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j.devcel.2017.09.017

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Article
The Spindle Assembly Checkpoint in Arabidopsis Is
Rapidly Shut Off during Severe Stress
Highlights
d
Arabidopsis thaliana has a functional spindle assembly
checkpoint (SAC)
d
Arabidopsis SAC architecture is different from that of yeast
and animals
d
SAC is switched off without checkpoint adaptation when
there is prolonged activation
d
Cells reset the cell cycle with duplicated chromosomes but
no nuclear division
Komaki & Schnittger, 2017, Developmental Cell 43, 172–185
October 23, 2017 ª 2017 Elsevier Inc.
https://doi.org/10.1016/j.devcel.2017.09.017
Authors
Shinichiro Komaki, Arp Schnittger
Correspondence
arp.schnittger@uni-hamburg.de
In Brief
Komaki and Schnittger investigate the
spindle assembly checkpoint (SAC) in
Arabidopsis thaliana. Plant SAC
architecture differs from yeast and
animals. Prolonged SAC activation
results in shutoff without checkpoint
adaptation and cell-cycle resetting with
no nuclear division, a possible plantspecific adaptation that could underlie
the ease of plant ploidy alterations.
Developmental Cell
Article
The Spindle Assembly Checkpoint in Arabidopsis
Is Rapidly Shut Off during Severe Stress
Shinichiro Komaki1,2 and Arp Schnittger1,3,*
1University of Hamburg, Biozentrum Klein Flottbek, Department of Developmental Biology, Ohnhorststrasse 18, D-22609 Hamburg, Germany
2Present address: Nara Institute of Science and Technology, Graduate School of Biological Sciences, 8916-5 Takayama, Ikoma, Nara
630-0192, Japan
3Lead Contact
*Correspondence: arp.schnittger@uni-hamburg.de
https://doi.org/10.1016/j.devcel.2017.09.017
SUMMARY
The spindle assembly checkpoint (SAC) in animals
and yeast assures equal segregation of chromosomes during cell division. The prevalent occurrence
of polyploidy in flowering plants together with the
observation that many plants can be readily forced
to double their genomes by application of microtubule drugs raises the question of whether plants
have a proper SAC. Here, we provide a functional
framework of the core SAC proteins in Arabidopsis.
We reveal that Arabidopsis will delay mitosis in a
SAC-dependent manner if the spindle is perturbed.
However, we also show that the molecular architecture of the SAC is unique in plants. Moreover, the
SAC is short-lived and cannot stay active for more
than 2 hr, after which the cell cycle is reset.
This resetting opens the possibility for genome duplications and raises the hypothesis that a rapid termination of a SAC-induced mitotic arrest provides
an adaptive advantage for plants impacting plant
genome evolution.
INTRODUCTION
Equal chromosome segregation is required for genome stability
and hence for proper growth, development, and reproduction.
The spindle assembly checkpoint (SAC) in animals and yeast
has been found to play a key role for the fidelity of chromosome
distribution in both mitosis and meiosis by preventing anaphase
onset until all chromosomes are correctly attached to the spindle
(London and Biggins, 2014b; Gorbsky, 2015). Reduction of SAC
activity in mammalian cells leads to missegregation of chromosomes resulting in aneuploidy and subsequently cell death or
cancer (Kops et al., 2005). Likewise, reduced SAC activity is lethal in yeast if the mitotic spindle is perturbed (Li and Murray,
1991; Hoyt et al., 1991).
The core proteins of the SAC are conserved from yeast to
mammals, including BUB1, BUB3, MAD1, MAD2, MAD3/
BUBR1, and MPS1, which are key components for SAC assembly (London and Biggins, 2014b). MPS1 localizes to kinetochores that are not yet attached to the microtubules of the
spindle (Ji et al., 2015; Hiruma et al., 2015). Once located at
the kinetochore MPS1 recruits BUB3, MAD2, MAD3, and
CDC20 to generate the mitotic checkpoint complex (MCC).
Until all kinetochores are properly attached to spindle microtubules, the MCC inhibits the activity of the anaphase promoting complex/cyclosome (APC/C), an E3 ubiquitin ligase
that targets securin and cyclins for degradation by the 26S
proteasome and thus, once active, promotes entry into
anaphase.
If the SAC cannot be satisfied for a prolonged period in animals
and yeast, the power of the checkpoint to delay mitosis will
vanish although the checkpoint is still active and cells will eventually divide, a phenomenon termed SAC adaptation or mitotic
slippage (Rossio et al., 2010; Rieder and Maiato, 2004). This
adaptation induces genomic instability because cells enter
anaphase without proper kinetochore-microtubule connections.
The resulting aneuploid daughter cells are typically removed by
apoptosis. The duration of the mitotic arrest induced by the
SAC prior to checkpoint adaptation varies by organism and
cell type, e.g., 5 hr in budding yeast, 4.5 hr in rat kangaroo
(PtK) cells, 15 hr in human RPE1 cells, and 18 hr in human
H261 cells (Rieder and Maiato, 2004).
Plants also have homologs for all core SAC components, i.e.,
in the case of Arabidopsis BUB1, BUB3;1, BUB3;2, BUB3;3,
MAD1, MAD2, MAD3;1, MAD3;2, and MPS1 (Komaki and
Schnittger, 2016). However, BUB1-type genes duplicated and
diverged many times during eukaryotic evolution. As a part of
this diversification process, the different domains of a presumed
ancestral BUB1 gene were re-shuffled and re-distributed over
the different BUB1 paralogs, remarkably following often a similar
pattern in different organisms (Suijkerbuijk et al., 2012; Murray,
2012; Tromer et al., 2016). In Arabidopsis thaliana, the three
BUB1-type paralogs, BUB1, MAD3;1, and MAD3;2, likely resulted from two duplication events that independently took place
of the ones that created Bub1 and BubR1/Mad3 in yeast and animals. Thus, the three BUB1-type paralogs in plants evolved
independently from the ones in animals and yeast and, for
instance, Bub1 in humans is not a homolog of BUB1 in plants.
Rather, all three Arabidopsis BUB1-type proteins share functional aspects with Bub1, Mad3, and BubR1 from animals and
yeast. Since the current nomination of Arabidopsis MAD3;1
and MAD3;2 as MAD3-type is misleading as it implies functional
conservation, we will refer in the following to these proteins
as BUB1/MAD3 FAMILY 1 (BMF1) for BUB1, as BMF2 for
MAD3;1, and BMF3 for MAD3;2.
172 Developmental Cell 43, 172–185, October 23, 2017 ª 2017 Elsevier Inc.
SAC activity has so far not been measured in plants. While the
accumulation of some of the SAC components, i.e., BMF1,
BMF2, BMF3, BUB3;1, and MAD2, at centromeres is consistent
with the presence of a SAC in plants, BUB3;1 has been found to
localize to other cellular compartment than its animal and yeast
homologs raising doubts about the presence of a canonical
SAC in plants (Caillaud et al., 2009; Yu et al., 1999; Wang
et al., 2012). Furthermore, plant SAC proteins have other/additional functions compared with their animal counterparts
(Komaki and Schnittger, 2016). For instance, Arabidopsis
MAD1 has been implicated in the control of flowering time and
endoreplication, a special cell cycle, which leads to cellular polyploidy (Bao et al., 2014; De Veylder et al., 2011). Notably,
polyploidization events are widespread in flowering plants and
microtubule-destabilizing drugs such as Colchicine easily
induce whole-genome duplications in plants, for instance in triticale, reinforcing the question whether plants have an effective
SAC (Hollister, 2015; Slusarkiewicz-Jarzina
et al., 2017).
Here, we provide a functional framework of SAC function and
regulation in the model plant Arabidopsis thaliana. We develop
an assay to measure SAC activity and demonstrate that Arabidopsis does have a SAC, which is in particular required at microtubule-disrupting conditions. However, we also find that SAC assembly and SAC signaling is different in Arabidopsis from the
situation found in other organisms. We further show that in Arabidopsis cells can quickly shut off their SAC surveillance mechanism without undergoing checkpoint adaptation. We speculate
that this plant-specific feature has key consequences for evolution and breeding.
RESULTS
SAC Genes Are Especially Required for Cell Survival and
Growth under Microtubule-Destabilizing Conditions
To address the function of the SAC in plants, we isolated and
analyzed transfer DNA insertion mutants of all putative core
SAC genes in Arabidopsis, i.e., BMF1, BMF2, BMF3, BUB3;1,
BUB3;2, BUB3;3, MAD1, MAD2, and MPS1. Loss of BUB3;1
has been previously reported to result in embryonic lethality (Lermontova et al., 2008), and consistently, we could not recover
homozygous bub3;1 mutants. However, a presumptive SAC
function did not appear to be essential for plant development
since homozygous null mutants of all remaining SAC components grew similar to wild-type plants, consistent with previous
findings (Wang et al., 2012; Ding et al., 2012) (Figures 1A
and 1B). This suggests that BUB3;1 has a specialized function
in addition or even instead of a possible contribution to the SAC.
