The Anaphase-Promoting Complex Is a Dual Integrator That
Regulates Both MicroRNA-Mediated Transcriptional
Regulation of Cyclin B1 and Degradation of Cyclin B1 during
Arabidopsis Male Gametophyte Development
Binglian Zheng,a,bXuemei Chen,band Sheila McCormicka,1
aPlant Gene Expression Center, U.S. Department of Agriculture/Agricultural Research Service and Department of Plant and
Microbial Biology, University of California-Berkeley, Albany, California 94710
bDepartment of Botany and Plant Sciences and Institute of Integrative Genome Biology, University of California, Riverside,
The anaphase-promoting complex/cyclosome (APC/C), an essential ubiquitin protein ligase, regulates mitotic progression
and exit by enhancing degradation of cell cycle regulatory proteins, such as CYCB1;1, whose transcripts are upregulated by
DUO POLLEN1 (DUO1). DUO1 is required for cell division in male gametophytes and is a target of microRNA 159 (miR159) in
Arabidopsis thaliana. Whether APC/C is required for DUO1-dependent CYCB1;1 regulation is unknown. Mutants in both
APC8 and APC13 had pleiotrophic phenotypes resembling those of mutants affecting microRNA biogenesis. We show that
these apc/c mutants had reduced miR159 levels and increased DUO1 and CYCB1;1 transcript levels and that APC/C is
required to recruit RNA polymerase II to MIR159 promoters. Thus, in addition to its role in degrading CYCB1;1, APC/C
stimulates production of miR159, which downregulates DUO1 expression, leading to reduced CYCB1;1 transcription. Both
MIR159 and APC8–yellow fluorescent protein accumulated in unicellular microspores and bicellular pollen but decreased in
tricellular pollen, suggesting that spatial and temporal regulation of miR159 by APC/C ensures mitotic progression.
Consistent with this, the percentage of mature pollen with no or single sperm-like cells increased in apc/c mutants and
plants overexpressing APC8 partially mimicked the duo1 phenotype. Thus, APC/C is an integrator that regulates both
microRNA-mediated transcriptional regulation of CYCB1;1 and degradation of CYCB1;1.
The gametes of flowering plants are formed within haploid
gametophytes (McCormick, 2004). In the male gametophyte,
each haploid microspore divides asymmetrically to produce a
larger vegetative cell that will form the pollen tube and a smaller
generative cell, which divides symmetrically to form twin sperm
cells. These sperm cells are delivered to the embryo sac via the
pollen tube, where they fuse with an egg and a central cell to
produce embryo and endosperm, respectively. The molecular
mechanisms underlying the production of male gametes remain
largely unknown, although a few required genes have been
2006; Mori et al., 2006; Nowack et al., 2006; Kim et al., 2008;
Brownfield et al., 2009b; Gusti et al., 2009; Ron et al., 2010).
Among these genes, DUO POLLEN1 (DUO1) encodes a male
germ cell–specific R2R3Myb protein(Rotman et al., 2005) thatis
required for expression of the Arabidopsis thaliana G2/M regu-
lator CyclinB1;1 (CYCB1;1) in the male germline (Brownfield
et al., 2009a), suggesting an integrative role for DUO1 in cell
sperm cell production. DUO1 mRNA is targeted directly by
microRNA159 (miR159) (Palatnik et al., 2007), which leads to its
Cell proliferation in all eukaryotes depends on the ubiquitin
ligase (E3) activity of the anaphase-promoting complex/cyclo-
at least 11 subunits (Capron et al., 2003a). Among them, APC3,
APC5, APC6, APC7, and APC8 each contain a tetratricopeptide
repeat (TPR) protein–protein interaction domain and are ex-
pected to function as receptors that interact with regulatory
proteins (Lima et al., 2010). Without APC/C, cells cannot sepa-
rate their sister chromatids in anaphase,cannot exit frommitosis
to divide into two daughter cells, and cannot initiate the steps
necessary for DNA replication in S phase (Peters, 2006). The
A- and B-type cyclins, thus facilitating exit from mitosis (Glotzer
et al., 1991). The genetic inactivation of APC/C has caused
lethality in all species in which it has been investigated so far (Yu
et al., 1998; Yamashita et al., 1999; Cullen et al., 2000; Bentley
et al., 2002; Garbe et al., 2004; Pa ´l et al., 2007; Jin et al., 2010).
1Address correspondence to email@example.com.
The author responsible for distribution of materials integral to the
findings presented in this article in accordance with the policy described
in the Instructions for Authors (www.plantcell.org) is: Sheila McCormick
CSome figures in this article are displayed in color online but in black
and white in the print edition.
WOnline version contains Web-only data.
The Plant Cell, Vol. 23: 1033–1046, March 2011, www.plantcell.org ã 2011 American Society of Plant Biologists
is a temperature-sensitive embryonic lethal due to defects in
germline meiosis and mitosis (Garbe et al., 2004).
In Arabidopsis, single-copy genes encode counterparts of all
known vertebrate APC/C subunits, except for APC3/CDC27,
which has two genes (Capron et al., 2003a). Plant APC/C is
involved in cell cycle regulation. For example, APC2 and APC6
are important for female gametophyte development (Capron
et al., 2003b; Kwee and Sundaresan, 2003), as plants with loss-
of-function alleles of either of these genes exhibited female
gametophytic lethality due to cell cycle arrest at an early stage of
embryo sac development. Reciprocal crosses with the apc6/
nomega mutant suggested that most defects came from the
female, although male transmission was somewhat reduced
(Kwee and Sundaresan, 2003). Interestingly, a mir159ab double
mutant (Allen et al., 2007) phenocopied the apc13 mutant (Saze
and Kakutani, 2007) and the apc6 and apc10 RNA interference
lines (Marrocco et al., 2009) that had pleiotropic morphological
defects, including small siliques, curled leaves, and abnormal
phylotaxy, suggesting that both APC/C and miR159 might be
required for similar developmental processes.
To identify new genes in miRNA biogenesis and function
pathways, we performed a forward genetic screen. One mutant
had pleiotrophic developmental phenotypes expected for mu-
tants in miRNA pathway genes, such as DICER-LIKE1 (DCL1)
(Jacobsen et al., 1999), but surprisingly it was a point mutation in
APC8. This point mutant, apc8-1, resulted in reduced MIR159
transcription and lessened accumulation of mature miR159. This
caused an increase in transcripts of both DUO1 and CYCB1;1 in
pollen. We further showed that APC/C is required for RNA
polymerase II (Pol II) recruitment to promoters of MIR genes.
