bundle cell size (Kumar et al., 2010). Reduction of APC6 transcript levels in Medicago truncatula causes a reduced lateral root number and root elongation (Kuppusamy et al., 2009) and, in Arabidopsis thaliana, a weak apc8 allele shows abnormal shoot, flower, and silique development (Zheng et al., 2011). Plants mutated in the HBT gene show a severe dwarfed phenotype (hence its name, HOBBIT), which results from severe cell differentiation defects in root and shoot meristems (Figure 2B) (Blilou et al., 2002). In hbt embryos, root and shoot primordia are correctly patterned, but the postembryonic formation of the meristematic regions is perturbed (Blilou et al., 2002), resulting in (1) a reduced mature root length because of a smaller root elongation zone and (2) leaves with impaired cell division and expansion, and reduced DNA ploidy levels, indicating impaired cell differentiation (Serralbo et al., 2006; Pérez-Pérez et al., 2008). Furthermore, HBT is also important for lateral root cap development during the late

4 Heyman & Veylder Control of Development by the APC/C 1185 Table 2. Known APC/C Substrates. Species Accession number Nomenclature Function Assay Reference Arabidopsis thaliana At4g37490 CYCB1;1 G2-M-specific cyclin CYCB1;1-D-box GUS accumulation in apc6 mutant embryo sacs Kwee and Sundaresan, 2003 In vitro ubiquitination using CDC27aOE plant extracts Rojas et al., 2009 CYCB1;1 YFP accumulation in apc8 mutant pollen grains Zheng et al., 2011 CYCB1;1-D-box GUS degradation APC10OE leaves Eloy et al., 2011 CYCB1;1-D-box GUS accumulation in apc4 mutant female gametophytes Wang et al., 2012 Arabidopsis thaliana At5g06150 CYCB1;2 G2-M-specific cyclin CYCB1;2-D-box YFP accumulation upon OSD1OE Iwata et al., 2011 Arabidopsis thaliana At1g15570 CYCA2;3 Regulation of G2-M transition CYCA2;3 GFP accumulation in ccs52a1 mutant plants Boudolf et al., 2009 CYCA2;3 GFP degradation in uvi4 mutant plants Heyman et al., 2011 CYCA2;3-HA accumulation in samba mutant plants Eloy et al., 2012 Arabidopsis thaliana At5g43080 CYCA3;1 G1-S-specific cyclin CYCA3;1 stabilization in apc2 mutant embryos Capron et al., 2003 Lycopersicon esculentum AJ CYCA3;1 CYCA3;1 stabilization in CCS52A1-silenced BY2 protoplasts Mathieu-Rivet et al., 2010 Arabidopsis thaliana At3g62800 dsrna-binding Protein 4 (DRB4) Involved in mirna biogenesis Reduced DRB4 levels in apc6 and apc10 mutant flowers Marrocco et al., 2012 Oryza sativa Os06g MONOCULM 1 (MOC1) Involved in axillary meristem initiation and formation In vitro ubiquitination using ccs52 mutant extracts Lin et al., 2012; Xu et al., 2012 Oryza sativa Os01g ROOT ARCHITECTURE ASSOCIATED 1 (RAA1) Oryza sativa Os02g RICE SALT SENSITIVE 1 (RSS1) M-phase-specific cell cycle inhibitor in roots S-phase-specific protein required for cell proliferation maintenance D-box-dependent stabilization upon proteasome inhibition D-box-dependent interaction with CDC20 Han et al., 2008 Ogawa et al., 2011

5 1186 Heyman & Veylder Control of Development by the APC/C Figure 2. Phenotypic Effects of APC/C Mutation on Plant Development. (A) Seed development in wild-type and apc4-1/+ transgenic plants, reproduced with permission from Wang et al. (2012). apc4 homozygous mutants are gametophytic lethal, resulting into undeveloped ovules (indicated by arrowheads) and aborted seeds (indicated by open stars) upon self-pollination of apc4/+ mutants. (B) One-week-old wild-type and hbt mutant seedlings, reproduced with permission from Willemsen et al. (1998). hbt mutants show a strong dwarfed (or HOBBIT) phenotype due to severe root and shoot meristem defects. The hbt mutant seedling is shown at 4 magnification of the wild-type seedling. (C) Effect of CDC20-1/CDC20-2 co-silencing on Arabidopsis thaliana plant growth, reproduced with permission from Kevei et al. (2011). From left to right, plants with reducing CDC20-1/CDC20-2 co-silencing levels are shown. (D) Red-ripe wild-type (left) and CCS52A-silenced (right) tomato fruits, respectively, reproduced with permission from Mathieu-Rivet et al. (2010). The CCS52A silenced tomato fruits are smaller because of reduced DNA ploidy levels. (E) Wild-type and ccs52 mutant rice plants, reproduced with permission from Xu et al. (2012). Plants mutated in CCS52 show a dwarfed growth and disrupted kernel development. Scale = 10 cm. (F) Three-week-old rosettes of wild-type Col-0 and ccs52a2 mutant plants show a severe growth reduction upon mutation of CCS52A2 compared to wild type. Scale = 1 mm. (G) Confocal image of 5-day-old propidium-iodide-stained wild-type Col-0 and ccs52a2 mutant root meristems (scale = 50 µm). ccs52a2 mutant root meristems are disorganized due to differentiation of the QC and stem cells. (H) Scanning electron microscope image of wild-type Col-0 and ccs52a1 mutant trichomes shows a reduced trichome branch number of ccs52a1 mutants compared to wild-type trichomes. Scale = 500 µm.

