NUCLEAR PORE ANCHOR, the Arabidopsis Homolog ofTpr/Mlp1/Mlp2/Megator, Is Involved in mRNA Exportand SUMO Homeostasis and Affects Diverse Aspectsof Plant Development W

Xianfeng Morgan Xu, Annkatrin Rose,1 Sivaramakrishnan Muthuswamy, Sun Yong Jeong,2

Sowmya Venkatakrishnan, Qiao Zhao, and Iris Meier3

Plant Biotechnology Center and Department of Plant Cellular and Molecular Biology, Ohio State University, Columbus,

Ohio 43210

Vertebrate Tpr and its yeast homologs Mlp1/Mlp2, long coiled-coil proteins of nuclear pore inner basket filaments, are

involved in mRNA export, telomere organization, spindle pole assembly, and unspliced RNA retention. We identified

Arabidopsis thaliana NUCLEAR PORE ANCHOR (NUA) encoding a 237-kD protein with similarity to Tpr. NUA is located at the

inner surface of the nuclear envelope in interphase and in the vicinity of the spindle in prometaphase. Four T-DNA insertion

lines were characterized, which comprise an allelic series of increasing severity for several correlating phenotypes, such as

early flowering under short days and long days, increased abundance of SUMO conjugates, altered expression of several

flowering regulators, and nuclear accumulation of poly(A)þ RNA. nua mutants phenocopy mutants of EARLY IN SHORT

DAYS4 (ESD4), an Arabidopsis SUMO protease concentrated at the nuclear periphery. nua esd4 double mutants resemble

nua and esd4 single mutants, suggesting that the two proteins act in the same pathway or complex, supported by yeast

two-hybrid interaction. Our data indicate that NUA is a component of nuclear pore-associated steps of sumoylation and

mRNA export in plants and that defects in these processes affect the signaling events of flowering time regulation and

additional developmental processes.

INTRODUCTION

The nuclear pore complex (NPC) is a large multiprotein complex

that is the sole gateway of macromolecular trafficking between

the cytoplasm and the nucleus. The mammalian and yeast NPC

consists of multiple copies of the ;30 different nucleoporins

(Nups). Together, they form a channel-like structure of eightfold

symmetry that has been roughly divided into three elements: a

nuclear basket, a central pore, and cytoplasmic fibrils. While a

small number of Nups are anchored to the nuclear envelope

membrane, others form a protein scaffold or line the central pore

cylinder with FG-repeat-containing hydrophobic domains. Nu-

clear import and export receptors traffic through the pore bound

to their cargos, and the Ran cycle provides spatial information on

the directionality of the transport (reviewed in Tran and Wente,

2006).

Recently, several reports have demonstrated that Nups are

involved in functions beyond being building blocks of the NPC.

Some Nups are highly dynamic and appear in locations away

from the pore (Griffis et al., 2002; Rabut et al., 2004). Several

Nups have mitotic functions, for example, involvement in kinet-

ochore assembly (reviewed in Chan et al., 2005). Possibly the

most exciting new function of Nups is their ability to dock specific

enzymatic activities to the NPC, thereby providing spatial reg-

ulation for the respective activities. An example that has been

known for several years is the docking of the mammalian Ran

GTPase activating protein RanGAP1 to the outer surface of the

NPC by the nucleoporin RanBP2, where it hydrolyzes the RanGTP

bound to export complexes (Matunis et al., 1998). Mammalian

RanGAP1 requires sumoylation to bind to RanBP2, and subse-

quently it was found that RanBP2 itself is a SUMO E3 ligase

(Matunis et al., 1998; Pichler et al., 2002).

At the nuclear side of the pore, the nuclear basket is also

involved in regulating SUMO modification. The Nups Mlp1/Mlp2

in yeast (Saccharomyces cerevisiae) and Nup153 in mammals

dock a SUMO protease to the NPC (Ulp1 in yeast and SENP2

in mammals) (Zhang et al., 2002; Panse et al., 2003). Mlp1 is

also a docking site for heterogeneous nuclear ribonucleoproteins

(hnRNPs) (Green et al., 2003), and mammalian hnRNPs have

been shown to be sumoylated (Vassileva and Matunis, 2004). It

has been proposed that Mlps act as a quality control checkpoint

for mRNA export (Galy et al., 2004).

At the outer pore surface, another nucleoporin acts as an

anchor/activator of a step in mRNA export. The nucleoporin Gle1

1 Current address: Department of Biology, Appalachian State University,Boone, NC 28608.2 Current address: Department of Biochemistry and Biophysics, Univer-sity of North Carolina, Chapel Hill, NC 27599.3 To whom correspondence should be addressed. E-mail meier.56@osu.edu; fax 614-292-5379.The author responsible for distribution of materials integral to thefindings presented in this article in accordance with the policy describedin the Instructions for Authors (www.plantcell.org) is: Iris Meier(meier.56@osu.edu).W Online version contains Web-only data.www.plantcell.org/cgi/doi/10.1105/tpc.106.049239

The Plant Cell, Vol. 19: 1537–1548, May 2007, www.plantcell.org ª 2007 American Society of Plant Biologists

binds the DEAD box helicase Dbp5 and together with the soluble

inositol polyphosphate InsP6 activates the ATPase activity of

Dbp5. This leads to a highly localized activation of mRNA

remodeling, likely involved in a release step of mRNA export

(Alcazar-Roman et al., 2006; Weirich et al., 2006). Together,

these examples support the emerging picture that precise spatial

regulation is crucial for a number of nuclear functions and em-

phasize that this spatial regulation can be provided by anchoring

enzymatic activities to nuclear pore proteins.

While the components of the animal and yeast NPC have been

identified in proteomic studies (Rout et al., 2000; Cronshaw et al.,

2002), the plant NPC is still largely a black box. Recently, putative

plant Nups have been identified by genetic screens for the

seemingly unrelated signaling pathways involved in plant-

microbe interactions, cold tolerance, and the action of the plant

hormone auxin (Zhang and Li, 2005; Dong et al., 2006; Kanamori

et al., 2006; Parry et al., 2006). The identified proteins have

convincing sequence similarity to Nup96, Nup160, and Nup133,

which are all components of the Nup107-160 complex, suggest-

ing its conservation during evolution. The Nup107-160 complex

is believed to provide a core scaffolding function for NPC

assembly in mammals (Harel et al., 2003; Walther et al., 2003).

