SAMBA, a plant-specific anaphase-promoting complex/cyclosome regulator is involved in early developmentand A-type cyclin stabilizationNubia 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 Biology, VIB, and bDepartment of Plant Biotechnology and Bioinformatics, Ghent University, 9052 Ghent, Belgium; cCenter forProteome Analysis and Mass Spectrometry and dDepartment of Biology, University of Antwerp, 2020 Antwerp, Belgium; eFlemish Institute for TechnologicalResearch (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 largemultiprotein E3 ubiquitin ligase involved in ubiquitin-dependentproteolysis of key cell cycle regulatory proteins, including thedestruction of mitotic cyclins at the metaphase-to-anaphase transi-tion. Despite its importance, the role of the APC/C in plant cells andthe regulation of its activity during cell division remain poorlyunderstood. Here, we describe the identification of a plant-specificnegative regulator of the APC/C complex, designated SAMBA. InArabidopsis thaliana, SAMBA is expressed during embryogenesisand early plant development and plays a key role in organ sizecontrol. Samba mutants produced larger seeds, leaves, and roots,which resulted from enlarged root and shoot apical meristems,and, additionally, they had a reduced fertility attributable to a ham-pered male gametogenesis. Inactivation of SAMBA stabilized A2-type cyclins during early development. Our data suggest thatSAMBA regulates cell proliferation during early development bytargeting CYCLIN A2 for APC/C-mediated proteolysis.

Plant organ size is determined by the total cell number and finalcell size, resulting from cell division and cell expansion, re-

spectively. In most, but not all, cases, the final organ size correlateswith cell number, rendering cell division the main driver thatcontrols growth (1).In all eukaryotes, unidirectional cell cycle progression requires

the coordinated destruction of essential cell cycle regulatoryproteins by ubiquitin-dependent proteolysis pathways (2–5).Specific E3 ubiquitin ligases mediate the recognition of targetproteins (6–8) that are subsequently polyubiquitinated and sub-jected to proteolysis by the 26S proteasome. One of the mostcomplex ubiquitin ligases involved in cell cycle control is theanaphase-promoting complex/cyclosome (APC/C), of whichthe composition can vary from 11 to 13 subunits depending on theorganism. The APC/C complex plays essential roles in mitosis,meiosis, and postmitotically differentiated cells (3, 9–12).The APC/C triggers the metaphase-to-anaphase transition and

the exit from mitosis by mediating the degradation of proteinssuch as securin and mitotic cyclins (13). In plants, the A- and B-type cyclins are subjected to proteolysis by APC/C through rec-ognition of specific amino acid motifs, the destruction (D) andKEN boxes (6, 14, 15). Plant A-type cyclins are produced anddegraded earlier in the cell cycle than B-type cyclins and havedistinct and nonredundant functions in the progression of celldivision (16). Based on their primary structures, the plant A-typecyclins are classified into A1, A2, and A3 groups (17). Thetranscriptional regulation of the CYCLIN A2 (CYCA2) groupcoordinates cell proliferation during plant development (18,19) and CYCA2;3 overexpression enhances cell division (20).The APC/C is regulated partly by two activating proteins

CELL DIVISION CYCLE 20 (CDC20) and CDC20 HOMOL-OGY1/CELL CYCLE SWITCH 52 (CDH1/CCS52), that also

determine substrate specificity (21). Recently, ULTRAVIOLET-B-INSENSITIVE4 (UVI4) and UVI4-like/OMISSION OFSECOND DIVISION1/GIGAS CELL1 (UVI4/OSD1/GIG1)were identified as plant-specific inhibitors of the APC/C that arerequired for proper mitotic progression in Arabidopsis (22, 23).In Arabidopsis, the genes encoding the subunits APC2, APC3a,

APC3b, APC4, APC6, APC8, and APC10 have been investigatedfunctionally. In all cases, except for APC8, the analysis of themutants revealed female gametophytic defects, probably as a con-sequence of the inability to degrade mitotic cyclins (24–28). APC8was shown to be involved in male gametogenesis (29). Moreover,reduced expression levels of APC6 (10) or APC10 (10, 28) ex-hibited several developmental abnormalities, including defects invascular development, whereas an APC4 loss-of-function mutantwas defective in embryogenesis (26). APC3b (CDC27b) has a rolein the maintenance of cell division in meristems during post-embryonic development (30), and overexpression of APC10 en-hances cell division and accelerates the degradation of the mitoticCYCB1;1, leading to increased leaf sizes (28).Here, we functionally analyzed an APC/C regulator of Arabi-

dopsis, designated SAMBA. The sambamutants have an enlargedmeristem size and show growth-related phenotypes, including theformation of large seeds, leaves, and roots; additionally, theirfertility is reduced because of a defect in male gametogenesis. Abiochemical analysis revealed that loss of function of SAMBAstabilizes CYCA2;3. We conclude that SAMBA is a plant-specificnegative regulator of APC/C involved in the degradation of A-type cyclins.