6 Heyman & Veylder Control of Development by the APC/C 1187 stages of embryogenesis, showing the pleiotropic effect of HBT during embryogenesis and whole-plant development (Willemsen et al., 1998). Contrary to the knockout or knockdown of APC/C activity, the overexpression of core APC/C subunits has often been observed to be beneficial for growth. Increased APC10 expression has been reported to result in an increased mature leaf size (Eloy et al., 2011). Likewise, overexpression of CDC27a results in an overall increased plant growth in both Arabidopsis and tobacco (Rojas et al., 2009). In both cases, increased growth has been attributed to an increase in cell number. THE APC/C AND HORMONE SIGNALING Failure of dark-grown hbt seedlings to maintain an apical hook suggested possible auxin signaling defects in hbt mutants. Accordingly, expression of the synthetic auxin responsive DR5 gene appears to be reduced in hbt seedlings, indicating a reduced auxin perception that correlates with the accumulation of the IAA17/ARX3 auxin repressor protein (Blilou et al., 2002). Given the fact that auxin is important for embryo development (Cooke et al., 1993; Liu et al., 1993), impaired auxin signaling might in part explain the observed embryo patterning defects. Also, apc4-defective embryos display disrupted auxin distributions (Wang et al., 2012), whereas roots of APC6-silenced Medicago truncatula plants show auxin insensitivity (Kuppusamy et al., 2009), suggesting again a connection between the APC/C and auxin signaling. In addition to its putative connection with auxin, other plant hormones can be related to APC/C activity. The reduced vascular bundle cell size caused by the weak apc6 allele in rice was found to involve gibberellin insensitivity (Ubeda-Tomás et al., 2009; Kumar et al., 2010). On the other hand, overexpression of APC10 results in increased transcription of the ethylene response factor 1 (ERF1) and leads to some of the typical ethylene triple response characteristics (Solano and Ecker, 1998), such as thickened hypocotyls and a reduced root elongation, suggesting that ethylene signaling might be linked with APC/C activity (Lindsay et al., 2011). These findings suggest that APC/C activity might be part of, or is being controlled by, hormone signaling cascades. However, the molecular mechanisms that link auxin, gibberellin, or ethylene signaling with APC/C activity remain unknown. CDC20 AND PLANT DEVELOPMENT The Arabidopsis genome encodes five CDC20 isoforms, named CDC20-1 to CDC20-5 (Kevei et al., 2011). The undetectable expression of CDC20-3 to CDC20-5 in nearly all plant tissues and the almost identical spatial temporal expression patterns of CDC20-1 and CDC20-2 suggest that these two subunits are predominantly activating the APC/C (Kevei et al., 2011). Due to their similar expression profiles, CDC20-1 and CDC20-2 are believed to work redundantly. Indeed, co-silencing of CDC20-1 and CDC20-2 results in dwarfed plants, due to a strong reduction in cell number (Figure 2C) (Kevei et al., 2011). The absence of transcripts, together with their unusual gene structure, suggests that CDC20-3 to CDC20-5 might be pseudogenes. Correspondingly, CDC20-3, CDC20-4, or CDC20-5 loss-of-function plants show no detectable phenotype. THE CCS52A ACTIVATOR SUBUNIT IS IMPORTANT FOR CELL DIFFERENTIATION AND ENDOCYCLE ONSET CCS52, the plant homolog of the animal CDH1 activator subunit, was first described for Medicago truncatula (Cebolla et al., 1999) but, soon after, it was identified also in other plant species. Similarly to its CDH1/FIZZY-RELATED counterpart in Drosophila, CCS52 is an important component controlling the timing of cell differentiation a correlation that was first observed in Medicago sativa nodules, where the highest CCS52 expression levels were found in endoreduplicating tissues (Cebolla et al., 1999). Endoreduplication is an alternative cell cycle following the exit of the mitotic cell cycle in many plant species, resulting in cellular polyploidy (De Veylder et al., 2011). Correspondingly, silencing of CCS52 reduced DNA ploidy levels in Medicago sativa leaves (Cebolla et al., 1999). Similar observations were