Arabidopsis thaliana BTB POZ-MATH proteins interact...

Arabidopsis thaliana BTB ⁄ POZ-MATH proteins interactwith members of the ERF ⁄AP2 transcription factor familyHenriette Weber1,2 and Hanjo Hellmann1

1 Washington State University, Pullman, WA, USA

2 Freie University Berlin, Germany

Keywords

APETALA; BPM; cullin; proteasome;

ubiquitin

Correspondence

H. Hellmann, Washington State University,

Pullman, WA 99164, USA

Fax: +1 509 335 3184

Tel: +1 509 335 2762

E-mail: [email protected]

(Received 17 July 2009, revised 7

September 2009, accepted 11 September

2009)

doi:10.1111/j.1742-4658.2009.07373.x

In Arabidopsis thaliana, the BTB ⁄POZ-MATH (BPM) proteins comprise a

small family of six members. They have been described previously to use

their broad complex, tram track, bric-a-brac ⁄POX virus and zinc finger

(BTB ⁄POZ) domain to assemble with CUL3a and CUL3b and potentially

to serve as substrate adaptors to cullin-based E3-ligases in plants. In this

article, we show that BPMs can also assemble with members of the ethyl-

ene response factor ⁄Apetala2 transcription factor family, and that this is

mediated by their meprin and TRAF (tumor necrosis factor receptor-asso-

ciated factor) homology (MATH) domain. In addition, we provide a

detailed description of BPM gene expression patterns in different tissues

and on abiotic stress treatments, as well as their subcellular localization.

This work connects, for the first time, BPM proteins with ethylene response

factor ⁄Apetala2 family members, which is likely to represent a novel

regulatory mechanism of transcriptional control.

Structured digital abstractl MINT-7262792: BPM1 (uniprotkb:Q8L765) physically interacts (MI:0915) with RAP2-4 (uni-

protkb:Q8H1E4) by two hybrid (MI:0018)l MINT-7262805: BPM1 (uniprotkb:Q8L765) physically interacts (MI:0915) with RAP2-13

(uniprotkb:Q9LM15) by two hybrid (MI:0018)l MINT-7262749: BPM3 (uniprotkb:Q2V416) physically interacts (MI:0915) with RAP2-4 (uni-

protkb:Q8H1E4) by two hybrid (MI:0018)l MINT-7262764: BPM3 (uniprotkb:Q2V416) physically interacts (MI:0915) with RAP2-13

(uniprotkb:Q9LM15) by two hybrid (MI:0018)l MINT-7262838, MINT-7262882, MINT-7262898, MINT-7263072: RAP2-4 (uni-

protkb:Q8H1E4) binds (MI:0407) to BPM1 (uniprotkb:Q8L765) by pull down (MI:0096)l MINT-7262911: RAP2-4 (uniprotkb:Q8H1E4) binds (MI:0407) to BPM2 (uni-

protkb:Q9M8J9) by pull down (MI:0096)l MINT-7262935: RAP2-4 (uniprotkb:Q8H1E4) binds (MI:0407) to BPM3 (uniprotkb:Q2V416)

by pull down (MI:0096)l MINT-7262945: RAP2-4 (uniprotkb:Q8H1E4) binds (MI:0407) to BPM4 (uni-

protkb:Q9SRV1) by pull down (MI:0096)l MINT-7262970: RAP2-4 (uniprotkb:Q8H1E4) binds (MI:0407) to BPM5 (uni-

protkb:Q1EBV6) by pull down (MI:0096)l MINT-7262992: RAP2-4 (uniprotkb:Q8H1E4) binds (MI:0407) to BPM6 (uni-

protkb:A1L4W5) by pull down (MI:0096)l MINT-7263095: RAP2-4 (uniprotkb:Q8H1E4) binds (MI:0407) to RAP2-4 (uniprotkb:

Q8H1E4) by pull down (MI:0096)

Abbreviations

BPM, BTB ⁄ POZ-MATH; BTB ⁄ POZ, broad complex, tram track, bric-a-brac ⁄ POX virus and zinc finger; ERF ⁄ AP2, ethylene response

factor ⁄ Apetala2; GFP, green fluorescent protein; GUS, b-glucuronidase; MATH, meprin and TRAF homology; proBPM, promoterBPM;

RAP2.4, related to Apetala2.4; TRAF, tumor necrosis factor receptor-associated factor; Y2H, yeast two-hybrid.