ResultsSAMBA Is a Plant-Specific APC/C Regulator. The SAMBA protein,encoded by AT1G32310, had been identified by tandem-affinitypurification (TAP) associated with the core APC/C and, morespecifically, with the subunits APC3b, APC7, and APC10 inprotein complexes purified from Arabidopsis cell suspensioncultures (31). In a TAP experiment on Arabidopsis cell cultureswith SAMBA as bait, 12 interacting proteins were identified,including the subunits APC1, APC2, APC3b, APC4, APC5,APC6, APC7, APC8, and APC10, except APC11, and the reg-ulators CCS52A2, UVI4, and UVI4-like/OSD1/GIG1 (31).

Author contributions: N.B.E., M.C.E.V.M., and D.I. designed research; N.B.E., N.G., J.V.L.,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.,H.R., G.D.J., and P.C.G.F. analyzed data; and N.B.E. and D.I. wrote the paper.

The authors declare no conflict of interest.1To whom correspondence may be addressed. E-mail: marc.vanmontagu@ugent.be ordirk.inze@psb.vib-ugent.be.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1211418109/-/DCSupplemental.

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To confirm that SAMBA forms a complex with APC/C inplanta, TAP was carried out with 6-d-old SAMBA-expressingseedlings C-terminally fused to the GS tag (see SI Materials andMethods) and proteins were identified by mass spectrometry(MS). In agreement with previous results, SAMBA interactedwith all APC/C subunits in planta, except APC11, which is dif-ficult to identify with the selected analytical approach because ofits small size (Table S1). Also, the interaction with the activatorCCS52A2 was found, but not with CCS52B, UVI4, and UVI4-like/OSD1/GIG1.Although TAP allowed the detection of the entire APC/C

complex, it did not provide insight into the direct interactionsbetween core subunits and associated proteins. Therefore, weperformed yeast two-hybrid (Y2H) assays with the SAMBAprotein against all APC/C subunits identified in Arabidopsis andits two coactivators, CCS52A2 and CCS52B. The results revealeda direct interaction of SAMBA with APC3b (Fig. S1A) but notwith APC1, APC2, APC3a, APC4, APC5, APC6, APC7, APC8,APC10, APC11, or the activators CCS52A2 and CCS52B. Takentogether, the protein interaction data indicate that SAMBA isa component of the APC/C complex in plants, by binding APC3b.SAMBA is 100 aa in length (10.8 kDa) and resembles APC16,

a recently described APC/C subunit conserved throughout meta-zoans (32–34) that is 110–120 residues in size and characterized byfour regions of sequence similarity, referred to as AH1 to AH4(33). Based on these regions of sequence similarity, several can-didate APC16 subunits can be found in plants, including in Ara-bidopsis (Fig. S1B), but except for some remote similarity at theAH2 and AH3 regions, SAMBA lacks sequence homology withAPC16, particularly in the extended homology region AH4 lo-cated toward its C terminus. Inversely, homology searches iden-tified single SAMBA-like proteins in various other plant genomes.Sequence alignments of SAMBA homologs revealed two regionsof sequence conservation [SAMBA homology region (SHR)1 andSHR2], spaced by a 10- to 20-residue-long low-complexity region(Fig. 1A). Because no homologous sequences were found inmetazoans or protista, SAMBA constitutes an APC/C regulatorunique to plants.

SAMBA Expression. Analysis of published microarray datasets anduse of the BioArray (http://www.bar.utoronto.ca) and Genevesti-gator (https://www.genevestigator.com) tools revealed that the

SAMBA expression was very weak in all tissues, except during seeddevelopment. To study the expression pattern of SAMBA, a 1.8-kbfragment upstream of the ATG codon of the SAMBA gene wasfused to a β-glucuronidase (GUS)-GFP tandem reporter cassetteand introduced into Arabidopsis plants. SAMBA expression washigh during embryogenesis (Fig. 1 B and C) but decreased grad-ually when seedlings germinated (Fig. 1 D and F). At 3 d afterstratification (DAS) (Fig. 1D), GUS staining was still well visible inall tissues; at 5 DAS, it diminished and became patchy in roottissues (Fig. 1E); finally, at 8 DAS, it was present only in thehypocotyls (Fig. 1F); and, at later stages of development, the ex-pression was only observed in pollen grains (Fig. 1G).