Next, we grew all homozygous SAC mutants on media containing the drug oryzalin to ask whether the respective genes
become important under microtubule-disrupting conditions.
Growth of bmf2, bmf3, bub3;3, mad1, mad2, and mps1 mutants
was severely compromised on media with oryzalin (Figures 1A
and 1B). In contrast, loss of BUB3;2 did not affect root growth
when exposed to microtubule drugs. Unexpectedly, we found
that root growth of bmf1 on oryzalin-containing media was
indistinguishable from that of the wild-type, although BMF1 is
a single copy gene in Arabidopsis and loss of BUB1 in other
organisms is lethal (Basu et al., 1999; Perera et al., 2007) (Figures 1A and 1B).
Given that loss of SAC function typically results in cell death in
mammals, we next analyzed cell survival of plants grown on media containing oryzalin. Matching the hypersensitivity toward oryzalin during root growth, we observed many dying cells, in
particular in the vasculature shortly above the root meristem, in
bmf2, bmf3, bub3;3, mad1, mad2, and mps1, but not in bmf1
and bub3;2 (Figures 1C–1G). A few dying cells were repeatedly
found on media without oryzalin, indicating that the SAC genes
are also required for cell survival under unperturbed growth
conditions.
Taken together, these data show that some but markedly not
all SAC genes in Arabidopsis are required for cell proliferation
and become especially important for cell survival and growth
when cells are exposed to oryzalin. This phenotype is hence intermediate between the strict requirement for cell survival of the
SAC in mammals and the importance of the SAC only under
microtubule-destabilizing conditions in yeast. Furthermore, as
seen in the case of BMF1, which is a central SAC component
in both animals and yeast but not in Arabidopsis, the regulation
of the APC/C during mitosis appears to be distinctive in plants.
Regulation of SAC Gene Expression
As a first step to investigate the regulation of SAC genes in Arabidopsis, we constructed SAC gene reporter lines in which the
GUS (b-glucuronidase) gene is under the control of approximately 2 kb upstream and 1 kb downstream sequences of
each SAC gene (PROSAC:GUS; Table S1). Contrary to our expectation based on the growth assays above, none of the PROSAC:
GUS transgenes but PROMAD2:GUS were expressed in the root
meristem although a few reporters showed activity in restricted
root tissues such as the quiescent center for BUB3;1 or the stele
in case of BMF2 (Figure 2A).
Next, we constructed genomic SAC gene reporter lines in
which a translational fusion of each SAC protein to GUS is produced by inserting the GUS gene before the STOP codon of
the respective gene (PROSAC:SAC:GUS; Table S1); notably,
these reporters contained all introns in contrast to our promoter
reporter lines. In contrast to the PROSAC:GUS lines, all PROSAC:
SAC:GUS lines showed strong GUS activity in the root tip
including the root meristem, suggesting that most of the major
regulatory sequences of SAC genes are located in introns (Figure 2B). In addition, the translational reporter for BMF2 revealed
a patchy pattern of GUS activity typically found in cell-cycleregulated genes, hinting at a posttranslational regulation of
BMF2 stability.
Spatial and Temporal Localization of the SAC
Components
Knowing that the entire genomic region of most SAC genes plays
an important role for their endogenous expression, we replaced
the GUS with the GFP gene in our translational reporters to
enable detailed subcellular localization studies of the SAC components during the cell cycle (Table S1). To test their functionality, we transformed the constructs into the respective mutants.
The BUB3;1:GFP construct could restore the embryonic
lethal phenotype of bub3;1 mutant plants (Table S1). The
remaining transgenic lines, with the exception of MAD1:GFP,
grew like wild-type plants on agar plates containing oryzalin
(Table S1). Remarkably, the MAD1:GFP construct did restore
Developmental Cell 43, 172–185, October 23, 2017 173
Figure 1. A Compromised SAC Leads to Hypersensitivity to Microtubule-Destabilizing Condition and Cell Death in Arabidopsis Roots
(A) Five-day-old seedlings grown on plates without oryzalin (DMSO control, upper row) and with 150 nM oryzalin (lower row).
(B) Quantification of the root growth on plates with and without oryzalin. The mean (±SD) of more than 52 seedlings per indicated genotype is shown. Level of
significance (p < 0.01; indicated by different letters) between the wild-type and SAC mutants were determined for each growth condition by one-way ANOVA
followed by Tukey-Kramer test.
(C) Five-day-old seedlings grown on plates without oryzalin (DMSO control, upper row) or 100 nM oryzalin (lower row). Roots were stained with 10 mg/mL
propidium iodide to visualize cell boundaries and dead cells. White arrowheads indicate the boundary between the meristem and the elongation zone based on
the cortex cell length. White brackets mark areas with dying cells visible by their red-stained cytoplasm.
(D–G) Quantification of meristem size and cell death phenotype in roots at 5 days after germination. Meristem sizes are provided by cortex cell numbers (D).
Meristem lengths were measured from the quiescent center to the first elongated cell in the cortical cell file (E). Cell death was quantified by the frequency of roots
of a given genotype that showed cell death and the area of dead cells per root (F and G). The mean (± SD) of more than 32 seedlings per genotype is shown. Level
of significance (p < 0.01; indicated by different letters) between the wild-type and SAC mutants were determined for each growth condition by one-way ANOVA
followed by Tukey-Kramer test.
174 Developmental Cell 43, 172–185, October 23, 2017
(legend on next page)
Developmental Cell 43, 172–185, October 23, 2017 175
the early-flowering phenotype of mad1 mutants, indicating that a
potential role in APC/C regulation can be uncoupled from the
previously published role in the control of flowering (Figures
S1A and S1B) (Bao et al., 2014). In addition, none of the other
SAC mutants showed an early-flowering phenotype, supporting
the view that flowering control by MAD1 is independent of SAC
action. Next, we generated an N-terminal genomic MAD1
GFP-reporter construct (GFP:MAD1). We found that this transgene could fully rescue both mutant phenotypes of mad1 plants
and have therefore used GFP:MAD1 plants for all subsequent
experiments concerning MAD1 localization (Table S1).
Unlike in mammalian and yeast cells, BMF1:GFP and
MPS1:GFP localized to centromeric regions throughout the cell
cycle, even in interphase (Figure 2C). However, while the intensity of BMF1:GFP at centromeres was constant during the cell
cycle, the MPS1:GFP signals decreased in strength just after
anaphase onset (Figure 2C; Movies S1A and S1G).
BUB3;1:GFP and BUB3;2:GFP accumulated in the nucleus
during interphase. After nuclear envelope breakdown (NEB),
both proteins were present in the cytoplasm. In telophase, they
localized to the middle part of the phragmoplast (Figure 2C;
Movie S1B, upper row; Movie S1B, middle row). In animals and
yeast, WD40-repeat domains in BUB3 act as the binding site
for other SAC components and are hence required for SAC function (Fraschini et al., 2001; Primorac et al., 2013). Although Arabidopsis BUB3;1 does not appear to be a SAC component, as
judged by its lack of hypersensitivity against oryzalin and its localization pattern, its WD40 domains are highly conserved (Figure 3A). To investigate the role of the WD40 domains in
BUB3;1, we exchanged W37 with G and W118 with G in the
two conserved WD40 domains of BUB3;1. Notably, both mutant
proteins lost their phragmoplast localization and could not
rescue the embryonic lethal phenotype of bub3;1 mutant plants
(Table S1; Figure 3B). Therefore, the function of WD40 domains
in BUB3;1 is conserved even outside of SAC function.
The reporter for the other BUB3-like protein, BUB3;3:GFP,
had a different localization pattern than BUB3;1 and BUB3;2
as it was present in the cytoplasm throughout the cell cycle (Figure 2C; Movie S1B, lower row). The two MAD3 proteins differed
in their localization patterns. First of all, the accumulation of the
BMF2:GFP (consistent with the BMF2:GUS reporter) appeared
to depend on the cell-cycle phase. Before NEB, BMF2:GFP
was present in the nucleus, while after NEB it was found
throughout the cytoplasm until anaphase onset (Figure 2C;
Movie S1E). In contrast, BMF3:GFP had a typical SAC localization pattern. In interphase, BMF3:GFP localized to the nucleus.
Just after NEB, BMF3:GFP started to accumulate at the kinetochores and disappeared from kinetochores as cells entered
anaphase (Figure 2C; Movie S1F).
To confirm the centromere localization of BMF3:GFP, we
generated transgenic plants expressing a TagRFP fused to the
centromeric histone variant CENH3, also called HTR12 or
CENPA (Table S1) (Talbert et al., 2002), and combined them
with plants producing BMF3:GFP. Indeed, we found a high level
of overlap between these two fluorescent reporters (Figure 2D).
