Functional interaction between the Arabidopsis orthologsof spindle assembly checkpoint proteins MAD1 and MAD2and the nucleoporin NUA

Dongfeng Ding • Sivaramakrishnan Muthuswamy •

Iris Meier

Received: 30 January 2012 / Accepted: 3 March 2012 / Published online: 29 March 2012

� Springer Science+Business Media B.V. 2012

Abstract In eukaryotes, the spindle assembly checkpoint

(SAC) ensures the fidelity of chromosome segregation

through monitoring the bipolar attachment of microtubules

to kinetochores. Recently, the SAC components Mitotic

Arrest Deficient 1 and 2 (MAD1 and MAD2) were found to

associate with the nuclear pore complex (NPC) during

interphase and to require certain nucleoporins, such as Tpr

in animal cells, to properly localize to kinetochores. In

plants, the SAC components MAD2, BUR1, BUB3 and

Mps1 have been identified, but their connection to the

nuclear pore has not been explored. Here, we show that

AtMAD1 and AtMAD2 are associated with the nuclear

envelope during interphase, requiring the Arabidopsis

homolog of Tpr, NUA. Both NUA and AtMAD2 loss-of-

function mutants have a shorter primary root and a smaller

root meristem, and this defect can be partially rescued by

sucrose. Mild AtMAD2 over-expressors exhibit a longer

primary root, and an extended root meristem. In BY-2

cells, AtMAD2 is associated with kinetochores during

prophase and prometaphase, but not metaphase, anaphase

and telophase. Protein-interaction assays demonstrate

binding of AtMAD2 to AtMAD1 and AtMAD1 to NUA.

Together, these data suggest that NUA scaffolds AtMAD1

and AtMAD2 at the nuclear pore to form a functional

complex and that both NUA and AtMAD2 suppress pre-

mature exit from cell division at the Arabidopsis root

meristem.

Keywords Spindle assembly checkpoint � Nuclear pore �Nucleoporin � Root meristem � Cell division

Introduction

Eukaryotic cells utilize the spindle assembly checkpoint

(SAC) to link progression into anaphase to the completion

of spindle assembly (Iouk et al. 2002; Zhou et al. 2002;

Suijkerbuijk and Kops 2008; De Souza et al. 2009). SAC

proteins, including Mad1, Mad2, and Mad3 (called BubR1

in mammalian systems) (Mitotic Arrest Deficient) and

Bub1, Bub2 and Bub3 (Budding Unperturbed by Benz-

imidazole) localize to kinetochores in prophase and gen-

erate a signal that inhibits the mitotic E3 ubiquitin ligase

anaphase promoting complex/cyclosome (APC/C) until all

kinetochores are properly attached to microtubules (Howell

et al. 2001; Musacchio and Salmon 2007). Upon proper

microtubule attachment, Mad1 and Mad2 are removed

from the kinetochore and the SAC is turned off. This

activates the APC/C and, as a result, the sister chromatids

are separated and cyclin B is degraded, leading to mitotic

exit (Suijkerbuijk and Kops 2008).

Of the SAC proteins, MAD2, BUBR1, and BUB3.1 have

been identified and characterized in plants. MAD2 was first

identified in maize, where it is associated with kinetochores

both in mitotic cells and during meiosis I and II (Yu et al.

Dongfeng Ding and Sivaramakrishnan Muthuswamy have equally

contributed to the study.

Electronic supplementary material The online version of thisarticle (doi:10.1007/s11103-012-9903-4) contains supplementarymaterial, which is available to authorized users.

D. Ding � S. Muthuswamy � I. Meier (&)

Department of Molecular Genetics, The Ohio State University,

520 Aronoff Laboratory, 318 W 12th Avenue, Columbus,

OH 43210, USA

e-mail: meier.56@osu.edu

Present Address:S. Muthuswamy

Weill Cornell Medical College, New York, NY, USA

123

Plant Mol Biol (2012) 79:203–216

DOI 10.1007/s11103-012-9903-4

1999). During mitosis, MAD2 localizes to kinetochores in

prometaphase and disappears from the kinetochores during

metaphase, in correlation with the attachment of k-fibers. In

meiosis, the disappearance correlates less with microtubule

attachment and more with the distance between the kine-

tochores, consistent with a tension-based mechanism.

Kinetochore association of MAD2 was also reported in

wheat (Kimbara et al. 2004). In addition, the localization

dynamics of Arabidopsis MAD2, BUB3.1 and BUBR1 in

tobacco suspension cultured cells has been described.

Arabidopsis MAD2, BUB3.1 and BURB1 are concentrated

at kinetochores if cells are arrested with either the micro-

tubule-destabilizing drug propyzamide or the proteasome

inhibitor MG132. Without arrest, all three proteins are

found diffusely distributed in the nucleus and cytoplasm. In

addition, BUB3.1 appears to have a specific location at the

phragmoplast/cell plate during cytokinesis (Caillaud et al.

2009). While AtMAD1 has not been experimentally

investigated, the Arabidopsis gene At5g49880 has been

annotated as putative MAD1 ortholog.

Recently, an intriguing connection between the SAC

proteins MAD1 and MAD2 and the nuclear pore complex

has emerged in several organisms (Buffin et al. 2005; Lee

et al. 2008; De Souza et al. 2009). The common theme of

these findings is that MAD1 and MAD2 are found asso-

ciated with the nuclear periphery during interphase and that

the inner nuclear basket nucleoporin Tpr (mammals), Mtor

(Drosophila), or Mlp1/2 (yeast, Aspergillus) is involved in

this association (Scott et al. 2005; Lee et al. 2008; De

Souza et al. 2009; Lince-Faria et al. 2009). In Drosophila,

Mtor depletion leads to a reduction of the Mad2 signal at

kinetochores and an accelerated entry into anaphase, con-

sistent with a weakened checkpoint (Lince-Faria et al.

2009). Depletion of Tpr leads to a loss of MAD2 from the

kinetochores, reduced Mad1 at kinetochores and to chro-

mosome segregation defects (Lee et al. 2008). These data

indicate that the presence of the SAC proteins at the

nuclear pore in interphase primes them for their mitotic

function by an unknown mechanism.

In plants, the connection between the nuclear pore and

the spindle assembly checkpoint has not been addressed.

Here, we show that the Arabidopsis protein NUA, the

ortholog of Tpr, Mtor, and Mlp1/2, binds to Arabidopsis

MAD1 (AtMAD1) and that AtMAD1 is associated with the

nuclear periphery. AtMAD1 binds to AtMAD2, which is

located in the nucleus and cytoplasm with some enrichment

at the nuclear envelope (NE). In a NUA mutant, AtMAD1

is displaced from the NE and the AtMAD2 NE signal also

disappears. Importantly, in the nua-4 null allele, root length

and meristematic size are affected in a way similar to the

AtMAD2 allele mad2-2, in which no full length AtMAD2

transcript was detected. These data indicate that the inter-

action of SAC proteins with the nuclear pore is conserved

beyond the opisthokonts, and that NUA and MAD2 may

play a role in meristematic cell division in plants.

Materials and methods

Plant materials

T-DNA insertion mutant lines nua-1 (SALK_057101), nua-

4 (WiscDsLox297300_17E), and atnup160-3 (SAIL_

877_B01) have been characterized previously (Xu et al.

2007a, b; Muthuswamy and Meier 2011). T-DNA

insertion mutant lines mad2-2 (SAIL_191_G06), mad2-3

(SALK_125904) and mad2-4 (SALK_136419) were

acquired from the Arabidopsis Biological Resource Center

(Alonso et al. 2003). Lines homozygous for the T-DNA

insertions were identified by PCR-based genotyping. Ara-

bidopsis thaliana wild type (Columbia ecotype, WT-Col)

and T-DNA insertion lines were grown in soil under

standard long-day condition (16 h light and 8 h dark) at

22 �C, or on Murashige and Skoog (MS) plates under

constant light at 22 �C. MS plates with 2 % sucrose were

used unless indicated otherwise.

Protein sequence analysis

To identify putative Arabidopsis orthologs of human Mad1

(GenBank ID: Q9Y6D9) and Mad2 (GenBank ID:

Q13257), GenBank non-redundant protein sequences of

Arabidopsis thaliana were searched using blastp. For

hMad1, At5g49880 encoded the ORF with highest simi-

larity to hMad1, with 28 % identity, 49 % similarity and an

e-value of 3e-20. At5g49880 was thus named AtMAD1.

Like hMad1, AtMAD1 has an extended coiled-coil domain,

and had been identified by that feature as one of the long

coiled-coil proteins in the Arabidopsis genome (Rose et al.

2004). The ORF with the second-highest similarity to

hMad1 had an e-value of 0.039 and was not further con-

sidered. For hMad2, the ORF with highest similarity was

encoded by At3g25980 (annotated as MAD2) with 47 %

identities, 68 % similarity and an e-value of 2e-66. The

ORF with the second-highest similarity was with the ORF

encoded by At1g16590 (annotated as MAD2b). With 23 %

identity, 49 % similarity and an e-value of 3e-06, this ORF

is unlikely to represent a second copy of Arabidopsis

MAD2 and was not further considered here.