CYCB1;1–green fluorescent protein (GFP) accumulated in both
the vegetative cell and sperm cells of tricellular pollen in apc8-1
but was absent in wild-type tricellular pollen. We also observed
an increased incidence of unicellular and bicellular pollen in
mature anthers of apc8-1 and increased ratio of bicellular pollen
in mature pollen by overexpressing APC8 in male germlines,
suggesting that disrupted balance of CYCB1;1 levels prevented
efficient transition of male gametophytes through the bicellular
and tricellular stages. Similar results were obtained by analyzing
a T-DNA insertion allele of APC13, another component of the
APC/C. Therefore, we propose a dual role for the APC/C both
in coordinating miRNA-mediated transcriptional regulation of
CYCB1;1 and in direct protein degradation of CYCB1;1.
Isolation and Characterization of a Weak APC8 Allele
et al., 1999), and seeds from dcl1 null alleles cannot be obtained
due to embryo lethality. However, a weak allele of dcl1, dcl1-14
(in which the T-DNA insertion is located in the promoter region,
SALK_056243; see Supplemental Figure 1A online), showed
curly leaves, abnormal reproductive development, and reduced
miRNA levels (seeSupplementalFigures1B and 1Conline) butis
fertile. We therefore performed a genetic screen after ethyl
of mutants with enhanced or suppressed phenotypes. We
isolated a mutant with much more severe developmental phe-
notypes than those of dcl1-14. The pleiotropic phenotypes
included distorted leaf shapes, abnormal shoot meristem devel-
opment, and a delay in the transition from the vegetative to the
reproductive stage (Figures 1A and 1B). Moreover, the mutant
bushy inflorescences and shorter siliques (Figures 1C and 1D).
Weshowedthatthesephenotypes weredcl1-14 independentby
backcrossing to wild-type plants. Therefore, the mutant without
dcl1-14 background was used for our subsequent analyses.
Collectively, these pleiotropic developmental defects suggested
that the mutated gene had roles at multiple stages of plant
To further understand the basis of these heritable pleiotropic
phenotypes, we examined their inheritance in F2 progeny from a
erecta plant. The genotype was determined for 661 F2 plants
F2 population. Characterization of Col/Landsberg erecta poly-
morphisms throughout the genome showed that all plants with
one region on chromosome 3, suggesting that a disrupted gene
in this region was responsible for the mutant phenotype. This
region was narrowed down to an interval between BAC clones
F18E5 and T8O5 (five recombinants and two recombinants,
respectively, out of 1322 chromosomes examined). We se-
quenced the genes in this region and found a point mutation (G
to A) in the coding region of APC8. This mutation resulted in the
conversion of a highly conserved Asp (Asp-309) to an Asn at a
position adjacent to the TPR domain (Figure 1F). To confirm that
this mutation was responsible for the phenotype, a genomic
fragment was fused to yellow fluorescent protein (YFP) and
introduced into the mutant. The morphological phenotypes were
fully rescued in 23 of 25 plants (Figure 1E). Therefore, we
concluded that the phenotypes were due to the mutation in
APC8 and designated this allele apc8-1.
Arabidopsis APC8 Can Complement an APC8 Mutant in
To determine whether Arabidopsis APC8 is a functional ortholog
of APC8 found in other eukaryotes, we tested whether Arabi-
dopsis APC8 was able to functionally complement an APC8
mutant in fission yeast. We introduced full-length cDNAs of both
APC8 and mutant apc8-1 (APC8 D to N) into a yeast expression
vector under the control of the Gal promoter. These constructs
were then transformed into the temperature-sensitive cut23-174
mutant, which has a lesion in the APC8 gene in S. pombe. This
mutant can growat 288Cbut not at 378C (Yamashita et al., 1999).
Expression of wild-type APC8 rescued the yeast mutant strain at
the restrictive temperature, whereas an empty vector or the
mutant apc8-1 did not (Figures 2A and 2B). Moreover, a con-
struct in which the Asp was changed to Glu (APC8 D to E) only
2B), which reinforces the idea that the conserved Asp is impor-
tant for APC8 function.
1034The Plant Cell
Subcellular Localization of APC8 Is Regulated during Male
Microarray analyses indicated that APC/C genes were ubiqui-
tously expressed in Arabidopsis tissues (Zimmermann et al.,
RT-PCR analyses using cDNA samples from different Arabi-
dopsis tissues. APC8 expression was detected in a variety of
online). To substantiate the RT-PCR analyses, we constructed
different promoter-reporter constructs. The b-glucuronidase
(GUS) reporter was expressed in almost all tissues examined,
including leaves, shoot apical meristems, inflorescences, and
siliques (see Supplemental Figure 2B online). During male ga-
metophyte development, the GFPreporter was detectable at the
unicellular and bicellular stages but appeared less intense at the
tricellular stage (see Supplemental Figure 2C online). We there-
fusion protein during male gametophyte development. This
protein fusion construct complemented the apc8-1 phenotypes
completely (Figure 1E), showing that YFP-tagged APC8 was
functional. Foci of APC8-YFP were visible in the nuclei and
cytoplasm of unicellular microspores (Figure 3A) and bicellular
pollen (Figure 3B), but only a very faint signal was detected in
tricellular pollen (Figure 3C), and no signal was detectable in
in different microspores and pollen grains (Figure 3).
APC/C Is Required for Efficient Male Transmission
Microscopy analysis showed that the siliques in apc8-1 had
led to obviously reduced seed set (Figure 4B). The mutant was
backcrossed to the wild type, and the F1 was allowed to self-
pollinate. Only ;10% of the F2 progeny had the mutant pheno-
types, instead of the expected 25%. These results suggested
that the mutant had transmission problems. Reciprocal crosses
Figure 1. Phenotypes and Complementation of the apc8-1 Mutant.
(A) A representative 5-week-old seedling of apc8-1.
(B) A representative 8-week-old apc8-1 plant.
(C) Close-up of fasciculate siliques in apc8-1.
(D) A representative 12-week-old apc8-1 plant.
(E) Complementation of apc8-1. A representative apc8-1 plant carrying the pAPC8:APC8-YFP transgene.
(F) Partial amino acid sequences of APC8 in various species. Identical residues are white on black background, and conserved residues are white on
gray background. Asterisk indicates the Asp (D309) that was mutated to Asn in apc8-1. At, Arabidopsis thaliana; Pt, Populus trichocarpa; Os, Oryza
sativa (japonica); Zm, Zea mays; Hs, Homo sapiens; Mm, Mus musculus; Xl, Xenopus laevis; Dm, Drosophila melanogaster; Sp, Schizosaccharomyces
Bars = 1 cm in (A), (B), (D), and (E), and 1 mm in (C).