6624 FEBS Journal 276 (2009) 6624–6635 ª 2009 The Authors Journal compilation ª 2009 FEBS

http://mint.bio.uniroma2.it/mint/search/interaction.do?interactionAc=MINT-7262792

http://www.ebi.uniprot.org/entry/Q8L765

http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0915

http://www.ebi.uniprot.org/entry/Q8H1E4





http://www.ebi.uniprot.org/entry/Q9LM15



http://www.ebi.uniprot.org/entry/Q2V416




















http://www.ebi.uniprot.org/entry/Q9M8J9










http://www.ebi.uniprot.org/entry/Q9SRV1





http://www.ebi.uniprot.org/entry/Q1EBV6





http://www.ebi.uniprot.org/entry/A1L4W5







Introduction

In recent years, a novel superfamily of proteins has

been described in plants that contains a conserved pro-

tein–protein interaction motif named broad complex,

tram track, bric-a-brac ⁄POX virus and zinc finger

(BTB ⁄POZ) [1–5]. This protein family is highly diverse

with, for example, 80 members in Arabidopsis and 149

in rice [4,5]. The BTB ⁄POZ domain has a length of

around 116 amino acids and mediates homophilic and

heterophilic interactions between the same or different

proteins, respectively [6,7]. The BTB ⁄POZ fold consists

of six a-helices and three b-sheets that form a tightly

interwound butterfly-shaped dimer with an extensive

hydrophobic interface [8,9]. BTB ⁄POZ proteins are

often transcriptional regulators containing a C2H2

domain for DNA binding, but can also be found in

combination with various other protein–protein inter-

action motifs, such as KELCH or meprin and TRAF

(tumor necrosis factor receptor-associated factor)

homology (MATH) motifs, indicating involvement in

various biological processes [5,10–13].

It has recently been demonstrated for animals and

plants that members of the BTB ⁄POZ family use their

BTB ⁄POZ domain to assemble with CUL3 proteins

[2–4,14–16]. Cullins are scaffolding subunits of multi-

meric E3-ligases that can polyubiquitinate their sub-

strates and thereby target them for degradation via the

26S proteasome [17]. Thus, plant BTB ⁄POZ proteins

potentially serve as substrate adaptors for CUL3-based

E3-ligases. Furthermore, self-assembly of BTB ⁄POZ

proteins has been established for Arabidopsis and rice

[2–4]. However, although plants encode for a large

number of BTB proteins, a functional role has only

been assigned for a few of them, including ETO1 (eth-

ylene biosynthesis [14]), NPH3 (blue light signal trans-

duction [18]), BOP1 (leaf development [19]), ARIA

(abscisic acid signaling [20]), NPY1 (auxin signaling

[21]) and NPR1 (salicylic acid signaling [22]).

Some BTB proteins from plants and animals contain

a secondary MATH domain which comprises around

150 amino acids forming eight b-sheets [23]. The motif

was noted on the basis of homology with the C-terminal

region of meprins A and B and the TRAF-C domain,

and, like the BTB domain, facilitates protein–protein

interaction [24]. Meprins are tissue-specific metalloen-

dopeptidases implicated in developmental and patho-

logical processes in animals by hydrolyzing a variety of

peptides and proteins [25–27]. In mammals, TRAFs

regulate cell growth signaling and apoptosis by interact-

ing with membrane-bound receptors through their

TRAF-C domains [28,29]. Although TRAFs and mep-

rins have not been described in plants, a variety of plant

proteins functionally unrelated to meprins and TRAFs

contain MATH domains [30], and proteins carrying

both BTB and MATH motifs are common in plants.

