SAMBA, a -specific anaphase-promoting complex/cyclosome regulator is involved in early development and A-type stabilization Nubia B. Eloy, Nathalie Gonzalez, Jelle van Leene, Katrien Maleux, Hannes Vanhaeren, Liesbeth de Milde, Stijn Dhondt, Leen Vercruysse, Erwin Witters, Raphaël Mercier, et al.

To cite this version:

Nubia B. Eloy, Nathalie Gonzalez, Jelle van Leene, Katrien Maleux, Hannes Vanhaeren, et al.. SAMBA, a plant-specific anaphase-promoting complex/cyclosome regulator is involved in early de- velopment and A-type cyclin stabilization. Proceedings of the National Academy of Sciences of the United States of America , National Academy of Sciences, 2012, 109 (34), pp.13853 - 13858. ￿10.1073/pnas.1211418109￿. ￿hal-01190736￿

HAL Id: hal-01190736 https://hal.archives-ouvertes.fr/hal-01190736 Submitted on 29 May 2020

HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. SAMBA, a plant-specific anaphase-promoting complex/ cyclosome regulator is involved in early development and A-type cyclin stabilization

Nubia B. Eloya,b, Nathalie Gonzaleza,b, Jelle Van Leenea,b, Katrien Maleuxa,b, Hannes Vanhaerena,b, Liesbeth De Mildea,b, Stijn Dhondta,b, Leen Vercruyssea,b, Erwin Wittersc,d,e, Raphaël Mercierf, Laurence Cromerf, Gerrit T. S. Beemsterd, Han Remautg, Marc C. E. Van Montagub,1, Geert De Jaegera,b, Paulo C. G. Ferreirah, and Dirk Inzéa,b,1 aDepartment of Plant Systems , VIB, and bDepartment of Plant Biotechnology and Bioinformatics, Ghent University, 9052 Ghent, Belgium; cCenter for Proteome Analysis and Mass Spectrometry and dDepartment of Biology, University of Antwerp, 2020 Antwerp, Belgium; eFlemish Institute for Technological Research (VITO), 2400 Mol, Belgium; fInstitut Jean-Pierre Bourgin, Unité Mixte de Recherche 1318, Institut National de la Recherche Agronomique– AgroParisTech, 78026 Versailles, France; gLaboratory of Structural and Molecular Microbiology, VIB–Vrije Universiteit Brussel, 1050 Brussels, Belgium; and hInstituto de Bioquímica Médica, Centro de Ciências da Saúde, Universidade Federal do Rio de Janeiro, CEP 21941-590, Rio de Janeiro, Brazil

Contributed by Marc C. E. Van Montagu, July 11, 2012 (sent for review February 23, 2012)

The anaphase-promoting complex/cyclosome (APC/C) is a large determine substrate specificity (21). Recently, ULTRAVIOLET- multiprotein E3 ligase involved in ubiquitin-dependent B-INSENSITIVE4 (UVI4) and UVI4-like/OMISSION OF of key cycle regulatory , including the SECOND DIVISION1/GIGAS CELL1 (UVI4/OSD1/GIG1) destruction of mitotic at the -to-anaphase transi- were identified as plant-specific inhibitors of the APC/C that are tion. Despite its importance, the role of the APC/C in plant cells and required for proper mitotic progression in Arabidopsis (22, 23). the regulation of its activity during remain poorly In Arabidopsis, the encoding the subunits APC2, APC3a, understood. Here, we describe the identification of a plant-specific APC3b, APC4, APC6, APC8,andAPC10 have been investigated negative regulator of the APC/C complex, designated SAMBA. In functionally. In all cases, except for APC8, the analysis of the Arabidopsis thaliana, SAMBA is expressed during embryogenesis mutants revealed female gametophytic defects, probably as a con- – APC8 and early plant development and plays a key role in organ size sequence of the inability to degrade mitotic cyclins (24 28). control. Samba mutants produced larger seeds, , and roots, was shown to be involved in male gametogenesis (29). Moreover, APC6 APC10 which resulted from enlarged root and shoot apical meristems, reduced expression levels of (10) or (10, 28) ex- and, additionally, they had a reduced fertility attributable to a ham- hibited several developmental abnormalities, including defects in SAMBA vascular development, whereas an APC4 loss-of-function mutant pered male gametogenesis. Inactivation of stabilized A2- APC3b CDC27b type cyclins during early development. Our data suggest that was defective in embryogenesis (26). ( ) has a role SAMBA regulates during early development by in the maintenance of cell division in meristems during post-