[See online article for color version of this figure.]
APC/C Regulates CYCB1;1 mRNA Levels1035
with wild-type plants showed that transmission through the
female was mildly affected but was strongly perturbed through
the male (Table 1). To rule out the possibility that disturbed
male transmission was specific to APC8, we analyzed the
T-DNA insertion allele of another APC/C component, APC13
(SALK_027397, here named apc13-2), which was reported to
have a bonsai phenotype (Saze and Kakutani, 2007) similar to
that of apc8-1. We found slightly reduced seed set in apc13-2
(Figure 4B), and reciprocal crosses with wild-type plants indi-
cated that male transmission was more affected than female
transmission (Table 1).
Because decreased APC/C function resulted in a reduced
seed set phenotype, we next tested whether increased APC/C
function had an effect on seed set. We generated transgenic
plants overexpressing APC8 under the control of the HISTONE
THREE RELATED 10 (HTR10) promoter. HTR10 is specifically
expressed in the male germ line, and HTR10 accumulates in
generative cell nuclei and subsequently in sperm cell nuclei
(Ingouff et al., 2007). We obtained 24 independent T1 lines; T2
plants fromfive T1 lines withsingle copy insertions were used for
further analyses. The T-DNA cassette carrying APC8 also con-
tained a pollen-specific marker (LAT52-GFP), which we used to
confirm homozygous plants. We measured the relative expres-
sion levels of APC8 RNA in these plants by quantitative RT-PCR
(qPCR) and compared the expression levels with those in wild-
type plants (see Supplemental Figure 3A online). We also scored
the seed set (see Supplemental Figure 3B online). All T2 prog-
enies tested showed higher (3.4- to 15.2-fold) APC8 expression
levels (see Supplemental Figure 3A online). These plants also
had, on average, 27% reduced seed set (see Supplemental
Figure 3B online), indicating that the higher APC8 levels in the
male germline disrupted seed set.
APC/C Is Necessary for Tricellular Pollen Formation
Given the importance of cell cycle regulation by APC/C (Peters,
2006) and the dynamic expression of APC8 during male game-
tophyte development (Figure 3), we suspected that disrupting
APC/C function might lead to disorganized cell divisions dur-
ing male gametophyte development. Indeed, when mature
pollen from apc8-1 plants was stained with 4’,6-diamidino-2-
Figure 2. Arabidopsis APC8 Complements the Fission Yeast APC8 Mutant cut23-174.
(A) Growth at the permissive (288C) or restrictive (378C) temperature of the cut23-174 yeast strain carrying an empty vector (pYPGE15), APC8
(pYPGE15-APC8), APC8 D to E (pYPGE15-APC8, D was mutated to E), or APC8 D to N (pYPGE15-APC8, cDNA from the apc8-1 mutant).
(B) Quantitative analysis of growth (OD600measurements) at the permissive (288C) or restrictive (378C) temperature of the cut23-174 yeast strain
carrying each plasmid as in (A) at 0, 12, and 24 h growth in liquid YEA media. Three colonies for each construct were analyzed, and all measurements
represent the average of three biological replicates with error bars representing the SE.
1036The Plant Cell
of wild-type APC8 into apc8-1 completely rescued these defects
of male gametophyte development (Figure 4C), confirming that
apc8-1 was responsible for these phenotypes. The defects in
pollenmaturation in apc13-2 showeda similar trend but were less
severe than those in apc8-1 (Figure 4C). A representative HTR10:
bicellular pollen, but no change in the incidence of unicellular
pollen (Figure 4C), consistent with the specific expression of
HTR10 in the male germline.
Ifthevegetativecellandthegenerative cellfailtodivide inapc/
c mutants and in APC8 overexpression lines, the expectation
might be that further DNA replication would not occur. We
therefore investigated DNA content in the vegetative cell nuclei
and the generative cell-like nuclei in apc8-1 and in the generative
cell-like nuclei in an HTR10:APC8 plant (line 8-2). Relative to the
nuclear DNA content in the wild type, nuclear DNA content in
both the vegetative cell nuclei and the generative cell-like nuclei
in apc8-1 was significantly increased, to nearly 2C (Figure 4D),
replication but cannot enter the subsequent mitosis. As ex-
pected, the nuclear DNA content in the generative cell-like nuclei
in HTR10:APC8-expressing plants increased, also to nearly 2C
(Figure 4D). To examine whether pollen cell fate was disturbed,
we introduced pHTR10:HTR10-mRFP (Ingouff et al., 2007) into
apc8-1. This marker was observed in sperm cell nuclei of apc8-1
mature pollen (see Supplemental Figure 3C online), indicating
that sperm cells in apc8-1 had not lost their gametic fate. Taken
together, our results indicate that APC/C plays an important role
in mitotic cell cycle progression during male gametophyte de-
velopment, promoting tricellular pollen formation.
APC/C Is Required for miR159 Accumulation, and APC/C
Promotes Pol II Recruitment to MIR159
Because apc8-1 was identified while looking for miRNA accu-
mulation mutants, we hoped to determine the molecular mech-
by examining whether apc8-1 was compromised in miRNA
accumulation. Of 10 tested miRNAs, miR159 accumulation
was obviously reduced (Figure 5A; see Supplemental Figure 4A
online), and this molecular phenotype was rescued by wild-type
APC8 (Figure 5A), suggesting that APC8 was involved in the
miR159 biogenesis pathway. We also found obvious reductions
detectable changes for miR156, miR164, miR167, miR168, and
miR171 (see Supplemental Figure 4Aonline), suggesting that the
role of APC8 in miRNA biogenesis is locus specific. Similar
Figure 3. Subcellular Localization of APC8 during Male Gametophyte Development.
Representative fluorescence microscopy images of unicellular pollen (A), bicellular pollen (B), tricellular pollen (C), and mature pollen (D) from at least 10
individual T1 apc8-1 plants harboring the pAPC8:APC8-YFP construct. Three examples for each developmental stage were shown. The left panel for
each represents YFP epifluorescence of pAPC8:APC8-YFP. The middle panel for each indicates the developmental stage determined by DAPI staining.
The right panel for each shows an overlay of the YFP and DAPI epifluorescence signals. Arrowheads show faint YFP signals. Bars = 10 mm.
[See online article for color version of this figure.]
APC/C Regulates CYCB1;1 mRNA Levels 1037
defects in mature miR159 accumulation were observed in
apc13-2 (see Supplemental Figure 4A online). miR159 is en-
coded by three genes (MIR159a, MIR159b, and MIR159c) (Park
et al., 2002; Rhoades et al., 2002).