Genotyping of T-DNA insertion lines

Genomic DNA was extracted according to a published

protocol (Krysan et al. 1999). Primers were designed using

IDT iSci tools (http://www.idtdna.com/analyzer/Applica

tions/OligoAnalyzer/). The T-DNA insertion sites were

204 Plant Mol Biol (2012) 79:203–216

123

determined by aligning the sequence of each T-DNA

insertion line as determined by the SIGnAL (Salk Institute

Genomic Analysis Laboratory) database (http://signal.salk.

edu) and the genomic sequence of AtMAD2. The T-DNA

insertion site of mad2-2 was also confirmed by sequencing

the product amplified with the T-DNA specific primer

pCSA110-LB and gene specific primer MAD2-2GpR. For

mad2-3 and mad2-4, primer pairs MAD2-3GpF/MAD2-

3GpR or MAD2-3GpF/MAD2-4GpR and MAD2-3GpF/

LBa4 were used to genotype the T-DNA insertion lines. For

nua-4, primer pairs CS850695FP/p745-primer and CS8506

95RP/p745-primer were used as previously described (Xu

et al. 2007b).

Root length and meristem size analysis

Seeds of WT-Col, nua-4, mad2-2, mad2-3, mad2-4, and lines

over-expressing GFP-AtMAD2 were grown on MS plates

with either 0 or 2 % sucrose under constant light at 22 �C.

After 8 days of growth, the primary root lengths of at least 45

seedlings were measured. To estimate the division zone (DZ)

size, 8-day old seedlings were stained with 10 lg/ll propi-

dium iodide (PI) for 7 min. Confocal images were taken with

a Nikon Eclipse 90i confocal microscope. The border

between the division zone and the elongation zone was

determined as described (Perilli and Sabatini 2010). The size

in the division zone was measured using the Nikon NIS-

Elements Microscope Imaging Software (Fig. 3d). Statistic

significance of differences was calculated by Student’s t test

using the Microsoft Excel software package.

RNA extraction and quantification

Total RNA was extracted with the RNeasy Plant Mini kit

(Qiagen) from 5-day-old Arabidopsis seedlings grown on

MS plates with 2 % sucrose. One microgram of the total

RNA was incubated with 1 ll DNase I (Invitrogen). After

digestion, cDNA synthesis was performed using oligo-dT

primers and the SuperScript III First-Strand Synthesis

System (Invitrogen). The cDNAs were used as templates in

PCR (RT-PCR) and Quantitative Real Time PCR (Q-PCR)

amplification with gene-specific primers (Table S1). For

RT-PCR, one microliter reverse-transcribed cDNA was

used as template in a total of 50 ll amplification mixture

and 30 PCR cycles were run. PCR products were separated

on 1 % agarose gels and band intensities were quantified

with the software ImageJ (Collins 2007). All band inten-

sities were first normalized against the Actin II control and

then expressed relative to the WT level set to one. The

experiment was repeated five times, with three biological

repeats. Statistic significance of differences was calculated

by Student’s t test using the Microsoft Excel software

package. Q-PCR was performed using the iQTM SYBR�

Green Supermix system (Bio-Rad). 1 ll cDNA solution

was used as template in 20 ll assay volume. Forty cycles

were run and the data were analyzed with the CFX96

TouchTM Real-Time PCR Detection System (Bio-Rad).

Standards were run in triplicate on each plate and two

biological repeats were conducted.

Plasmid vector construction

The AtMAD2 cDNA was obtained from the ABRC and

cloned into the pENTR/D-TOPO vector (Invitrogen). The

AtMAD1 cDNA was cloned by RT-PCR from RNA isolated

from 2-week old seedlings and inserted into pENTR/D-

TOPO. The inserts were confirmed by sequencing. To con-

struct the N-terminal GFP fusion proteins, each cDNA was

inserted into the Gateway binary vector pGWB6 (Nakagawa

et al. 2007) by LR recombination cloning (Invitrogen). To

construct the vector expressing HA-AtMAD1, the AtMAD1

full coding region was moved from pENTR/D-TOPO into

pEarlyGate 102 by LR recombination. For Bimolecular

Fluorescence Complementary (BiFC) constructs (NUA-

CGFP, AtMAD1-NGFP, AtMAD2-NGFP, and RanGAP-

NGFP) driven by CaMV 35S promoter, a multisite Gateway

reaction was conducted to generate translational fusions

driven by the CaMV 35S promoter in the pH7m34GW and

pK7m34GW destination vectors according to available

protocols (Boruc et al. 2010).

Yeast two-hybrid assays

Yeast two-hybrid (Y2H) vectors pDEST22 and pDEST32

(Invitrogen) harboring AtMAD1, AtMAD2, and AtYRA1

(At5g59950) full coding region were constructed according

to published protocols (Dohmen et al. 1991; James et al.

1996) and Y2H assays were conducted as described in the

Clonetech Yeast Protocols Handbook (1996).

Generation of transgenic plants

Expression vectors were transformed into Agrobacterium

tumefaciens (Agrobacterium) strain GV3101 by electropor-

ation. Arabidopsis WT-Col were transformed by floral dip

(Clough and Bent 1998) with pGWB6 harboring AtMAD1 or

AtMAD2 and selected by kanamycin (50 lg/ml) resistance

for primary transformants. The T1 generation of transfor-

mants was subjected to further analysis, such as immunola-

beling, primary root growth and fluorescence analysis.

Agrobacterium infiltration, BiFC

and immunoprecipitation

Agrobacterium strains containing constructs expressing NUA-

CGFP, AtMAD1-NGFP, AtMAD2-NGFP, and RanGAP-

Plant Mol Biol (2012) 79:203–216 205

123

NGFP were incubated overnight in LB liquid cultures with the

corresponding antibiotic at 28 �C. Agrobacterium was col-

lected by centrifugation and re-suspended with infiltration

buffer (50 mM MES, 2 mM MgCl2, and 100 mM acetosy-

ringone) with a final OD600 of 0.4. For BiFC analysis, Agro-

bacterium mixtures containing constructs NUA-CGFP and

AtMAD1-NGFP, NUA-CGFP and AtMAD2-NGFP or NUA-

CGFP and RanGAP1-NGFP were infiltrated into Nicotiana

benthamiana epidermal leaves as described (Yang et al. 2000;

Boruc et al. 2010). After 3 days, the epidermal cells were

examined for GFP fluorescence by Nikon Eclipse 90i confocal

microscope. RanGAP1-NGFP was used as an unrelated pro-

tein to serve as negative control.

For immunoprecipitation (IP), combinations of GFP-

AtMAD2 and HA-AtMAD1 (GFP-AtMAD2/HA-At-

MAD1) or GFP and HA-AtMAD1 (GFP/HA-AtMAD1)

were transiently co-expressed in N. benthamiana epidermal

leaves for 3 days together with P19 as silencing suppressor

(Zhao et al. 2006). The following steps were conducted at

4 �C. Four hundred microliter of tissue powder from the

N. benthamiana leaves containing combinations of GFP-

AtMAD2/HA-AtMAD1 or GFP/HA-AtMAD1 were sus-

pended with 1 ml IP buffer (50 mM Tris–Cl, 150 mM

NaCl, 0.5 % Nonidet P-40, 1 mM EDTA, 3 mM DTT,

1 mM PMSF, and protease inhibitor cocktail (Sigma-

Aldrich, St. Louis, USA). After centrifugation at

16,000g for 10 min, the supernatant was for 3 h incubated

with anti-HA antibody (Sigma-Aldrich) (1:200 dilution) or

anti-GFP antibody (Invitrogen) (1:100 dilution) bound to

protein A-Sepharose. After centrifugation at 1,000g for

3 min, the immunoprecipitates were washed for 3 times

with IP buffer and suspended in 50 ll 1 9 SDS-PAGE

loading buffer and subjected to 12 % SDS-PAGE and

immunoblotting with anti-GFP antibody (A11122, 1:2,000;

Molecular Probes, Eugene, OR) and HRP-conjugated anti-

HA antibody (1:2,000; Sigma-Aldrich). The HRP-conju-

gated anti-rabbit secondary antibody (1:15,000, GE

healthcare, Piscataway, NJ) was used against anti-GFP.

The Supersignal West Pico Chemiluminescent Substrate

for the HRP system was used for detection.

BY-2 cell culture and transformation

Bright Yellow-2 (BY-2) tobacco (Nicotiana tabacum) cell

suspension cultures were maintained and transformed

according to protocol (Joubes et al. 2004) with slight

modifications. BY-2 cells were grown in modified

Murashige and Skoog medium (BY-2 medium) (David and

Perrot-Rechenmann 2001) at 25 �C in the dark and were

diluted 1:10 in fresh medium every 7 days. To generate

BY-2 cells stably expressing GFP-AtMAD2 driven by the

35S promoter, 5 ml of a suspension of stationary phase

cells grown for 7 days were transferred to 50 ml of fresh

medium. After 2 days, 5 ml of BY-2 cells and 500 ll of

Agrobacterium (OD600 = 0.8) bearing the plasmid

pGWB6 containing AtMAD2 were co-inoculated in Petri

dishes in the dark for 3 days without agitation. Then, the

cells were plated on a BY-2 medium with 0.8 % agar

containing 50 mg/ml kanamycin and 500 mg/ml carbeni-

cillin. After 3 weeks, individual resistant calli were trans-

ferred into fresh solid BY-2 medium for maintenance or

were re-suspended in liquid BY-2 medium to obtain liquid

suspension of transgenic cells expressing GFP-AtMAD2.