Arabidopsis, for example, expresses six members of this

BTB subfamily [referred to as the BPM (BTB ⁄POZ-

MATH) family] [2,16], whereas, in rice, 74 members are

annotated [5]. Although it has been established that the

BTB domain is employed to facilitate assembly with

CUL3 and other BTB proteins [2,16], it remains unclear

what kind of interactions are mediated by the MATH

domain in plants.

In this article, we show that the MATH domain of

BPM proteins is used to assemble with members of the

ethylene response factor ⁄Apetala2 (ERF ⁄AP2) tran-

scription factor family. We also provide a detailed

description of the Arabidopsis thaliana BPM family

expression and subcellular localization profile, including

promoter:b-glucuronidase (promoter:GUS) and green

fluorescent protein (GFP) fusion protein studies for all

six members. Overall, the work demonstrates a novel role

for BPMs as potential regulators that affect transcrip-

tional activities of ERF ⁄AP2 proteins in higher plants.

Results

BPM proteins assemble with members of the

ERF ⁄ AP2 transcription factor family

Because it has been shown previously that Arabidopsis

BPM proteins use their BTB ⁄POZ domain to interact

with the cullins CUL3A and CUL3B [3,4,16], we inves-

tigated what kind of protein–protein interactions were

facilitated by their MATH domains by performing two

l MINT-7262855: RAP2-13 (uniprotkb:Q9LM15) binds (MI:0407) to BPM1 (uniprotkb:

Q8L765) by pull down (MI:0096)l MINT-7263015: BPM1 (uniprotkb:Q8L765) binds (MI:0407) to At4g13620 (uniprotkb:

Q9SVQ0) by pull down (MI:0096)l MINT-7263047: BPM1 (uniprotkb:Q8L765) binds (MI:0407) to At4g39780 (uniprotkb:

O65665) by pull down (MI:0096)

H. Weber and H. Hellmann Arabidopsis thaliana BPM–ERF ⁄ AP2 interaction

FEBS Journal 276 (2009) 6624–6635 ª 2009 The Authors Journal compilation ª 2009 FEBS 6625









http://www.ebi.uniprot.org/entry/Q9SVQ0





http://www.ebi.uniprot.org/entry/O65665


yeast two-hybrid (Y2H) screens on a root-specific

cDNA library. One screen was performed with a full-

length BPM3 (At2g39760), whereas, for the other, we

used a BPM1 (At5g19000) fragment that lacked the

BTB ⁄POZ domain [denoted as BPM1(1–189); Fig. 1B].

As both the MATH and BTB domains mediate assem-

bly with other proteins, we speculated that this dual

approach would not only identify specific interactors

for the MATH domain, but would also provide infor-

mation on to what extent the two different MATH

domains of BPM1 and BPM3 target the same group

of proteins (see Table S1A,B for identity ⁄ similarity

comparisons of BPM proteins and their MATH

domains, respectively).

In total, 250 yeast clones were analyzed as primary

positives and, consistent with earlier studies [2], BPM4

was found using BPM3 as bait (data not shown). How-

ever, predominantly, we identified RAP2.4 (At1g78080;

related to Apetala2.4), which was found 15 times with

BPM3 and 18 times with the BPM1(1–189) fragment

(Fig. 1A). RAP2.4 belongs to the ERF ⁄AP2 family of

transcription factors and contains a single AP2 domain.

The protein has been described previously in context

with abiotic stress tolerance, red light response and eth-

ylene signaling [31]. We also identified once At1g22190

in the BPM3 screen, which is the closest relative of

RAP2.4 [32]. It should be noted that both RAP2.4 and

At1g22190 were isolated as partial clones lacking the

first 60 amino acids (Fig. 1B), demonstrating that this

region is not essential for assembly with BPM proteins.