To investigate which step in miR159 biogenesis was affected,
we used qPCR to examine whether primary (pri) miRNA tran-
scription of each MIR159 was affected in the pollen of apc/c
mutants. We detected reduced pri-miR159 transcription from all
three MIR159 genes in apc8-1 and apc13-2, most notably an
;70% decrease for MIR159c in apc8-1 (Figure 5B). As ex-
pected, overexpression of APC8 resulted in increased pri-
miR159 transcription (Figure5B).Tofurtherconfirmtheseresults
and to determine the expression patterns of each MIR159 during
male gametophyte development, we generated constructs in
which the promoter regions of MIR159a, b, or c were fused to
GFP. We introduced these constructs into wild-type and apc/c
mutants and examined multiple transgenic lines for each con-
struct. In the wild type, MIR159a-GFP, MIR159b-GFP, and
Figure 4. Seed Set Analysis and Pollen Development of apc8-1.
(A) Dissected mature siliques of wild-type (Col-0, top) and apc8-1 plants (bottom). Undeveloped ovules are indicated with arrows. The undeveloped
ovules are tiny and white, whereas developing seeds are large and green. Bar = 500 mm.
(B) Percentage of normal seeds (light gray), aborted seeds (dark gray), and undeveloped ovules (black) from self-pollinated Col-0, apc8-1, and apc13-2
plants. Error bars represent SD from the mean of 10 siliques from each of 20 individual plants.
(C) Distribution of uninucleate microspores (UP; light gray), bicellular pollen (BP; dark gray), and tricellular pollen (TP; black) in mature anthers. Pollen
was stained with DAPI, and the percentage of each stage was determined for Col-0, apc8-1, APC8-YFP apc8-1, and HTR10:APC8 (line 8-2). All
measurements represent the average of three biological replicates with error bars representing the SE. For each independent replicate for each
genotype, 600 to 800 pollen grains from 5 to 10 plants were analyzed.
(D) DNA content of vegetative cell nuclei (VCN; light gray), generative cell-like nuclei/sperm cell nuclei (GCLN/SCN; dark gray) in pollen from different
genotypes. The relative C values of apc8-1 (n = 108) and HTR10:APC8 (n = 126 from line 8-2) were calculated from DAPI fluorescence values normalized
to the mean fluorescence of wild-type cells (n = 98 for vegetative cell nuclei; n = 112 for sperm cell nuclei). n indicates the number of pollen grains that
was measured. All measurements represent the average of three biological replicates, with error bars representing the SE.
[See online article for color version of this figure.]
Table 1. Genetic Analysis of apc8-1 and apc13-2
Parental GenotypesGenotype of F1 Plants
$ 3 #
apc/+ Wild Type
apc8-1/+ 3 wild type
Wild type 3 apc8-1/+
apc13-2/+ 3 wild type
Wild type 3 apc13-2/+
136/172 3 100% = 79.1%
61/238 3 100% = 25.2%
158/187 3 100% = 84.5%
109/261 3 100% = 41.8%
1038 The Plant Cell
MIR159c-GFP were strongly expressed at the unicellular micro-
spore stage, detectable at the bicellular stage, but undetectable
at the tricellular stage; their expression was restricted to vege-
reporter gene fusions was detectable at any stage of male
gametophyte development. Consistent with the much weaker
phenotypes in apc13-2 than in apc8-1 described above, we
observed only a slight reduction of expression of the three
MIR159-GFPs in apc13-2 (see Supplemental Figure 4B online).
Since GFP signal intensity was not a quantitative measure
of reporter gene expression, we performed qPCR to determine
the levels of GFP mRNA in inflorescences. Indeed, GFP mRNA
levels were lower in apc/c mutants than in the wild type
(see Supplemental Figure 4C online). These results indicate
that APC/C is required for miR159 biogenesis during male
gametophyte development and that it acts at the transcriptional
miR159 and the activities of the MIR159 promoters were com-
could be an indirect effect of reduced expression of certain
known genes in the miRNA biogenesis pathway. To evaluate this
possibility, we first examined the expression of several miRNA
biogenesis pathway genes in apc8-1 by qPCR. None of these
genes was affected in apc8-1 (see Supplemental Figure 5A
online). Furthermore, the protein levels of these genes were
comparable to those in the wild type (see Supplemental Figures
miRNA levels in the apc/c mutants are unlikely due to reduced
expression of miRNA biogenesis genes. This result prompted us
to examine whether APC/C plays a direct role in the transcription
and plants (Lee et al., 2004; Xie et al., 2005; Kim et al., 2011).
Therefore, we next examined Pol II occupancy at promoters of
several MIR genes by chromatin immunoprecipitation (ChIP)
using anantibody against the largest subunit of Pol II (RPB1). We
monitored MIR159a, MIR159b, MIR159c, MIR166a, MIR167a,
and MIR171a using Actin2 as a positive control (Figures 6A and
6B). PolII occupancy was enrichedatregions encompassing the
represent two regions flanking two transcription start sites in
MIR159b loci, respectively), over the no antibody ChIP that
served as a negative control (Figures 6A and 6B). Pol II occu-
pancy at these loci was significantly reduced in apc8-1 (Figure
6B). These results indicate that APC/C promotes Pol II recruit-
ment to MIR genes.
Figure 5. APC8 Is Required for miR159 Biogenesis during Male Gametophyte Development.
(A) miR159 accumulation in Col-0, apc8-1, and APC8 apc8-1 (the pAPC8:APC8-YFP construct was introduced into apc8-1). Total RNAs were extracted
from inflorescences. U6 was the loading control.
(B) Expression of MIR159a (light gray), MIR159b (dark gray), and MIR159c (black) in pollen from Col-0, apc8-1, apc13-2, and HTR10:APC8 (line 8-2).
UBIQUITIN5 (UBQ5) was the loading control. All measurements represent the average of three biological replicates with error bars representing the SE.
(C) Expression patterns of ProMIR159a, b, c-GFP during pollen development in wild-type and apc8-1 plants. Fifteen individual T1 plants in the wild type
or the apc8-1 background were examined. Panels (top to bottom) show three progressive stages: UP, uninucleate microspores; BP, bicellular pollen;
TP, tricellular pollen. A representative DAPI-stained image for each stage is shown. Bar = 10 mm.
[See online article for color version of this figure.]