To investigate the localization of GFP-AtMAD2 during

mitosis, 5 ml BY-2 cells expressing GFP-AtMAD2 were

transferred to fresh BY-2 medium for 3 days and cells were

stained for 30 min with DRAQ5 (1:50 dilution, Biostatus

Limited, UK), a live cell DNA dye. For the detection of

GFP in the green channel, the 488-nm excitation line of an

Argon laser was used in combination with a 515/530-nm

band-pass emission filter. To detect the DNA stained with

DRAQ5 in the far-red channel, the 635-nm excitation of a

modulated diode laser was used in combination with a

650LP long-pass emission filter. The Nikon EZ-C1 soft-

ware was used for image capture.

Results

Characterization of AtMAD2 T-DNA insertion lines

Three Arabidopsis lines with T-DNA insertions in AtMAD2

were selected from the SIGnAL T-DNA insertion line

collection (Sessions et al. 2002; Alonso et al. 2003)

(Fig. 1a) and given the mutant allele names mad2-2, mad2-

3, and mad2-4. The allele mad2-2 has a T-DNA insertion at

nucleotide ?579 (with ?1 being the A of the start codon

ATG), which is within exon #4 (Fig. 1a). Flanking

sequences of the mad2-2 T-DNA insertion site were

amplified by PCR and sequenced to confirm the insertion

site (data not shown). Although the insertion sites of mad2-

3 and mad2-4 were mapped to the same site at -392 (with

?1 being the A of the start codon ATG), mad2-3 and

mad2-4 are treated as two separate alleles by SIGnAL and

TAIR. Because the upstream flanking sequences are not

known, it can not be excluded that the insertions are

physically different. Therefore, in this study we charac-

terized them as separate alleles.

To identify whether full-length transcripts of AtMAD2

were present in these lines, RT-PCR was performed with

primer pair 3 (Fig. 1a). The results showed that no full-

length AtMAD2 transcript was detected in mad2-2 after 30

PCR cycles (Fig. 1b, c) or 40 PCR cycles (data not shown),

indicating that most likely no full-length AtMAD2 protein

is present in mad2-2. When using quantitative RT-PCR

(Q-PCR) to quantify the amount of AtMAD2 transcripts

206 Plant Mol Biol (2012) 79:203–216

123

with a primer pair amplifying exon #6 and exon #7

(Fig. 1a), a significantly higher amount of transcript was

detected in mad2-2 than in WT (Fig. S1). Together, these

data indicate that—consistent with the site of the T-DNA

insertion—no full length AtMAD2 transcript can be

detected but that the region of the AtMAD2 gene 30 of the

T-DNA insertion is transcribed, likely as a run-through

transcript initiated in the T-DNA.

For mad2-3 and mad2-4, slightly higher AtMAD2

transcript levels were detected with RT-PCR using primer

pair 3 (Fig. 1b, c). Quantification of band intensities from

5 experiments suggests that the relative AtMAD2 mRNA

levels in mad2-3 and mad2-4 are indeed higher than in

WT (p \ 0.01) (Fig. 1c). Consistent with these results,

Q-PCR analysis also detected higher levels of AtMAD2

transcripts in mad2-3 and mad2-4 (Fig. S1). In summary,

we conclude that no full-length AtMAD2 mRNA accu-

mulates in mad2-2 and that somewhat increased levels of

full-length AtMAD2 mRNA accumulate in mad2-3 and

mad2-4.

Phenotypic analysis of AtMAD2 T-DNA mutants

No gross phenotypic alterations were identified in the

AtMAD2 mutant and overexpressor lines described here

when soil-grown plants were observed. It has been shown

that in fungi and animals MAD2 regulates cell cycle pro-

gression through inhibiting anaphase entry until all chro-

mosomes are bidirectionally attached to microtubules

(Musacchio and Salmon 2007; Skinner et al. 2008). In

mice, MAD2 down-regulation initiates apoptosis (Dobles

et al. 2000), whereas MAD2 over-expression triggers

tumor formation (Sotillo et al. 2007). To investigate the

potential roles of AtMAD2 in Arabidopsis cell division and

development, the length of the primary root of AtMAD2

T-DNA insertion alleles that are either null for AtMAD2

(mad2-2) or slightly over-accumulating AtMAD2 mRNA

(mad2-3 and mad2-4) was measured. At 8 days post ger-

mination, roots of mad2-2 seedlings grown in the absence

of sucrose were significantly stunted (Fig. 2a, g), whereas

mad2-3 and mad2-4 roots were slightly longer that WT

seedling roots (Fig. 2c, e, g). Addition of 2 % sucrose

partially rescued the mad2-2 growth inhibition (Fig. 2b, g),

while it had only a marginal effect on mad2-3 and mad2-4

(Fig. 2d, f, g). Sucrose promotes cell division in the root,

and the current model in the field is that this is mediated

through the up-regulation of CycD expression (Riou-Kha-

mlichi et al. 2000; Eveland and Jackson 2011).

To investigate whether the small, but significant

increase in root growth observed in mad2-3 and mad2-4 is

due to the overexpression of AtMAD2, lines expressing

GFP-AtMAD2 driven by a CaMV 35S promoter were also

(A)

(B) (C)

*

*

**

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

Ban

d R

elat

ive

Inte

nsity

Fig. 1 Characterization of AtMAD2 T-DNA insertion lines.

a Position of T-DNA insertions and PCR primers. The confirmed

insertion sites of mad2-2, mad2-3, and mad2-4 are shown by verticalarrows above and below the schematic genomic structure of the

AtMAD2 gene. Primer pairs 1, 2 and 3 were used for mad2-3, mad2-4and mad2-2 mutant genotyping. Primer pair 4 was used for full length

AtMAD2 RT-PCR. Primer pair 5 was used for quantitative real-time

PCR. b RT-PCR products amplified with primer pair 3 indicated in

(a) and Actin II primer pair (Table S1). The Arabidopsis genotypes

are indicated on the top. WT, Arabidopsis ecotype Col-0 wild type.

c Quantification of band intensity of five RT-PCR experiments [as

shown in (b)] using the software ImageJ. Asterisks indicate a

significant difference from WT (at least p \ 0.05)

Plant Mol Biol (2012) 79:203–216 207

123

investigated. T1 transgenic seedlings from five independent

lines expressing GFP-AtMAD2 (shown by GFP fluores-

cence, data not shown) also had longer roots after growth

on MS plates with 2 % sucrose for 8 days (Fig. S2), similar

to mad2-3 and mad2-4. These data suggest that overex-

pression of AtMAD2 might promote primary seedling root

growth, while down-regulation of AtMAD2 clearly inhibits

primary seedling root growth in Arabidopsis.

The postembryonic growth of the plant root occurs from

localized regions, the meristems. In the primary Arabid-

opsis root, a small stem cell niche (the quiescent center)

generates cells that divide a finite number of times in the

proximal meristem, the division zone (DZ). They then

enter a zone of rapid cell elongation and differentiation in

the elongation-differentiation zone (EDZ). Elongation of

the primary root is thus regulated by the rate of cell divi-

sion in the DZ and the rate of elongation/differentiation in

the EDZ (Dello Loio et al. 2008). The altered root length of

AtMAD2 T-DNA insertion alleles could thus be attributed

either to altered meristematic division or to root elongation

abnormalities. Since AtMAD2 was shown to be primarily

expressed in meristematic tissues in Arabidopsis (Caillaud

et al. 2009), it was most likely that a meristematic abnor-

mality would be the primary cause for the altered primary

root length in the AtMAD2 T-DNA insertion alleles. In

addition, no sign of increased cell elongation or increased

endoreduplication in the EZ, as assessed by the size and

patterning of stained nuclei, was observed (data not

shown).

The size of the Arabidopsis seedling root meristem can

be quantified by measuring the length from the root tip to

the cell wall immediately below the first cortex cell

undergoing cell elongation (see Fig. 3d for illustration), or,

alternatively, by counting one row of cortex cells up to this

point (Perilli and Sabatini 2010). As shown in Fig. 2,

mad2-2 has a shorter DZ, while mad2-3 and mad2-4 have

slightly longer DZs, both in the absence and in the presence

of 2 % sucrose (Fig. 2h). These data suggest that the pri-

mary effect of AtMAD2 on seedling root growth occurs in

the DZ, consistent with the fact that in Arabidopsis roots,

AtMAD2 is only expressed in the DZ (Caillaud et al.

2009).