APC10 PLANT BIOLOGY targeting for APC/C-mediated proteolysis. (30), and overexpression of en- hances cell division and accelerates the degradation of the mitotic fi CYCB1;1, leading to increased sizes (28). lant organ size is determined by the total cell number and nal Here, we functionally analyzed an APC/C regulator of Arabi- cell size, resulting from cell division and cell expansion, re- dopsis samba P fi , designated SAMBA. The mutants have an enlarged spectively. In most, but not all, cases, the nal organ size correlates meristem size and show growth-related phenotypes, including the with cell number, rendering cell division the main driver that formation of large seeds, leaves, and roots; additionally, their controls growth (1). fertility is reduced because of a defect in male gametogenesis. A In all eukaryotes, unidirectional progression requires biochemical analysis revealed that loss of function of SAMBA the coordinated destruction of essential cell cycle regulatory stabilizes CYCA2;3. We conclude that SAMBA is a plant-specific proteins by ubiquitin-dependent proteolysis pathways (2–5). Specific E3 ubiquitin ligases mediate the recognition of target negative regulator of APC/C involved in the degradation of A- proteins (6–8) that are subsequently polyubiquitinated and sub- type cyclins. jected to proteolysis by the 26S . One of the most Results complex ubiquitin ligases involved in cell cycle control is the anaphase-promoting complex/cyclosome (APC/C), of which SAMBA Is a Plant-Specific APC/C Regulator. The SAMBA , fi fi the composition can vary from 11 to 13 subunits depending on the encoded by AT1G32310, had been identi ed by tandem-af nity organism. The APC/C complex plays essential roles in , purification (TAP) associated with the core APC/C and, more , and postmitotically differentiated cells (3, 9–12). specifically, with the subunits APC3b, APC7, and APC10 in The APC/C triggers the metaphase-to-anaphase transition and protein complexes purified from Arabidopsis cell suspension the exit from mitosis by mediating the degradation of proteins cultures (31). In a TAP experiment on Arabidopsis cell cultures such as and mitotic cyclins (13). In , the A- and B- with SAMBA as bait, 12 interacting proteins were identified, type cyclins are subjected to proteolysis by APC/C through rec- including the subunits APC1, APC2, APC3b, APC4, APC5, ognition of specific amino acid motifs, the destruction (D) and APC6, APC7, APC8, and APC10, except APC11, and the reg- KEN boxes (6, 14, 15). Plant A-type cyclins are produced and ulators CCS52A2, UVI4, and UVI4-like/OSD1/GIG1 (31). degraded earlier in the cell cycle than B-type cyclins and have distinct and nonredundant functions in the progression of cell division (16). Based on their primary structures, the plant A-type Author contributions: N.B.E., M.C.E.V.M., and D.I. designed research; N.B.E., N.G., J.V.L., cyclins are classified into A1, A2, and A3 groups (17). The K.M., H.V., L.D.M., L.V., and L.C. performed research; N.B.E., N.G., S.D., E.W., R.M., G.T.S.B., transcriptional regulation of the CYCLIN A2 (CYCA2) group H.R., G.D.J., and P.C.G.F. analyzed data; and N.B.E. and D.I. wrote the paper. coordinates cell proliferation during plant development (18, The authors declare no conflict of interest. 19) and CYCA2;3 overexpression enhances cell division (20). 1To whom correspondence may be addressed. E-mail: [email protected] or The APC/C is regulated partly by two activating proteins [email protected]. CELL DIVISION CYCLE 20 (CDC20) and CDC20 HOMOL- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. OGY1/CELL CYCLE SWITCH 52 (CDH1/CCS52), that also 1073/pnas.1211418109/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1211418109 PNAS | August 21, 2012 | vol. 109 | no. 34 | 13853–13858 To confirm that SAMBA forms a complex with APC/C in SAMBA expression was very weak in all tissues, except during seed planta, TAP was carried out with 6-d-old SAMBA-expressing development. To study the expression pattern of SAMBA, a 1.8-kb seedlings C-terminally fused to the GS tag (see SI Materials and fragment upstream of the ATG codon of the SAMBA was Methods) and proteins were identified by mass spectrometry fused to a β-glucuronidase (GUS)-GFP tandem reporter cassette (MS). In agreement with previous results, SAMBA interacted and introduced into Arabidopsis plants. SAMBA expression was with all APC/C subunits in planta, except APC11, which is dif- high during embryogenesis (Fig. 1 B and C) but decreased grad- ficult to identify with the selected analytical approach because of ually when seedlings germinated (Fig. 1 D and F). At 3 d after its small size (Table S1). Also, the interaction with the activator stratification (DAS) (Fig. 1D), GUS staining was still well visible in CCS52A2 was found, but not with CCS52B, UVI4, and UVI4- all tissues; at 5 DAS, it diminished and became patchy in root like/OSD1/GIG1. tissues (Fig. 1E); finally, at 8 DAS, it was present only in the Although TAP allowed the detection of the entire APC/C hypocotyls (Fig. 1F); and, at later stages of development, the ex- complex, it did not provide insight into the direct interactions pression was only observed in pollen grains (Fig. 1G). between core subunits and associated proteins. Therefore, we performed two-hybrid (Y2H) assays with the SAMBA samba Mutants Develop Enlarged Organs. To assess the role of the protein against all APC/C subunits identified in Arabidopsis and SAMBA gene in plant growth and development, we analyzed its its two coactivators, CCS52A2 and CCS52B. The results revealed loss-of-function phenotype in two independent T-DNA insertion a direct interaction of SAMBA with APC3b (Fig. S1A) but not lines. Both lines were inserted into the first intron (Fig. S1C), with APC1, APC2, APC3a, APC4, APC5, APC6, APC7, APC8, completely abolishing gene expression. Because both mutants had APC10, APC11, or the activators CCS52A2 and CCS52B. Taken the same phenotype, they will be further referred to as samba, together, the protein interaction data indicate that SAMBA is unless specified. Interestingly, the analysis of samba showed an a component of the APC/C complex in plants, by binding APC3b. increased seed size [up to 143% of the wild type (WT)] (Fig. 2A SAMBA is 100 aa in length (10.8 kDa) and resembles APC16, and Fig. S2A) and size (Fig. 2B). Additionally, samba a recently described APC/C subunit conserved throughout meta- rosettes were visibly larger (Fig. 2C) and roots were longer (Fig. zoans (32–34) that is 110–120 residues in size and characterized by 2D and Fig. S2B) than those of WT plants. As a consequence of the four regions of sequence similarity, referred to as AH1 to AH4 increased root length, the number of lateral roots in samba (33). Based on these regions of sequence similarity, several can- mutants was higher than that in the WT (Fig. S2C). The increased didate APC16 subunits can be found in plants, including in Ara- growth of samba mutants also led to a significant increase in fresh bidopsis (Fig. S1B), but except for some remote similarity at the and dry weight of shoots and roots (Fig. S2 D–G). AH2 and AH3 regions, SAMBA lacks sequence homology with To understand the samba growth phenotype, we analyzed both APC16, particularly in the extended homology region AH4 lo- root and shoot meristem sizes. The root apical meristem (RAM), cated toward its C terminus. Inversely, homology searches iden- measured from the quiescent center (QC) toward the point where tified single SAMBA-like proteins in various other plant . cortical cells start to elongate, was on average 26% larger in samba Sequence alignments of SAMBA homologs revealed two regions than in WT (Fig. 2E and Fig. S2H). The shoot apical meristem of sequence conservation [SAMBA homology region (SHR)1 and (SAM) of seedlings at 12 DAS was analyzed by means of a 3D SHR2], spaced by a 10- to 20-residue-long low-complexity region reconstruction made from serial sections. Volumetric measure- (Fig. 1A). Because no homologous sequences were found in ments of seedlings at 12 DAS revealed that the mean SAM volume metazoans or protista, SAMBA constitutes an APC/C regulator of samba was ∼40% higher (139 × 103 μm3; n = 19) than that of the unique to plants. WT (99 × 103 μm3; n = 16) (Fig. 2F and Fig. S2I). Also when 3D reconstructions of the first leaf initials were compared at 4 DAS, SAMBA Expression. Analysis of published microarray datasets and leaf primordia of samba plants were on average 80% larger than use of the BioArray (http://www.bar.utoronto.ca) and Genevesti- those of the WT (Fig. 2G and Fig. S2J). In conclusion, SAMBA gator (https://www.genevestigator.com) tools revealed that the appears to negatively regulate early seedling growth.