also reported for other plant species (González-Sama et al., 2006; Lammens et al., 2008; Mathieu-Rivet et al., 2010; Su udi et al., 2012). During tomato fruit development, CCS52A expression peaks when endoreduplication-driven cell expansion occurs, whereas, in red-ripe tomato fruits, decreased CCS52A expression causes a striking reduction in DNA ploidy levels and hence fruit size (Figure 2D) (Joubès and Chevalier, 2000; Mathieu-Rivet et al., 2010). Mutation of the rice CCS52A homolog (Figure 2E), annotated as TILLER ENHANCER (TE) or TILLERING AND DWARF 1 (TAD1), results in disrupted kernel development, due to a reduced DNA content in developing endosperm, which is known to undergo endoreduplication (Lin et al., 2012; Su udi et al., 2012; Xu et al., 2012). In Arabidopsis thaliana, two different A-type CCS52 proteins are encoded by the genome, namely CCS52A1 and CCS52A2 (Tarayre et al., 2004). Both CCS52A1 and CCS52A2 isoforms regulate endoreduplication onset in leaves, as evidenced by reduced DNA ploidy levels in knockout plants (Lammens et al., 2008; Kasili et al., 2010). In the case of CCS52A2, a peak in expression can be observed at the moment leaf cells exit their cell division program, suggesting its importance for controlling the appropriate timing of cell differentiation (Figure 2F) (Lammens et al., 2008). The presence of an E2F consensus cis-regulatory element in the CCS52A2 promoter, combined with chromatin immunoprecipitation (ChIP) experiments, has shown that the increased CCS52A2 transcript levels at the cell division cycle-to-endocycle transition is controlled by the E2Fe/ DEL1 transcriptional repressor (Lammens et al., 2008). E2Fe/ DEL1 represents an atypical E2F transcription factor that is

7 1188 Heyman & Veylder Control of Development by the APC/C able to bind consensus E2F cis-regulatory promoter elements in order to repress transcription of its target genes (reviewed in Lammens et al., 2009). E2Fe/DEL1 expression is restricted to dividing tissues, where it inhibits endocycle onset through repression of CCS52A2. In E2Fe/DEL1 knockout plants, repression of CCS52A2 is not maintained, which triggers cells to enter the endoreduplication program prematurely (Vlieghe et al., 2005; Lammens et al., 2008). Whereas in leaves both CCS52A1 and CCS52A2 control the timing of cell cycle exit, in roots, it is mainly CCS52A1 that steers the onset of cell differentiation, as evidenced by its restricted expression in the root elongation zone, where cells start to differentiate. Furthermore, mutation of CCS52A1 results in longer roots that originate from an increased meristem length, suggesting that CCS52A1 plays an active role in promoting cell cycle exit in root cells (Vanstraelen et al., 2009). CCS52A2 activity rather appears to play a role in root meristem maintenance, as ccs52a2 mutants display a disorganized root apical meristem due to an inability to repress the mitotic activity of the quiscent center (QC) (Figure 2G) (Vanstraelen et al., 2009). Because expression of CCS52A1 under control of the CCS52A2 promoter can complement the ccs52a2 mutant phenotype, it might be the differential expression patterns of the CCS52A1 and CCS52A2 isoforms, rather than their differences in substrate specificity, that account for the distinct root phenotypes (Vanstraelen et al., 2009). Besides controlling endoreduplication onset in Arabidopsis leaves and roots (Lammens et al., 2008; Vanstraelen et al., 2009), the CCS52A1 activator subunit is an important regulator of endoreduplication in Arabidopsis trichomes, which have a DNA content up to 32C (Melaragno et al., 1993; Hülskamp et al., 1994). Mutation of CCS52A1 results in a decreased trichome branching that correlates with a decreased DNA ploidy level of trichome nuclei (Figure 2H) (Kasili et al., 2010). On the other hand, ccs52a2 mutants do not show an altered trichome phenotype, possibly explained by the absence of detectable CCS52A2 expression in trichomes (Marks et al., 2009; Kasili et al., 2010). However, APC/C CCS52A1 activity does not appear to be the only ubiquitin ligase involved in controlling the DNA ploidy levels in trichomes: the CULLIN 4-RING FINGER-LIGASE