Partial in vivo depletion of this complex from HeLa cells via RNA

interference or immunodepletion from in vitro Xenopus laevis

nuclear assembly reactions leads to defects in the assembly of

the NPC, which suggests their pivotal roles (Harel et al., 2003;

Walther et al., 2003). The various phenotypes associated with the

mutations of putative plant Nups underline that many cellular and

developmental processes involve the communication between

the nucleus and the cytoplasm via the NPC.

A nuclear rim-associated activity with developmental func-

tions is desumoylation mediated by the SUMO protease EARLY

IN SHORT DAYS4 (ESD4). esd4 mutants flower extremely early

and have pleiotropic alterations in shoot development (Reeves

et al., 2002). The early flowering phenotype is consistent with

reduced expression of the flowering repressor FLOWERING

LOCUS C (FLC). In line with the in vitro SUMO protease activity of

ESD4, esd4 mutants accumulate more SUMO conjugates and

have less free SUMO than wild-type plants (Murtas et al., 2003).

These data suggest a connection in Arabidopsis thaliana be-

tween SUMO homeostasis and flowering-time regulation.

Here, we show that mutants of an Arabidopsis protein,

NUCLEAR PORE ANCHOR (NUA), with similarity to the inner

nuclear basket proteins Tpr (for Translocated Promoter Region),

Mlp1/Mlp2 (for Myosin-like proteins 1 and 2), and Megator flower

extremely early and have several phenotypic characteristics in

common with esd4 mutants. NUA is located at the inner nu-

clear envelope, and nua esd4 double mutants resemble nua and

esd4 single mutants, indicating that the two proteins might act in

a shared pathway or complex, supported by their interaction

in yeast two-hybrid assays. nua mutant alleles show an increase

in SUMO conjugates and reduction of free SUMO, an increase in

nuclear poly(A)þ RNA, and altered expression of several genes

involved in flowering-time regulation. We propose that NUA acts

as a docking site at the inner nuclear pore for activities required

for desumoylation and mRNA export and that disruption of this

docking affects the expression of key regulators of plant devel-

opment.

RESULTS

Identification of NUA, an Arabidopsis Protein Similar to

Mammalian Tpr, Drosophila Megator, and Yeast Mlp1/Mlp2

NUA was identified in a targeted phenotypic screen of 36 T-DNA

insertion mutants in Arabidopsis genes coding for long coiled-

coil proteins that might play a structural-organizational role in

the nucleus or the endomembrane system. The genes were

selected by the following criteria from the ARABI-COIL Arabi-

dopsis coiled-coil protein database (Rose et al., 2004). The pro-

teins should be at least 500 amino acids long, with a coiled-coil

coverage of at least 50% and either a nuclear localization signal

(NLS) or at least one predicted transmembrane domain. For all

selected open reading frames, T-DNA insertion lines generated

by the Salk Institute Genomic Analysis Laboratory (SIGnAL; Alonso

et al., 2003) were acquired from the ABRC (Ohio State Univer-

sity), and segregating populations were screened for visible phe-

notypes to identify proteins with an experimentally approachable

biological role. nua-1 was identified as an extreme early-flowering

mutant that was stunted in growth and had phyllotaxy defects in

the inflorescence.

The translated open reading frame of NUA has significant

sequence similarity to mammalian Tpr, an inner nuclear pore-

associated long coiled-coil protein. Figure 1A shows the coiled-

coil domains in NUA in comparison to human Tpr, Drosophila

melanogaster Megator, and yeast Mlp1 and Mlp2. The size of the

predicted protein, the length of the coiled-coil domain, the pres-

ence of a non-coiled-coil C-terminal tail, and the distribution of

predicted NLS are very similar in NUA and the known Tpr-like

proteins (Kuznetsov et al., 2002). Using the full-length NUA pro-

tein in WU-BLAST against the plant Gene Indices, homologous

partial protein sequences with a significant identity and similarity

(>35 and 50%, respectively) can be recovered from various plant

species, including rice (Oryza sativa), maize (Zea mays), wheat

(Triticum aestivum), and potato (Solanum tuberosum). Hence,

NUA is evolutionarily conserved both among and within different

kingdoms.

Based on the existing EST and microarray data (Zimmermann

et al., 2004), NUA appears to be expressed ubiquitously in all tis-

sues and during all developmental stages. This was also con-

firmedbyRT-PCRwithdifferent tissues, including root, stem,flower,

silique, and cauline leaf (see Supplemental Figure 1A online).

Characterization of NUA T-DNA Insertion Lines

Three additional T-DNA insertion alleles of NUA were identified

(Figure 1B). Flanking sequences of the T-DNA insertion sites

were amplified by PCR and sequenced. The original allele nua-1

(SALK_057101) has an insertion at nucleotide þ1855 (with þ1

being the A of the ATG) (within intron 8), the nua-2 (SALK_069922)

insertion causes a 45-bp deletion between nucleotides þ5657

and þ5902, and nua-3 (SAIL_505_H11) has an insertion at nu-

cleotide þ7531 (within exon 29). The insertion site of nua-4

(WiscDsLox297300_17E) was mapped to nucleotide þ2145

(within exon 9).

The NUA cDNA was cloned by RT-PCR (see Methods), and the

partial cDNA fragment encoding amino acids 518 to 1248 (see

1538 The Plant Cell

Figure 1. Characterization of NUA T-DNA Insertion Lines.

(A) The coiled-coil domain prediction of NUA (At1g79280) using COILS, and comparison with human Tpr, Drosophila Megator, and yeast Mlp1 and

Mlp2. Black bars show predicted coiled-coil domains. The dimerization and NPC association domain of Tpr are also depicted. Bar ¼ 200 amino acids

(aa).