GFP:MAD1 localized to the nucleus and the nuclear envelope
in interphase. Right after NEB, GFP:MAD1 started to accumulate
at kinetochores. With the onset of anaphase, GFP:MAD1 disappeared from kinetochores. In telophase, GFP:MAD1 localized to
the nuclear envelope and was weakly present at the middle part
of the phragmoplast (Figure 2C; Movie S1C, upper row).
MAD2:GFP localization was similar to that of BMF3:GFP (Figure 2C; Movie S1D, upper row).
Taken together, the localization of BMF1:GFP, BMF3:GFP,
GFP:MAD1, MAD2:GFP, and MPS1:GFP in Arabidopsis resembles the typical pattern of SAC components, although BMF1
and MPS1 display different dynamics in Arabidopsis when
compared with other organisms. At the same time, especially
BUB3;1:GFP, BUB3;2:GFP, and BUB3;3:GFP showed a plantspecific accumulation pattern that does not correspond to established SAC function in animal and yeast cells.
Kinetochore Recruitment of the SAC Components
In mammalian cells, all SAC components are recruited to the
kinetochore to activate the checkpoint. MPS1 is known as the
initiator of this activation process and if MPS1 is lost in animals
the entire SAC cannot be assembled (Maciejowski et al., 2010).
Our finding that, in Arabidopsis, MPS1 is present at the kinetochores throughout the cell cycle (Figure 2C) raised the question
whether MPS1 is needed for SAC activation. To address this
question, we monitored the localization of the SAC:GFP
reporters in mps1 mutants. We focused here on those SAC
components that show the typical localization pattern of
SAC proteins in other species, i.e., BMF1:GFP, BMF3:GFP,
GFP:MAD1, and MAD2:GFP. Indeed, we found that the kinetochore localization of MAD2:GFP is lost in mps1 mutants
(Figure 3C; Movie S1D, middle row), indicating that MPS1 is
also required for the recruitment of MAD2 to kinetochores in Arabidopsis. However, BMF1:GFP, BMF3:GFP, and GFP:MAD1
localized to kinetochores in mps1 mutants in the same manner
as in the wild-type (Figure 3C). Thus, we conclude that MPS1
is not the central organizer of SAC assembly in Arabidopsis.
Next, we focused on the localization of the MAD1-MAD2 complex. In other organisms, BUB1 is responsible for the kinetochore
localization of MAD1. However, its wild-type-like growth
behavior on media containing oryzalin and its localization suggested that BMF1 in Arabidopsis might have lost its SAC function
and might not be required for the correct localization of MAD1.
Indeed, the kinetochore localization of GFP:MAD1 was not
altered in bmf1 mutants. In addition, GFP:MAD1 was correctly
localized in bmf2 and mad2 mutants. However, GFP:MAD1
was not present at the kinetochores in bmf3 mutants, indicating
that BMF3 instead of BMF1 is responsible for the recruitment
of MAD1 in Arabidopsis (Figure 3D; Movie S1C, lower row).
Figure 2. Expression and Subcellular Localization of SAC Proteins during the Cell Cycle
(A and B) Comparison of the expression pattern between PROSAC:GUS and PROSAC:SAC:GUS in 5-day-old seedlings (see the STAR Methods). For each
construct, three independent lines were analyzed and representative images are shown. Scale bars, 100 mm.
(C) Functional SAC protein reporter lines in which the SAC gene of interest is fused with GFP (see also the STAR Methods) were crossed with TagRFP:TUA5expressing plants to visualize the microtubule structures. For live imaging, root tips of 5-day-old seedlings were used (see STAR Methods).
(D) BMF3:GFP co-localized with the centromere marker TagRFP:CENH3.
176 Developmental Cell 43, 172–185, October 23, 2017
Figure 3. Arabidopsis Has a Unique SAC Architecture
(A) Multiple sequence alignment of WD40 domains in the BUB3 from yeast and Arabidopsis. Asterisks indicate identical, hence highly conserved residues, colons stand
for conserved and periods for a less conserved residues between the different sequences. Red arrowheads indicate the amino acids previously shown to be important
for interaction with other SAC components (Fraschini et al., 2001). The sequences were aligned with the Clustal Omega program (Sievers et al., 2011).
(B) The mutations in either one of the WD40 domains of BUB3;1 disrupt the phragmoplast localization. Microtubules are visualized by TagRFP:TUA5.
(C) The kinetochore localization of BMF1, BMF3, and MAD1 is not altered in mps1 mutants. In contrast, MAD2 requires the presence of MPS1 for kinetochore
localization.
(D) GFP:MAD1 localization is not affected in bmf1, bmf2, and mad2, but depends on the presence of BMF3.
(E) Kinetochore localization of MAD2:GFP is lost in mad1 mutants. Microtubules are visualized by TagRFP:TUA5. For live imaging, root tips of 5-day-old seedlings
were used (see the STAR Methods).
(F) Scheme of the structural organization of the BUB1 and MAD3/BMFs with four important domains high-lighted by colors.
(G) Interaction between MAD1 and itself, as well as with MAD1 and MAD2, and MAD1 and BMF3 were revealed by yeast two-hybrid assay. Monomeric GFP
(mGFP) was used as a negative control. Each strain was spotted on SD plates without tryptophan and leucine ( TL; control media) or without tryptophan, leucine,
and histidine ( TLH; selection media) and photographed after incubation at 30 C for 2 days.
(H) The kinetochore localization of AUR3 is not altered in bmf1 mutants. Scale bar, 10 mm.
Developmental Cell 43, 172–185, October 23, 2017 177
Interestingly, the accumulation of MAD1 at the phragmoplast
could still be observed in bmf3 mutants, and hence the regulation of MAD1 at the kinetochores can be uncoupled from an
alleged role at the phragmoplast. Finally, we found that MAD1
is required for the kinetochore localization of MAD2 implying a
step-by-step recruitment of the SAC in plants (Figure 3E;
Movie S1D, lower row). The principle of a stepwise requirement
process resembles the situation in animals and yeast. However,
at least for some of the SAC components, i.e., MPS1, BMF3,
and BUB3, the order of the recruitment steps and/or their position in the SAC are different in plants compared with animals
and yeast.
To further dissect the kinetochore recruitment process of the
SAC in plants, we tested the interaction of SAC components
by a yeast two-hybrid assay (Figures 3F and 3G). Similar to the
interaction profile obtained in other organisms (Chen et al.,
1999; Kim et al., 2012), we observed that MAD1 interacts with
itself and directly binds to MAD2 (Figure 3G). As a possible
explanation for the plant-specific requirement of BMF3 for the
localization of MAD1, we found that MAD1 directly binds to
BMF3 (Figure 3G).
In animals and yeast, the interaction of MAD1 with BUB1 is
mediated by a conserved domain, called CD1, in BUB1 (Klebig et al., 2009; London and Biggins, 2014a). Interestingly,
three CD1-like sequences have been identified in Arabidopsis
BMF3, while Arabidopsis BMF1 does not possess any CD1
domain (Tromer et al., 2016). To evaluate the importance of
these CD1-like domains for the above-observed interaction
between MAD1 and BMF3, we created BMF3 mutants in
which all three single-CD1 domains were eliminated individually (DCD1a, DCD1b, and DCD1c) (Figure 3F). The deletion of
CD1a and CD1b apparently did not or only slightly reduced
the interaction of BMF3 with MAD1, respectively (Figure 3G).
In contrast, the DCD1c version of BMF3 strongly decreased
the interaction with MAD1. A similar reduction in binding
capability of BMF3 to MAD1 was found when both CD1a
and CD1b were deleted concomitantly (DCD1ab). Any combination of the deletion of CD1a and CD1b with DCD1c, i.e.,
DCD1ac, DCD1bc, and DCD1abc, completely abolished the
interaction of BMF3 with MAD1 (Figure 3G). Thus, CD1c appears to be most important for the interaction between
BMF3 and MAD1, with CD1a and CD1b having an axillary
function.
In animals and yeast, Bub1 phosphorylates histone H2A and
by that promotes the kinetochore localization of Aurora kinase
(Yamagishi et al., 2010; Kawashima et al., 2010), which activity
is in turn needed to correct the attachment of microtubules at
kinetochores. Arabidopsis has three Aurora kinase homologs,
but only Aurora kinase 3 (AUR3) shows kinetochore localization
(Demidov et al., 2005). To test the necessity of BMF1 for AUR3
localization, we constructed a genomic AUR3 reporter
construct in which GFP was fused to the C terminus of the
AUR3 (Table S1). Analyzing the GFP fluorescence in these
transgenic plants expressing this construct, revealed the previously found AUR3 localization pattern (Demidov et al., 2005).