Fig. 1. SAMBA orthologs and in vivo expression profile. (A) ClustalW2 multiple sequence alignment of putative SAMBA orthologs identified in selected plant genomes. A low-complexity region (LCR) that separates two regions of increased sequence similarity is marked as SHR1 and SHR2. (B) Expression of the pSAMBA- GUS-GFP reporter gene at different developmental stages. Embryo at heart stage visualized by confocal microscopy. (C) GUS activity in mature embryo. (D) Seedling at 3 DAS. (E) Seedling at 5 DAS. (F) Seedling at 8 DAS. (G) Mature pollen grains. [Scale bars: 20 μm(B); 500 μm(C and D); 1 mm (E and F); 100 μm(G).]

13854 | www.pnas.org/cgi/doi/10.1073/pnas.1211418109 Eloy et al. number over time, the average cell division rate for the entire leaf in samba and control plants was similar (Fig. 3D), meaning that the difference observed in samba mutants happened already before analysis of the first time point (4 DAS). An additional parameter involved in leaf development is the onset of endoreduplication that marks the transition between cell division and cell differentiation. Cells from leaves 1 and 2 divide up to 10 DAS, after which they gradually exit the cell cycle and start to endoreduplicate (35). At 8 DAS, leaves 1 and 2 of samba and control lines had an equally low endoreduplication index (EI), representing the mean number of endoreduplication cycles per nucleus. The EI of samba leaves was significantly higher between 12 and 20 DAS than that of equivalent control leaves, particularly because of an increase in the 8C and 16C cell population (Fig. 3E, Inset).