has been shown to be indispensable for successive endocycles in trichome nuclei, whereas the APC/C CCS52A1 is suggested to control only the onset of endoreduplication (Roodbarkelari et al., 2010). In contrast to the A-type CCS52 activators, currently only limited information is available on the biological relevance of CCS52B. Overexpression of CCS52B in tobacco cell suspension cultures results in a dramatically increased number of cells with a 4C DNA content, suggesting that mitosis is blocked and cells fail to re-enter the G1 phase (Tarayre et al., 2004). It is speculated that the complementary cell cycle-dependent expression of the A- and B-type CCS52 activators might allow a fine-tuned regulation of APC/C activity during the cell cycle (Tarayre et al., 2004). Further detailed investigation of CCS52B function during plant development is required to support this hypothesis. THE CCS52A ACTIVATOR SUBUNIT IS IMPORTANT FOR PLANT BIOTIC INTERACTIONS Apart from their involvement in the developmentally programmed onset of endoreduplication, CCS52A subunits also play an important role in different types of plant-symbiotic interactions. For instance, the interaction between roots of leguminous plants such as Medicago truncatula and M. sativa and the soil bacterium Sinorhizobium meliloti results in the formation of nitrogen-fixing nodules, which arise from differentiated root cortical cells that re-enter the cell cycle. In M. truncatula, transcripts of CCS52A are absent in dividing cortical cells, even though CCS52A expression is activated strongly in the central region of the establishing nodule (Vinardell et al., 2003). In later stages of nodule development, CCS52A is expressed in the dividing and endoreduplicating zones of the nodule, but not in the differentiated nitrogen-fixing zone (Vinardell et al., 2003). CCS52A2 silencing resulted in a severe reduction or absence of nodule formation. DNA content measurements of the aborted nodules showed a reduction in DNA ploidy levels (Vinardell et al., 2003), indicating that CCS52A-mediated APC/C activity is indispensable to initiate and develop symbiotic root nodules. Similarly to Medicago, initiation of nodule formation, as well as nodule primordial formation in Lupinus albus and Lotus japonicas, requires CCS52A expression (González-Sama et al., 2006). In addition to nodule formation, Medicago plants can also engage parasitic interactions with the root-knot nematode Meloidogyne incognita. Meloidogyne infections induce nematode feeding sites (NFS), a group of giant cells that develop from root cells (Williamson and Hussey, 1996). In response to the parasite, infected cells undergo several rounds of DNA replication accompanied by nuclear and cell expansion, resulting in large multinucleated and polyploid cells (de Almeida Engler et al., 1999). Upon nematode infection, CCS52A expression is triggered at the NFS, especially in cells that become polyploid, suggesting a role for CCS52A in NFS establishment (Favery et al., 2002; de Almeida et al., 2012). Correspondingly, NFS development is strongly affected in CCS52 silenced lines, illustrating the need for APC/C activity for feeding site maturation and demonstrating that endoreduplication is potentially essential to maintain a high metabolic activity of the NFS to assure maturation and reproduction of the nematodes. PLANT-SPECIFIC REGULATORS OF APC/C ACTIVITY Given the importance of CCS52A in controlling cell cycle activity and cell differentiation, strict regulation of APC/C activity is essential. In the animal field, APC/C CDH1 regulators have been investigated thoroughly. In Homo sapiens, Mus musculus, and Xenopus laevis, the Early mitotic inhibitor 1 (Emi1), known