However, the localization of AUR3 at the kinetochores was
not affected in bmf1 mutants representing a marked difference
between animals and yeast versus Arabidopsis (Table S1;
Figure 3H).
178 Developmental Cell 43, 172–185, October 23, 2017
SAC-Dependent Mitotic Delay under MicrotubuleDestabilizing Conditions
With functional SAC reporter lines in hand, we were finally able to
ask whether plants employ a SAC to delay anaphase onset. To
this end, we monitored the progression from NEB to anaphase
onset by live imaging of root cells co-expressing TagRFP:TUA5
and BMF3:GFP to concomitantly visualize microtubules and
SAC complexes. In wild-type cells, the average time from NEB
to anaphase onset was determined to be 556 ± 30 s (n = 15) under normal growth condition. After NEB, chromosomes were
rapidly aligned in the metaphase plate, then BMF3:GFP disappeared from kinetochores, followed by anaphase onset (Figure 4A; Movie S2A, upper row). When treated with 100 nM oryzalin, cells took significantly more time to align their chromosomes
in the metaphase plate, and the BMF3:GFP residual time at the
kinetochores was prolonged resulting in an average time from
NEB to anaphase onset of 835 ± 112 s (n = 16) (Figure 4A; Movie
S2A, middle row). This duration was further extended to 1,266 ±
453 s (n = 13) when cells were treated with 200 nM oryzalin, indicating that the delay of anaphase onset correlates with the level
of microtubule destabilization (Figure 4A; Movie S2A, lower row).
To address whether this delay is SAC dependent, we
observed the time from NEB to anaphase onset in mad2 mutants. In mad2 cells, the average time from NEB to anaphase
onset was 422 ± 24 s (n = 10; p < 0.01 according to Student’s
t test) under the normal growth condition (Figure 4B; Movie
S2B, upper row). This finding indicated that, even under externally non-perturbed conditions, a SAC delays mitosis in Arabidopsis by around 100 s. An extension of the time from NEB to
anaphase onset was not observed in mad2 cells when treated
with oryzalin, i.e., 434 ± 27 s (n = 11; p < 0.01 according to Student’s t test) for 100 nM oryzalin (Figure 4B; Movie S2B, middle
row), and 430 ± 31 s (n = 13; p < 0.01 according to Student’s
t test) for 200 nM oryzalin (Figure 4B; Movie S2B, lower row).
Interestingly, we often observed kinetochores with a persistent
BMF3:GFP signal in anaphase and telophase that normally disappears from kinetochores before anaphase onset, demonstrating that cells divided although the SAC was not satisfied
(Figure 4B arrow head; Movie S2B, middle row).
Thus, Arabidopsis possesses a SAC that can delay anaphase
onset if the chromosomes are not properly attached to the
spindle. The SAC appears to operate already during a normal
cell cycle, i.e., in the absence of microtubule drugs. However,
the delay imposed by the plant SAC is relatively short in comparison with animals and yeast, indicating that in plants the attachment of microtubule to kinetochores represents a rather robust
mechanism.
Arabidopsis Cells Do Not Show Symptoms of Checkpoint
Adaptation
After having identified the existence of a SAC in plants, we
wondered whether this checkpoint is subject to adaptation under continuous stress, similar to what has been observed in animals and yeast. To this end, we treated seedlings expressing
TagRFP:TUA5 and BMF3:GFP with 1,000 nM oryzalin. Under
this condition, cells cannot form a proper spindle but rather a
mass of collapsed microtubules is built to which BMF3:GFP
localized (Figure 5A; Movie S3). Surprisingly, after 4,148 ±
787 s (n = 5) from NEB, all cells exited from mitotic arrest without
Figure 4. Arabidopsis Cells Have a Proper SAC
(A) Duration from NEB to anaphase onset in the wild-type grown on plates without oryzalin (DMSO control), 100 and 200 nM oryzalin.
(B) Duration from NEB to anaphase onset of mad2 mutants grown on plates without oryzalin (DMSO control), 100 and 200 nM oryzalin. Microtubules and SAC
complexes are visualized by TagRFP:TUA5 and BMF3:GFP, respectively. For live imaging, root tips of 5-day-old seedlings were used (see the STAR Methods).
White arrow head indicates the persistence of BMF3:GFP after anaphase onset indicative for an unsatisfied SAC.
subsequent cell division but rather rebuilt a nuclear envelope
(Figure 5A; Movie S3). Moreover, while SAC components retain
their typical localization pattern during checkpoint adaptation
in animals, we found that the BMF3:GFP signal disappeared
from kinetochores, suggesting that Arabidopsis cells shut off
the SAC and terminate mitotic arrest (Figure 5A; Movie S3).
The disappearance of the BMF3:GFP signal and, with this presumably SAC activity, also implied that the APC/C becomes
Developmental Cell 43, 172–185, October 23, 2017 179
Figure 5. SAC Is Actively Shut Off within 90 Min in Arabidopsis Cells Even under Microtubule-Destabilizing Conditions
(A) Microtubules and SAC complexes are visualized by TagRFP:TUA5 and BMF3:GFP, respectively. The BMF3:GFP signal disappeared from kinetochores at
3,000 s after NEB. The TagRFP:TUA5 signal disappeared 3,280 s after NEB.
(B) Timing of the CYCB1;2:GFP degradation. The degradation is completed just before a nuclear envelope is rebuilt under microtubule-destabilizing conditions.
Microtubules are visualized by TagRFP:TUA5. For live imaging, root tips of 5-day-old seedlings were used (see the STAR Methods).
(C) Kinetics of CYCB1;2:GFP degradation. The maximum and basal intensity of CYCB1;2:GFP in each cells were set to 100 and 0, respectively. Time point zero for
the control experiment is the moment when chromosomes are fully separated in late anaphase. Time point zero for the cells treated with 1,000 nM oryzalin is the
moment when the nuclear envelope is fully reformed.
(D) Analysis of polyploidization after mitotic exit. Microtubules and centromeres are visualized by TagRFP:TUA5 and GFP:CENH3, respectively. White circles
indicate the cells after mitotic exit.
activated. In animals, B1-type cyclins are a major substrate of
the APC/C at anaphase onset (Peters, 2002). To evaluate the
APC/C activity under continuous stress, we used seedlings expressing both TagRFP:TUA5 and a GFP-tagged version of
CYCB1;2, which shows a typical accumulation pattern of an
APC/C substrate in plants (Schnittger et al., 2002). Under normal
growth conditions, the CYCB1;2:GFP signal immediately started
180 Developmental Cell 43, 172–185, October 23, 2017
to disappear with anaphase onset (n = 10). The moment of fully
separated chromosomes, when the CYCB1;2:GFP fluorescence
reached background levels, was set at time point zero, and
approximately 6 min before half of the fluorescence already disappeared (t1/2 = 560 ± 107 s), indicating that we are able to
monitor APC/C activity using this reporter (Figures 5B and 5C;
Movie S4A). When we treated seedlings with 1,000 nM oryzalin,
the CYCB1;2:GFP signal also rapidly disappeared just prior to
rebuilding of a nuclear envelope (n = 10). The CYCB1;2:GFP
levels reached background intensities when the nuclear envelope was fully formed, and this moment was set at time point
zero for this experiment. Approximately 6 min before the
nuclear envelope was fully restored, half of the fluorescence
disappeared with a similar kinetics as under unperturbed conditions (t1/2 = 594 ± 41 s; p > 0.05 according to Student’s t test;
Figures 5B and 5C; Movie S4B). The similar kinetics of cyclin
degradation suggests that the APC/C is indeed activated when
the SAC is shut off under continuous stress. Our results also
stand in marked contrast to the slow and gradual decay curves
observed in animals cells when undergoing mitotic slippage
(Gascoigne and Taylor, 2008; Rieder and Maiato, 2004; Brito
and Rieder, 2006).
A second major substrate of the APC/C is securing, which
keeps separase in an inactive state. After removal of securin,
separase cleaves the alpha-kleisin subunit of cohesion and
hence allows the separation of the sister chromatids as individual
chromosomes (Peters, 2002). To monitor the integrity of cohesion, we followed seedlings expressing both TagRFP:TUA5
and a GFP fusion to CENH3. Under unperturbed growth condition, we found 9.72 ± 0.73 GFP dots (n = 18) in one cell reflecting
the diploid chromosome number of Arabidopsis (2N = 10; Figure 5D). When exposed to 1,000 nM oryzalin, we detected
19.0 ± 0.93 GFP dots (n = 15) in one individual cell demonstrating
that cohesion was released and that the chromosome number
duplicated after mitotic exit (Figure 5D).
DISCUSSION
The SAC, as a surveillance system ensuring equal chromosome
segregation during mitosis and meiosis, has been well studied in
animals and yeast. In this work, we have addressed the question
of whether there is a functional SAC in the model plant Arabidopsis thaliana, and what its molecular and cytological properties
are. Previously, the localization of some of the Arabidopsis homologs for the core SAC components has been determined. In
addition, a few components have been functionally analyzed.