SAMBA Loss of Function Affects a Diversity of Transcripts. To un- derstand the molecular mechanisms associated with the vegeta- tive samba mutant phenotype, we extracted RNA from leaves 1 and 2 at 10 DAS for microarray transcript profiling. At 10 DAS, leaves 1 and 2 of samba mutants were clearly larger than the WT leaves. A total of 298 genes were differentially expressed (fold change 1.5; false discovery rate < 0.05): 287 were up-regulated, and 11 down-regulated. PageMan (36) analysis indicated that among the 287 up-regulated genes, the most overrepresented classes were ethylene signal transduction, stress response, tran- scription, and calcium signaling (Dataset S1). Because the samba mutants were affected in cell number, as well as endoreduplication, we analyzed the expression profile of a set of 28 cell cycle marker and growth-related genes (Table S3). WT and samba plants were grown in vitro for 8, 10, and 12 DAS, and RNA levels in dissected leaves 1 and 2 were measured with the nCounter system that allows a direct multiplexed analysis of selected transcripts (37). Most of the G2/M-phase–related genes analyzed, such as CYCB1;1, cyclin-dependent CDKB2;1, 3xHMG-box2 – fi Fig. 2. Representative pictures of the phenotypical analysis of samba.(A) and (38 40), were signi cantly down-regulated at all Seed size of WT and samba.(B) Mature of WT and samba.(C) three developmental time points in samba mutants, whereas genes PLANT BIOLOGY Seedlings of WT and samba at 15 DAS. (D) Root phenotype of WT and samba encoding cell cycle inhibitors, such as KIP-RELATED PROTEIN 1, at 10 DAS. (E) Propidium iodide–stained root meristems of WT and samba at SIAMESE RELATED 1 (SMR1), SMR2, and SMR3, were up- 5 DAS. Arrowheads mark the QC position and the meristem end, defined by regulated, in particular at 8 DAS. A similar expression profile was the position where cells start elongating. (F) Side view of a reconstructed found for the SHOOT MERISTEMLESS and EXPANSIN11 samba and WT SAM at 12 DAS. (G) Adaxial view of reconstructed primordia (EXP11) genes (Fig. 3F). In conclusion, the expression analysis is of leaves 1 and 2 of WT and samba at 4 DAS. [Scale bars: 100 μm(A and B); consistent with the hypothesis that loss of SAMBA advances early 1mm(C and D); 20 μm(E); 50 μm(F and G).] The number of samples ana- development as seen by the down-regulation of mitotic cell cycle lyzed and the statistical analysis are provided in Fig. S2. genes and the up-regulation of genes encoding cell cycle inhib- itors, expansin, and ethylene-responsive genes.

To demonstrate that the observed growth phenotype is caused by Loss of SAMBA Stabilizes CYCA2;3. Recently, APC10 had been the samba-1 and samba-2 , we engineered a rescue con- SAMBA SAMBA shown to regulate the CYCB1;1 abundance (28), and the two APC/ struct (p : ) consisting of 1,817 bp of the promoter C inhibitors UVI4 and UVI-like/OSD1/GIG1 stabilize CYCA2;3 SAMBA sequence fused to the full-length genomic sequence. and CYCB1;2, respectively (22, 23). Therefore, we tested whether Transgenic lines expressing this construction complemented the SAMBA interacts with mitotic cyclins and affect their stability. samba A growth phenotype in both mutant lines analyzed (Fig. S3 ). Y2H assays between SAMBA and six different mitotic cyclins (CYCA1;1, CYCA2;2, CYCA2;3, CYCA3;1, CYCB1;2, and Cellular Analysis of the Samba Leaf Phenotype. To investigate the CYCB2;2) revealed that SAMBA interacted with CYCA2;2 and SAMBA effect of inactivation at later stages of vegetative de- CYCA2;3, weakly with CYCA1;1, and not with CYCA3;1, velopment, we analyzed leaf growth kinematically. From 4 until 24 CYCB1;2, or CYCB2;2 (Fig. 4A). When the D-box in CYCA2;3 DAS, the first leaf pair of samba and WT plants, grown side by was deleted, no interaction with SAMBA was observed (Fig. 4B). side, was harvested and leaf blade area, cell number, cell area, and To substantiate the Y2H results, WT and samba mutant plants stomatal index were measured. In agreement with the measure- were stably transformed with a hemagglutinin (HA)-tagged ments of the shoot apex by 3D reconstructions, the leaf size of the CYCA2;3 under control of the CaMV35S promoter. Subsequently, first leaf pair of samba plants was already larger during the initial the CYCA2;3 abundance was analyzed with a commercially stages of development (Fig. 3A, Inset). At 4 DAS, the size of the available anti-HA antibody. Because SAMBA is strongly expressed samba leaf initials was on average 94% larger than that of the WT during early developmental stages, protein extracts of etiolated (Table S2), but, during further development, it became relatively plants at 5 DAS, were used (Fig. S4A), avoiding interference with less pronounced and at maturity (24 DAS) had increased ∼36% the large subunit of Rubisco. After fractionation by SDS/PAGE, (Fig. 3A). Cellular measurements demonstrated that cell number Western blotting revealed a 51-kDa band specific to CYCA2;3- (Fig. 3B) and, to a lesser extent, cell size (Fig. 3C) contributed to HA only in the samba mutant (Fig. 4C), indicating the stabili- the increased leaf area. At 4 DAS, samba leaf initials already zation of CYCA2;3 in samba plants. Quantitative (q)RT-PCR contained ∼56% more cells than the WT (Fig. 3B Inset and Table analysis confirmed the overexpression of CYCA2;3 and the ex- S2). However, calculated on the basis of the increase in cell pression levels of SAMBA in the lines analyzed (Fig. S4 B and C).