samba Mutants Develop Enlarged Organs. To assess the role of theSAMBA gene in plant growth and development, we analyzed itsloss-of-function phenotype in two independent T-DNA insertionlines. Both lines were inserted into the first intron (Fig. S1C),completely abolishing gene expression. Because both mutants hadthe same phenotype, they will be further referred to as samba,unless specified. Interestingly, the analysis of samba showed anincreased seed size [up to 143% of the wild type (WT)] (Fig. 2Aand Fig. S2A) and embryo size (Fig. 2B). Additionally, sambarosettes were visibly larger (Fig. 2C) and roots were longer (Fig.2D and Fig. S2B) than those ofWTplants. As a consequence of theincreased root length, the number of lateral roots in sambamutants was higher than that in theWT (Fig. S2C). The increasedgrowth of sambamutants also led to a significant increase in freshand dry weight of shoots and roots (Fig. S2 D–G).To understand the samba growth phenotype, we analyzed both

root and shoot meristem sizes. The root apical meristem (RAM),measured from the quiescent center (QC) toward the point wherecortical cells start to elongate, was on average 26% larger in sambathan in WT (Fig. 2E and Fig. S2H). The shoot apical meristem(SAM) of seedlings at 12 DAS was analyzed by means of a 3Dreconstruction made from serial sections. Volumetric measure-ments of seedlings at 12DAS revealed that themean SAMvolumeof sambawas∼40% higher (139× 103 μm3; n= 19) than that of theWT (99 × 103 μm3; n = 16) (Fig. 2F and Fig. S2I). Also when 3Dreconstructions of the first leaf initials were compared at 4 DAS,leaf primordia of samba plants were on average 80% larger thanthose of the WT (Fig. 2G and Fig. S2J). In conclusion, SAMBAappears 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 plantgenomes. 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).]

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Todemonstrate that the observed growth phenotype is caused bythe samba-1 and samba-2 mutations, we engineered a rescue con-struct (pSAMBA:SAMBA) consisting of 1,817 bp of the promotersequence fused to the SAMBA full-length genomic sequence.Transgenic lines expressing this construction complemented thesamba growth phenotype in both mutant lines analyzed (Fig. S3A).

Cellular Analysis of the Samba Leaf Phenotype. To investigate theeffect of SAMBA inactivation at later stages of vegetative de-velopment, we analyzed leaf growth kinematically. From 4 until 24DAS, the first leaf pair of samba and WT plants, grown side byside, was harvested and leaf blade area, cell number, cell area, andstomatal index were measured. In agreement with the measure-ments of the shoot apex by 3D reconstructions, the leaf size of thefirst leaf pair of samba plants was already larger during the initialstages of development (Fig. 3A, Inset). At 4 DAS, the size of thesamba leaf initials was on average 94% larger than that of the WT(Table S2), but, during further development, it became relativelyless pronounced and at maturity (24 DAS) had increased ∼36%(Fig. 3A). Cellular measurements demonstrated that cell number(Fig. 3B) and, to a lesser extent, cell size (Fig. 3C) contributed tothe increased leaf area. At 4 DAS, samba leaf initials alreadycontained ∼56% more cells than the WT (Fig. 3B Inset and TableS2). However, calculated on the basis of the increase in cell

number over time, the average cell division rate for the entire leafin samba and control plants was similar (Fig. 3D), meaning that thedifference observed in samba mutants happened already beforeanalysis of the first time point (4 DAS).An additional parameter involved in leaf development is the