However, the lack of a molecular framework of these components has restricted further insights. With a complete set of functional reporter lines for the presumptive core components of the
SAC in Arabidopsis we have now addressed central aspects of
SAC function and regulation in plants and reveal that the plantspecific features of the SAC likely have important implications
for evolution and breeding.
Importance of SAC Genes in Plants
While the SAC is essential in many animals including mammals, it
is not vital in Drosophila and yeast. Previous studies and our data
indicate that SAC genes are also nonessential in Arabidopsis.
The reason why some species require more SAC activity than
others is not clear yet. However, we observed here that SAC mutants show cell death in roots even under normal growth condition, indicating that the SAC is usually required for accurate
mitosis in Arabidopsis. Currently, it is unclear whether this cell
death is active or passive. However, it is interesting to note
that, after exposure to DNA damaging drugs or in mutants that
have a high propensity for cell death, such as retinoblastoma-
related 1, a similar pattern of dying cells can be found that is preferentially appearing in the vascular stem cells and their progeny
(Fulcher and Sablowski, 2009; Biedermann et al., 2017; Horvath
et al., 2017). Hence, these cells might be sensitized to die by special cell-cycle properties.
We found that all SAC mutants but two were hypersensitive to
oryzalin. Thus, the SAC is especially important for Arabidopsis
growth under microtubule-destabilizing condition, a situation
reminiscent to SAC action in yeast (Li and Murray, 1991; Hoyt
et al., 1991). One of the SAC mutants that was not sensitive to
oryzalin is bub3;2. Arabidopsis has three BUB3 homologs and
two of them, BUB3;1 and BUB3;2, localize to the phragmoplast.
Mutants in BUB3;1 have been previously found to be embryo lethal (Lermontova et al., 2008). In contrast, all other mutants in
SAC core components develop and grow as the wild-type
(although mad1 mutants flower earlier that the wild-type), suggesting that BUB3;1, and likely the closely related BUB3;2
have additional functions outside of the SAC. Noteworthy, mutations in the conserved WD40 domains of BUB3;1 abrogated its
phragmoplast localization, and expression of the mutant version
of BUB3;1 could not rescue the embryonic lethal phenotype of
bub3;1. While BUB3;1 and BUB3;2 share 88% amino acid identity, BUB3;3 has only 37% sequence similarity with both BUB3;1
and BUB3;2 and was previously not regarded as a core SAC
component in Arabidopsis. Nonetheless, we observed that
bub3;3 single mutants are as hypersensitive to oryzalin as other
SAC mutants, indicating that BUB3;3 could also play a role in
SAC function (see also below).
The other mutant in a SAC gene, which is not hypersensitive to
microtubule-destabilizing drugs, is bmf1. This was surprising
since Bub1 not only functions as a central SAC component in animals and yeast but also phosphorylates histone H2A to direct
the targeting of Aurora B and the chromosomal passenger complex to centromeres (Yamagishi et al., 2010; Kawashima et al.,
2010). Consistent with its wild-type like growth and development, our experiments revealed that AUR3 localization was not
perturbed in bmf1. Hence, it remains an interesting question to
understand how Aurora kinase localization is controlled in plants.
SAC Architecture in Arabidopsis Is Different Compared
with Animals and Yeast
The kinetochore accumulation of SAC components is essential
for SAC activation. MPS1 is the key protein to initiate this assembly in animals and yeast. When cells enter mitosis, MPS1 re-localizes from the cytoplasm to the nucleus, where it becomes
tethered to the kinetochores through NDC80, a member of the
KMN (Knl1-Mis12-Ndc80) kinetochore complex (Zhang et al.,
2011; Kemmler et al., 2009) (Figure 6). MPS1 then phosphorylates KNL1, a member of the kinetochore protein complex, by
which the BUB3-BUB1 complex is recruited to the kinetochores
(Shepperd et al., 2012; London et al., 2012; Yamagishi et al.,
2012). In addition, Mps1 phosphorylates Bub1 and Mad1, which
are required for SAC function in human cells (Ji et al., 2017). The
BUB3-BUB1 complex then mediates the kinetochore localization of BUBR1 (Millband and Hardwick, 2002). BUB1 and
BUBR1 bind to BUB3 through their GLEBS domain (Larsen
et al., 2007). BUB1 also facilitates the recruitment of MAD1MAD2 complex at the kinetochores (London and Biggins,
2014a; Mora-Santos et al., 2016). The kinetochore-localized
Developmental Cell 43, 172–185, October 23, 2017 181
Figure 6. Schematic Comparison of the SAC in Animals/Yeast
versus Plants
Model of the typical SAC architecture at kinetochores and MCC formation in
animals and yeast compared with the structure and function of the SAC in
Arabidopsis as derived from this study. How a putative plant MCC complex is
formed and what its constituents are, is currently not clear. Although BMF1
localizes to kinetochores reflecting the localization pattern in animals and
yeast, it does not appear to be required for SAC function in Arabidopsis.
Possibly, BMF1 participates in other kinetochore related functions, such as the
recruitment of SHUGOSHIN (SGO) to kinetochores, as seen for the rice BMF1
homolog BRK1. Furthermore, BUB3;1 and BUB3;2 are apparently not involved
in SAC function. Instead, they localize to the phragmoplast and might have a
function in controlling microtubule dynamics during cytokinesis in plants.
MAD1-MAD2 complex finally promotes the formation of the
MCC, the actual APC/C inhibitor comprising BUB3, BUBR1,
MAD2, and CDC20 (Figure 6) (De Antoni et al., 2005).
Although Arabidopsis has homologs of all the core SAC
components, we found that the SAC is differently assembled
(Figure 6). First, MPS1 is always localized to kinetochores
throughout the cell cycle. Since the kinetochore accumulation
of MPS1 is the first step for SAC activation in many other organisms, a crucial question is how SAC assembly is controlled in
plants. One alternate way of SAC activation is found in the nematode C. elegans, in which the Polo-like kinase functionally substitutes for MPS1 to trigger SAC formation (Espeut et al., 2015).
However, a homolog of Polo has not been identified in any plant
species so far.
While we found that MPS1 is required for the correct localization of MAD2 it is not required for the kinetochore localization of
MAD1. However, our finding that MAD2 localization to kinetochores does depend on the presence of MAD1 indicates that a
MPS1-dependent activity is required for the interaction between
MAD1 and MAD2 at kinetochores. Given that MPS1 is a kinase,
an obvious hypothesis is that either MAD1 and/or MAD2 needs
182 Developmental Cell 43, 172–185, October 23, 2017
to be phosphorylated to allow binding of MAD2 to the kinetochore. Interestingly, BMF3 is correctly localized in bmf1 and
mps1 mutants, suggesting that BMF3 directly binds to the kinetochore protein complex (Figure 6).
BMF2 is apparently subject to proteolytic control since it
shows a patchy pattern in GUS staining. After NEB, nuclearlocalized BMF2-GFP is released in the cytoplasm and then
rapidly disappears after anaphase onset. A motif search revealed the presence of several putative degrons in BMF2, i.e.,
several KEN and one destruction box. This feature resembles
BubR1 in animals, which shows two distinct localization patterns. On the one hand it is localized to unattached kinetochores
to recruit other SAC components (Vleugel et al., 2015), and on
the other hand it is present in the cytoplasm as a component
of the MCC to inhibit the APC/C (Tang et al., 2001; Han et al.,
2013; Chao et al., 2012; Alfieri et al., 2016). It has been shown
that two KEN boxes in BUBR1 are necessary for the APC/C inhibition. However, BMF2 does not localize to unattached kinetochores as BubR1. In contrast, BMF3 localizes to unattached
kinetochores but does not have the typical pattern of KEN boxes
as BubR1 in animals. Therefore, the BubR1 function appears to
be divided into BMF2 and BMF3 in plants. Furthermore, the
recruitment of MAD1 by BMF3 in Arabidopsis resembles Bub1
in animals and yeast, although BMF3 does not possess a kinase
domain (London and Biggins, 2014a). Instead, BMF1 in plants
has maintained the conserved kinase domain of BUB1-type proteins, but appears not to be needed for SAC activity as judged by
the similar growth performance on media containing oryzalin of
bmf1 mutants in comparison with the wild-type and in contrast
to other mutants of core SAC components. Our work also
showed that none of the BUB3 homologs is localized to kinetochores, consistent with the fact that in Arabidopsis neither
BMF1, BMF2, nor BMF3 has a GLEBS domain through which
they interact with BUB3 in animals and yeast cells (Figure 3F).