Eloy et al. PNAS | August 21, 2012 | vol. 109 | no. 34 | 13855 Fig. 3. Kinematic analysis of leaf growth in samba and WT plants. (A) Leaf blade area of the first leaf pair of samba (gray) and WT (black) plants grown in vitro from 4 to 24 DAS. (Inset) Measurement at 4 and 5 DAS. (B) Number of cells on the abaxial side of leaves. (Inset) Cell numbers at 4 and 5 DAS. (C) Cell area. (D) Cell division rate. (E) distribution of the first leaf pair of samba and WT plants. (Inset) Ploidy distribution measured by flow cytometry of the first leaves from 20- d-old samba and WT plants. (F) Transcript analysis of selected cell cycle genes in samba mutants measured by nCounter analysis. All values were normalized against the expression level of the housekeeping genes and expression was compared with the expression data in the WT. Data are means ± SE (n = 3).

This result, together with the Y2H experiments, implies that which were small, whitish, and shrunken (Fig. S5A). This phenotype SAMBA targets CYCA2;3 for degradation. was probably attributable to malfunctioning of the male gameto- To assess the specificity of the CYCA2;3 stabilization in genesis, because samba mutants produced considerably less viable samba mutant plants, we crossed transgenic plants carrying the pollen than WT plants. Indeed, in anthers of samba mutants, only CYCB1;1 promoter fused to the CYCB1;1 D-box-GUS/GFP 29% of pollen grains colored red after Alexander’s staining, in- construct with samba plants. Expression of the pCYCB1;1: dicating viability (n = 1,606; Fig. 5 A and B). + CYCB1;1 D-box-GUS/GFP construct allowed us to estimate the Reciprocal crosses between samba-2/ and WT plants were rate of CYCB1;1 degradation in proliferating cells (28, 41). The carried out to determine the T-DNA transmission. The trans- CYCB1;1 degradation rate did not differ in the leaf primordia mission rate was severely reduced only when heterozygous samba or in the RAM of WT and samba mutant plants (Fig. S4 D and mutants were used as paternal donor, confirming the dysfunction E), suggesting that SAMBA specifically targets A2-type and not of the male gametogenesis (Fig. S5B). Furthermore, the con- B-type cyclins for degradation. struct pSAMBA:SAMBA fully complemented the ovule abortion and the pollen viability phenotypes, showing that they were SAMBA Is Required for Male Gametogenesis. Although homozygous caused by the SAMBA inactivation (Fig. S3 B and C). To samba mutants could be obtained, immature siliques after self- understand which stage of pollen development is affected in the fertilization of the samba-1 and samba-2 mutants contained ∼46– samba mutants, we analyzed the progression through male meiosis. 47% (403 of 875 and 249 of 531, respectively) of aborted ovules, In both samba-1 and samba-2 homozygous mutants, meiosis was