(B) Position of T-DNA insertions and PCR primers. The confirmed insertion sites in nua-1, nua-2, nua-3, and nua-4 are indicated by vertical arrows above

and below the schematic exon-intron structure of the NUA gene. Exons are depicted as gray bars and introns as black lines. Horizontal arrows indicate

the positions of PCR primers, and brackets indicate the positions of the RT-PCR products shown in (D). The area surrounding the nua-2 insertion site is

shown enlarged, and the structure of the ;200 and ;300-bp fragments derived from RT-PCR with NUA-D primers in nua-2 is indicated. ex, exon; int,

intron.

(C) Immunoprecipitation of NUA from Arabidopsis wild-type and nua-1, nua-2, and nua-3 seedlings. Arrowheads depict NUA bands, and asterisks mark

unspecific IgG bands also seen without plant extract (data not shown). Molecular weights are indicated on the left.

(D) RT-PCR products derived from primer pairs indicated on the right (as shown in [B]) and plant tissues as indicated on the top. Approximate sizes of

the fragments amplified with the primer pair for NUA-D in nua-2 are indicated on the left. Two exposures are shown for the fragments amplified with

NUA-D primers. The primer combinations in each reaction were as follows: NUA-A, F1 þ LP115409; NUA-B, F1 þ SALK_057101LP; NUA-C,

RP057101.1 þ R1(1599); NUA-D, RP069922 þ LP069922; NUA-E, CS821281FP þ R2(3798); NUA-F, RP079795 þ R3(4993). All primer sequences are

listed in Supplemental Table 2 online. Tub, tubulin 2 (At5g62690).

Arabidopsis Homolog of Tpr/Mlp1/Mlp2/Megator 1539

Supplemental Figure 4 online) was used to express His-tagged

protein in Escherichia coli and generate an antiserum (anti-NUA)

that detects a protein of an extrapolated molecular mass of 220

kD. Figure 1C shows that after immunoprecipitation no 220-kD

protein was detected in nua-1 (note that the short N-terminal

fragment potentially expressed in nua-1 would not be detected

by anti-NUA). A weak band of approximately full-length size was

detected in nua-2 and a band corresponding to a truncated

protein of ;100 kD in nua-3 (Figure 1C). NUA protein was also

absent in nua-4 (see Supplemental Figure 3C online).

Analysis of NUA transcripts by RT-PCR showed that the locus

is transcribed both upstream and downstream of the T-DNA

insertion in nua-1, nua-3, and nua-4 but that no transcript was

detected across the insertion (Figure 1D; see Supplemental

Figure 3B online). By contrast, a small amount of full-length tran-

script in addition to truncated transcripts was detected across

the nua-2 insertion (Figure 1D). The truncated bands were se-

quenced and indicated that alternative splicing leads to in-frame

deletions of 135 bp (exon 22) or 135 þ 99 bp (exons 22 and 23)

(Figure 1B). All PCR products were derived from specific cDNA

templates since they were not present in the control reactions in

which reverse-transcriptase was omitted (data not shown) and

since all primer pairs span introns. We speculate that either

mRNA is synthesized through the T-DNA insertion (which is

confirmed in the nua-2 mutant) or that a transcript reads out of

the T-DNA into the NUA gene. In any case, no protein in the NUA

open reading frame is made downstream of the T-DNA insertions

of nua-1 and nua-4 because it would be detectable with our

antibody. In summary, we conclude that nua-1 and nua-4 are

likely null mutations. In nua-2, a small amount of full-length

protein and/or almost-full-length proteins with short internal

deletions are present. In nua-3, a partial protein of ;100 kD is

made.

NUA T-DNA Insertion Alleles Flower Early in Long Days and

Short Days and Have Pleiotropic Developmental Defects

All four lines flowered early in long days (Figure 2A, Table 1). nua-1

and nua-4 were the most extreme alleles and bolted with 4 to

5 rosette leaves in long days, while nua-2 and nua-3 bolted with

8 to 10 and 6 to 8 leaves, respectively (wild-type Columbia: 10 to

12 leaves). nua-1, nua-2, and nua-3 also flowered early in short

days (Table 1), with nua-1 again being the most severe, nua-3 be-

ing moderate, and nua-2 being a mild allele (nua-4 was not tested).

In addition to early flowering, nua-1, nua-4, and nua-3 also showed

stunted inflorescences (Figure 2B; see Supplemental Figure 3A

online) and smaller, narrower rosette leaves (Figure 2A), while

adult nua-2 plants were indistinguishable from the wild type.

nua-1 and nua-4 showed several additional developmental

alterations not found in nua-2 and nua-3 (Figures 2C to 2E; see

Supplemental Figure 3A online). The inflorescence of the mu-

tants showed some abnormalities with a reduced number of

flower buds on the top of the main inflorescence (Figure 2C).

Siliques were found at unexpected positions; two or three si-

liques were positioned at one node or at the top of the main

inflorescence. In addition, indeterminate shoots were found in

one node together with a silique and a cauline leaf. The size of

siliques of the mutants was shorter and more stunted compared

with the wild type (Figure 2D), with fewer developed seeds per

silique. Furthermore, the majority of flowers did not set seeds.

The stamens of the mutants were shorter and the size of petals

was slightly smaller than the wild type (Figure 2E).

Together, we conclude that loss of NUA leads to severe

developmental defects and that the isolated T-DNA insertions

comprise an allelic series of increasing severity in the order

nua-2, nua-3, and nua-1/nua-4.

Figure 2. Phenotypic Characteristics of nua Mutant Alleles.

(A) Seedlings at 25 d after germination, grown under long-day condi-

tions.

(B) Plants at 34 d after germination, grown under long-day conditions.

(C) Phyllotaxy defects of nua-1.

(D) Silique phenotype of nua-1.

(E) Flower phenotype of nua-1.