Although our localization studies reveal the plant-specific architecture of the SAC at kinetochores, it is still unclear which
components are included in a putative plant MCC. Since only
bub3;3 mutants show oryzalin hypersensitivity in the BUB3 family and BMF2 has two KEN boxes, which are necessary for the
inhibition of APC/C activity in animal and yeast, we speculate
that the plant MCC consists of BUB3;3, CDC20, MAD2, BMF2,
and/or BMF3 (Figure 6). However, further studies are needed
to understand the actual APC/C inhibitory function in plants.
Termination of Mitotic Arrest versus Checkpoint
Adaptation
In animals and yeast, the SAC can cause metaphase arrest for
several hours. However, after a prolonged time, cells eventually
either undergo apoptosis or divide even if the SAC is not satisfied, which is called SAC adaptation or mitotic slippage (Rieder
and Maiato, 2004). The molecular mechanisms of SAC adaptation are still not fully understood and appear to be complex, since
even cells in a homogeneous cell population can follow one of
several paths in an as yet not predicable manner, i.e., death during mitotic arrest, mitotic exit, and cell survival or death after
mitotic exit (Gascoigne and Taylor, 2008). Mitotic slippage appears to be linked to Cdk-Cyc activity (Brito and Rieder, 2006)
and, possibly, its relationship to an alleged cell death factor
(Gascoigne and Taylor, 2008). A hallmark of mitotic slippage is
the slow and linear decline of cyclin B levels presumably due to
degradation even under active SAC conditions.
We found that Arabidopsis cells also experience a SACdependent mitotic delay when treated with oryzalin. However,
the mitotic arrest could not be maintained for a long time, i.e.,
longer than 90 min, after which a nuclear enveloped reformed.
Notably, this response was uniform in all root cells analyzed.
While the SAC complex does not dissociate from kinetochores
during adaptation in animals (Brito and Rieder, 2006), the level
of BMF3, a core SAC component was strongly reduced at kinetochores when Arabidopsis cells exited from metaphase arrest
and prior to nuclear envelope reappearance.
Moreover, we found that CYCB1;2, as monitored by a
CYCB1;2-GFP fusion, suddenly disappeared prior to the
rebuilding of a nuclear envelope, suggesting that the APC/C
became active (Figures 5B and 5C). This disappearance of
CYCB1;2 had a similar kinetics as under unperturbed conditions
when the SAC was satisfied indicating the termination of the SAC
and an activation of the APC/C under continuous stress. Consistent with the release of the APC/C inhibition, we observed the
apparent opening of cohesion and the release of sister chromatids visualized by the duplication of centromeric histone foci.
Why the activation of the APC/C and the associated drop in
CDK-cyclin activity do not initiate a mitotic exit program leading
to cytokinesis is not clear as yet, especially since earlier work
suggested that high levels of APC/C activity are associated
with cell division and cytokinesis in Arabidopsis (Roodbarkelari
et al., 2010). In any case, our observations indicate that, in Arabidopsis, unlike in animals, exit from metaphase arrest after
prolonged stress is not due to a slow decay of mitotic cyclins.
Importantly, we found that Arabidopsis appears to reset the
cell cycle thereby paving the road for a new round of DNA replication and subsequent cellular polyploidy (endoreplication),
which, when occurring in or propagated to germ cells, results
in polyploid offspring (De Veylder et al., 2011; Del Pozo and Ramirez-Parra, 2015; Fox and Duronio, 2013).
It is currently not clear what degree of variation in terms of
SAC activity and function exists among Arabidopsis cells or in
different plant species. However, treatment of Arabidopsis shoot
meristem cells also resulted in polyploidization (Grandjean et al.,
2004). Moreover, application of microtubule drugs is often
used in plant breeding to duplicate the genomic content, for
instance in hybrid breeding (Slusarkiewicz-Jarzina
et al., 2017).
A compromised microtubule organization also triggered a new
round of DNA replication without cell division in the single-celled
algae Chlamydomonas (Tulin and Cross, 2014), suggesting that
SAC action under severe stress conditions is conserved
throughout the plant lineage. A quick and active escape from
metaphase arrest might be beneficial since a cell is presumably
not physiologically fully functional with condensed chromosomes and consequential limitations in transcription (Gottesfeld
and Forbes, 1997). At the same time, resetting the cell cycle to
pre-S-phase and not continuing with mitosis prevents aneuploidies, which might be more dangerous for cellular function,
as indicated by our finding of prevalent cell death in SAC
mutants.
The ease by which plant cells can be made polyploid is also
exploited in agriculture, e.g., for diploidization of pollen and the
subsequent generation of entire plants that are completely ho-
mozygous (Younis et al., 2014). It is tempting to speculate that
the here-identified quick shut down of a SAC-induced metaphase arrest provides the functional base for these breeding applications. Moreover, it has become clear over the last decade
that whole-genome duplications regularly took place during the
evolution of the plant lineage. For instance, Arabidopsis has undergone at least two rounds of whole-genome duplications
(Panchy et al., 2016). At the same time, polyploidization has
emerged as a major driving force in plant evolution (Comai,
2005; Soltis and Soltis, 2009), raising the question whether there
was a selective advantage for plants to deliberately reset the cell
cycle and become polyploid.
Taken together, we have shown that the model plant Arabidopsis has a SAC that becomes especially important under
microtubule-destabilizing conditions. This SAC, although apparently comprised of the same core components as in animals and
yeast, has a distinct architecture. Importantly, we did not find
signs of checkpoint deterioration/adaptation as in animals after
severe microtubule stress, which would lead to prolonged activation of the checkpoint in animals and yeast. Instead, the
checkpoint appeared to be actively shut down in Arabidopsis
and plants exited mitosis without nuclear division. This checkpoint behavior has important implications for plant evolution by
affecting ploidy levels, which has been likely exploited in agricultural breeding programs.
STAR+METHODS
Detailed methods are provided in the online version of this paper
and include the following:
d
d
d
d
d
KEY RESOURCES TABLE
CONTACT FOR REAGENT AND RESOURCE SHARING
EXPERIMENTAL MODEL AND SUBJECT DETAILS
B Plant Materials
B Accession Numbers
METHOD DETAILS
B Plasmid Construction and Transgenic Plants
B Oryzalin Treatment and Phenotypic Analysis
B Confocal Microscopy and Sample Preparation
B GUS Histochemical Analysis
B Yeast Two-Hybrid Assay
QUANTIFICATION AND STATISTICAL ANALYSIS
SUPPLEMENTAL INFORMATION
Supplemental Information includes one figure, two tables, and four movies and
can be found with this article online at https://doi.org/10.1016/j.devcel.2017.
09.017.
AUTHOR CONTRIBUTIONS
S.K. and A.S. conceived the experiments. S.K. performed all the experiments
and statistical analyses. S.K. and A.S. analyzed the data. S.K. and A.S. wrote
the manuscript.
ACKNOWLEDGMENTS
We thank Frederick R. Cross, Katja Wassmann, Maren Heese, and Peter Bommert for critical reading and helpful comments on the manuscript. We
acknowledge the GABI-Kat T-DNA collection, the SALK T-DNA collection,
Developmental Cell 43, 172–185, October 23, 2017 183
and the NASC stock center for providing material used in this study. We thank
the Salk Institute Genomic Analysis Laboratory for providing the sequence-indexed Arabidopsis T-DNA insertion mutants. We are grateful to the University
of Hamburg for core funding. This work was supported through a DFG grant
(SCHN 736/8-1) to A.S.
Received: October 31, 2016
Revised: July 18, 2017
Accepted: September 21, 2017
Published: October 23, 2017
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Lechner, J. (2009). Mimicking Ndc80 phosphorylation triggers spindle assembly checkpoint signalling. EMBO J. 28, 1099–1110.
Kim, S., Sun, H., Tomchick, D.R., Yu, H., and Luo, X. (2012). Structure of human Mad1 C-terminal domain reveals its involvement in kinetochore targeting.