13856 | www.pnas.org/cgi/doi/10.1073/pnas.1211418109 Eloy et al. regulator of APC/C also unique to plants. TAP of protein com- plexes with SAMBA as bait allowed in planta isolation of the entire APC/C complex. The SAMBA protein associates with the APC/C through direct interaction with APC3b that also binds to the CCS52 and CDC20 proteins throughout the common C-box and IR motifs (25). Al- though SAMBA does not have these motifs, it might interact with APC3b via the highly conserved SHR region found in all putative SAMBA orthologs. We speculate that SAMBA competes through interaction with APC3b with CDC20/CCS52 in targeting sub- strates for APC/C-mediated proteolysis. The SAMBA gene is highly expressed in developing seeds and during early plant development, indicating a specific regulatory role at these early developmental stages. In concert, loss of func- tion of SAMBA increases seed, embryo, and seedling sizes, sug- gesting that SAMBA acts as a negative regulator of growth. Later Fig. 4. CYCA2;3 stabilization. (A and B) Y2H interactions between SAMBA samba and different cyclins and CYCA2;3ΔD-box (D-box–mutated), respectively. (C) in development, the size differences between mutants and WT plants decrease. No size difference can be observed during Detection of CYCA2;3-HA protein levels in etiolated seedlings of WT fl CYCA2;3-HA (control line) and samba CYCA2;3-HA at 5 DAS. ower development. Other APC/C regulators might act at differ- ent developmental stages. For example, APC10 is involved in CYCB1;1 degradation and has a role during vascular development normal and formed balanced spore-containing tetrads (Fig. S5C). (10, 28), whereas the APC/C inhibitor UVI4 is important for tri- Also, analysis of spreads revealed no abnormalities chome branching and UVI4-like/OSD1/GIG1 is required for the (Fig. S5D). We conclude that SAMBA is not required for meiosis, proper development of guard cells (22, 23). and, therefore, the defective male gametogenesis must be A detailed kinematics analysis revealed that the increased postmeiotic. Indeed, analysis of the pollen grains at the mature growth in samba plants results from enhanced cell proliferation stage revealed that WT pollen contained one vegetative nucleus during embryogenesis and very early, postembryogenic develop- and two sperm nuclei (Fig. 5C), whereas ∼21% (n = 682) of the ment, noticeable by the difference in leaf size and cell number at samba pollen contained only one vegetative nucleus (Fig. 5D). the first time point analyzed (4 DAS). The size of the SAM and These results indicate that the lack of SAMBA expression early leaf primordia in the samba plants was on average 40% and interferes with mitosis I of pollen microspore development. 94% larger than that of the WT plants, respectively. Moreover, neither cell division rates nor timing were affected in the samba Discussion mutants. We interpret that the loss of function of SAMBA stim- Although the APC/C plays an important regulatory role in the ulates cell proliferation in developing seeds and early seedlings, eukaryotic cell cycle and has been subject to numerous studies (12), and this initial size difference is maintained throughout leaf de- still much has to be learned on its biochemical structure and reg- velopment until maturity. fi Whereas differences in cell number already occurred at early PLANT BIOLOGY ulation. For example, only recently, APC16 had been identi ed as fi a functional APC/C component in animals (33, 34) and, similarly, developmental stages, differences in cell size became signi cant fi only at later stages of leaf development. Moreover, the endor- UVI4 and UVI4-like/OSD1/GIG1 as plant-speci c inhibitors of samba APC/C (22, 23). Here, we identified SAMBA as a negative eduplication rate of is higher, resulting from increased fractions of 8C, 16C, and 32C nuclei, suggesting that despite the increased cell number cells exit the division cycle faster. This hy- pothesis is supported by the expression analysis of cell cycle marker genes that revealed a more rapid down-regulation of mi- totic genes and an up-regulation of the cell cycle inhibitory genes in the samba mutants. The EXP11 gene expression was also up- regulated, in agreement with data showing that the ectopic ex- pression of EXPANSIN genes stimulate plant growth and sup- pression by gene silencing reduces it (42, 43). Furthermore, microarray analysis showed that in samba knockout plants, the ethylene signal transduction was clearly overrepresented, which plays a prominent role in cell division regulation during leaf development by arresting the cell cycle (44). SAMBA expression does not appear to be cell cycle–regulated in cell cultures (45) or in root tips synchronized with hydroxyurea (HU) treatment. SAMBA interacts with A-type cyclins, and, at least for CYCA2;3, this interaction depends on the presence of the D-box, suggesting that SAMBA might target A-type cyclins for the APC/C-mediated proteolysis. In support of this hypothesis, samba mutants highly accumulate the A2-type cyclins. This CYCA2 ac- cumulation during early seed and seedling development is likely responsible for the stimulation of cell division, as corroborated by the enhanced cell proliferation in plants with increased CYCA2;3 levels, attributable to a in the D-box (20). Also, other Fig. 5. Pollen phenotype of samba mutants and WT. The viability of pollen grains was tested by coloration with Alexander’s stain in WT (A) and samba APC/C components were shown to control cyclin stabilization. UVI4 negatively regulates the endoreduplication onset in Ara- (B) plants. The red staining indicates that the pollen is viable. (C and D)WT bidopsis and samba pollen stained with DAPI and observed under UV fluorescence, by restraining the activity of the CCS52A1 activator respectively. Two densely stained sperm nuclei and one large diffuse vege- subunit. As a result, uvi4 mutants fail to accumulate CYCA2;3 tative nucleus are visible in the WT but frequently only one single vegetative during the (22). Conversely, UVI4-like/OSD1/GIG1 may nucleus in samba (arrows). [Scale bars: 500 μm(A and B); 10 μm(C and D).] prevent the occurrence of endoreduplication by selective

Eloy et al. PNAS | August 21, 2012 | vol. 109 | no. 34 | 13857 inhibition of CCS52A2 activity during mitosis, thereby pro- Kinematic Analysis. The complete kinematics was analyzed on leaves 1 and 2 moting the accumulation of CYCB1;2 (23). of 8–10 samba and WT plants grown in vitro and harvested daily from 4 to Mutations in most APC/C subunits affect female and/or male 24 DAS. gametogenesis (24–29). Here, we show that samba is defective in samba mitosis I of pollen development, resulting in pollen without Phenotypical Analysis of . For measurements of different parameters of the samba mutant phenotype, see SI Materials and Methods. sperm nuclei. Mutants of APC8 and APC13 (29, 46) show phe- samba notypes similar to those described here for the plants. Immunoprecipitation and Protein Gel Blotting. Whole etiolated plant tissue (5 Both mutants are affected in pollen development, leading to an DAS) was ground with 4-mm metal beads. Proteins were extracted with increased proportion of uninucleated mature pollen, indicating homogenization buffer. After centrifugation at 20,000 × g, 300 μg of total that the APC/C is required during the first mitosis of the male protein extract was incubated overnight with Anti-HA Affinity Matrix gametophytic development. Also, in agreement with the samba according to the instructions of the manufacturer (Roche). male phenotype, SAMBA was expressed strongly in pollen grains but not in female organs. ACKNOWLEDGMENTS. We thank Moritz Nowack for advice with pollen anal- ysis and Martine De Cock for help in preparing the manuscript. This work was supported by Ghent University (“Bijzonder Onderzoeksfonds Methusalem” Materials and Methods Project Grant BOF08/01M00408 and Multidisciplinary Research Partnership A full discussion of materials and methods can be found in SI Materials “Biotechnology for a Sustainable Economy” Project 01MRB510W), the Belgian and Methods. Science Policy Office (BELSPO) [Interuniversity Attraction Poles Programme (IUAP VI/33) and a postdoctoral fellowship to N.B.E.], and the European Union Sixth Framework Programme (“AGRON-OMICS” Grant LSHG-CT-2006-037704). Plant Material and Production of Transgenic Plants. Samba mutant plants S.D. and H.V. were supported by a predoctoral fellowship from the Agency for (seed code SALK_018488 and SALK_048833) were obtained from the SALK Innovation by Science and Technology. J.V.L. is a postdoctoral fellow of the collection (http://signal.salk.edu). Research Foundation-Flanders.