8 Heyman & Veylder Control of Development by the APC/C 1189 as Regulator of CyclinA1 (Rca1) in Drosophila melanogaster, inhibits APC/C CDH1 activity during the mitotic cell cycle by operating as a pseudo-substrate (Di Fiore and Pines, 2007). Similarly, in budding yeast (Saccharomyces cerevisiae), APC/ C CDH1 modulator 1 (Acm1) acts as a pseudo-substrate of APC/ C CDH1, suppressing the degradation of mitotic cyclins (Burton et al., 2011). In addition, the Emi1 homolog Erp1/Emi2 functions specifically in the meiotic cell cycle, and is essential for the transition from meiose I to meiose II (Tung et al., 2005; Ohe et al., 2007). Based on sequence identity, no orthologs of Emi1/Rca1, Acm1, or Erp1/Emi2 have been identified in plants. However, TAP of the Arabidopsis APC/C resulted in the identification of a new putative plant-specific APC/C subunit, namely ULTRAVIOLET-B-INSENSITIVE 4 (UVI4)/POLYCHOME (PYM) (Van Leene et al., 2010). Despite the absence of conserved sequence homology, UVI4 shows a strikingly similar domain organization to the animal Emi1 and Erp1/Emi2. Correspondingly, it has been shown that UVI4 is a plant-specific inhibitor of APC/C CCS52A1 activity through direct interaction with CCS52A1 (Heyman et al., 2011; Iwata et al., 2011). UVI4 was originally shown to be involved in endoreduplication and its mutation conveys increased UV-B tolerance to plants, also explaining its name (Perazza et al., 1999; Hase et al., 2006). Mutation of UVI4 leads to a loss of APC/C CCS52A1 inhibition at the G1-to-S transition, triggering increased degradation of the downstream CYCA2;3 that is required for mitotic cell divisions, and that ultimately causes a premature endocycle onset (Imai et al., 2006; Boudolf et al., 2009; Heyman et al., 2011). The Arabidopsis genome encodes a UVI4 homolog, namely UVI4-Like/OMISSION OF SECOND DIVISION 1 (OSD1)/GIGAS CELL 1 (GIGAS) (Hase et al., 2006; d Erfurth et al., 2009; Heyman et al., 2011; Iwata et al., 2011). Loss of OSD1 function was originally shown to cause defects in the second mitotic division during the meiotic cell cycle, resulting in diploid male and female gametes, giving rise to tetraploid progeny (d Erfurth et al., 2009, 2010; Cromer et al., 2012). UVI4 plays an important role in determining the meristem size in roots and the cell number and size in leaves, whereas the diploid osd1 mutant shows no obvious defects in meristem and leaf size (Heyman et al., 2011). However, OSD1 might not function exclusively during the meiotic cell cycle, since osd1 cotelydons show abnormal, large guard cells and round cells with increased DNA ploidy levels that originate from endomitotic events (Iwata et al., 2011). In addition, some redundancy between UVI4 and OSD1 is proposed, since the uvi4 osd1 double mutant is difficult to obtain and displays severe compromised developmental defects (Iwata et al., 2011; Cromer et al., 2012). Corresponding with the hypothesis that OSD1 interacts and inhibits APC/C CDC20 activity during mitosis (Iwata et al., 2011), OSD1 is expressed during the mitotic cell cycle, peaking at the G2-to-M transition (data not published; Figure 3A). On the contrary, the expression of UVI4 peaks at the G1-to-S transition (Figure 3A) (Heyman et al., 2011). It suggests that UVI4 and OSD1 operate sequentially, with UVI4 securing the G2-to-M transition by inhibition of the endocycle, whereas OSD1 operates during the M phase in order to prevent endomitosis (Figure 3B). However, the molecular details on how OSD1 represses endomitosis remain to be revealed. Like UVI4 and OSD1, the recently identified APC/C subunit SAMBA appears to be plant-specific and functions as a negative regulator of growth (Eloy et al., 2012). SAMBA mutation increases seed, embryo, and seedling size. Together with the strong expression of SAMBA in young seedlings, these data suggest that its gene product functions as an early-in-development regulator of the APC/C that might operate in a different developmental time frame than UVI4 and OSD1. However, in contrast to UVI4, mutation of SAMBA results in increased CYCA2;3 levels, suggesting that SAMBA is rather a potential activator of the APC/C during early plant development (Eloy et al., 2012). THE PLANT SUBSTRATES OF THE APC/C Compared to the number of characterized animal and yeast APC/C substrates, only little is known about the plant targets. Due to the lack of good in vitro or in vivo APC/C ubiquitination assays, only indirect data on putative APC/C substrates are currently available (Table 2). Only three reported in vitro biochemical assays measure the level of ubiquitination of potential targets using