Orthologs of NUA Tpr/Megator/Mlp1p/Mlp2/An-Mlp1

have been demonstrated as NE anchors of SAC proteins

in mammalian cells, Drosophila, yeast and Aspergillus,

suggesting a functional involvement in a SAC protein

role (Lee et al. 2008; De Souza et al. 2009; Lince-Faria

et al. 2009). Thus, the primary root growth and root DZ

size were also checked in nua-4, a knock-out line of

NUA (Xu et al. 2007b). Consistent with the implication

of their functional connection, nua-4 and mad2-2 have

such similar phenotypes as stunted primary root devel-

opment in the absence of sucrose, shorter primary

roots in the presence of 2 % sucrose, and smaller DZ

size in the absence and presence of 2 % sucrose

(Fig. 3a–c, e).

Roo

t Len

gth

(cm

)

WT mad2-2 mad2-3 mad2-4 WT mad2-2 mad2-3 mad2-4

0% sucrose

WT mad2-2 mad2-3 mad2-4 WT mad2-2 mad2-3 mad2-4

(A)

(C)

(E) (F)

(G)

(H)

WT 4-2dam4-2dam

(B)

(D)

WT

WT

TWTW

WT

mad2-2 mad2-2

mad2-3 mad2-3

*

*

* *

0

50

100

150

200

250

300

350

400

450

Div

isio

n Z

one

Leng

th (

µm)

2% sucrose

0% sucrose 2% sucrose

Fig. 2 Phenotypic characteristics of AtMAD2-mutant seedling roots.

a, c, and e Seedlings at 8 days after germination on MS plate with

0 % sucrose, grown under long-day conditions. b, d and f Seedlings at

8 days after germination on MS plate with 2 % sucrose, grown under

long-day conditions. g Length quantification of seedling roots grown

as shown in a–f. A minimum of 45 seedlings each were measured.

h Length quantification of the division zone of seedling roots grown

as shown in a–f. Ten seedlings each were randomly picked from the

seedlings used in (g) to measure the length of the division zone.

Asterisks indicate significant difference from WT (p \ 0.05)

208 Plant Mol Biol (2012) 79:203–216

123

Physical interaction among NUA, AtMAD2

and AtMAD1

The mammalian homolog of NUA (Tpr) directly interacts

with MAD1 and MAD2 in immunoprecipitation assays and

MAD1 interacts with MAD2 at the nuclear rim and at the

kinetochore (Lee et al. 2008). To investigate the physical

interactions between Arabidopsis NUA, AtMAD1, and

AtMAD2, yeast two-hybrid (Y2H) assays were performed.

The data shown in Fig. 4 support AtMAD1-AtMAD1,

AtMAD1-NUA, and AtMAD1-AtMAD2 interactions, but

no NUA-AtMAD2 interaction. To confirm the interaction

between AtMAD1 and AtMAD2 in planta, co-immuno-

precipitation (CoIP) assays were carried out. Either GFP-

AtMAD2 and HA-AtMAD1 or GFP and HA-AtMAD1

were transiently expressed in the Nicotiana benthamiana

leaf epidermis (Fig. 5). After immunoprecipitation with a

GFP antibody, both GFP-AtMAD2 and GFP were detected

in the precipitates from GFP-AtMAD2 & HA-AtMAD1

and GFP & HA-AtMAD1 infiltrated leaves. However, HA-

AtMAD1 was only detected in the precipitates from GFP-

WT nua-4

0% sucrose

WT nua-4

2% sucrosse

(A) (B)

(C) (D)

(E)

0

50

100

150

200

250

300

350

400

Div

isio

n Z

one

Leng

th (

µm)

0

1

2

3

4

5

Roo

t Len

gth

(cm

)

WT nua-4 WT nua-4

0% sucrose 2% sucrose

WT nua-4 WT nua-4

0% sucrose 2% sucrose

*

*

*

*

Fig. 3 Phenotypic characteristics of nua-4 seedling roots. a and

b Representative 8-day old seedlings grown on MS with 0 or 2 %

sucrose under long-day conditions. c Primary root length of 8-day old

seedlings grown on MS with 0 or 2 % sucrose under long-day

conditions. Mean values and standard deviation for at least 45

seedlings each are shown. d An 8-day old WT seedling stained with

propidium iodide. Length of the division zone and position of the first

elongating cell in the cortex (inset) are indicated. e Length of the

division zone of 8-day old seedlings grown on MS with 0 or 2 %

sucrose under long-day conditions. Ten seedlings each were randomly

picked from the experiment shown in (c). Asterisks indicate

significant difference compared to WT (p \ 0.05)

BD:AtY

RA1

BD:AtM

AD2

BD AD:AtY

RA1

AD:AtM

AD2

BD

AD:AtMAD1

AD:AtMAD1

BD:AtMAD1

BD:AtMAD1

-L-T-H

-L-T

AD

AD

BD:AtM

AD1

BD:AtM

AD1

BD:AtM

AD2

AD:AtM

AD1

AD:AtM

AD2

AD:AtMAD1

AD:AtMAD1

AD:NUA

AD:NUA

BD:NUA

BD:NUA

-L-T-H

-L-T

(A) (B) (C)

(D) (E) (F)

Fig. 4 Yeast two-hybrid interactions between NUA, AtMAD1, and

AtMAD2. a, b AtMAD1 interacts with AtMAD2, but not with the

unrelated protein AtYRA1. c Empty vector control. d AtMAD1 self-

interacts. e, f NUA interacts with AtMAD1 but not AtMAD2. AD,

GAL4 activation domain; BD, GAL4 DNA-binding domain; –L–T–

H, dropout medium without leucine, tryptophan, and histidine; –L–T,

dropout medium without leucine and tryptophan

Input α HA α GFP

IP

HA-AtMAD1 &GFP-AtMAD2

PF

G α :BI

AH α :

BI

HA-AtMAD1 &GFP

Input α HA

IP

α GFP

(A)

(B)

HA-AtMAD1 &GFP-AtMAD2

HA-AtMAD1 &GFP

Fig. 5 AtMAD2 interacts with AtMAD1 in a co-immunoprecipita-

tion assay. HA-AtMAD1 and GFP-AtMAD2 or HA-AtMAD1 and

GFP were co-expressed in epidermal cells of Nicotiana benthamianaleaves, as indicated on the right of each panel (a, b). Immunoblots

(IB) in (a) were detected with an anti-HA antibody (aHA).

Immunoblots in (b) were detected with anti-GFP antibody (aGFP).

Input indicates that protein extract was directly detected for

HA-AtMAD1, GFP-AtMAD2 and GFP. ‘‘IP’’ indicates which anti-

body was used for immunoprecipitation

Plant Mol Biol (2012) 79:203–216 209

123

AtMAD2 & HA-AtMAD1 infiltrated leaves, but not from

GFP & HA-AtMAD1 infiltrated leaves. When the anti-HA

antibody was used to precipitate proteins from GFP-At-

MAD2 & HA-AtMAD1 and GFP & HA-AtMAD1 infil-

trated leaves, GFP-AtMAD2 was detected in the

precipitate, but not GFP. These data indicate that AtMAD1

and AtMAD2 associate in planta.

Full-length GFP-NUA or NUA-GFP cannot be immu-

noprecipitated to a detectable level in our hands, likely due

to extreme protein instability during the IP protocol (data

not shown). Therefore, the in planta interactions between

NUA and AtMAD1 or NUA and AtMAD2 were investi-

gated by BiFC analysis (Fig. 6). Co-expression of NUA-

CGFP (NUA fused to the C-terminal half of GFP) and

AtMAD1-NGFP (AtMAD1 fused to the N-terminal half of

GFP) resulted in the reconstituted GFP complexes in the

cytoplasm of epidermal cells (Fig. 6a). No GFP fluores-

cence was detected in the negative-control combination

NUA-CGFP and RanGAP1-NGFP (RanGAP1 fused to the

N-terminal half of GFP; Fig. 6b). Additionally, no GFP

fluorescence was detected in co-expression of NUA-CGFP

and NGFP-AtMAD2 (AtMAD2 fused to the N-terminal

half of GFP; Fig. 6c). These suggest that, consistent with

the yeast two-hybrid data, NUA interacts with AtMAD1

but not AtMAD2. The fact that reconstituted GFP fluo-

rescence was detected only in the cytoplasm of N. benth-

amiana epidermal cells suggests that the NUA-AtMAD1

complex does not associate with the nuclear envelope in

this heterologous system.

In summary, NUA interacts with AtMAD1, but not

AtMAD2 in an Y2H assay and in BiFC analysis. AtMAD1

interacts with AtMAD2 in both Y2H and CoIP assays.

These data are consistent with a complex consisting of

NUA, AtMAD1 and AtMAD2 (with AtMAD1 acting as a

bridge between NUA and AtMAD2) or with two separate

complexes, one containing AtMAD1 and NUA and the

second containing AtMAD1 and AtMAD2.

Subcellular localization of AtMAD1 and AtMAD2

Mad1 and Mad2 in mammalian cells are associated with

the nuclear envelope (NE) in interphase (Lee et al. 2008).