1. Breuninger H, Lenhard M (2010) Control of tissue and organ growth in plants. Curr 25. Pérez-Pérez JM, et al. (2008) Specialization of CDC27 function in the Arabidopsis Top Dev Biol 91:185–220. thaliana anaphase-promoting complex (APC/C). Plant J 53:78–89. 2. Zachariae W, Schwab M, Nasmyth K, Seufert W (1998) Control of cyclin ubiquitination 26. Wang Y, et al. (2012) The Arabidopsis APC4 subunit of the anaphase-promoting by CDK-regulated binding of Hct1 to the anaphase promoting complex. Science 282: complex/cyclosome (APC/C) is critical for both female gametogenesis and embryo- 1721–1724. genesis. Plant J 69:227–240. 3. Capron A, Ökrész L, Genschik P (2003) First glance at the plant APC/C, a highly con- 27. Kwee H-S, Sundaresan V (2003) The NOMEGA gene required for female gametophyte served ubiquitin-protein ligase. Trends Plant Sci 8:83–89. development encodes the putative APC6/CDC16 component of the Anaphase Pro- 4. Peters J-M (2006) The anaphase promoting complex/cyclosome: A machine designed moting Complex in Arabidopsis. Plant J 36:853–866. to destroy. Nat Rev Mol Cell Biol 7:644–656. 28. Eloy NB, et al. (2011) The APC/C subunit 10 plays an essential role in cell proliferation 5. Marrocco K, Bergdoll M, Achard P, Criqui M-C, Genschik P (2010) Selective proteolysis during leaf development. Plant J 68:351–363. sets the tempo of the cell cycle. Curr Opin Plant Biol 13:631–639. 29. Zheng B, Chen X, McCormick S (2011) The anaphase-promoting complex is a dual 6. Glotzer M, Murray AW, Kirschner MW (1991) Cyclin is degraded by the ubiquitin integrator that regulates both microRNA-mediated transcriptional regulation of cy- – pathway. Nature 349:132 138. clin B1 and degradation of during Arabidopsis male gametophyte de- 7. Hershko A, et al. (1994) Components of a system that ligates cyclin to ubiquitin and velopment. Plant Cell 23:1033–1046. – their regulation by the protein kinase cdc2. J Biol Chem 269:4940 4946. 30. Blilou I, et al. (2002) The Arabidopsis HOBBIT gene encodes a CDC27 homolog that 8. Pickart CM, Fushman D (2004) Polyubiquitin chains: Polymeric protein signals. Curr links the plant cell cycle to progression of cell differentiation. Genes Dev 16: – Opin Chem Biol 8:610 616. 2566–2575. 9. Eloy NB, Coppens F, Beemster GTS, Hemerly AS, Ferreira PCG (2006) The Arabidopsis 31. Van Leene J, et al. (2010) Targeted interactomics reveals a complex core cell cycle anaphase promoting complex (APC): Regulation through subunit availability in plant machinery in Arabidopsis thaliana. Mol Syst Biol 6:397. – tissues. Cell Cycle 5:1957 1965. 32. Hutchins JRA, et al. (2010) Systematic analysis of human protein complexes identifies 10. Marrocco K, Thomann A, Parmentier Y, Genschik P, Criqui MC (2009) The APC/C E3 chromosome segregation proteins. Science 328:593–599. ligase remains active in most post-mitotic Arabidopsis cells and is required for proper 33. Kops GJPL, et al. (2010) APC16 is a conserved subunit of the anaphase-promoting vasculature development and organization. Development 136:1475–1485. complex/cyclosome. J Cell Sci 123:1623–1633 [Erratum J Cell Sci 123:1875]. 11. Yang Y, Kim AH, Bonni A (2010) The dynamic ubiquitin ligase duo: Cdh1-APC and 34. Shakes DC, Allen AK, Albert KM, Golden A (2011) emb-1 encodes the APC16 subunit Cdc20-APC regulate neuronal and connectivity. Curr Opin Neurobiol of the Caenorhabditis elegans anaphase-promoting complex. Genetics 189:549–560. 20:92–99. 35. Beemster GTS, et al. (2005) -wide analysis of gene expression profiles asso- 12. McLean JR, Chaix D, Ohi MD, Gould KL (2011) State of the APC/C: Organization, ciated with cell cycle transitions in growing organs of Arabidopsis. Plant Physiol 138: function, and structure. Crit Rev Biochem Mol Biol 46:118–136. 734–743. 