extracts of APC/C mutants or overexpressors (Rojas et al., 2009; Lin et al., 2012; Xu et al., 2012). Other indirect studies measure protein abundance of putative targets in APC/C mutant versus wild-type extracts (Mathieu-Rivet et al., 2010; Heyman et al., 2011) or make use of the CYCB1;1-D-box GUS fusion, consisting of the D-box containing aminoterminal part of the CYCB1;1 cyclin fused to the GUS reporter. Increased CYCB1;1-D-box GUS signals are observed in apc4 mutant female gametophytes and during embryogenesis (Wang et al., 2012) and in apc6 mutant embryo sacs and leaves (Kwee and Sundaresan, 2003; Marrocco et al., 2009), whereas reduced and increased CYCB1;1-D-box GUS signals are detected in young and more mature leaves upon APC10 overexpression and silencing, respectively (Marrocco et al., 2009; Eloy et al., 2011). Alternative assays make use of CYCB1;1 YFP (Zheng et al., 2011) or other D-box-reporter fusion constructs (Capron et al., 2003) and identified the tomato CYCA3;1 and the Arabidopsis CYCB2;1 and CYCA2;3 cyclins as substrates (Boudolf et al., 2009; Mathieu-Rivet et al., 2010; Iwata et al., 2011). However, in all cases, it should be kept in mind that stabilization of the D-box markers might also be due to an indirect effect of an altered cell cycle activity, instead of direct protein turnover by the APC/C. In addition to the core cell cycle substrates, cell division-independent APC/C targets have been identified as well. The dsrna-binding Protein 4 (DRB4), involved in RNA silencing, has been shown to interact with the APC10 subunit. Moreover, silencing of APC6 or APC10 resulted in a strong DRB4 accumulation in flowers, suggesting that DRB4 indeed represents an APC/C substrate (Marrocco et al., 2012). In rice, APC/C CCS52 mediates MONOCULM 1 (MOC1) degradation, a protein involved in

9 1190 Heyman & Veylder Control of Development by the APC/C Figure 3. Action of Plant-Specific Regulators on APC/C Activity during the Cell Cycle. (A) Cell cycle phase-specific expression of the UVI4 and OSD1 APC/C inhibitors. Relative expression levels in hydroxyurea-synchronized root tips show maximum transcription of UVI4 and OSD1 at the G1-to-S and the G2-to-M transition, respectively. The transcript level of non-synchronized root tips (0 h) was arbitrarily set to 1. Data represent mean ± SE (n = 2). (B) Schematic representation of APC/C activity during the cell cycle, together with its inhibitors UVI4 and OSD1, and putative substrates CYCB1;2 and CYCA2;3. During late G2 phase, OSD1 allows CYCB1;2 accumulation by APC/C CDC20 inhibition. Activation of APC/C CDC20 in early mitosis degrades CYCB1;2 until anaphase, when the APC/C is activated by CCS52A. During late G1 phase, APC/C CCS52A activity is inhibited by UVI4, ensuring accumulation of CYCA2;3 to allow progression into the G2/M phase. P, prophase; M, metaphase; A, anaphase; T, telophase. regulating axillary meristem initiation and formation. Stabilization of MOC1 upon ccs52 mutation increases shoot branching and tillering, causing a dwarfed phenotype and decreased grain yield (Lin et al., 2012; Xu et al., 2012). Another described APC/C substrate in rice is the ROOT ARCHITECTURE-ASSOCIATED 1 (RAA1), a putative cell cycle inhibitor that controls the metaphase-to-anaphase transition during root growth (Ge et al., 2004; Han et al., 2008; Xu et al., 2010). Finally, the RICE SALT SENSITIVE 1 (RSS1) protein, required for meristematic cell vigor under salt stress conditions, interacts with CDC20, making it a putative APC/C CDC20 target (Ogawa et al., 2011). FUTURE OUTLOOK: UNRAVELING THE PLANT APC/C MYSTERIES Only in the past decade, major advances in unraveling the mysteries of the plant APC/C have been achieved. Phenotypical and molecular analyses have revealed that the APC/C is essential for plant development, control of cell proliferation, and onset of cell differentiation. However, the APC/C might also fulfill an important role in differentiated tissues. For instance, D-box GUS constructs do not accumulate in differentiated leaves, suggesting the presence of active APC/C complexes in postmitotic tissues (Marrocco et al., 2009). In mammals, a role for the APC/C