onset of endoreduplication that marks the transition betweencell division and cell differentiation. Cells from leaves 1 and 2divide up to 10 DAS, after which they gradually exit the cell cycleand start to endoreduplicate (35). At 8 DAS, leaves 1 and 2 ofsamba and control lines had an equally low endoreduplicationindex (EI), representing the mean number of endoreduplicationcycles per nucleus. The EI of samba leaves was significantlyhigher between 12 and 20 DAS than that of equivalent controlleaves, particularly because of an increase in the 8C and 16C cellpopulation (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 1and 2 at 10 DAS for microarray transcript profiling. At 10 DAS,leaves 1 and 2 of samba mutants were clearly larger than the WTleaves. A total of 298 genes were differentially expressed (foldchange 1.5; false discovery rate < 0.05): 287 were up-regulated,and 11 down-regulated. PageMan (36) analysis indicated thatamong the 287 up-regulated genes, the most overrepresentedclasses 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 ofa 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 withthe nCounter system that allows a direct multiplexed analysis ofselected transcripts (37). Most of the G2/M-phase–related genesanalyzed, such as CYCB1;1, cyclin-dependent kinase CDKB2;1,and 3xHMG-box2 (38–40), were significantly down-regulated at allthree developmental time points in sambamutants, whereas genesencoding cell cycle inhibitors, such asKIP-RELATED PROTEIN 1,SIAMESE RELATED 1 (SMR1), SMR2, and SMR3, were up-regulated, in particular at 8 DAS. A similar expression profile wasfound for the SHOOT MERISTEMLESS and EXPANSIN11(EXP11) genes (Fig. 3F). In conclusion, the expression analysis isconsistent with the hypothesis that loss of SAMBA advances earlydevelopment as seen by the down-regulation of mitotic cell cyclegenes and the up-regulation of genes encoding cell cycle inhib-itors, expansin, and ethylene-responsive genes.

Loss of SAMBA Stabilizes CYCA2;3. Recently, APC10 had beenshown to regulate theCYCB1;1 abundance (28), and the twoAPC/C inhibitors UVI4 and UVI-like/OSD1/GIG1 stabilize CYCA2;3and CYCB1;2, respectively (22, 23). Therefore, we tested whetherSAMBA interacts with mitotic cyclins and affect their stability.Y2H assays between SAMBA and six different mitotic cyclins(CYCA1;1, CYCA2;2, CYCA2;3, CYCA3;1, CYCB1;2, andCYCB2;2) revealed that SAMBA interacted with CYCA2;2 andCYCA2;3, weakly with CYCA1;1, and not with CYCA3;1,CYCB1;2, or CYCB2;2 (Fig. 4A). When the D-box in CYCA2;3was deleted, no interaction with SAMBA was observed (Fig. 4B).To substantiate the Y2H results, WT and samba mutant plants

were stably transformed with a hemagglutinin (HA)-taggedCYCA2;3 under control of the CaMV35S promoter. Subsequently,the CYCA2;3 abundance was analyzed with a commerciallyavailable anti-HA antibody. Because SAMBA is strongly expressedduring early developmental stages, protein extracts of etiolatedplants at 5 DAS, were used (Fig. S4A), avoiding interference withthe large subunit of Rubisco. After fractionation by SDS/PAGE,Western blotting revealed a 51-kDa band specific to CYCA2;3-HA only in the samba mutant (Fig. 4C), indicating the stabili-zation of CYCA2;3 in samba plants. Quantitative (q)RT-PCRanalysis confirmed the overexpression of CYCA2;3 and the ex-pression levels of SAMBA in the lines analyzed (Fig. S4 B and C).

Fig. 2. Representative pictures of the phenotypical analysis of samba. (A)Seed size of WT and samba. (B) Mature embryos of WT and samba. (C)Seedlings of WT and samba at 15 DAS. (D) Root phenotype of WT and sambaat 10 DAS. (E) Propidium iodide–stained root meristems of WT and samba at5 DAS. Arrowheads mark the QC position and the meristem end, defined bythe position where cells start elongating. (F) Side view of a reconstructedsamba and WT SAM at 12 DAS. (G) Adaxial view of reconstructed primordiaof leaves 1 and 2 of WT and samba at 4 DAS. [Scale bars: 100 μm (A and B);1 mm (C and D); 20 μm (E); 50 μm (F and G).] The number of samples ana-lyzed and the statistical analysis are provided in Fig. S2.

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This result, together with the Y2H experiments, implies thatSAMBA targets CYCA2;3 for degradation.To assess the specificity of the CYCA2;3 stabilization in

samba mutant plants, we crossed transgenic plants carrying theCYCB1;1 promoter fused to the CYCB1;1 D-box-GUS/GFPconstruct with samba plants. Expression of the pCYCB1;1:CYCB1;1 D-box-GUS/GFP construct allowed us to estimate therate of CYCB1;1 degradation in proliferating cells (28, 41). TheCYCB1;1 degradation rate did not differ in the leaf primordiaor in the RAM of WT and samba mutant plants (Fig. S4 D andE), suggesting that SAMBA specifically targets A2-type and notB-type cyclins for degradation.