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Klebig, C., Korinth, D., and Meraldi, P. (2009). Bub1 regulates chromosome
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Developmental Cell 43, 172–185, October 23, 2017 185
STAR+METHODS
KEY RESOURCES TABLE
REAGENT or RESOURCE
SOURCE
IDENTIFIER
Critical Commercial Assays
pENTR/D-TOPO cloning kit
Thermo Fisher Scientific
Cat# K240020
Gateway LR Clonase II Enzyme mix
Thermo Fisher Scientific
Cat# 11791020
Experimental Models: Organisms/Strains
Arabidopsis thaliana: WT Col-0
N/A
Arabidopsis thaliana: bmf1
NASC
SALK_122554
Arabidopsis thaliana: bmf2
NASC
SAIL_303_E05
Arabidopsis thaliana: bmf3
NASC
GABI_084G06
Arabidopsis thaliana: bub3;1
Lermontova et al., 2008
GABI_362D05
Arabidopsis thaliana: bub3;2
Lermontova et al., 2008
SALK_151687
Arabidopsis thaliana: bub3;3
NASC
SALK_041372
Arabidopsis thaliana: mad1
Bao et al., 2014
SALK_073889
Arabidopsis thaliana: mad2
Ding et al., 2012
SAIL_191G06
Arabidopsis thaliana: mps1
NASC
GABI_663H07
Arabidopsis thaliana: PROBMF1:GUS in WT Col-0
This paper
N/A
Arabidopsis thaliana: PROBMF2:GUS in WT Col-0
This paper
N/A
Arabidopsis thaliana: PROBMF3:GUS in WT Col-0
This paper
N/A
Arabidopsis thaliana: PROBUB3;1:GUS in WT Col-0
This paper
N/A
Arabidopsis thaliana: PROBUB3;2:GUS in WT Col-0
This paper
N/A
Arabidopsis thaliana: PROBUB3;3:GUS in WT Col-0
This paper
N/A
N/A
Arabidopsis thaliana: PROMAD1:GUS in WT Col-0
This paper
Arabidopsis thaliana: PROMAD2:GUS in WT Col-0
This paper
N/A
Arabidopsis thaliana: PROMPS1:GUS in WT Col-0
This paper
N/A
Arabidopsis thaliana: PROBMF1:BMF1:GUS in WT Col-0
This paper
N/A
Arabidopsis thaliana: PROBMF2:BMF2:GUS in WT Col-0
This paper
N/A
Arabidopsis thaliana: PROBMF3:BMF3:GUS in WT Col-0
This paper
N/A
Arabidopsis thaliana: PROBUB3;1:BUB3;1:GUS in WT Col-0
This paper
N/A
Arabidopsis thaliana: PROBUB3;2:BUB3;2:GUS in WT Col-0
This paper
N/A
Arabidopsis thaliana: PROBUB3;3:BUB3;3:GUS in WT Col-0
This paper
N/A
Arabidopsis thaliana: PROMAD1:MAD1:GUS in WT Col-0
This paper
N/A
Arabidopsis thaliana: PROMAD2:MAD2:GUS in WT Col-0
This paper
N/A
Arabidopsis thaliana: PROMPS1:MPS1:GUS in WT Col-0
This paper
N/A
Arabidopsis thaliana: PROBMF1:BMF1:GFP in bmf1
This paper
N/A
Arabidopsis thaliana: PROBMF1:BMF1:GFP + PRORPS5A:TagRFP:
TUA5 in mps1
This paper
N/A
Arabidopsis thaliana: PROBMF1:BMF1:GFP + PRORPS5A:TagRFP:
TUA5 in WT Col-0
This paper
N/A
Arabidopsis thaliana: PROBMF2:BMF2:GFP in bmf2
This paper
N/A
Arabidopsis thaliana: PROBMF2:BMF2:GFP + PRORPS5A:TagRFP:
TUA5 in WT Col-0
This paper
N/A
Arabidopsis thaliana: PROBMF3:BMF3:GFP + PRORPS5A:TagRFP:
TUA5 in bmf1
This paper
N/A
Arabidopsis thaliana: PROBMF3:BMF3:GFP in bmf3
This paper
N/A
Arabidopsis thaliana: PROBMF3:BMF3:GFP + PRORPS5A:TagRFP:
TUA5 in mps1
This paper
N/A
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REAGENT or RESOURCE
SOURCE
IDENTIFIER
Arabidopsis thaliana: PROBMF3:BMF3:GFP + PRORPS5A:TagRFP:
TUA5 in WT Col-0
This paper
N/A
Arabidopsis thaliana: PROBUB3;1:BUB3;1:GFP in bub3;1
This paper
N/A
Arabidopsis thaliana: PROBUB3;1:BUB3;1:GFP + PRORPS5A:
TagRFP:TUA5 in WT Col-0
This paper
N/A
Arabidopsis thaliana: PROBUB3;1:BUB3;1W37G:GFP in bub3;1
This paper
N/A
Arabidopsis thaliana: PROBUB3;1: BUB3;1W37G:GFP + PRORPS5A:
TagRFP:TUA5 in WT Col-0
This paper
N/A
Arabidopsis thaliana: PROBUB3;1:BUB3;1W118G:GFP in bub3;1
This paper
N/A
Arabidopsis thaliana: PROBUB3;1: BUB3;1W118G:GFP + PRORPS5A:
TagRFP:TUA5 in WT Col-0
This paper
N/A
Arabidopsis thaliana: PROBUB3;2:BUB3;2:GFP in bub3;2
This paper
N/A
Arabidopsis thaliana: PROBUB3;2:BUB3;2:GFP + PRORPS5A:
TagRFP:TUA5 in WT Col-0
This paper
N/A
Arabidopsis thaliana: PROBUB3;3:BUB3;3:GFP in bub3;3
This paper
N/A
Arabidopsis thaliana: PROBUB3;3:BUB3;3:GFP + PRORPS5A:
TagRFP:TUA5 in WT Col-0
This paper
N/A
Arabidopsis thaliana: PROMAD1:MAD1:GFP in mad1
This paper
N/A
Arabidopsis thaliana: PROMAD1:GFP:MAD1 + PRORPS5A:TagRFP:
TUA5 in bmf1
This paper
N/A
Arabidopsis thaliana: PROMAD1:GFP:MAD1 + PRORPS5A:TagRFP:
TUA5 in bmf2
This paper
N/A
Arabidopsis thaliana: PROMAD1:GFP:MAD1 + PRORPS5A:TagRFP:
TUA5 in bmf3
This paper
N/A
Arabidopsis thaliana: PROMAD1:GFP:MAD1 in mad1
This paper
N/A
Arabidopsis thaliana: PROMAD1:GFP:MAD1 + PRORPS5A:TagRFP:
TUA5 in mad2
This paper
N/A
Arabidopsis thaliana: PROMAD1:GFP:MAD1 + PRORPS5A:TagRFP:
TUA5 in mps1
This paper
N/A
Arabidopsis thaliana: PROMAD1:GFP:MAD1 + PRORPS5A:TagRFP:
TUA5 in WT Col-0
This paper
N/A
Arabidopsis thaliana: PROMAD2:MAD2:GFP in mad2
This paper
N/A
Arabidopsis thaliana: PROMAD2:MAD2:GFP + PRORPS5A:TagRFP:
TUA5 in mps1
This paper
N/A
Arabidopsis thaliana: PROMAD2:MAD2:GFP + PRORPS5A:TagRFP:
TUA5 in WT Col-0
This paper
N/A
Arabidopsis thaliana: PROMPS1:MPS1:GFP in mps1
This paper
N/A
Arabidopsis thaliana: PROMPS1:MPS1:GFP + PRORPS5A:TagRFP:
TUA5 in WT Col-0
This paper
N/A
Arabidopsis thaliana: PRORPS5A:TagRFP:TUA5 in WT Col-0
This paper
N/A
Arabidopsis thaliana: PROCENH3:GFP:CENH3 + PRORPS5A:
TagRFP:TUA5 in WT Col-0
This paper
N/A
Arabidopsis thaliana: PROCENH3:TagRFP:CENH3 + PROBMF3:
BMF3:GFP in WT Col-0
This paper
N/A
Arabidopsis thaliana: PROAUR3:AUR3:GFP + PRORPS5A:TagRFP:
TUA5 in WT Col-0
This paper
N/A
Arabidopsis thaliana: PROAUR3:AUR3:GFP + PRORPS5A:TagRFP:
TUA5 in bmf1
This paper
N/A
Yeast: pGBT9/mGFP in AH109
This paper
N/A
Yeast: pGBT9/MAD1 in AH109
This paper
N/A
Yeast: pGAD424/mGFP in AH109
This paper
N/A
Yeast: pGAD424/ MAD1 in AH109
This paper
N/A
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REAGENT or RESOURCE
SOURCE
IDENTIFIER
Yeast: pGAD424/ MAD2 in AH109
This paper
N/A
Yeast: pGAD424/ BMF3 in AH109
This paper
N/A
Yeast: pGAD424/BMF3DCD1a in AH109
This paper
N/A
Yeast: pGAD424/BMF3DCD1b in AH109
This paper
N/A
Yeast: pGAD424/BMF3DCD1c in AH109
This paper
N/A
Yeast: pGAD424/BMF3DCD1ab in AH109
This paper
N/A
Yeast: pGAD424/BMF3DCD1ac in AH109
This paper
N/A
Yeast: pGAD424/BMF3DCD1bc in AH109
This paper
N/A
Yeast: pGAD424/BMF3DCD1abc in AH109
This paper
N/A
This paper
N/A
PROBMF1:GUS
This paper
N/A
PROBMF2:GUS
This paper
N/A
PROBMF3:GUS
This paper
N/A
PROBUB3;1:GUS
This paper
N/A
PROBUB3;2:GUS
This paper
N/A
PROBUB3;3:GUS
This paper
N/A
PROMAD1:GUS
This paper
N/A
Oligonucleotides
Primers for genotyping and plasmid construction, see Table S1
Recombinant DNA
PROMAD2:GUS
This paper
N/A
PROMPS1:GUS
This paper
N/A
PROBMF1:BMF1:GUS
This paper
N/A
PROBMF2:BMF2:GUS
This paper
N/A
PROBMF3:BMF3:GUS
This paper
N/A
PROBUB3;1:BUB3;1:GUS
This paper
N/A
PROBUB3;2:BUB3;2:GUS
This paper
N/A
PROBUB3;3:BUB3;3:GUS
This paper
N/A
PROMAD1:MAD1:GUS
This paper
N/A
PROMAD2:MAD2:GUS
This paper
N/A
PROMPS1:MPS1:GUS
This paper
N/A
PROBMF1:BMF1:GFP
This paper
N/A
PROBMF2:BMF2:GFP
This paper
N/A
PROBMF3:BMF3:GFP
This paper
N/A
PROBUB3;1:BUB3;1:GFP