As 12 RAP2 proteins have been annotated previously

[33], we retained this nomenclature and denoted

At1g22190 as RAP2.13.

To further confirm the interaction of RAP2.4 and

RAP2.13 with BPM proteins, we cloned the corre-

sponding full-length cDNAs for both genes, generated

GST fusion proteins and tested these in pulldown

assays with BPM1(1–189). As shown in Fig. 1C,

BPM1(1–189) coprecipitated with both GST:RAP2

proteins, but not with GST alone, further corroborat-

ing the Y2H data. Because RAP2.4 and RAP2.13 are

closely related to each other, we mainly focused on

RAP2.4 as a representative example in subsequent

experiments. Here, RAP2.4 also interacted with a full-

length BPM1 (using a GST:BPM1 protein) and with

itself (using GST:RAP2.4) (Fig. 1D). Additional pull-

down assays positively confirmed binding to BPM2,

BPM4, BPM5 and BPM6 (Fig. 1E). To exclude non-

specific assembly with BPM proteins, we decided to

test At1g65050. This protein has no BTB motif, but

contains a MATH domain that is most closely related

to those from BPMs [30]. In these experiments,

RAP2.13fragment

RAP2.4fragment

33460

AP2

214150

26160

AP2

81 142

MATHBPM11–189

1 38 150 189BPM11–189

RAP2.4

RAP2.13

Empty vector

BPM3

Input

GSTGST:R

AP2.4

GST:RAP2.13

BPM11–189

BA

C E

Input

GSTGST:R

AP2.4

BPM1

BPM2

BPM3

BPM4

BPM5

BPM6

At1g65050

D

Input

GSTGST

GST:RAP2.4

GST:BPM

1

RAP2.4

Fig. 1. BPM proteins interact with members of the ERF ⁄ AP2 family. (A) Y2H assays demonstrate the assembly of BPM1(1–189) and BPM3

with RAP2.4 and RAP2.13, respectively. (B) Schematic drawing of the BPM1 fragment used for the Y2H screen and the shortest RAP2.4

and RAP2.13 fragments retrieved from the screens. (C) In vitro-translated BPM1(1–189) coprecipitates with full-length GST:RAP2.4 and

GST:RAP2.13. (D) In vitro-translated RAP2.4 coprecipitates with GST:BPM1 and GST:RAP2.4. (E) In vitro-translated BPM1–6 co-precipitate

with GST:RAP2.4, but the MATH protein At1g65050 does not. In this and all subsequent figures, the input represents 1–3 lL of the coupled

in vitro transcription ⁄ translation reaction mixture, and GST was used as a negative control. All experiments in this and subsequent figures

were repeated at least three times.

Arabidopsis thaliana BPM–ERF ⁄ AP2 interaction H. Weber and H. Hellmann


RAP2.4 did not interact with At1g65050 (Fig. 1E),

which is a critical finding as it suggests that RAP2.4–

BPM assembly is specific.

BPM1–RAP2.4 interaction requires a complete

MATH domain and the N-terminal region of

RAP2.4

The use of a truncated version of BPM1 in the Y2H

screens demonstrated that the BTB domain is not

involved in the assembly with RAP2.4. However,

BPM1(1–189) still contains nearly 80 amino acids that

are not part of the MATH domain and which could rep-

resent possible interaction sites for RAP2.4. To further

confirm that a full-length MATH domain is sufficient for

binding to RAP2.4, we generated a new truncated BPM1

version of 151 amino acids [BPM1(1–151)] comprising

the first 38 amino acids of BPM1 followed by the com-

plete MATH domain. As shown in Fig. 2A, BPM1(1–

151) is entirely capable of binding to GST:RAP2.4, mak-

ing it highly probable that only the MATH domain is

required for RAP2.4–BPM1 assembly.