1540 The Plant Cell

Subcellular Localization of NUA

We used immunofluorescence with anti-NUA to characterize the

subcellular localization of NUA in root tip cells of Arabidopsis

seedlings. In interphase cells, NUA is clearly located at the

nuclear envelope (Figure 3). During prometaphase, at the onset

of spindle formation, the antibody decorated a structure in the

vicinity of the spindle, which did not directly colocalize with

tubulin (Figures 3A to 3D). During metaphase, this signal was less

obvious (Figures 3E to 3H). During cytokinesis, NUA reappeared

at the nuclear envelope (Figures 3I to 3L). Supplemental Figure 2

online shows that the NE signal is absent in nua-1, indicating that

anti-NUA specifically decorates NUA at the nuclear envelope. To

determine if NUA is associated with the inner nuclear envelope,

we performed double labeling in plants expressing a green

fluorescent protein (GFP) fusion of WIP1, an Arabidopsis outer

nuclear envelope/NPC-associated protein (X.M. Xu and I. Meier,

unpublished data). Figure 4 shows that the NUA signal could be

clearly resolved inside the GFP-WIP1 signal, indicating that NUA

is indeed associated with the inner surface of the nuclear

envelope.

nua-1 esd4-2 Double Mutant Analysis

The observed pleiotropic phenotype of nua-1 and nua-4 is rem-

iniscent of the esd4-1 and esd4-2 mutations (Reeves et al., 2002;

Murtas et al., 2003). To investigate their genetic interaction, homo-

zygote nua-1 plants were crossed with esd4-2 (SALK_032317)

plants, resulting in a wild-type phenotype for all seedlings in the

F1 generation. Double mutants in the F2 generation were indis-

tinguishable from nua-1 and esd4-2 in terms of flowering time

under long-day conditions (Figures 5A and 5B). The only differ-

ence observed was that nua-1 esd4-2 had even shorter stamens

and reduced fertility than the single mutants (Figure 5C; data not

shown). These data suggest that nua-1 and esd4-2 might act in a

shared pathway or complex that affects flowering time as well as

Table 1. Flowering Time of Wild-Type Columbia and nua Mutants, Given as Number of Rosette Leaves at Time of Bolting

Columbia nua-1 nua-2 nua-3 nua-4

Long day 12.2 6 1.0 (45 plants) 4.0 6 0.0 (53 plants) 8.0 6 0.9 (16 plants) 6.1 6 0.6 (16 plants) 4.2 6 0.4 (53 plants)

Short day 50.6 6 2.0 (11 plants) 7.4 6 0.5 (53 plants) 45.6 6 1.8 (10 plants) 33.6 6 1.2 (6 plants) ND

The 6 indicates SD. Long day: 16 h light/8 h dark, 75 to 125 mmol s�1 m�2. Short day: 8 h light/16 h dark, 85 to 95 mmol s�1 m�2. ND, not determined.

Figure 3. NUA Is Located at the Nuclear Envelope during Interphase and in the Vicinity of the Spindle during Prometaphase.

Immunofluorescence images of root tip cell files in interphase ([A] to [D] and [I] to [L]), prometaphase ([A] to [D]), metaphase ([E] to [H]), and late

cytokinesis ([I] to [L]). Green, anti-NUA; red in (D), (H), and (L) and magenta in (B), (C), (F), (G), (J), and (K), anti-tubulin; blue in (D), (H), and (L),

49,6-diamidino-2-phenylindole. (A), (E), and (I) show the green channel only, and (B), (F), and (J) show the red channel only (false colored in magenta).

(C), (G), and (K) show the red and green channels, and (D), (H), and (L) show the red, green, and blue channels. The arrowhead in (A) indicates

the interphase nuclear envelope, and the arrow in (A) indicates the spindle-like structure in prometaphase. Bars ¼ 10 mm in (D), (H), and (L).

Arabidopsis Homolog of Tpr/Mlp1/Mlp2/Megator 1541

vegetative and inflorescence development but that they act

additively in stamen development. This notion is supported by

the interaction of NUA and ESD4 in yeast two-hybrid assays,

indicating that ESD4 binds specifically to the N-terminal 533

amino acids of NUA (see Supplemental Figure 4 online).

Effects of nua Mutants on Sumoylation and Flowering

Gene Expression

ESD4 is a SUMO protease associated with the nuclear envelope

(Reeves et al., 2002; Murtas et al., 2003). Mutations in ESD4 lead

to an increase in SUMO conjugates and a decrease in free SUMO

(Murtas et al., 2003). To test if mutations in NUA have a similar

molecular phenotype, we investigated the SUMO conjugation

pattern in nua-1, nua-2, nua-3, and nua-4, compared with the

T-DNA insertion allele esd4-2 and wild-type Columbia plants

(Figure 6A; see Supplemental Figure 3D online). In esd4-2, the

level of high molecular weight SUMO conjugates was increased,

while the free SUMO level was reduced, as described previously

(Murtas et al., 2003). We found that nua-1 and nua-4 phenocopy

esd4-2 and that nua-3 leads to an intermediate and nua-2 to the

least increase of SUMO conjugates. The level of free SUMO was

reciprocally altered in all alleles.

ESD4 mutations have been shown to decrease the mRNA level

of the floral repressor FLC and increase mRNA levels of the floral

activators FLOWERING LOCUS T (FT) and SUPPRESSOR OF

OVEREXPRESSION OF CO1 (SOC1) (Reeves et al., 2002). To

test whether the nua mutants would also phenocopy these

effects, we tested mRNA abundance of FLC, FT, and SOC1 by

semiquantitative RT-PCR. Indeed, as shown in Figure 6B, FLC

mRNA level is strongly reduced in nua-1 and nua-4, comparable

to esd4-2, and is somewhat reduced in nua-2 and nua-3, with

nua-2 showing the weakest effect. Consistently, FT and SOC1

mRNA levels were found inversely affected.

Since nua-1 and nua-4 flower significantly earlier than the flc-3

null mutant (four to five compared with 10 to 11 rosette leaves in

long-day conditions; see Supplemental Table 1 online) (Michaels

and Amasino, 1999, 2001), additional factors involved in flowering-

time regulation are likely affected in nua mutants. Additionally,

Figure 4. NUA Is Localized at the Inner Side of the Nuclear Envelope,

Shown by Colocalization with the Outer Nuclear Envelope/NPC-Local-

ized Protein GFP-WIP1.

NUA and GFP-WIP1 were detected by rabbit anti-NUA antibody (A) and

mouse anti-GFP antibody (B), respectively, with appropriate secondary

antibodies. The overlay image is shown in (C) (green, GFP-WIP1; ma-

genta, NUA). The dashed box in (C) was enlarged, and the green and

magenta fluorescence profiles were analyzed in (D). Bar ¼ 5 mm.