APC/C Regulates CYCB1;1 mRNA Levels 1039
APC/C-Mediated miR159 Accumulation Led to
Because miR159 biogenesis was perturbed in these apc/c
mutants, we next asked whether this perturbation had biological
significance. Since DUO1, an miR159 target (Palatnik et al.,
2007), was reported to play a role in twin sperm cell formation
(Brownfield et al., 2009a), we first used qPCR to examine
whether DUO1 expression was affected in the pollen of apc/c
mutants. The mRNA level of DUO1 in apc8-1 was increased;2-
fold (Figure 7A), consistent with the notion that DUO1 is nega-
tively regulated by miR159. By contrast, DUO3, which has a
function similar to that of DUO1 but is not a miR159 target, had
similar expression levels in apc8-1 and wild-type pollen (Figure
7A). A similar trend was observed for apc13-2 (Figure 7A). As
expected, DUO1 levels were reduced in HTR10:APC8 pollen
(Figure 7A). To substantiate the above result, we compared the
expression levelofDUO1-mRFP atdifferentstages duringpollen
development in wild-type or apc8-1 plants containing the
pDUO1:DUO1-mRFP construct. We consistently observed a
slightly increased intensity of DUO1-mRFP signals through early
bicellular stagetomature polleninapc8-1(Figure7B).Therefore,
our results demonstrate that APC/C plays a role in miR159
biogenesis that is important for miR159-dependent DUO1 re-
APC/C Regulates CYCB1;1 mRNA Levels
As increased DUO1 expression was observed in apc/c mutants
by DUO1 (Brownfield et al., 2009a), we speculated that APC/C
of CYCB1;1. To test this hypothesis, we compared CYCB1;1
mRNA levels in wild-type and apc8-1 pollen byqPCR. Therewas
(Figure 8A). Similarly, increased CYCB1;1 mRNA was also ob-
served in apc13-2 (Figure 8A). Taken together, these results
indicate that APC/C can regulate CYCB1;1 expression.
To determine if CYCB1;1 accumulates during male gameto-
genesis in these apc/c mutants, we introduced pCYCB1;1:
CYCB1;1-GFP into wild-type, apc8-1, and apc13-2 plants. In
the wild type, CYCB1;1-GFP was detected only in the nucleus of
pollen, but not in tricellular or mature pollen (Figure 8B), consis-
CYCB1;1-GFP accumulated in nuclei of both the vegetative cell
and sperm cells of tricellular and mature pollen from apc8-1
(Figure 8B) and apc13-2 (see Supplemental Figure 6A online).
The CYCB1;1-GFP accumulation in vegetative nuclei and ex-
panded expression in sperm cell nuclei at the tricellular stage in
these apc/c mutants suggest that APC/C is necessary for re-
moval of CYCB1;1 during male gametophyte development.
To further support the observation of accumulated CYCB1;1-
GFP in apc8-1, we used immunoblots to examine the total
protein levels of CYCB1;1-GFP in inflorescences from multiple
individual wild-type and apc8-1 transgenic plants expressing
in apc8-1 (see Supplemental Figure 6B online), further indicating
that disruption of APC8 function caused the failure of CYCB1;1
degradation. Taken together, our data demonstrate that APC/C,
in addition to its known role in regulating CYCB1;1 degradation,
can regulate CYCB1;1 at the mRNA level.
We obtained evidence for a mechanism by which APC/C regu-
lates CYCB1;1 at the transcriptional level and directly degrades
CYCB1;1. To explore this dual regulation in cell cycle progres-
sion, we focused on the well-defined cell lineages in male
gametophyte development and showed that disruption of APC/
C resulted in increased incidence of uninucleate or binucleate
pollen (Figure 4C).
Our results showed that the transcription of all three MIR159
genes was reduced in apc/c mutants (Figures 5B and 5C; see
the functional redundancy shown among MIR159a, MIR159b,
and MIR159c (Allen et al., 2007). Interestingly, although these
three geneshadnearly identical expression patternsduring male
gametophyte development, the MIR159c expression level was
higher than the expression levels of MIR159a and MIR159b, and
a more obvious reduction of MIR159c transcription was seen in
apc/c mutants(Figures 5B and5C;seeSupplementalFigures4B
and 4C online). Deep sequencing showed that miR159c was
much less abundant in leaves, seedlings (Rajagopalan et al.,
Figure 6. Pol II Occupancy at miRNA Loci.
(A) ChIP performed with Col-0 (light gray bars) or apc8 (dark gray bars)
with no antibody as the negative control.
(B) ChIP performed with anti-RPB1 antibody. The results were repro-
ducible in two biological replicates. The mean and SD were determined
from one representative biological replicate.
Error bars in (A) and (B) show SD calculated from three technical
1040The Plant Cell
2006), and inflorescences (Fahlgren et al., 2007), consistent with
the hypothesis that miR159c is pollen specific and accordingly
plays a predominant role during male gametophyte develop-
The temporal expression patterns of APC8 (Figure 3), MIR159
(Figure 5C), and CYCB1;1 (Brownfield et al., 2009a; Figure 8B)
are similar during male gametophyte development and are
reciprocal to the temporal expression pattern of DUO1. Why is
it necessary that expression patterns of these genes are regu-
lated? One of the well-known functions of the APC/C complex is
to degrade cyclins to allow progression through the cell cycle.
Enrichment of APC/C at the early stages of male gametophyte
development correlates precisely with the period of active and
continuous cell division. The most probable consequence of
MIR159 expression is to restrict the expression of its target
genes. DUO1, one of its target genes, functions in the formation
Thus, the abundance of miR159 at early stages of male game-
tophyte development might be mainly to limit the expression of
DUO1, which is also consistent with reduced seed set of
miR159ab double mutant (Allen et al., 2007). However, the
expression of MIR159 and DUO1 overlaps at the bicellular stage
(Brownfield et al., 2009a; Figure 5C). Other miR159 targets might
play a role in male gametophyte development when their ex-
pression patterns differ from that of DUO1 (Schwab et al., 2005).
Another possibility is that DUO1 expression is regulated by
factors other than miR159.
DUO1 expression is initiated at late stages (Rotman et al.,
2005), and DUO1 can promote CYCB1;1 expression by an
unknown mechanism (Brownfield et al., 2009a), which suggests
that the relationship between DUO1 and CYCB1;1 might be im-
portant and specific to the second mitosis. Moreover, overexpres-
sion of CYCB1;1 in male germlines rescued only the cell division
defects of duo1/+ heterozygous plants (Brownfield et al., 2009a),
indicating that CYCB1;1 is the output of a DUO1-dependent
pathway during male gametophyte development. The dynamic
requirements for CYCB1;1 expression in the second mitosis can
be accounted for, as CYCB1;1 was expressed in the generative
cell at the early bicellular stage and the late bicellular stage, but
not at the middle bicellular stage or at the tricellular stage
Figure 7. Increased DUO1 Expression in apc8-1 during Male Gametophyte Development.