Likewise, we were interested in the RAP2.4 region

that mediates the assembly with BPM1 proteins. Its

AP2 domain stretches from amino acid residue 150 to

214. To test whether a functional AP2 domain is criti-

cal for assembly with BPMs, we took advantage of an

earlier description of ap2-1 and ap2-5 mutants, in

which mutation of a glycine residue in the AP2

domain disrupts the protein’s DNA-binding affinity

[33,34]. This glycine is highly conserved and can also

be found in RAP2.4 at position 179 [35]. However, the

introduction of a point mutation that changed the gly-

cine residue to serine [RAP2.4(G179S)] did not affect

assembly with GST:BPM1, indicating that a functional

AP2 domain is not required for this type of interac-

A

B

347150381 203 442

BTBHTAMBPM1

1891–151

F

RAP2.4

1

150 214

AP2

334

60

125–251

C

D

BPM1

BPM11–151 BPM11–189

GSTGST:RAP2.4

– – – – ––+ + +– – – – –+ – –+

BPM1T7 input BPM11–

151

BPM11–

189

– +– – + –– –– + – +

GSTGST:BPM1

T7 input RAP2.41–251RAP2.4

1–25

1

RAP2.41–

295

RAP2.41–295

E

RAP2.4G179S

RAP2.4134–END

RAP2.4116–END

RAP2.4125–END

Input

GSTGST:B

PM1

RAP2.4134–END

RAP2.4125–END

RAP2.4

Input

GSTGST:R

AP2.4Fig. 2. Mapping of the interactive sites in

BPM1 and RAP2.4. (A) In vitro-translated

BPM1(1–151) can interact with GST:RAP2.4.

(B) Schematic drawing of BPM1. The trian-

gle indicates the fragment used for the Y2H

screen and for pulldowns in (A). (C) In vitro-

translated RAP2.4(1–251) is able to interact

with GST:BPM1. (D) GST:BPM1 can assem-

ble with in vitro-translated RAP2.4(G179S),

RAP2.4(116–END) and RAP2.4(125–END),

but not with RAP2.4(135–END). (E)

GST:RAP2.4 can interact with in vitro-

translated RAP2.4(125–END), but not with

RAP2.4(135–END). (F) Schematic drawing of

RAP2.4. Triangle indicates the fragment

found in the Y2H screen.



tion. Next, we generated several truncated versions of

RAP2.4 that were translated in vitro and tested for

interaction with GST:BPM1. As we originally found

truncated versions of RAP2.4 and RAP2.13 that were

missing the first 60 amino acids in the Y2H screens,

we started out with further reduced versions that

lacked the first 116, 125 and 134 amino acids.

Although complete deletion of the first 116 and 125

amino acids [RAP2.4(125–END)] did not affect copre-

cipitation with GST:BPM1, we could not detect inter-

action with a truncated version that lacked the first

134 amino acids [RAP2.4(134–END)] (Fig. 2D). In

addition, deletion of amino acid residues C-terminal

from the AP2 domain [RAP2.4(1–251)] did not affect

the interaction with GST:BPM1 (Fig. 2C). We there-

fore conclude that a critical region for assembly with

BPM proteins is located within amino acid residues

125–251 of RAP2.4.

Detailed expression analysis of BPM and RAP2.4

shows distinct patterns for the different genes

Although the interaction studies presented demonstrate

that all BPM proteins can assemble with RAP2.4, and

even provide strong evidence for the assembly of the

transcription factor in planta, it is still unclear whether

BPM and RAP2.4 genes are expressed in the same tis-

sues. Consequently, we analyzed the tissue-specific

expression patterns of all BPM genes and RAP2.4 via

semiquantitative RT-PCR, and further described their

expression in greater detail using promoter:GUS lines

(referred to as proBPM:GUS and proPRAP2.4:GUS,

respectively).