SAMBA Is Required for Male Gametogenesis. Although homozygoussamba mutants could be obtained, immature siliques after self-fertilization of the samba-1 and samba-2 mutants contained ∼46–47% (403 of 875 and 249 of 531, respectively) of aborted ovules,

which were small, whitish, and shrunken (Fig. S5A). This phenotypewas probably attributable to malfunctioning of the male gameto-genesis, because samba mutants produced considerably less viablepollen than WT plants. Indeed, in anthers of samba mutants, only29% of pollen grains colored red after Alexander’s staining, in-dicating viability (n = 1,606; Fig. 5 A and B).Reciprocal crosses between samba-2/+ and WT plants were

carried out to determine the T-DNA transmission. The trans-mission rate was severely reduced only when heterozygous sambamutants were used as paternal donor, confirming the dysfunctionof the male gametogenesis (Fig. S5B). Furthermore, the con-struct pSAMBA:SAMBA fully complemented the ovule abortionand the pollen viability phenotypes, showing that they werecaused by the SAMBA inactivation (Fig. S3 B and C). Tounderstand which stage of pollen development is affected in thesambamutants, we analyzed the progression through male meiosis.In both samba-1 and samba-2 homozygous mutants, meiosis was

Fig. 3. Kinematic analysis of leaf growth in samba andWT plants. (A) Leaf blade area of the first leaf pair of samba (gray) andWT (black) plants grown in vitrofrom 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) Celldivision rate. (E) Ploidy distribution of thefirst leaf pair of samba andWTplants. (Inset) Ploidy distributionmeasured byflow cytometry of thefirst 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 normalizedagainst the expression level of the housekeeping genes and expression was compared with the expression data in the WT. Data are means ± SE (n = 3).

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normal and formed balanced spore-containing tetrads (Fig. S5C).Also, analysis of chromosome spreads revealed no abnormalities(Fig. S5D). We conclude that SAMBA is not required for meiosis,and, therefore, the defective male gametogenesis must bepostmeiotic. Indeed, analysis of the pollen grains at the maturestage revealed that WT pollen contained one vegetative nucleusand two sperm nuclei (Fig. 5C), whereas ∼21% (n = 682) of thesamba pollen contained only one vegetative nucleus (Fig. 5D).These results indicate that the lack of SAMBA expressioninterferes with mitosis I of pollen microspore development.

DiscussionAlthough the APC/C plays an important regulatory role in theeukaryotic cell cycle and has been subject to numerous studies (12),still much has to be learned on its biochemical structure and reg-ulation. For example, only recently, APC16 had been identified asa functional APC/C component in animals (33, 34) and, similarly,UVI4 and UVI4-like/OSD1/GIG1 as plant-specific inhibitors ofAPC/C (22, 23). Here, we identified SAMBA as a negative

regulator of APC/C also unique to plants. TAP of protein com-plexes with SAMBA as bait allowed in planta isolation of the entireAPC/C complex.The SAMBA protein associates with the APC/C through direct

interaction with APC3b that also binds to the CCS52 and CDC20proteins throughout the common C-box and IR motifs (25). Al-though SAMBA does not have these motifs, it might interact withAPC3b via the highly conserved SHR region found in all putativeSAMBA orthologs. We speculate that SAMBA competes throughinteraction 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 regulatoryrole 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. Laterin development, the size differences between samba mutants andWT plants decrease. No size difference can be observed duringflower development. Other APC/C regulators might act at differ-ent developmental stages. For example, APC10 is involved inCYCB1;1 degradation and has a role during vascular development(10, 28), whereas the APC/C inhibitor UVI4 is important for tri-chome branching and UVI4-like/OSD1/GIG1 is required for theproper development of guard cells (22, 23).A detailed kinematics analysis revealed that the increased

growth in samba plants results from enhanced cell proliferationduring embryogenesis and very early, postembryogenic develop-ment, noticeable by the difference in leaf size and cell number atthe first time point analyzed (4 DAS). The size of the SAM andearly leaf primordia in the samba plants was on average 40% and94% larger than that of the WT plants, respectively. Moreover,neither cell division rates nor timing were affected in the sambamutants. We interpret that the loss of function of SAMBA stim-ulates cell proliferation in developing seeds and early seedlings,and this initial size difference is maintained throughout leaf de-velopment until maturity.Whereas differences in cell number already occurred at early