(A) Expression of DUO1 and DUO3 in mature pollen from Col-0, apc8-1, apc13-2, and HTR10:APC8 as determined by qPCR. UBIQUITIN5 (UBQ5) was
the loading control. All measurements represent the average of three biological replicates with error bars representing the SE.
(B) Expression of pDUO1:DUO1-mRFP in the wild type and apc8-1. Plants homozygous for pDUO1:DUO1-mRFP were crossed into apc8-1 and then F3
plants homozygous for both apc8-1 and pDUO1:DUO1-mRFP were analyzed. One example for each developmental stage is shown. All images were
acquired using the same exposure times. UP, unicellular pollen; EBP, early bicellular pollen; MBP, middle bicellular pollen; LBP, late bicellular pollen;
TP, tricellular pollen. Bar = 10 mm.
(C) Quantitative data of RFP signal intensity at the early bicellular pollen and middle bicellular pollen stages in (B) is shown. Each x axis represents a
5-mm distance centered on the fluorescent focus. WT, wild type.
[See online article for color version of this figure.]
APC/C Regulates CYCB1;1 mRNA Levels 1041
(Brownfield et al., 2009a). By contrast, we showed that
CYCB1;1-GFP was detected only in the vegetative nuclei of
bicellular pollen and not in tricellular or mature pollen (Figure 8B).
The differences in these two studies might be due to the different
constructs used: Brownfield et al. (2009a) used a possible
transcriptional reporter (Glotzer et al., 1991), which included
the mitotic destruction box in the first exon fused to GUS,
whereas we used a fusion protein construct that encodes the
entire protein. This might indicate that the remaining coding
region of CYCB1;1 contains posttranscriptional information that
regulates translation or protein stability. Alternatively, the possi-
ble transcriptional reporter might be more sensitive than the
protein fusion construct.
Mitotic cyclins were the first demonstrated APC/C substrates
(Glotzer et al., 1991), and CYCB1;1 was shown to accumulate in
several apc/c mutants (this study; Capron et al., 2003b; Kwee
and Sundaresan, 2003; Pe ´rez-Pe ´rez et al., 2008). CYCB1;1
expression was reported to be promoted by DUO1 (Brownfield
et al., 2009a). What is the relationship between APC/C-directed
CYCB1;1 degradation and DUO1-mediated CYCB1;1 regula-
tion? We propose a model in Figure 9 to explain the dual
regulation of CYCB1;1 by APC/C during male gametophyte
development. We showed that two pathways, integrated
by APC/C, regulate CYCB1;1 expression and CYCB1;1 degra-
dation. One outcome is that disrupted APC/C function directly
of APC/C (Glotzer et al., 1991). The other outcome is that
reduced miR159 accumulation in apc/c mutants, accompanied
with increased DUO1 expression, promotes CYCB1;1 upregu-
lation in a direct or indirect manner because DUO1 is a tran-
scription factor (Rotman et al., 2005), and reduced CYCB1;1
transcription was observed in duo1 heterozygous plants
(Brownfield et al., 2009a). In this model, we propose that the
CYCB1;1 expression promoted by DUO1 is transient, which only
ensures a proper G2/M transition during pollen maturation; once
that is fulfilled, CYCB1;1 must be removed immediately by
APC/C. This hypothesis is consistent with the observation of
dynamic expression of CYCB1;1 (Brownfield et al., 2009a). The
existence of CYCB;1 transcripts at the early bicellular stage
implies that CYCB1;1 from the first mitosis has not been com-
pletely degraded by APC/C. The absence of CYCB1;1 tran-
scripts at the middle bicellular stage implies that APC/C has
Figure 8. CYCB1;1 Expression Is Increased in Male Gametophytes of
(A) Expression of CYCB1;1 in mature pollen from Col-0, apc8-1, and
apc13-2. UBIQUITIN5 (UBQ5) was the loading control. All measurements
represent the average of three biological replicates with error bars
representing the SE.
(B) Subcellular localization of CYCB1-GFP during pollen development in
wild-type and apc8-1 plants harboring pCYCB1;1:CYCB1-GFP. Fifteen
individual T1 plants in the wild type or the apc8-1 background were
examined. DAPI staining of uninucleate microspores (UP; [a] and [c]),
bicellular pollen (BP; [e] and [g]), and tricellular pollen (TP; [i] and [k]). GFP
was examined in uninucleate microspores ([b] and [d]), bicellular pollen
([f] and [h]), and tricellular pollen ([j] and [l]). CYCB1;1-GFP in apc8-1
was detected weakly in generative cell nuclei (arrow) of bicellular pollen (i)
and obviously accumulated in both the vegetative nucleus (arrowhead)
and sperm cell nuclei (arrow) of tricellular pollen (l). Bar = 10 mm.
[See online article for color version of this figure.]
Figure 9. A Model for the Dual Roles of APC/C in Regulating Cyclin B1;1
during Male Gametophyte Development.
APC/C not only degrades CYCB1;1 directly but also directly or indirectly
regulates miR159 transcription by recruiting Pol II to promoter regions.
The positive regulation of miR159 by APC/C causes the correspondingly
negative regulation of a miR159 target, DUO1, which ultimately leads to
reduced CYCB1;1 expression. The dual role of APC/C in CYCB1;1
function ensures proper progression of the two mitoses. VN, vegetative
nucleus; SC, sperm cells.
1042 The Plant Cell
degraded all the CYCB1;1 that was required for the first mitosis.
The reappearance of CYCB1;1 transcripts at the late bicellular
stage is presumably due to the miR159-mediated DUO1 repres-
sion, which facilitates progression of the second mitosis.