The results from RT-PCR showed that BPM2 and

BPM5 were strongly expressed in all tested tissues

(roots, rosette and cauline leaves, stems and flowers)

(Fig. 3A). Although BPM6 was also strongly

expressed, its expression level was weaker overall in

comparison with BPM2 and BPM5. For BPM1 and

BPM3, we could hardly detect expression in the differ-

ent tissues, and had to load double the amount of RT-

PCR products on the gels to visualize any PCR prod-

ucts (Fig. 3A). BPM1 showed only slightly higher

expression levels in root and flower, and BPM3 expres-

sion levels showed little variation between the different

tissues (Fig. 3A). BPM4 also showed little variation,

and expression was lower than that of the other BPM

genes. In this case, we had to load triple the amount

of RT-PCR product relative to that used for BPM2

and BPM5. Finally, RAP2.4 was expressed strongly in

roots, rosette and cauline leaves, and flowers, with

slightly weaker expression levels in the stem (Fig. 3A).

Rosette

leaf

Cauline l

eaf

Stem Flower

Root

BPM1

Control

Control

NaClSorb

itol

BPM2

BPM3

BPM4

BPM5

BPM6

RAP2.4

actin2

Drought

CBA

*

*

(2x)

(2x)

(3x)

Fig. 3. Expression profiles of BPMs and RAP2.4 genes in Arabidopsis thaliana analyzed by semiquantitative RT-PCR. (A) Total RNA (100 ng),

extracted from roots, rosette and caulin leaves, sections of stems and open flowers of mature plants grown in soil, was used for RT-PCR.

The expression of all tested genes was detected in all tested tissues, but with considerable differences in expression strength ⁄ intensity: For

BPM1 and BPM3 twofold, and for BPM4 threefold, the amount of RT-PCR product was loaded (compared with actin2 control reaction).

(B) RT-PCR analysis showing BPM1, BPM2, BPM5 and RAP2.4 up-regulated by salt (200 mM NaCl for 6 h) and osmotic stress (200 mM sor-

bitol for 6 h). Sorbitol treatment also induced BPM3 and BPM4. (C) On drought treatment (drying for 6 h on a laboratory bench), only BPM1

and BPM4 showed up-regulation in expression. Numbers in parentheses indicate the fold amount of RT-PCR loaded in comparison with

actin2. Asterisks indicate correct RT-PCR products.



Because RAP2.4 has been described previously to

play a role in abiotic stress tolerance, we tested

whether expression of the different BPM genes was

regulated by salt (NaCl), osmotic (sorbitol) and

drought stress. Treatment of Col0 wild-type plants

with 200 mm NaCl for 6 h resulted in a clear up-regu-

lation of BPM1 and BPM5 expression (Fig. 3B). We

also observed an up-regulation of BPM1 and BPM5

after treatment with sorbitol for 6 h, together with

increased BPM4 levels (Fig. 3B). RAP2.4 also

responded to both treatments with enhanced expres-

sion, which is in agreement with earlier findings from

Lin et al. [31] (Fig. 3B). Drought stress only induced

the expression of BPM1 and BPM4; all other BPM

genes and RAP2.4 remained unchanged (Fig. 3B).

Overall, these data indicate that BPM1, BPM4 and

BPM5 are involved in the abiotic stress response.

The analysis of transgenic plants carrying the differ-

ent promoter:GUS constructs showed, for proB-

PM1:GUS lines, GUS expression in pollen, but also in

stipules and leaf hydathodes (Fig. 4A). Rosette leaves

showed staining within the vascular tissue at the end

of the leaf blade, whereas the basal parts close to the

petiole remained almost unstained. We observed clear

GUS expression in the primary root of 7-day-old seed-

lings, strongest at the base of emerging lateral roots,

but no expression at detectable levels in the tips of pri-

mary and budding lateral roots. proBPM2:GUS lines

(Fig. 4B) showed strong expression in the vascular tis-

sue of cotyledons and rosette leaves, and in most parts

of the flower. Similar to proBPM1:GUS, strong stain-

ing was detectable in the stipules, pollen and at the

base of siliques. Expression in roots was detectable

along the primary but not lateral roots, with strongest

staining present at the budding lateral root primordia.