This paper
N/A
PROBUB3;1:BUB3;1W37G:GFP
This paper
N/A
PROBUB3;1:BUB3;1W118G:GFP
This paper
N/A
PROBUB3;2:BUB3;2:GFP
This paper
N/A
PROBUB3;3:BUB3;3:GFP
This paper
N/A
PROMAD1:MAD1:GFP
This paper
N/A
PROMAD1:GFP:MAD1
This paper
N/A
PROMAD2:MAD2:GFP
This paper
N/A
PROMPS1:MPS1:GFP
This paper
N/A
PRORPS5A:TagRFP:TUA5
This paper
N/A
PROCENH3:GFP:CENH3
This paper
N/A
PROCENH3:TagRFP:CENH3
This paper
N/A
PROAUR3:AUR3:GFP
This paper
N/A
pGBT9/mGFP
This paper
N/A
pGBT9/MAD1
This paper
N/A
pGAD424/mGFP
This paper
N/A
(Continued on next page)
e3 Developmental Cell 43, 172–185.e1–e5, October 23, 2017
Continued
REAGENT or RESOURCE
SOURCE
IDENTIFIER
pGAD424/MAD1
This paper
N/A
pGAD424/MAD2
This paper
N/A
pGAD424/BMF3
This paper
N/A
pGAD424/BMF3DCD1a
This paper
N/A
pGAD424/BMF3DCD1b
This paper
N/A
pGAD424/BMF3DCD1c
This paper
N/A
pGAD424/BMF3DCD1ab
This paper
N/A
pGAD424/BMF3DCD1ac
This paper
N/A
pGAD424/BMF3DCD1bc
This paper
N/A
pGAD424/BMF3DCD1abc
This paper
N/A
NIH
https://imagej.nih.gov/ij/
Software and Algorithms
ImageJ
CONTACT FOR REAGENT AND RESOURCE SHARING
Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Arp
Schnittger (arp.schnittger@uni-hamburg.de).
EXPERIMENTAL MODEL AND SUBJECT DETAILS
Plant Materials
Arabidopsis thaliana accession Columbia (Col) was used as the wildtype in this study. All mutants are in the Col background. Plants
were growth on a solid medium containing half-strength Murashige and Skoog (MS) salts, 1% sucrose and 1% (w/v) agar in a growth
chamber (16h of light; 21 C/8h of dark; 18 C cycles).
The T-DNA insertion lines SALK_122554 (bmf1), SAIL_303_E05 (bmf2), GABI_084G06 (bmf3), GABI_362D05 (bub3;1),
SALK_151687 (bub3;2), SALK_041372 (bub3;3), SALK_073889 (mad1), SAIL_191G06 (mad2), GABI_663H07 (mps1) were obtained
through the Nottingham Arabidopsis Stock Center. Primer pairs for genotyping are described in Table S2 (Kleinboelting et al., 2012;
Alonso et al., 2003).
Accession Numbers
Sequence data from this study can be found in the Arabidopsis Genome Initiative under the following accession numbers:
BMF1/BRK1 (At2g20635), BMF2/BUBR1 (At2g33560), BMF3 (At5g05510), BUB3;1 (At3g19590), BUB3;2 (At1g49910), BUB3;3
(At1g69400), MAD1 (At5g49880), MAD2 (At3g25980), MPS1 (At1g77720).
METHOD DETAILS
Plasmid Construction and Transgenic Plants
To create PROSAC:SAC:GUS constructs, the genomic fragment of each gene was amplified by PCR and cloned into pENTR-D-TOPO
or pDONR221. The SnaBI site was inserted in front of the stop codon of BUB3;2 construct. The SmaI site was inserted in front of the
stop codon of the other constructs. All the constructs were linearized by SnaBI or SmaI digestion and were ligated to the GUS gene,
followed by LR recombination reactions with the destination vector pGWB501 (Nakagawa et al., 2007). To create PROSAC:GUS constructs, the coding region of each gene was removed from PROSAC:SAC:GUS constructs by inverse PCR. For localization analysis of
the SAC proteins, the GUS gene in PROSAC:SAC:GUS constructs was replaced by the gene for GFP. Primer pairs for plasmid construction are described in Table S2. Transgenic plants were generated by the floral dip method (Clough and Bent, 1998).
The Agrobacterium tumefaciens strain GV3101 (pMP90) harboring the gene of interest on a binary plasmid was grown in 3 ml of LB
media at 28 C. The Agrobacterium was resuspended in 3 ml of transformation buffer containing 5% sucrose and 0.05% silwet L-77,
and used for plant transformation.
Oryzalin Treatment and Phenotypic Analysis
A stock solution of oryzalin in dimethyl sulfoxide (DMSO) was prepared and kept at -20 C. Plates containing oryzalin were poured
adding the appropriate stock solution to a half-strength MS medium. The final concentration of DMSO in the medium was 0.05%.
For quantification of root growth, five-day-old seedlings grown on plates with and without oryzalin were photographed and the primary root length was measured by ImageJ. For quantification of meristem size and cell death phenotype in roots, five-day-old seedlings grown on plates with and without oryzalin were stained with 10 mg/ml propidium iodide to visualize cell boundaries and dead
Developmental Cell 43, 172–185.e1–e5, October 23, 2017 e4
cells, and imaged by confocal microscopy. Meristem sizes were determined by the number of cortex cells and the length of the meristem. Meristem lengths were defined as the region from the quiescent center to the first elongated cell in the cortical cell file. Cell
death was quantified by the frequency of roots of a given genotype that showed cell death and the area of dead cells per root. ImageJ
was used for the quantification of dead cell.
Confocal Microscopy and Sample Preparation
For live imaging, root tips of five-day-old seedlings were put on glass bottom dishes and covered with a solid medium containing halfstrength MS salts, 1% sucrose and 1% (w/v) agar with 0.05% DMSO as a control or oryzalin (prepared in DMSO; Sigma-Aldorich) for
1 h. Confocal images were acquired using a Leica TCS SP8 inverted confocal microscopy. GFP and TagRFP were excited by using
488-and 561-nm laser, respectively. Images were obtained at 20-second intervals and corrected for image drift by the StackReg plugin (Rigid Body option) for ImageJ version 1.49.
GUS Histochemical Analysis
The explants were fixed in 90% acetone for 15 min and were washed in 50 mM sodium phosphate buffer. Then, the explants were
incubated in GUS solution (50 mM sodium phosphate buffer, pH 7.0, 0.5 % Triton X-100, 0.5 mM K3Fe(CN)6, 0.5 mM K4Fe(CN)6 and
0.5 mg ml-1 X-gluc) for 1 h at 37 C.
Yeast Two-Hybrid Assay
For yeast two-hybrid assay, all of the cDNAs tested were amplified by PCR with gene specific primers, followed by PCR with universal
attB primers, and cloned into pDONR221. The subcloned cDNAs were recombined into the destination vector pGBT9 (DNA-BD) or
pGAD424 (AD) by LR recombination reactions. The resulting constructs were transformed into the yeast strain AH109. Transformants
were spotted on SD plates without Tryptophan and Leucine (-TL; control media) or without Tryptophan, Leucine and Histidine (-TLH;
selection media) and photographed after incubation at 30 C for 2 days. Primer pairs for plasmid construction are described
in Table S2.
QUANTIFICATION AND STATISTICAL ANALYSIS
The significance of the difference between two groups was determined by Student’s t-test. The significance of the difference in more
than two groups was determined by One-way ANOVA followed by Tukey-Kramer test. Level of significance is shown by different letters. The numbers of samples are indicated in the Figure legends of Results.
e5 Developmental Cell 43, 172–185.e1–e5, October 23, 2017
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