Figure 5. The nua-1 esd4-2 Double Mutant Resembles nua-1 and

esd4-2 in Flowering Time and Stunted Growth Characteristics, whereas

Additive Effects Exist for Stamen Length.

(A) Wild-type, nua-1, esd4-2, and nua-1 esd4-2 seedlings at 21 d after

germination, gown under long-day conditions.

(B) Plants after 34 d in long-day conditions.

(C) Close-up for open flowers. Arrows indicate top of stamens.

1542 The Plant Cell

the ESD4 mutation was also suggested to promote flowering

both through and independently of FLC (Reeves et al., 2002). To

probe into additional effects of NUA and ESD4 mutations on the

flowering pathways, we tested mRNA levels of several additional

floral repressors and floral integrators. MAF4, one of the FLC

paralogs also implied in floral repression (Ratcliffe et al., 2003),

was decreased in nua-1, nua-3, nua-4, and esd4-2 (Figure 6B).

Furthermore, the phytohormone gibberellin is known to promote

floral transition strongly under short-day conditions via activating

the floral integrator LEAFY (LFY) by GAMYB transcription factors

(Achard et al., 2004 and references therein). When the expres-

sion levels of LFY, MYB33, and MYB65 were analyzed, elevation

in nua-1 and nua-4 mutants was apparent (Figure 6B), again

supporting the notion that several flowering pathways were

affected in the nua mutants. We noted that LFY, MYB33, and

MYB65 expression levels were not significantly altered in esd4-2,

supporting an overlapping, but not identical, effect of the two

gene knockouts on flowering regulator expression.

GFP-ESD4 Localization Is Not Altered in nua Mutants

If NUA acted as a nuclear pore anchor for ESD4, as predicted for

the interaction between Ulp1 and Mlp1 in yeast, depletion of NUA

should lead to a release of ESD4 from the nuclear periphery. We

tested this model by investigating the localization of GFP-ESD4

in wild-type Columbia and the nua mutant alleles. Figure 7A

shows that GFP-ESD4 has a nuclear location with enrichment at

the nuclear envelope in root cell files, consistent with previous

reports for ESD4-GFP (Murtas et al., 2003). Figures 7B to 7D and

Supplemental Figure 3E online show that no significant changes

in this pattern were seen in nua-1, nua-2, nua-3, and nua-4,

suggesting that the increase in SUMO conjugates in nua mutants

is unlikely to be based on delocalization of ESD4.

nua Mutants Accumulate Nuclear Poly(A)1 RNA

Both mammalian Tpr and yeast Mlp1/Mlp2 have been implicated

in affecting mRNA export. While poly(A)þ RNA accumulates in

nuclei immunodepleted of Tpr (Shibata et al., 2002) and in cells

that overexpress nuclear Tpr fragments (Bangs et al., 1998),

Figure 6. nua Mutant Alleles Lead to Increasing Accumulation of SUMO

Conjugates and Altered Expression of Genes Involved in Different

Flowering Pathways.

(A) Protein extracts from 10-d-old seedlings were probed with an anti-

SUMO1 antibody. Free SUMO is reduced (arrowhead), and the amount

of SUMO conjugates increased (bracket) in nua mutants, like previously

shown for esd4 mutants. The effect increases in severity in the order of

nua-2, nua-3, and nua-1. The asterisk indicates the putative SUMO

dimer.

(B) RT-PCR of tubulin 2 (Tub), FLC, MAF4, FT, SOC1, LFY, MYB33, and

MYB65 mRNAs in Arabidopsis wild type, nua-1, nua-2, nua-3, nua-4, and

esd4-2.

Figure 7. The Concentration of ESD4 on the Nuclear Periphery Is Not

Abolished in nua Mutants.

GFP fluorescence was observed at the nuclear periphery, in root cell files

of Arabidopsis seedlings transformed with GFP-ESD4. Bars ¼ 10 mm.

(A) Wild-type background.

(B) nua-1 background.

(C) nua-2 background.

(D) nua-3 background.

Arabidopsis Homolog of Tpr/Mlp1/Mlp2/Megator 1543

double mutants of Mlp1 and Mlp2 show no nuclear mRNA

accumulation (Kosova et al., 2000). However, they are affected in

the nuclear retention of mis-spliced RNAs (Galy et al., 2004), and

Mlp1 binds hnRNPs (Green et al., 2003). Using in situ hybridiza-

tion with an oligo(dT) probe, we tested if the nua mutant alleles

accumulate more nuclear poly(A)þ RNA compared with the wild

type. Figure 8 and Supplemental Figure 3F online show that,

indeed, all four mutant alleles show higher nuclear signals than

the wild type. Significantly, the amount of nuclear signal detected

correlated well with the severity of the mutant alleles in terms of

flowering time, SUMO conjugate abundance, flowering regulator

expression, and developmental defects. In all assays employed,

nua-1 and nua-4 were the most severe alleles, followed by nua-3

as intermediate and nua-2 as the mildest allele.

DISCUSSION

Mammalian Tpr (Park et al., 1986) and its closest structural re-

latives in Drosophila (Zimowska et al., 1997) and yeast (Strambio-

de-Castillia et al., 1999) are large coiled-coil proteins located

on the nucleoplasmic side of the NPC (Cordes et al., 1997;

Zimowska et al., 1997; Bangs et al., 1998; Strambio-de-Castillia

et al., 1999). While Mlp1/Mlp2 deletion mutants in yeast are

viable, knockout mutants of Megator are embryonic lethal (Qi

et al., 2004). Megator depletion via RNAi leads to a reduction of

cells going through mitosis (Qi et al., 2004). To our knowledge, no

Tpr knockout mutants have so far been reported in vertebrates.

We show here that a likely knockout of the single Arabidopsis

Tpr/Megator/Mlp1/Mlp2 homolog NUA is viable. Loss of NUA

leads to complex developmental phenotypes, including early

flowering, stunted growth, defects in stamen and silique devel-

opment, and changes in phyllotaxy. Our data indicate that loss of

NUA affects a number of regulatory pathways, likely by affecting

the expression level of key regulators, as shown here for the

example of flowering-time regulators.