13. Harper JW, Burton JL, Solomon MJ (2002) The anaphase-promoting complex: It’s not 36. Usadel B, et al. (2006) PageMan: An interactive ontology tool to generate, display, just for mitosis any more. Genes Dev 16:2179–2206. and annotate overview graphs for profiling experiments. BMC Bioinformatics 7:535. 14. Pfleger CM, Kirschner MW (2000) The KEN box: An APC recognition signal distinct 37. Geiss GK, et al. (2008) Direct multiplexed measurement of gene expression with color- from the D box targeted by Cdh1. Genes Dev 14:655–665. coded probe pairs. Nat Biotechnol 26:317–325. 15. Criqui MC, et al. (2000) Cell cycle-dependent proteolysis and ectopic overexpression of 38. Hemerly A, Bergounioux C, Van Montagu M, Inzé D, Ferreira P (1992) Genes regu- cyclin B1 in tobacco BY2 cells. Plant J 24:763–773. lating the plant cell cycle: Isolation of a mitotic-like cyclin from Arabidopsis thaliana. 16. De Veylder L, Joubès J, Inzé D (2003) Plant cell cycle transitions. Curr Opin Plant Biol 6: – 536–543. Proc Natl Acad Sci USA 89:3295 3299. 17. Vandepoele K, et al. (2002) Genome-wide analysis of core cell cycle genes in Arabi- 39. Segers G, et al. (1996) The Arabidopsis cyclin-dependent kinase gene cdc2bAt is pref- – dopsis. Plant Cell 14:903–916. erentially expressed during S and G2 phases of the cell cycle. Plant J 10:601 612. fi 18. Vanneste S, et al. (2011) Developmental regulation of CYCA2s contributes to tissue- 40. Pedersen DS, et al. (2011) The plant-speci c family of DNA-binding proteins con- specific proliferation in Arabidopsis. EMBO J 30:3430–3441. taining three HMG-box domains interacts with mitotic and meiotic . – 19. Yoshizumi T, et al. (2006) INCREASED LEVEL OF POLYPLOIDY1, a conserved repressor New Phytol 192:577 589. of CYCLINA2 , controls endoreduplication in Arabidopsis. Plant Cell 18: 41. Genschik P, Criqui MC, Parmentier Y, Derevier A, Fleck J (1998) Cell cycle -dependent fi 2452–2468. proteolysis in plants. Identi cation of the destruction box pathway and metaphase – 20. Imai KK, et al. (2006) The A-type cyclin CYCA2;3 is a key regulator of ploidy levels in arrest produced by the proteasome inhibitor mg132. Plant Cell 10:2063 2075. Arabidopsis endoreduplication. Plant Cell 18:382–396. 42. Pien S, Wyrzykowska J, McQueen-Mason S, Smart C, Fleming A (2001) Local expres- 21. Kramer ER, Scheuringer N, Podtelejnikov AV, Mann M, Peters J-M (2000) Mitotic sion of expansin induces the entire process of leaf development and modifies leaf regulation of the APC activator proteins CDC20 and CDH1. Mol Biol Cell 11: shape. Proc Natl Acad Sci USA 98:11812–11817. 1555–1569. 43. Choi D, Lee Y, Cho HT, Kende H (2003) Regulation of expansin gene expression affects 22. Heyman J, et al. (2011) Arabidopsis ULTRAVIOLET-B-INSENSITIVE4 maintains cell di- growth and development in transgenic rice plants. Plant Cell 15:1386–1398. vision activity by temporal inhibition of the anaphase-promoting complex/cyclosome. 44. Skirycz A, et al. (2011) Pause-and-stop: The effects of osmotic stress on cell pro- Plant Cell 23:4394–4410. liferation during early leaf development in Arabidopsis and a role for ethylene sig- 23. Iwata E, et al. (2011) GIGAS CELL1, a novel negative regulator of the anaphase-pro- naling in cell cycle arrest. Plant Cell 23:1876–1888. moting complex/cyclosome, is required for proper mitotic progression and cell fate 45. Menges M, Hennig L, Gruissem W, Murray JA (2003) Genome-wide gene expression in determination in Arabidopsis. Plant Cell 23:4382–4393. an Arabidopsis cell suspension. Plant Mol Biol 53:423–442. 24. Capron A, et al. (2003) The Arabidopsis anaphase-promoting complex or cyclosome: 46. Saze H, Kakutani T (2007) Epigenetic mutation of a transposon-flanked gene. EMBO J Molecular and genetic characterization of the APC2 subunit. Plant Cell 15:2370–2382. 26:3641–3652.

13858 | www.pnas.org/cgi/doi/10.1073/pnas.1211418109 Eloy et al.