in differentiated cells has already been proven, with APC/C CDC20 and APC/C CDH1 playing a role in neuronal morphogenesis and axon growth, respectively (Harmey et al., 2009; Puram et al., 2010). In addition, APC/C CDH1 is found in pre- and post-synaptic compartments to regulate the synaptic strength of mature neurons in Drosophila melanogaster and Caenorhabditis elegans (reviewed by Manchado et al., 2010; Yang et al., 2010), highlighting that the importance of the APC/C exceeds its known cell cycle-dependent role. For instance, in APC6 and APC10-silenced seedlings, increased vascular tissue in 5-week-old plants arises from postmitotic events, whereas no root meristem defects can be detected (Marrocco et al., 2009). To shed light on the mitotic and postmitotic processes involving the plant APC/C, identification of its specific substrates will be indispensable. A first proteome-wide search for ubiquitinated proteins was performed in Arabidopsis cell suspension cultures (Maor et al., 2007), followed by the identification of total ubiquitinated proteins in Arabidopsis seedlings (Saracco et al., 2009). However, these strategies present a rather general view on the spectrum of ubiquitinated plant proteins and do not allow the identification of APC/C-specific substrates. The latter might be elucidated through a more specialized in-depth purification and quantification of the ubiquitinated proteins in wild-type versus apc mutant plants. Mutants that might be helpful in this approach are those lacking the recently

10 Heyman & Veylder Control of Development by the APC/C 1191 identified APC/C inhibitors UVI4 and OSD1 (Heyman et al., 2011; Iwata et al., 2011; Cromer et al., 2012), although a lot still needs to be learned on their APC/C inhibitory activities. Direct interaction studies have shown that these inhibitors interact with the APC/C, mainly through direct binding with the CDC20 and CCS52 activator subunits, but no functional inhibitory domains or the mode of inhibition have been elucidated so far. To determine the actual inhibitory domains and how APC/C inhibition is achieved, elucidation of the UVI4 and OSD1 protein structures, alone or in complex with the APC/C holocomplex, is desired. In addition, OSD1 is subjected to CDK phosphorylation, even though the significance of this posttranslational control is unclear. More is known on the control of APC/C activity through the phosphorylation of its activating subunits. Phosphorylation renders CDC20 unable to bind and activate the APC/C core complex, and mutation of these phosphorylation sites results in a hyperactivated APC/C by CDC20 in M. truncatula (Tarayre et al., 2004). These results confirm the observation that CDC20 is phosphorylated by the mitogen-activated protein kinase (MAPK) during the spindle checkpoint in Xenopus laevis, rendering it unable to activate the APC/C (Chung and Chen, 2003). In human cells, CDC20 phosphorylation is additionally controlled by BUBR1 (Tang et al., 2004), and direct interaction between CDC20 and MAD2 impedes activation of the APC/C (Mondal et al., 2006). Both BUBR1 and MAD2 are components of the Mitotic Checkpoint Complex that prevents premature APC/C activation during mitosis (reviewed in Sudakin et al., 2001; Elowe, 2011; Tipton et al., 2011). A similar spindle checkpoint complex is present in Arabidopsis and might contribute to preventing premature APC/C CDC20 activation during the mitotic cell cycle (Caillaud et al., 2009). However, how APC/C activity is regulated by the plant spindle checkpoint still remains to be elucidated. Finally, as discussed above, APC/C activity appears to be partially controlled by hormone signaling pathways, which is not surprising, since the plant hormones auxin, gibberellin, and ethylene are important cues during embryogenesis, meristem maintenance, and cell expansion (Cooke et al., 1993; Ubeda- Tomás et al., 2009). Apart from hbt and apc6 mutants that show reduced or complete auxin sensitivity, nothing is known about the involvement of hormone signaling pathways that regulate APC/C activity (Blilou et al., 2002; Kuppusamy et al., 2009). 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