Given that AtMAD2 and NUA mutants have similar root

growth defects and NUA, AtMAD1, and AtMAD2 physi-

cally interact, we questioned if AtMAD1 and AtMAD2 are

associated with the NE and whether this interaction

requires NUA. To determine the localization of AtMAD1

in interphase, transgenic Arabidopsis lines expressing GFP-

AtMAD1 driven by the CaMV 35S promoter were gener-

ated. When roots of the T1 seedlings were imaged by

confocal microscopy, GFP-AtMAD1 was concentrated at

the NE (Fig. 7a, b). Co-immunolocalization utilizing anti-

GFP and anti-NUA antibodies to simultaneously detect

GFP-AtMAD1 and NUA showed clear co-localization of

the two signals at the NE (Fig. 7c, top panel). Consistent

with the interaction between AtMAD1 and NUA in Y2H

and BiFC assays, this suggests that AtMAD1 associates

with NUA at the NE in Arabidopsis.

To investigate the localization of AtMAD2, transgenic

Arabidopsis Col-0 lines were created that express GFP-

AtMAD2 driven by the CaMV 35S promoter. Fluorescence

signals from roots of live T1 transgenic seedlings contain-

ing GFP-AtMAD2 showed a distribution between the

nucleus and the cytoplasm and a concentration at the NE

(Fig. 8a). GFP-AtMAD2 was also predominantly nuclear in

the epidermal cells of N. benthamiana transiently express-

ing GFP-AtMAD2 (Fig. 8b, c) and in BY-2 cells stably

expressing GFP-AtMAD2 (Fig. 9a–c). In both cases, no

NUA-CGFP/AtMAD1-NGFP DIC Overlay

NUA-CGFP/RanGAP1-NGFP DIC Overlay

NUA-CGFP/NGFP-AtMAD2 DIC Overlay

(A)

(B)

(C)

(D)

Fig. 6 NUA and AtMAD1 interact in an in planta BiFC assay.

Imaging of epidermal leaf cells of N. benthamiana infiltrated with A.tumefaciens co-expressing NUA-CGFP and AtMAD1-NGFP (a),

NUA-CGFP and RanGAP-NGFP (b), NA-CGFP and NGFP-

AtMAD2 (c), or no exogenous protein (d). Left panels GFP

fluorescence, middle panel, differential interference contrast (DIC),

right panel merge of GFP and DIC images. Arrows in (a) point at

areas showing specific BiFC signal; green signal in (b–d) is

background autofluorescence. CGFP, C-terminal fragment of GFP;

NGFP, N-terminal fragment of GFP. The confocal images were

recorded at the same gain setting. Scale bars 50 lm

210 Plant Mol Biol (2012) 79:203–216

123

clear concentration at the NE was observed. This is con-

sistent with the prior report of AtMAD2 localization in the

nucleus and cytoplasm in epidermal cells of N. benthami-

ana and BY-2 cells (Caillaud et al. 2009). To further

address the localization of AtMAD2 in Arabidopsis, the

MAD2 (C19) (anti-hMAD2) antibody against human Mad2

(SC-6329; Santa Cruz Biotechnology Inc.) was used in an

immunofluorescence experiment using wildtype Col-0

seedlings. Using blastp analysis, the alignment between

Human Mad2 and AtMAD2 showed 47 % identity and

68 % similarity at the amino acid level over the entire

length of the protein, suggesting that this polyclonal serum

might successfully detect AtMAD2. The anti hMAD2

antibody decorated the nuclear rim co-labeled by the anti-

NUA antibody (Fig. 8d–f), suggesting that a fraction of

AtMAD2 and NUA co-localizes at the NE. However, the

anti-hMAD2 antibody did not detect any protein in immu-

noblots of whole Arabidopsis seedlings extracts (data not

shown). It is possible that the concentration of AtMAD2 is

too dilute in a whole-seedling protein extract to be observed

by immunoblotting, since the promoter activity of AtMAD2

is primarily confined to the meristematic regions (Caillaud

et al. 2009). Alternatively, the antibody might detect

AtMAD2 in fixed cells but not after SDS-PAGE, or

AtMAD2 was not sufficiently extracted from the nuclei by

our protocol. Additionally, using immunofluorescence

microscopy with the anti-GFP and anti-NUA antibodies,

co-localization of AtMAD2 and NUA at the NE could also

be shown in the root tip cells of transgenic seedlings

expressing GFP-AtMAD2 (Fig. 8m–o). These data indicate

that AtMAD2 is located in the nucleus and the cytoplasm

and that a fraction of AtMAD2 co-localizes with NUA at

the Arabidopsis NE. For reasons not fully explored, this

fraction is more clearly revealed by immunofluorescence

microscopy than by live imaging of GFP.

Kinetochore localization of AtMAD2

In metazoan cells, when spindle assembly was inhibited

with microtubule poisons, MAD2 was associated with the

GF

P-A

tMA

D1

in W

TG

PF

-AtM

AD

1 in

nua

-1

anti-GFP

GF

P-A

tMA

D1

in W

T

GF

P-A

tMA

D1

in W

T

(A) (B)

(C) Overlayanti-NUA

Fig. 7 Localization of GFP-AtMAD1 in the Arabidopsis root divi-

sion zone. a, b Live fluorescence images of GFP-AtMAD1 in root tip

cells of Arabidopsis. a Root tip. b A single root-tip cell. c Immuno-

fluorescence images of root tip cell files. The green and red channelswere probed with first antibodies anti-GFP and anti-NUA,

respectively, and with corresponding second antibodies. White arrowsin (a), (b), and top panel of (c) indicate nuclear envelope. Whitearrow in bottom panel of (c) indicates the location of the nucleus.

Scale bar in (a) = 50 lm. Scale bar in (b) = 5 lm. Scale bars in

(c) = 2 lm

Plant Mol Biol (2012) 79:203–216 211

123

kinetochores (Chen et al. 1996; Lee et al. 2008). In maize,

wheat and Arabidopsis, MAD2 was also shown to localize

to kinetochores when the SAC was chemically activated

(Yu et al. 1999; Kimbara et al. 2004; Caillaud et al. 2009).

However, maize MAD2 was also shown to localize to

kinetochores in prometaphase without any microtubule

poison treatment (Yu et al. 1999). To further gain insights

into the dynamic localization of AtMAD2 during mitosis, a

BY-2 suspension culture cell line was created that stably

expressed GFP-AtMAD2 under the control of the CaMV

35S promoter. This orientation of fusion was shown to not

disrupt the function of MAD2 in human cells (Howell et al.

2000).

The mitotic stages were determined based on the con-

densation of DNA visualized by live staining with DRAQ5.

GFP-AtMAD2 showed a strong nucleoplasmic and a

weaker cytoplasmic signal in interphase cells (Fig. 9a–c).

In prophase, GFP-AtMAD2 was found concentrated in

bright spots when DNA condensation could be observed

(Fig. 9d–f). In prometaphase (DNA aligning in the

metaphase plane, but trailing chromosomes still visible)

GFP-AtMAD2 was concentrated in bright spots on the

condensed chromosomes (Fig. 9g–i). When cells were in

metaphase (chromosomes perfectly aligned at the meta-

phase plane, no trailing chromosomes detected), GFP-

AtMAD2 was diffuse, with no bright spots detected

(Fig. 9j–l). No further concentration of GFP-AtMAD2 was

observed in cells during anaphase (not shown) and telo-

phase (Fig. 9m–o), defined by the beginning and pro-

gressing separation of the chromosomes. The same

localization of GFP-AtMAD2 in bright spots resembling

kinetochores was also observed in cells not counterstained

Fig. 8 Localization of AtMAD2. Live fluorescence images of GFP-

AtMAD2 in root tip cells of Arabidopsis (a) and epidermal cells of N.benthamiana (b, c). b Shows a whole cell and c shows the nucleus of

the cell in (b). Immunofluorescence images of root tip cell files in

interphase (d–o). Green in (d), (g), and (j) indicate proteins decorated

by anti-hMAD2 antibody; green in (m) indicate proteins detected by

anti-GFP antibody; red in (e), (h), (k) and (n) indicate proteins

detected by anti-NUA antibody. d, g, j, and m show the greenchannel only. e, h, k, and n show the red channel only. f, i, l, and

o show the green and red channels combined. The arrows in (f) and

(i) indicate the nuclear envelope. Scale bars (a, c) = 5 lm. Scale bar(b) = 50 lm. Scale bars (d–o) = 2 lm

Fig. 9 Localization of GFP-AtMAD2 during various stages of mitosis

in live tobacco BY-2 cells. The GFP signal from GFP-AtMAD2 is

shown in green and the signal from DNA stained is false-colored in bluewith DRAQ5. Fluorescence images of GFP-AtMAD2 in interphase (a–

c), prophase (d–f), prometaphase (g–i), metaphase (j–l), and anaphase/

telophase (m–o). (a), (d), (g), (j), and (m), GFP signal; (b), (e), (h), (k),

and (n), DRAQ5 signal; (c), (f), (i), (l), and (o), overlay. The arrows in

(d) and (g) point at the enrichment of GFP-AtMAD2 in the vicinity of

the kinetochores. Scale bars 5 lm

212 Plant Mol Biol (2012) 79:203–216

123

with DRAQ5 (data not shown), suggesting that it is not due

to DRAQ5 treatment. These data suggest that AtMAD2 is

associated with the kinetochores from prophase to early

metaphase, consistent with the predictions based on the

other model organisms.