Regulation of CYCB1;1 by APC/C at the transcriptional level
was unexpected. Based on the observation that APC/C is
necessary for MIR159 transcription (Figure 5C), perhaps APC/
C, as an ubiquitin ligase, is recruited to the transcriptional
complex and acts to degrade a component specific for MIR
gene transcription. It will be interesting to determine how APC/C
affects the transcription of MIR genes and especially to examine
whether the transcriptional machinery for MIR genes is different
from that of most coding genes, although MIR genes are also
transcribed by Pol II (Lee et al., 2004; Xie et al., 2005; Kim et al.,
2011). Recent studies showed that a human mediator of DNA
damage checkpoint protein 1 (hMDC1), which is associated with
one of the core subunits of APC/C (Townsend et al., 2009), is
required for normal metaphase-to-anaphase transition (Coster
et al., 2007). hMDC1 is a transcription factor that belongs to the
BRAC1 complex (Goldberg et al., 2003; Lou et al., 2003; Stewart
et al., 2003). BRAC1 was shown to be a component of the Pol II
holoenzyme (Scully et al., 1997). These relationships suggest
that APC/C could be directly or indirectly involved in the tran-
scription of MIRgenes and that Arabidopsis orthologs of hMDC1
might be a bridge to recruit APC/C to transcriptional sites.
from fungi to mammals, we suspect that the dual regulation of
CYCB1;1 expression by APC/C and the involvement of miRNAs
in this process could also be widely conserved. A recent study
showed that human immunodeficiency virus (HIV)-encoded Tat
protein regulates cyclin B1 by promoting both its expression and
degradation in HIV-infected lymphocytes (Zhang et al., 2010). In
however, the mechanism remains unknown. It will be important
to understand how APC/C coordinates its proteasome activity
and its role in regulating a miRNA pathway.
We showed that the Arabidopsis APC/C is a key regulatory
factor in male gametophyte development, in addition to its
documented role in female gametophyte development (Capron
et al., 2003b; Kwee and Sundaresan, 2003). Given that APC/C is
a protein complex, it is unknown why mutations in different APC/
C subunits can affect one sex more profoundly than the other,
and this phenomenon appears to be conserved in yeast and
animals (Garbe et al., 2004; Pa ´l et al., 2007; Jin et al., 2010).
Perhaps this can be explained by target specificity of the APC/C
subunits. APC/C is an E3 ubiquitin ligase, but it can only
ubiquinate substrates with the help of two cofactors, the
ubiquitin-activating (E1) enzyme and a ubiquitin-conjugating
(E2) enzyme. The impact of a particular mutation on male or
female transmission might be related to structural similarities
among subunits. For example, APC6 and APC8 both have a TPR
domain, while APC2 does not; mutants of APC6 and APC8 both
showed defects in male transmission (Kwee and Sundaresan,
These results indicate that while the overall structure of the
APC/C is conserved among eukaryotes, this E3 ligase might
have assumed specialized functions in different kingdoms (Lima
et al., 2010). Another important layer of APC/C regulation in
plants could be through subunit availability in specific tissues
and/or cellular compartment, as it is known that APC subunits
are differentially expressed in Arabidopsis organs (Eloy et al.,
availability and the different subunits could play unique regula-
tory roles (Eloy et al. 2006).
Seeds, Strains, and apc8-1 Genotyping
The pDUO1:DUO1-mRFP and pHTR10:HTR10-mRFP seeds were kindly
provided by David Twell and Fred Berger, respectively. The pDCL1:
DCL1-YFP and pHYL1:HYL1-YFP plasmids were kindly provided by
David L. Spector. T-DNA insertion mutants (SALK_027397, apc13-2;
SALK_056243, dcl1-14) were obtained from the ABRC. The Schizo-
saccaromyces pombe temperature-sensitive apc8 strain (Cut23-174,
FY9688) was obtained from the Yeast Genetic Resource Center in Japan
(YGRC/NBRP). The Arabidopsis thaliana apc8-1 mutant was genotyped
by ApoI digestion of the PCR fragment generated using primers APC8F2
and APC8R2; the fragment in the mutant is 25 bp smaller than that in the
wild type, which is detectable when separated on a 3% agarose gel.
APC8 was amplified from Col-0 genomic DNA with primers APC8F1 and
APC8R1 (see Supplemental Table 1 online), cloned into pENTR-D/TOPO
(Invitrogen), and then transferred into the plant expression destination
vector pGWB40 (Nakagawa et al., 2007). The resulting plasmid was
introduced into apc8-1 plants by agroinfiltration for complementation
(Clough and Bent, 1998). For APC8 promoter analysis, a 2.4-kb fragment
upstream of the ATG was amplified using primers APC8F1 and APC8R7
and was subcloned into pENTR-D/TOPO and then transferred into the
plant expression vectors pGII-NLS3XGFP (Hellens et al., 2000) and
pMDC164 (Curtis and Grossniklaus, 2003). For yeast complementation,
full-length cDNAs of APC8 and APC8-1 were amplified from leaves of
Col-0 and apc8-1 plants, respectively, using RT-PCR and primers
APC8F3 and APC8R3, and then cloned into the pYPGE15 yeast expres-
sion vector (Brunelli and Pall, 1993). The amino acid Asp was changed to
Glu by site-directed mutagenesis using primers APC8F11 and APC8R11.
Cut23-174 mutant (Cullen et al., 2000). For the HTR10:APC8 overexpres-
pB7WG2 (Karimi et al., 2002) was digested with SacI and SpeI to replace
the 35S promoter with the HTR10 promoter and then digested with KpnI
and ApaI to insert the LAT52-GFP cassette. ALR reaction was performed
between the TOPO vector containing the full-length APC8 cDNA and
For CYCB1;1 expression analysis, a genomic fragment of CYCB1;1,
including a 2.5-kb fragment upstream of the ATG and downstream
sequences to the stop codon, was amplified with primers CYCB1;1F1
and CYCB1;1R1 and subcloned into pENTR-D/TOPO and then trans-
ferred into the plant expression destination vector pMDC107 (Curtis and
Grossniklaus, 2003). For MIR159 expression analysis, fragments of 1.9,
2.4, or 1.6 kb upstream of the first nucleotide of MIR159a, MIR159b, and
MIR159c, respectively, were subcloned into pENTR-D/TOPO and then
2000). The primer sequences are listed in Supplemental Table 1 online.
A single colony of cut23-174 was grown in YEA medium (5 g/L yeast
extract, 30 g/L Glc, 225 mg/L adenine, 225 mg/L His, 225 mg/L Leu,
APC/C Regulates CYCB1;1 mRNA Levels1043
225 mg/L Lys hydrochloride) for 2 to 3 d at 288C. One microgram of
DNA of pYPGE15, pYPGE15-APC8, pYPGE15-APC8DDE, or pYPGE15-
APC8DDN was transformed into 100 mL freshly made competent cells.
divided and plated on YEA plates, one incubated at 288C and the other at
378C. Colonies were observed after 2 to 3 d. A single colony from each
plate wasdiluted1:10, 1:100,or1:1000andspottedontoYEAplates for2
single colony from each plate was cultured in YEA liquid medium for 2 to
3 d at 288C and then inoculated into fresh YEA liquid medium to a starting
OD600of 0.2 to 0.3 and then OD600was measured after growth for 12 or
24 h. Three colonies were analyzed from each genotype, with two
Microscopy Analysis and Seed Set Analysis
Pollen dissected from closed buds and open flowers from 10 to 15
individual transgenic T1 plants was stained with DAPI for 30 min at room
temperature in the dark. Images were acquired with an Axiovert micro-
scope (Zeiss) under the epifluorescence channel (the excitation/emission
wavelengths are 365 nm/397 nm [DAPI], 470 nm/505 to 530 nm [GFP],
500 nm/535 nm [YFP], and 545 nm/605 nm [RFP]) using an AxioCamRM
camera and AxioVision 4.8.1 software. Images were processed using
Adobe Photoshop CS2 (Adobe). Seed set analysis was according to Ron
et al. (2010).