Several immuno-electron microscopy studies have suggested

that Tpr is located at the nuclear basket of the NPC (Cordes et al.,

1997; Frosst et al., 2002; Krull et al., 2004). Nevertheless, on the

light microscopy level, a continuous nuclear envelope staining

was seen in several recent studies of human Tpr (Hase et al.,

2001; Hase and Cordes, 2003; Krull et al., 2004). Similarly, in our

system, we do not detect a punctate pattern for NUA. This does

not contradict nuclear pore localization but simply indicates that

a higher level of resolution (such as immunogold labeling)

is required to investigate the precise ultrastructural location

of NUA.

We have identified an allelic series of four T-DNA insertion

alleles that have increasing severity of several correlating whole-

plant and molecular phenotypes. In the order of nua-2, nua-3,

and nua-1/nua-4, we have observed an increase in severity of

early flowering, accumulation of sumoylated proteins, nuclear

accumulation of poly(A)þ RNA, and altered expression of several

flowering-time regulators. This suggests that these events are

connected by the activity of NUA. Molecular characterization of

the NUA insertion alleles indicates that nua-1 and nua-4 are likely

null alleles, that nua-2 is a knockdown allele, and that nua-3

expresses an ;100-kD N-terminal fragment of NUA. nua-3 acts

as a functional knockdown of intermediate severity, indicating

that part of the NUA activity involved in SUMO homeostasis,

RNA metabolism, and flowering-time regulation resides in the

N-terminal fragment expressed in nua-3.

Yeast Mlp1 and Mlp2 function in anchoring the SUMO prote-

ase Ulp1 to the NPC (Zhao et al., 2004). Mlp1/Mlp2 deletion

mutants in yeast exhibit a clonal lethality phenotype caused by

an increase of extrachromosomal 2-mm circle DNA and show an

enhanced sensitivity to DNA-damaging drugs (Galy et al., 2000;

Kosova et al., 2000; Zhao et al., 2004). This phenotype is con-

sistent with SUMO pathway mutants and can be suppressed by

overexpression of Ulp1, whereas deletion of Ulp1 or delocaliza-

tion of Ulp1 through deletion of its targeting domain mimics

Mlp1/Mlp2 deletion mutants (Zhao et al., 2004). These data

indicate that Mlp1/Mlp2 are required for Ulp1 function, most

likely through its anchoring at the NPC. By contrast, the mam-

malian SUMO protease SENP2 is anchored to the inner nuclear

pore by binding Nup153, the same nucleoporin that is involved in

anchoring Tpr to the nuclear basket (Hang and Dasso, 2002;

Zhang et al., 2002).

Our data show that in Arabidopsis, NUA and ESD4 are both

associated with the nuclear periphery, and the accumulation of

sumoylated proteins in nua mutants indicates that NUA is in-

volved in desumoylation. The close similarity of the whole-plant

and molecular phenotypes of nua and esd4 knockout mutants,

together with their ability to interact in yeast two-hybrid assays,

are consistent with a model in which the two proteins interact at

the nuclear periphery, thereby affecting mRNA accumulation,

desumoylation, and alterations in gene expression. If the inter-

action of NUA and ESD4 were analogous to Mlp1/Mlp2 and Ulp1,

Figure 8. Poly(A)þ RNA Accumulates in Nuclei of nua Mutants.

Whole-mount in situ hybridization of 10-d-old seedling petioles with

fluoresceine-labeled oligo(dT) probe shows increasing severity of nu-

clear poly(A)þ mRNA retention in nua-2, nua-3, and nua-1. Insets show

single nuclei at a higher magnification. All images were taken at identical

gain settings. The experiment was repeated twice. Bars ¼ 20 mm.

1544 The Plant Cell

we would expect that ESD4 lost nuclear rim localization in a nua

null mutant. However, our data show that GFP-ESD4 is still

targeted to the nuclear rim in nua-1 and nua-4, suggesting that

the desumoylation defect observed in nua mutants is not caused

by the delocalization of ESD4. In addition, the interaction be-

tween NUA and ESD4 in yeast two-hybrid assays might be

indirect or additional proteins might tether ESD4 to the NPC.

Further experiments are needed to test these hypotheses. Our

current working model for the functional interaction between

NUA and ESD4 is therefore that ESD4 and possibly other

Arabidopsis SUMO proteases and NUA have to be in close

proximity at the NPC for wild-type-level desumoylation to occur.

This could, for example, be envisioned if sumoylated proteins

bind to NUA, thereby being presented to localized SUMO pro-

tease activity.

Our finding that nua mutants accumulate nuclear poly(A)þRNA

indicates a function of NUA in mRNA export and/or nuclear

mRNA turnover. There are several lines of evidence for a role of

Tpr-like proteins in mRNA export. Overexpression of mammalian

Tpr and its depletion through the injection of antibodies leads

to an accumulation of poly(A)þ RNA in the nucleus, partially in

enlarged clusters containing splicing factors (Bangs et al., 1998;

Shibata et al., 2002). Overexpression of Mlp1 in yeast causes

nuclear accumulation of poly(A)þ RNA, while no nuclear poly(A)þ

RNA accumulation was seen in double deletion mutants of Mlp1/

Mlp2 (Kosova et al., 2000). The C-terminal domain of yeast Mlp1

interacts with hnRNPs via Nab2, and a model has been proposed

for Mlp1 to act as a checkpoint for RNA export (Green et al.,

2003). Mlp1 is responsible for the nuclear retention of unspliced

mRNAs, and deletion of Mlp1 leads to a leakage of intron-

containing RNAs into the cytoplasm (Galy et al., 2004). Our data

demonstrate an effect on mRNA export by a likely null mutant of a

Tpr-like protein and contrast the yeast data that show no effect in

an Mlp1/Mlp2 null mutant. This indicates that while homologs of

Tpr in different kingdoms all appear to function in an aspect of

mRNA export, their precise role probably differs.

SUMO modification has been discussed as a possible mech-

anism to control nucleocytoplasmic transport of proteins (for

review, see Pichler and Melchior, 2002). Recently, hnRNPs, RNA

helicases, and other proteins involved in RNA metabolism were

identified as substrates for SUMO modification in mammals (Li

et al., 2004; Vassileva and Matunis, 2004; Vertegaal et al., 2004).