Requirement of NUA for the nuclear envelope

localization of AtMAD1 and AtMAD2

In HeLa cells, the nuclear rim localization of human Mad1

and Mad2 requires the nucleoporin Tpr. Tpr directly binds to

Mad1 and Mad2, and Tpr depletion disrupts NE localization

of Mad1 and Mad2 (Lee et al. 2008). To investigate whether

NUA is also required for the NE localization of AtMAD1 in

Arabidopsis, we attempted to create transgenic nua-1 and

nua-4 lines expressing GFP-AtMAD1 under the control of

the CaMV 35S promoter. For nua-1, primary transformants

were obtained, however, they did not develop past the cot-

yledon stage. For nua-4, no primary transformants express-

ing GFP-AtMAD1 were obtained. When heterozygous nua-

4 plants expression GFP-AtMAD1 were self-pollinated, no

homozygous nua-4 stably expressing GFP-AtMAD1 could

be obtained in the progeny. These data suggest that over-

expression of GFP-AtMAD1 in NUA null mutants might be

lethal. Immunolabeling with anti-GFP and anti-NUA anti-

bodies was performed on root-tip cells of primary transfor-

mants of nua-1 stably expressing GFP-AtMAD1 (Fig. 7c,

bottom panel). These experiments were performed at the

cotyledon stage of T1 seedlings that eventually died without

developing the first pair of true leaves. GFP-MAD1 was

detected in the nucleus, not associated with the NE.

Co-labeling with the anti-NUA antibody showed only a

diffuse background signal, as previously reported for this

null allele (Xu et al. 2007b). These data suggest that the NE

association of GFP-AtMAD1 requires NUA.

Because AtMAD2 might indirectly interact with NUA

through AtMAD1, AtMAD2 NE localization might also

depend on NUA. Immunolabeling analysis using anti-

hMAD2 and anti-NUA indicated that the anti-hMAD2

antibody did not decorate the NE in nua-4 (Fig. 8g–i) or

nua-1 (data not shown). To test if the disappearance of

AtMAD2 from the NE was specific for the loss of NUA, or

was caused by a more generic defect in mutant nuclear

pores, we also tested AtMAD2 localization in nup160-3, a

mutant of the scaffold nucleoporin Nup160, which has very

similar developmental and molecular phenotypes as nua-1

and nua-4 (Muthuswamy and Meier 2011). Both anti-NUA

and anti-hMAD2 still decorated the NE in nup160-3

(Fig. 8j–l). Together, these data suggest that NUA is

required for the proper NE localization of both AtMAD1

and AtMAD2 and that this requirement does not

reflect gross alterations of the nuclear pore complex in the

mutant.

Discussion

The SAC protein MAD1 is conserved in mammalians,

Drosophila, C. elegans, Aspergillus, and yeast (Iouk et al.

2002; Scott et al. 2005; Katsani et al. 2008; Kitagawa 2009;

Lince-Faria et al. 2009; Fava et al. 2011). It has been

shown that MAD1 is associated with the nuclear envelope

in interphase and re-locates to the kinetochores when the

SAC is inactivated (Iouk et al. 2002; Scott et al. 2005; Lee

et al. 2008; De Souza et al. 2009). In mice, MAD1 is an

essential protein and a MAD1 null mutant is embryo lethal

(Iwanaga et al. 2007). MAD1 has not yet been investigated

in plants. Our data now show that Arabidopsis MAD1 is

also associated with the NE in interphase. Its association

with the NE requires NUA, the Arabidopsis homolog of the

nuclear basket protein Tpr. Consistent with results from

metazoans, AtMAD1 interacts with NUA and AtMAD2.

These data indicate that MAD1 proteins are conversed

across species, including plants.

MAD2 proteins have also been shown to be conserved

in mammalians, Drosophila, yeast, C. elegans, and plants

(Kitagawa and Rose 1999; Orr et al. 2007; Katsani et al.

2008; Caillaud et al. 2009; Barnhart et al. 2011; Fava et al.

2011). As a key SAC component, MAD2 delays the onset

of anaphase until all kinetochores are bidirectionally

attached to microtubules by inactivating CDC20, a regu-

latory subunit of the APC/C (Elledge 1998; Fang et al.

1998). Down-regulation of MAD2 proteins promotes

transition from metaphase to anaphase in several model

organisms and increases the chromosome segregation

errors in mammalian cells, which initiates apoptosis (Do-

bles et al. 2000; Michel et al. 2001; Orr et al. 2007;

Barnhart et al. 2011). Interestingly, over-expression of

MAD2 has been observed in various tumor cells and even a

transient over-expression of MAD2 is sufficient for trig-

gering tumor formation (Alizadeh et al. 2000; Garber et al.

2001; Chen et al. 2002; Sotillo et al. 2007).

Although AtMAD2 was first identified in 2009, its effect

on Arabidopsis development has not been characterized

(Caillaud et al. 2009). We have investigated here three

T-DNA insertion alleles of AtMAD2, which affect the size

of the seedling primary root. mad2-2, an allele lacking the

full-length AtMAD2 transcript, has a shorter primary root

and a smaller root division zone. These defects can be

partially rescued by supplying sucrose to the medium, an

effect implicated to reveal a cell-cycle defect (Eveland and

Jackson 2011; Hirano et al. 2011). Sucrose promotes cell

division in the root, which has been suggested to be med-

iated through the up-regulation of CycD expression (Riou-

Khamlichi et al. 2000; Eveland and Jackson 2011). This

phenotype is very similar to that of nua-4, a null mutant of

the nucleoporin NUA, suggesting that the two proteins are

both required for a step in root meristem growth the

Plant Mol Biol (2012) 79:203–216 213

123

disturbance of which can be overridden by exogenous

sucrose supplementation.

In contrast, the two promoter insertions mad2-3 and

mad2-4 lead to an increased abundance of AtMAD2 mRNA,

somewhat longer primary roots and an increased meristem

size. This effect is particularly revealed in the presence by

exogenous sucrose, suggesting that an overabundance of

AtMAD2 facilitates a sucrose-based stimulation of growth

(Rolland et al. 2006; Hirano et al. 2011). Consistent with

these data, the 35S promoter-driven expression of a GFP-

AtMAD2 fusion protein also leads to increased root length in

the presence of sucrose, supporting the above notion and

suggesting that GFP-AtMAD2 is functional. Together, these

data would be consistent with a role of AtMAD2 (and NUA)

in modulating meristem size and restricting the transition to

differentiation in Arabidopsis, in line with the finding that

AtMAD2 is expressed predominantly in meristematic tissues

(Caillaud et al. 2009). One established function of non-plant

MAD2 is to act as an inhibitor of the regulatory subunit

CDC20 of the APC/C. The APC/C is a large cell-cycle-

regulated E3 ligase. The Cdh1-driven APC/C targets cyclin

B for degradation and controls exit from mitosis and G1

maintenance. In addition, it targets securin for degradation,

leading to chromosome separation. Several mutants of APC/

C subunits have recently been described in plants. Interest-

ingly, APC/C appears to be expressed also in non-cycling,

differentiated cells and has been functionally implied in

hormone regulation, vascular development, and cell fate

determination in Arabidopsis (Marrocco et al. 2009, 2010;

Lindsay et al. 2011). A mutant of a recently described neg-

ative regulator of Arabidopsis APC/C, ULTRAVIOLET-B-

INSENSITIVE4 (UVI4) affects the primary root meristem

very similarly to mad2-2 and nua-4 (Heyman et al. 2011).

Uvi4 mutants prematurely exit the cell cycle and trigger the

onset of the endocycle. While UVI4 acts as a regulator of

APC/C during S phase, it is conceivable that lack of temporal

inhibition of APC/C both during S and M phase triggers exit

from the cell cycle and entry into differentiation, thereby

regulating meristem size and plant growth rate.

NUA is the Arabidopsis ortholog of a long coiled-coil

nucleoporin named Tpr, Mtor, and Mlp1/Mlp2 in mamma-

lian systems, Drosophila and yeast, respectively (Xu et al.

2007b). In HeLa cells and yeast, interactions of Tpr and

Mlp1/Mlp2, respectively, with the SAC proteins MAD1 and

MAD2 have been demonstrated (Scott et al. 2005; Lee et al.

2008). In HeLa cells, Tpr directly interacts with MAD1 and

MAD2 (Lee et al. 2008). Although no physical interaction of

Mlp1p and Mlp2p with yeast Mad1p and Mad2p was shown,

Mad1p requires Mlp1p and Mlp2p for nuclear rim associa-

tion (Scott et al. 2005). Based on our Y2H, BiFC and CoIP

assays, the physical interactions among the Tpr-like proteins

and MAD1/MAD2 vary between the different organisms

investigated. In Arabidopsis, NUA interacts with AtMAD1,

but not AtMAD2, while AtMAD1 interacts with AtMAD2.

Thus, the physical attachment of the MAD1/MAD2 complex

with the nuclear pore might be based on different affinities of

the SAC proteins to nucleoporins in different organisms that

lead to the same outcome: an association of MAD1/MAD2

with the NE in interphase. In Arabidopsis, NUA, AtMAD1

and AtMAD2 can be in the same complex if NUA interacts

with AtMAD2 through its association with AtMAD1 as

intermediate. Consistent with this hypothesis, AtMAD1 and

AtMAD2 co-localize with NUA at the nuclear rim in inter-

phase and disappear from the NE in NUA null mutants,

suggesting that NUA functions as a NE scaffold for

AtMAD1 and AtMAD2. It is currently not resolved whether

the disappearance of AtMAD1 and AtMAD2 from the NE in

NUA null mutants is also accompanied with a reduction in

protein abundance or is solely a delocalization effect.