Analysis of Nuclear DNA Content
DNA content was determined as described by Rotman et al. (2005) with
modifications. Mature pollen from various genotypes was stained by
DAPI. Relative DNA content of vegetative cell nuclei (Col-0 plants),
vegetative cell-like nuclei (apc8-1), sperm cell nuclei (Col-0 plants), and
generative cell-like nuclei (apc8-1 and HTR10:APC8 plants) were mea-
sured by DAPI fluorescence. Images were analyzed for fluorescence with
AxioVision 4.8.1 software. A net value for each nucleus was obtained by
subtracting a corresponding background reading from the cytoplasm. To
standardize the relative fluorescence per C value of DNA, we first set the
average net value of vegetative cell nuclei and sperm cell nuclei using
wild-type mature pollen as 1C.
miRNA RNA Gel Blotting
Total RNA was isolated from inflorescences of Col-0, apc8-1, apc13-2,
and APC8-YFP apc8-1. Small RNA hybridization for miRNAs was
performed as described (Park et al., 2002). The 59 end-labeled (32P)
antisense oligonucleotides were used to detect U6 and miRNAs. Radio-
active signals were detected with a phosphor imager.
Total RNA was extracted using a RNeasy plant mini kit (Qiagen) and
reverse transcribed with the SuperScript III first-strand synthesis system
(Invitrogen) according to the manufacturer’s instructions. Real-time
qPCR was performed with three technical replicates for each of three
biological replicates using a MyIQ Real-Time PCR detection system (Bio-
Rad) with SYBR Green PCR Master Mix (Bio-Rad). The difference
between the cycle threshold (Ct) of target genes and the Ct of control
primers (DCt = Cttargetgene – Ctcontrol) was used to obtain the normalized
ChIP was performed according to Zheng et al. (2009). Pol II occupancy at
several miRNA loci was determined by ChIP using anti-RPB1 antibody
and 2-week-old seedlings from Col-0 and apc8-2, respectively. DNA
present in the immunoprecipitates was quantified by qPCR relative to
total input DNA. The results shown were consistent in two biological
replicates. Commercial anti-RPB1 (Abcam) was used. The primer sets
used for the PCR are listed in Supplemental Table 1 online.
Tissue from 10 to 15 individual PAPC8-GUS transgenic T1 plants was
fixed in 90% acetone for 2 to 3 h and then stained for 12 h in 50 mM
sodium phosphate buffer, pH 7.0, containing 0.2% Triton X-100, 5 mM
then washed in 70% ethanol three times. Images were taken with a Nikon
SMZ800 stereoscope. Images were processed using Adobe Photoshop
Inflorescences were ground into powder in liquid nitrogen, agitated
vigorously after adding 23 SDS-PAGE loading buffer, boiled for 5 min,
SDS-PAGE gels, electrophoresed for 2 to 3 h at 80 V, and transferred to
nitrocellulose membrane. Mouse anti-GFP (Clontech) and anti-Hsc70
(Stressgen) antibodies were used at 1:2000 and 1:10,000 dilutions,
respectively. HEN1 antibody and AGO1 antibody were made by X.C.’s
lab. Detection was with an ECL plus detection kit (GE).
Sequence data from this article can be found in the GenBank/EMBL data
libraries under the following accession numbers: APC8 (At3g48150),
APC13 (At1g73177), MIR159a (At1g73687), MIR159b (At1g8075),
MIR159c (At2g46255), CYCB1;1 (At4g37490), DUO1 (At3g60460),
DUO3 (At1g64570), UBQ5 (At3g62250), DCL1 (At1g01040), HYL1
(At1g09700), HEN1 (At4g20910), AGO1 (At1g48410), MIR166a (At2g46685),
MIR167a (At3g22886), MIR171a (At3g51375), and Actin2 (At3g18780).
The following materials are available in the online version of this article.
Supplemental Figure 1. Isolation and Characterization of dcl1-14.
Supplemental Figure 2. Expression Analysis of APC8.
Supplemental Figure 3. Overexpression of APC8 in Male Germline
Disrupts Seed Set Development and HTR10 Expression in apc8
Supplemental Figure 4. Mature miRNA Levels and miRNA Tran-
scription in apc/c Mutants.
Supplemental Figure 5. Expression of Known miRNA Biogenesis
Genes in apc8-1.
Supplemental Figure 6. Increased CYCB1;1 Expression in apc8-1
Supplemental Table 1. Primers Used for This Study.
We thank Guang Wu for comments on the manuscript, Peng Qin and
Hua Jiang for discussions and advice on phenotypic characterizations,
Rebecca Haussmann for microscope assistance, and David Hantz and
1044The Plant Cell
Julie Calfas for dedicated greenhouse maintenance. We also thank
University of California-Berkeley undergraduates Daniel Park and Hugo
Hua for technical assistance. We thank David Twell for pDUO1:DUO1-
mRFP transgenic seeds, Fred Berger for pHTR10:HTR10-mRFP trans-
genic seeds, ABRC for SALK_056243 and SALK_027397 seeds, the
YGRC/NBRP for providing the yeast apc8 mutant, Yuefeng Guan for
modifying the pGWB7*, and Mily Ron for modifying the pGII-NLS3XGFP
into a Gateway cassette vector. This work was supported by grants
from the National Institutes of Health (GM61146) and the National
Science Foundation (MCB-0718029) to X.C. and the USDA Current
Research Information System (5335-21000-030-00D) to S.M.
Received February 2, 2011; revised February 2, 2011; accepted March 9,
2011; published March 25, 2011.
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1046The Plant Cell
; originally published online March 25, 2011; 2011;23;1033-1046
Binglian Zheng, Xuemei Chen and Sheila McCormick
Male Gametophyte Development
and Degradation of Cyclin B1
MicroRNA-Mediated Transcriptional Regulation of
The Anaphase-Promoting Complex Is a Dual Integrator That Regulates Both
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