This suggests a previously unrecognized link between the SUMO

pathway and mRNA metabolism. Our data, showing that nua

mutants are affected both in SUMO homeostasis and nuclear

RNA accumulation, indicate that such a link also exists in plants.

Loss of NUA leads to a number of developmental defects, the

most striking one being extreme early flowering under both long-

day and short-day conditions. Early flowering is consistent with

the reduction of FLC expression and a concomitant increase of

expression of FT and SOC1, a reduction of MAF4 expression,

and an increase of MYB33, MYB65, and LFY. Additional un-

tested factors involved in flowering-time regulation might also be

affected in nua mutants. If the observed effect on mRNA in nua

mutants also included a perturbation of microRNA (miRNA)

export or turnover, it is conceivable that miRNA targets are

misexpressed in nua mutants. Interestingly, both flowering-time

regulation and anther development include miRNA-regulated

steps (Achard et al., 2004; Millar and Gubler, 2005). In the future,

it will be important to determine if miRNA export is indeed

affected in nua mutants and how the observed defects in SUMO

and mRNA homeostasis are molecularly connected.

METHODS

Plant Material and Growth Conditions

For T-DNA insertion mutants nua-1 (SALK_057101), nua-2 (SALK_069922),

nua-3 (SAIL_505_H11), nua-4 (WiscDsLox297300_17E), and esd4-2

(SALK_032317), T3 or T4 bulk seeds were acquired from the ABRC.

The nua-1 esd4-2 double mutant was identified in the F2 generation from

crosses between nua-1 and esd4-2. Arabidopsis thaliana wild-type and

T-DNA lines were grown on soil under standard long-day conditions (16 h

light/8 h dark) or short-day conditions (8 h light/16 h dark) or on Murashige

and Skoog plates under constant light.

Flowering-Time Measurements

Plants were grown on soil in short-day or long-day conditions. Flowering

time was measured by counting the total number of rosette leaves at the

time of bolting. Data shown are the mean value and SD of 11 to 53 samples

per line.

PCR-Based Genotyping of T-DNA Insertion Lines

Genomic DNA was extracted as described (Krysan et al., 1999). Primers

were designed using SIGnAL iSect tools (http://signal.salk.edu/tdnaprimers.

html). All primer sequences are summarized in Supplemental Table 2

online. The exact T-DNA insertion sites were determined by sequenc-

ing the PCR products derived from primer combinations of gene-

specific primers plus T-DNA-specific primers. In nua-1 and nua-3, primer

combinations LP057101.1/LBa1 and CS821281RP/pCSA110-LB were

used, respectively. In nua-2, primer combinations RP069922/LBa1 and

LP069922/New-RB-primer were used. In nua-4, the insertion site was

mapped to nucleotide þ2145 (within exon 9) using primer combinations

CS850695FP/p745-primer and CS850695RP/p745-primer.

Cloning of NUA Full-Length cDNA and Plasmid Construction

Due to the large size of the predicted NUA coding sequence (> 6 kb), the

NUA cDNA was cloned in four fragments by RT-PCR using the Thermo-

Script RT-PCR system (Invitrogen). Partial fragments were cloned into the

pENTR/D-TOPO vector and confirmed by sequencing. The full-length

cDNA was assembled using the unique AatII, ScaI, and XmaI restriction

enzyme sites and moved into the Gateway destination vectors pDEST17,

pDEST22, and pDEST32 (Invitrogen). To validate the assembled >6-kb

cDNA, full-length NUA transcript was reverse-transcribed with gene-

specific primers. After amplification using the BD Advantage 2 PCR

enzyme system (BD Biosciences Clontech), the 6282-bp cDNA was fully

sequenced and found identical to the assembled cDNA (see Supple-

mental Figure 1 online). The ESD4 cDNA (Reeves et al., 2002) in the

pENTR/SD/D-TOPO vector was received from the ABRC. The gene was

sequenced for confirmation and moved into the GFP vector pK7WGF2

(Karimi et al., 2002).

Arabidopsis Transformation

Arabidopsis Columbia wild type, nua-1, nua-2, and nua-3 were trans-

formed by floral dipping (Clough and Bent, 1998) with Agrobacterium

tumefaciens strain ABI harboring plasmid pK7WGF2-ESD4. Primary

transformants were selected for kanamycin resistance.

Arabidopsis Homolog of Tpr/Mlp1/Mlp2/Megator 1545

RT-PCR

Total RNA was extracted with the RNeasy plant mini kit (Qiagen) from

10-d-old seedlings (a stage at which none of the mutants had bolted yet)

grown on Murashige and Skoog plates under long-day conditions. After

digestion with DNase I (amplification grade; Invitrogen), cDNA was syn-

thesized with oligo(dT) primers from ;3 mg total RNA using the Thermo-

script RT-PCR system (Invitrogen). cDNA templates were PCR amplified

with gene-specific primers (see Supplemental Table 2 online) for quan-

tification of mRNA levels of flowering time integrator genes. Twenty-eight

cycles were used for tubulin 2, 35 cycles for FLC, 30 cycles for FT, 26

cycles for SOC1, 35 cycles for LFY, and 33 cycles for MYB33 and MYB65.

Sequence Analysis

Coiled-coil domains were predicted using the Protean software from the

Lasergene package (DNASTAR) and the algorithm MultiCoil (Wolf et al.,

1997).

Protein Expression, Purification, and Antibody Production

The anti-NUA antibody (OSU156) was generated against a partial recom-

binant protein (amino acids 518 to 1248). The N-terminal 6xHis-tagged

protein was purified from Escherichia coli BL21-AI using Ni-NTA resin

according to the QIAexpressionist manual (Qiagen) and preparative SDS-

PAGE. The rabbit antiserum was generated by Cocalico Biologicals.

Immunoprecipitation and Sumoylation Assays

For immunoprecipitation, all steps were performed at 48C. One milliliter of

tissue powder from 12-d-old seedlings was suspended in 1 mL of buffer

containing 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). After centrifugation at 16,000g, the supernatant was incubated

with anti-NUA (1:500 dilution) bound to protein A-sepharose for 3 h. The

immunoprecipitates were resuspended in 50 mL 33 SDS-PAGE loading

buffer and subjected to SDS-PAGE. For the analysis of sumoylation

profiles, total protein from 2-week-old seedlings was extracted as de-

scribed (Thompson et al., 2005). Approximately 100 mg of protein was

separated on a two-layer SDS-PAGE gel to resolve both the free SUMO

and high molecular weight conjugates (8% top half and 15% bottom half).