AtMAD2 was detected in different locations based on

different methods. Live imaging of GFP-AtMAD2 showed

an enrichment in the nucleus in root tip cells of Arabidopsis,

in epidermal cells of N. benthamiana and in BY-2 cells.

However, the NE was decorated predominantly when

AtMAD2 was detected with the anti-hMAD2 antibody or

when GFP-AtMAD2 was detected with the anti-GFP anti-

body. This discrepancy is probably due to the technical dif-

ference between immunofluorescence microscopy and live

imaging. When whole-mount immunofluorescence was used

to detect AtMAD2 localization, unbound AtMAD2 in the

nucleus and cytoplasm might be washed out by the MTSB

buffer (Friml et al. 2003) and only the bound AtMAD2 in the

NUA/AtMAD1/AtMAD2 complex might remain at the NE.

The NE-bound AtMAD2 could then be detected with the

anti-hMAD2 antibody and anti-GFP antibody.

MAD2 has been demonstrated to be a key sensor for the

attachment of microtubules to kinetochores (Fang et al.

1998; Nasmyth 2005). In live PtK1 cells, GFP fusions of

human MAD2 (GFP-hMAD2) and Xenopus MAD2 (GFP-

XMAD2) begin to concentrate at the kinetochore during

prophase and early prometaphase (Howell et al. 2000).

Once cells progress to metaphase, GFP-hMAD2 and GFP-

XMAD2 signals disappear from kinetochores and remain

off the kinetochores during the following anaphase and

telophase (Howell et al. 2000). Consistent with the locali-

zation dynamics of GFP-hMAD2 and GFP-XMAD2, GFP-

AtMAD2 was here also found at kinetochore-like spots

along with the condensation of DNA in prophase/promet-

aphase, but not once chromosomes were fully aligned in

the metaphase plane. These results differ from the reported

localization of AtMAD2-GFP in prometaphase in live

BY-2 cells (Caillaud et al. 2009), where no AtMAD2-GFP

concentration at kinetochores was seen during the entire

cell cycle. This discrepancy might be due to the orientation

of the GFP fusions used in the two studies. It is conceivable

that only GFP-AtMAD2, but not AtMAD2-GFP can

214 Plant Mol Biol (2012) 79:203–216

123

associate with the kinetochores to a level detectable by

fluorescence microscopy during undisturbed cell cycle.

GFP at the C-terminus of MAD2 might interfere somewhat

with MAD2 function because it has been shown in mam-

malian cells, Xenopus and yeast that the C-terminus of

MAD2 is required to bind MAD1, Tpr and Cdc20 (Fang

et al. 1998; Chen et al. 1999; Lee et al. 2008).

Together, our data suggest that aspects of MAD1/MAD2

function are conserved in plants. Specifically, these include

the interaction of AtMAD1 and AtMAD2 with the NE in

interphase, the dependence of this interaction on the

nuclear pore protein NUA as well as a functional role of

AtMAD2 and NUA in meristematic cell division. It will be

interesting to now further compare the developmental

defects of NUA and MAD2 mutants with those of mutants

in APC/C subunits and confirmed APC/C regulators and to

expand studying the ability of sucrose to rescue these

phenotypes to a larger group of cell-cycle implicated

mutants.

Acknowledgments We would like to thank Drs. Stephen Osmani,

Rebecca Lamb, and Keith Slotkin for helpful discussions and critical

reading of the original draft of this manuscript. This work was sup-

ported by a grant from the National Science Foundation (MCB-

0641271) to I. M.

References

Alizadeh AA, Eisen MB, Davis RE, Ma C, Lossos IS, Rosenwald A,

Boldrick JG, Sabet H, Tran T, Yu X, Powell JI, Yang LM, Marti

GE, Moore T, Hudson J, Lu LS, Lewis DB, Tibshirani R,

Sherlock G, Chan WC, Greiner TC, Weisenburger DD, Armitage

JO, Warnke R, Levy R, Wilson W, Grever MR, Byrd JC,

Botstein D, Brown PO, Staudt LM (2000) Distinct types of

diffuse large B-cell lymphoma identified by gene expression

profiling. Nature 403:503–511

Alonso JM, Stepanova AN, Leisse TJ, Kim CJ, Chen HM, Shinn P,

Stevenson DK, Zimmerman J, Barajas P, Cheuk R, Gadrinab C,

Heller C, Jeske A, Koesema E, Meyers CC, Parker H, Prednis L,

Ansari Y, Choy N, Deen H, Geralt M, Hazari N, Hom E, Karnes

M, Mulholland C, Ndubaku R, Schmidt I, Guzman P, Aguilar-

Henonin L, Schmid M, Weigel D, Carter DE, Marchand T,

Risseeuw E, Brogden D, Zeko A, Crosby WL, Berry CC, Ecker

JR (2003) Genome-wide Insertional mutagenesis of Arabidopsisthaliana. Science 301:653–657

Barnhart EL, Dorer RK, Murray AW, Schuyler SC (2011) Reduced

Mad2 expression keeps relaxed kinetochores from arresting

budding yeast in mitosis. Mol Biol Cell 22:2448–2457

Boruc J, Van den Daele H, Hollunder J, Rombauts S, Mylle E, Hilson

P, Inze D, De Veylder L, Russinova E (2010) Functional

modules in the Arabidopsis core cell cycle binary protein–

protein interaction network. Plant Cell 22:1264–1280

Buffin E, Lefebvre C, Huang J, Gagou ME, Karess RE (2005)

Recruitment of Mad2 to the kinetochore requires the Rod/Zw10

complex. Curr Biol 15:856–861

Caillaud MC, Paganelli L, Lecomte P, Deslandes L, Quentin M,

Pecrix Y, Le Bris M, Marfaing N, Abad P, Favery B (2009)

Spindle assembly checkpoint protein dynamics reveal conserved

and unsuspected roles in plant cell division. PLoS ONE 4:e6757

Chen RH, Waters JC, Salmon ED, Murray AW (1996) Association of

spindle assembly checkpoint component XMAD2 with unat-

tached kinetochores. Science 274:242–246

Chen RH, Brady DM, Smith D, Murray AW, Hardwick KG (1999)

The spindle checkpoint of budding yeast depends on a tight

complex between the Mad1 and Mad2 proteins. Mol Biol Cell

10:2607–2618

Chen X, Cheung ST, So S, Fan ST, Barry C, Higgins J, Lai KM, Ji JF,

Dudoit S, Ng IOL, van de Rijn M, Botstein D, Brown PO (2002)

Gene expression patterns in human liver cancers. Mol Biol Cell

13:1929–1939

Clough SJ, Bent AF (1998) Floral dip: a simplified method for

Agrobacterium-mediated transformation of Arabidopsis thali-ana. Plant J 16:735–743

Collins TJ (2007) ImageJ for microscopy. Biotechniques 43:25

David KM, Perrot-Rechenmann C (2001) Characterization of a

tobacco bright yellow 2 cell line expressing the tetracycline

repressor at a high level for strict regulation of transgene

expression. Plant Physiol 125:1548–1553

De Souza CP, Hashmi SB, Nayak T, Oakley B, Osmani SA (2009)

Mlp1 acts as a mitotic scaffold to spatially regulate spindle

assembly checkpoint proteins in Aspergillus nidulans. Mol Biol

Cell 20:2146–2159

Dello Loio R, Linhares FS, Sabatini S (2008) Emerging role of

cytokinin as a regulator of cellular differentiation. Curr Opin

Plant Biol 11:23–27

Dobles M, Liberal V, Scott ML, Benezra R, Sorger PK (2000)

Chromosome missegregation and apoptosis in mice lacking the

mitotic checkpoint protein Mad2. Cell 101:635–645

Dohmen RJ, Strasser AW, Honer CB, Hollenberg CP (1991) An

efficient transformation procedure enabling long-term storage of

competent cells of various yeast genera. Yeast 7:691–692

Elledge SJ (1998) Mitotic arrest: Mad2 prevents sleepy from waking

up the APC. Science 279:999–1000

Eveland AL, Jackson DP (2011) Sugars, signalling, and plant

development. J Exp Bot PMID 22140246

Fang GW, Yu HT, Kirschner MW (1998) The checkpoint protein

MAD2 and the mitotic regulator CDC20 form a ternary complex

with the anaphase-promoting complex to control anaphase

initiation. Genes Dev 12:1871–1883

Fava LL, Kaulich M, Nigg EA, Santamaria A (2011) Probing the in

vivo function of Mad1:C-Mad2 in the spindle assembly check-

point. EMBO J 30:3322–3336

Friml J, Benkova E, Mayer U, Palme K, Muster G (2003) Automated

whole mount localisation techniques for plant seedlings. Plant J

34:115–124

Garber ME, Troyanskaya OG, Schluens K, Petersen S, Thaesler Z,

Pacyna-Gengelbach M, van de Rijn M, Rosen GD, Perou CM,

Whyte RI, Altman RB, Brown PO, Botstein D, Petersen I (2001)

Diversity of gene expression in adenocarcinoma of the lung.