developmental stages, differences in cell size became significantonly at later stages of leaf development. Moreover, the endor-eduplication rate of samba is higher, resulting from increasedfractions of 8C, 16C, and 32C nuclei, suggesting that despite theincreased cell number cells exit the division cycle faster. This hy-pothesis is supported by the expression analysis of cell cyclemarker genes that revealed a more rapid down-regulation of mi-totic genes and an up-regulation of the cell cycle inhibitory genesin 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, theethylene signal transduction was clearly overrepresented, whichplays a prominent role in cell division regulation during leafdevelopment 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, atleast for CYCA2;3, this interaction depends on the presence of theD-box, suggesting that SAMBA might target A-type cyclins for theAPC/C-mediated proteolysis. In support of this hypothesis, sambamutants highly accumulate the A2-type cyclins. This CYCA2 ac-cumulation during early seed and seedling development is likelyresponsible for the stimulation of cell division, as corroborated bythe enhanced cell proliferation in plants with increased CYCA2;3levels, attributable to a mutation in the D-box (20). Also, otherAPC/C components were shown to control cyclin stabilization.UVI4 negatively regulates the endoreduplication onset in Ara-bidopsis by restraining the activity of the CCS52A1 activatorsubunit. As a result, uvi4 mutants fail to accumulate CYCA2;3during the S phase (22). Conversely, UVI4-like/OSD1/GIG1 mayprevent the occurrence of endoreduplication by selective

Fig. 4. CYCA2;3 stabilization. (A and B) Y2H interactions between SAMBAand different cyclins and CYCA2;3ΔD-box (D-box–mutated), respectively. (C)Detection of CYCA2;3-HA protein levels in etiolated seedlings of WTCYCA2;3-HA (control line) and samba CYCA2;3-HA at 5 DAS.

Fig. 5. Pollen phenotype of samba mutants and WT. The viability of pollengrains was tested by coloration with Alexander’s stain in WT (A) and samba(B) plants. The red staining indicates that the pollen is viable. (C and D) WTand samba pollen stained with DAPI and observed under UV fluorescence,respectively. Two densely stained sperm nuclei and one large diffuse vege-tative nucleus are visible in the WT but frequently only one single vegetativenucleus in samba (arrows). [Scale bars: 500 μm (A and B); 10 μm (C and D).]

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inhibition of CCS52A2 activity during mitosis, thereby pro-moting the accumulation of CYCB1;2 (23).Mutations in most APC/C subunits affect female and/or male

gametogenesis (24–29). Here, we show that samba is defective inmitosis I of pollen development, resulting in pollen withoutsperm nuclei. Mutants of APC8 and APC13 (29, 46) show phe-notypes similar to those described here for the samba plants.Both mutants are affected in pollen development, leading to anincreased proportion of uninucleated mature pollen, indicatingthat the APC/C is required during the first mitosis of the malegametophytic development. Also, in agreement with the sambamale phenotype, SAMBA was expressed strongly in pollen grainsbut not in female organs.

Materials and MethodsA full discussion of materials and methods can be found in SI Materialsand Methods.

Plant Material and Production of Transgenic Plants. Samba mutant plants(seed code SALK_018488 and SALK_048833) were obtained from the SALKcollection (http://signal.salk.edu).

Kinematic Analysis. The complete kinematics was analyzed on leaves 1 and 2of 8–10 samba and WT plants grown in vitro and harvested daily from 4 to24 DAS.

Phenotypical Analysis of samba. For measurements of different parameters ofthe samba mutant phenotype, see SI Materials and Methods.

Immunoprecipitation and Protein Gel Blotting. Whole etiolated plant tissue (5DAS) was ground with 4-mm metal beads. Proteins were extracted withhomogenization buffer. After centrifugation at 20,000 × g, 300 μg of totalprotein extract was incubated overnight with Anti-HA Affinity Matrixaccording to the instructions of the manufacturer (Roche).

ACKNOWLEDGMENTS. We thank Moritz Nowack for advice with pollen anal-ysis and Martine De Cock for help in preparing the manuscript. This work wassupported by Ghent University (“Bijzonder Onderzoeksfonds Methusalem”

Project Grant BOF08/01M00408 and Multidisciplinary Research Partnership“Biotechnology for a Sustainable Economy” Project 01MRB510W), the BelgianScience Policy Office (BELSPO) [Interuniversity Attraction Poles Programme(IUAP VI/33) and a postdoctoral fellowship to N.B.E.], and the European UnionSixth Framework Programme (“AGRON-OMICS” Grant LSHG-CT-2006-037704).S.D. and H.V. were supported by a predoctoral fellowship from the Agency forInnovation by Science and Technology. J.V.L. is a postdoctoral fellow of theResearch Foundation-Flanders.

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