After SDS-PAGE, proteins were then transferred to a polyvinylidene

difluoride membrane (Bio-Rad), probed with anti-NUA (1:2000) or

anti-AtSUMO1 (Abcam), and subsequently probed with peroxidase-

conjugated anti-rabbit secondary antibody (GE Healthcare; 1:15,000).

For detection, the Supersignal West Pico Chemiluminescent Substrate

for the HRP system (Pierce) was used.

Immunolocalization

Whole-mount immunolocalization in Arabidopsis root tip cells was

performed as described (Friml et al., 2003) using anti-NUA (1:100),

monoclonal anti-a-tubulin (1:100, DM1A; Sigma-Aldrich), monoclonal

anti-NPC (1:250, QE5, recognizing Nup214, Nup153, and p62 in mam-

mals; Covance), monoclonal anti-GFP (2.5 mg/mL; Molecular Probes),

primary antibodies, and appropriate secondary antibodies conjugated

to Alexa Fluor 488 or 568 (Invitrogen). DNA was counterstained by

49,6-diamidino-2-phenylindole (Sigma-Aldrich). The images in Figure 3

were collected from a Leica TCS SP2 AOBS confocal laser scanning

microscope equipped with four lasers (red helium neon 633 nm, green

helium neon 543 nm, argon 458/476/488/496/514 nm, and argon UV). The

images in Figure 4 and Supplemental Figure 2 online were collected on a

PCM 2000/Nikon Eclipse 600 confocal laser scanning microscope as

described (Rose and Meier, 2001).

In Situ Hybridization

The poly(A)þ RNA in situ hybridization was conducted essentially as

described (Gong et al., 2005) with minor modifications. Briefly, samples

were taken from equivalent portions of young leaves at similar develop-

mental stages (2-week-old, four true leaves) from Columbia wild type or

nua mutants and were fixed in glass vials by adding 8 to 10 mL of fixation

cocktail containing a mixture of 50% fixation buffer (120 mM NaCl, 7 mM

Na2HPO4, 3 mM NaH2PO4, 2.7 mM KCl, 0.1% Tween 20, 80 mM EGTA,

5% formaldehyde, and 10% DMSO) and 50% heptane to completely

immerse the sample. Samples were shaken gently for 30 min at room

temperature. After dehydration twice for 5 min each in absolute methanol

and three times for 5 min each in absolute ethanol, the samples were

incubated for 30 min in 1:1 (v/v) ethanol:xylene and then washed twice for

5 min each with absolute ethanol, twice for 5 min each with absolute

methanol, and once for 5 min with 1:1 (v/v) methanol:fixation buffer with-

out formaldehyde. The samples were post-fixed in fixation buffer for

30 min at room temperature. After fixation, the samples were rinsed twice

with fixation buffer without formaldehyde and once with 1 mL of perfect

Hyb Plus hybridization buffer (Sigma-Aldrich; H-7033). To each glass vial,

1 mL of hybridization buffer was added, and samples were prehybridized

for 1 h at 508C. After prehybridization, 10 pmol 45-mer oligo(dT) labeled

with one molecule of fluoresceine at the 59-end (synthesized by MWG

Company) was used for hybridization at 508C in darkness for at least 8 h.

After hybridization, the samples were washed once for 60 min in 23 SSC

(0.3 M NaCl and 0.03 M sodium citrate) and 0.1% SDS at 508C and once

for 20 min in 0.23 SSC and 0.1% SDS at 508C in darkness. Confocal

images were acquired as described (Rose and Meier, 2001).

Yeast Two-Hybrid Assays

Yeast strain PJ69-4A was used. Competent cell preparation and yeast

transformation was performed as described (Dohmen et al., 1991).

Accession Number

Sequence data from this article can be found in the GenBank/EMBL data

libraries under accession number EF426860.

Supplemental Data

The following materials are available in the online version of this article.

Supplemental Figure 1. The Expression Pattern of NUA and the

Validation of the Full-Length NUA cDNA.

Supplemental Figure 2. Immunofluorescence in Wild-Type and

nua-1 Root Tip Cells.

Supplemental Figure 3. Characterization of the nua-4 Mutant.

Supplemental Figure 4. NUA Self-Interacts and Interacts with ESD4

in Yeast Two-Hybrid Assays.

Supplemental Table 1. Flowering Time of Wild Type Columbia, nua

Mutants, and flc-3 Null Mutant, Given as Number of Rosette Leaves at

the Time of Bolting.

Supplemental Table 2. Primer Sequences Used in This Study.

ACKNOWLEDGMENTS

We thank SIGnAL and the ABRC for providing the sequence-indexed

Arabidopsis T-DNA insertion mutants, Richard Amasino for the flc-3 line,

Biao Ding for the use of his confocal microscope, Jelena Brkljacic for

fruitful discussions, David E. Somers for critical reading of the manu-

script, and Chao (Sylvia) He and Kyle Kennedy for technical assistance.

1546 The Plant Cell

Financial support by the National Science Foundation is greatly ac-

knowledged.

Received November 28, 2006; revised April 18, 2007; accepted May 2,

2007; published May 18, 2007.

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1548 The Plant Cell

DOI 10.1105/tpc.106.049239; originally published online May 18, 2007; 2007;19;1537-1548Plant Cell

Venkatakrishnan, Qiao Zhao and Iris MeierXianfeng Morgan Xu, Annkatrin Rose, Sivaramakrishnan Muthuswamy, Sun Yong Jeong, Sowmyain mRNA Export and SUMO Homeostasis and Affects Diverse Aspects of Plant Development

Homolog of Tpr/Mlp1/Mlp2/Megator, Is InvolvedArabidopsisNUCLEAR PORE ANCHOR, the

 This information is current as of January 16, 2021

 

Supplemental Data /content/suppl/2007/05/17/tpc.106.049239.DC1.html

References /content/19/5/1537.full.html#ref-list-1

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