Proc Natl Acad Sci USA 98:13784–13789

Heyman J, Van den Daele H, De Wit K, Boudolf V, Berckmans B,

Verkest A, Kamei CL, De Jaeger G, Koncz C, De Veylder L

(2011) Arabidopsis ULTRAVIOLET-B-INSENSITIVE4 main-

tains cell division activity by temporal inhibition of the

anaphase-promoting complex/cyclosome. Plant Cell 23:4394–

4410

Hirano H, Shinmyo A, Sekine M (2011) Arabidopsis G1 cell cycle

proteins undergo proteasome-dependent degradation during

sucrose starvation. Plant Physiol Biochem 49:687–691

Howell BJ, Hoffman DB, Fang G, Murray AW, Salmon ED (2000)

Visualization of Mad2 dynamics at kinetochores, along spindle

fibers, and at spindle poles in living cells. J Cell Biol

150:1233–1249

Howell BJ, McEwen BE, Canman JC, Hoffman DB, Farrar EM,

Rieder CL, Salmon ED (2001) Cytoplasmic dynein/dynactin

Plant Mol Biol (2012) 79:203–216 215

123

drives kinetochore protein transport to the spindle poles and has

a role in mitotic spindle checkpoint inactivation. J Cell Biol

155:1159–1172

Iouk T, Kerscher O, Scott RJ, Basrai MA, Wozniak RW (2002) The

yeast nuclear pore complex functionally interacts with compo-

nents of the spindle assembly checkpoint. J Cell Biol

159:807–819

Iwanaga Y, Chi YH, Miyazato A, Sheleg S, Haller K, Peloponese JM

Jr, Li Y, Ward JM, Benezra R, Jeang KT (2007) Heterozygous

deletion of mitotic arrest-deficient protein 1 (MAD1) increases

the incidence of tumors in mice. Cancer Res 67:160–166

James P, Halladay J, Craig EA (1996) Genomic libraries and a host

strain designed for highly efficient two-hybrid selection in yeast.

Genetics 144:1425–1436

Joubes J, De Schutter K, Verkest A, Inze D, De Veylder L (2004)

Conditional, recombinase-mediated expression of genes in plant

cell cultures. Plant J 37:889–896

Katsani KR, Karess RE, Dostatni N, Doye V (2008) In vivo dynamics

of Drosophila nuclear envelope components. Mol Biol Cell

19:3652–3666

Kimbara J, Endo TR, Nasuda S (2004) Characterization of the genes

encoding for MAD2 homologues in wheat. Chromosome Res

12:703–714

Kitagawa R (2009) The spindle assembly checkpoint in Caenorhab-ditis elegans—one who lacks Mad1 becomes mad one. Cell

Cycle 8:338–344

Kitagawa R, Rose AM (1999) Components of the spindle-assembly

checkpoint are essential in Caenorhabditis elegans. Nat Cell

Biol 1:514–521

Krysan PJ, Young JC, Sussman MR (1999) T-DNA as an insertional

mutagen in Arabidopsis. Plant Cell 11:2283–2290

Lee SH, Sterling H, Burlingame A, McCormick F (2008) Tpr directly

binds to Mad1 and Mad2 and is important for the Mad1-Mad2-

mediated mitotic spindle checkpoint. Genes Dev 22:2926–2931

Lince-Faria M, Maffini S, Orr B, Ding Y, Claudia F, Sunkel CE,

Tavares A, Johansen J, Johansen KM, Maiato H (2009)

Spatiotemporal control of mitosis by the conserved spindle

matrix protein Megator. J Cell Biol 184:647–657

Lindsay DL, Bonham-Smith PC, Postnikoff S, Gray GR, Harkness

TA (2011) A role for the anaphase promoting complex in

hormone regulation. Planta 233:1223–1235

Marrocco K, Thomann A, Parmentier Y, Genschik P, Criqui MC

(2009) The APC/C E3 ligase remains active in most post-mitotic

Arabidopsis cells and is required for proper vasculature devel-

opment and organization. Development 136:1475–1485

Marrocco K, Bergdoll M, Achard P, Criqui MC, Genschik P (2010)

Selective proteolysis sets the tempo of the cell cycle. Curr Opin

Plant Biol 13:631–639

Michel LS, Liberal V, Chatterjee A, Kirchwegger R, Pasche B,

Gerald W, Dobles M, Sorger PK, Murty VVVS, Benezra R

(2001) MAD2 haplo-insufficiency causes premature anaphase

and chromosome instability in mammalian cells. Nature

409:355–359

Musacchio A, Salmon ED (2007) The spindle-assembly checkpoint in

space and time. Nat Rev Mol Cell Biol 8:379–393

Muthuswamy S, Meier I (2011) Genetic and environmental changes

in SUMO homeostasis lead to nuclear mRNA retention in plants.

Planta 233:201–208

Nakagawa T, Kurose T, Hino T, Tanaka K, Kawamukai M, Niwa Y,

Toyooka K, Matsuoka K, Jinbo T, Kimura T (2007) Develop-

ment of series of gateway binary vectors, pGWBs, for realizing

efficient construction of fusion genes for plant transformation.

J Biosci Bioeng 104:34–41

Nasmyth K (2005) How do so few control so many? Cell

120:739–746

Orr B, Bousbaa H, Sunkel CE (2007) Mad2-independent spindle

assembly checkpoint activation and controlled metaphase-ana-

phase transition in Drosophila S2 cells. Mol Biol of the Cell

18:850–863

Perilli S, Sabatini S (2010) Analysis of root meristem size develop-

ment. Methods Mol Biol 655:177–187

Riou-Khamlichi C, Menges M, Healy JM, Murray JA (2000) Sugar

control of the plant cell cycle: differential regulation of

Arabidopsis D-type cyclin gene expression. Mol Cell Biol

20:4513–4521

Rolland F, Baena-Gonzalez E, Sheen J (2006) Sugar sensing and

signaling in plants: conserved and novel mechanisms. Ann Rev

Plant Biol 57:675–709

Rose A, Manikantan S, Schraegle SJ, Maloy MA, Stahlberg EA,

Meier I (2004) Genome-wide identification of Arabidopsis

coiled-coil proteins and establishment of the ARABI-COIL

database. Plant Physiol 134:927–939

Scott RJ, Lusk CP, Dilworth DJ, Aitchison JD, Wozniak RW (2005)

Interactions between Mad1p and the nuclear transport machinery in

the yeast Saccharomyces cerevisiae. Mol Biol Cell 16:4362–4374

Sessions A, Burke E, Presting G, Aux G, McElver J, Patton D,

Dietrich B, Ho P, Bacwaden J, Ko C, Clarke JD, Cotton D, Bullis

D, Snell J, Miguel T, Hutchison D, Kimmerly B, Mitzel T,

Katagiri F, Glazebrook J, Law M, Goff SA (2002) A high-

throughput Arabidopsis reverse genetics system. Plant Cell

14:2985–2994

Skinner JJ, Wood S, Shorter J, Englander SW, Black BE (2008) The

Mad2 partial unfolding model: regulating mitosis through Mad2

conformational switching. J Cell Biol 183:761–768

Sotillo R, Hernando E, Diaz-Rodriguez E, Teruya-Feldstein J,

Cordon-Cardo C, Lowe SW, Benezra R (2007) Mad2 overex-

pression promotes aneuploidy and tumorigenesis in mice. Cancer

Cell 11:9–23

Suijkerbuijk SJE, Kops GJPL (2008) Preventing aneuploidy: the

contribution of mitotic checkpoint proteins. Biochim Biophys

Acta 1786:24–31

Xu XM, Rose A, Meier I (2007a) NUA activities at the plant nuclear

pore. Plant Signal Behav 2:553–555

Xu XM, Rose A, Muthuswamy S, Jeong SY, Venkatakrishnan S,

Zhao Q, Meier I (2007b) NUCLEAR PORE ANCHOR, the

Arabidopsis homolog of Tpr/Mlp1/Mlp2/megator, is involved in

mRNA export and SUMO homeostasis and affects diverse

aspects of plant development. Plant Cell 19:1537–1548

Yang Y, Li R, Qi M (2000) In vivo analysis of plant promoters and

transcription factors by agroinfiltration of tobacco leaves. Plant J

22:543–551

Yu HG, Muszynski MG, Kelly Dawe R (1999) The maize homologue

of the cell cycle checkpoint protein MAD2 reveals kinetochore

substructure and contrasting mitotic and meiotic localization

patterns. J Cell Biol 145:425–435

Zhao Q, Leung S, Corbett AH, Meier I (2006) Identification and

characterization of the Arabidopsis orthologs of nuclear transport

factor 2, the nuclear import factor of ran. Plant Physiol 140:869–

878

Zhou J, Yao J, Joshi HC (2002) Attachment and tension in the spindle

assembly checkpoint. J Cell Sci 115:3547–3555

216 Plant Mol Biol (2012) 79:203–216

123