proBPM3:GUS lines showed clear GUS expression in

the root tips, but also in the stipules, anthers and in

the central veins and petioles of rosette leaves

(Fig. 4C). proBPM4:GUS showed GUS staining very

similar to that of proBPM3:GUS in the stipules, the

central veins of rosette leaves and in the anthers of dif-

ferentiated flowers. We also detected meagre expres-

sion along the root, with most obvious staining

present at the lateral root primordia and the base of

the lateral roots, and also in the columella (Fig. 4D).

Like proBPM2:GUS, proBPM5:GUS plants showed a

wide range of expression patterns in all tested organs

(Fig. 4E). Both cauline and rosette leaves showed

strong GUS expression, as did the primary root tips

and the stem, whereas, in the flower, expression was

detectable in the petals, stamen and stigmata. In proB-

PM6:GUS lines, we saw GUS expression in the vascu-

lar tissue of cotyledons and mature leaves, whereas, in

the flowers, the anthers, connectives and filaments and

the base and tip of the stigmata were stained. Similar

to BPM2, BPM3 and BPM5 promoter:GUS lines, the

root tips were strongly stained, with the exception of

columella cells which remained nearly white (Fig. 4F).

Finally, proPRAP2.4:GUS lines showed blue staining

in cotyledons of 3-day-old seedlings, but not in parts

of the hypocotyls (Fig. 4G). In rosette leaves, we

observed expression in the vascular tissue of the leaf

blade, whereas, interestingly, in older parts of the mid-

rib, no GUS expression was detectable. This was dif-

ferent from cauline leaves, in which all vascular tissue

was stained. In the flower, we detected GUS expres-

sion exclusively in the pollen. Siliques were stained at

the base and at the tip, with overall very weak staining

of the fused carpels. In the root, the central cylinder

was stained, whereas the primary root tips and tips of

emerging lateral roots showed no blue staining

(Fig. 4G, part f, marked by arrows).

Subcellular localization analysis of BPM proteins

and their interactors

Using a GFP:RAP2.4 fusion protein, it has recently

been established that RAP2.4 is primarily located in

the nucleus [31]. Accordingly, one would expect that

this organelle would be the most likely location for the

assembly of BPM proteins with RAP2.4. We generated

expression constructs for all BPM genes; however,

only for BPM4 were we able to obtain GFP:BPM4

overexpressing plants. As an alternative approach to

investigate the subcellular localization of the different

BPM proteins, we transiently expressed them in

tobacco leaves. We also included GFP:RAP2.4 and

GFP:CUL3a in these experiments to compare their

localization with that of BPM proteins.

Transient expression of GFP:BPM1 and GFP:BPM2

revealed that both proteins, like GFP:RAP2.4, were

primarily localized to the nucleus (Fig. 5 and Fig. S2).

The predominantly nuclear localization of BPM1 and

BPM2 GFP fusion proteins contrasted with all other

BPMs, as GFP:BPM3, GFP:BPM5 and GFP:BPM6

were found inside as well as outside the nucleus.

Remarkably, GFP:BPM4 was the only BPM protein

that was excluded from the nucleus, suggesting that

BPM4 and RAP2.4 are not present in the same cellular

compartments. We observed this in transient expres-

sion assays, but also in Arabidopsis plants that stably

expressed GFP:BPM4 (Fig. 5 and Fig. S3). Also note-

worthy was the observation that GFP:CUL3a showed

a subcellular localization pattern similar to

GFP:BPM3, GFP:BPM5 and GFP:BPM6. Overall,

these analyses revealed a very distinct and different



proBPM6:GUS

proBPM1:GUS proBPM2:GUS

proBPM4:GUSproBPM3:GUS

proBPM5:GUS proRAP2.4:GUS

BA

DC

GFE

a

b

e

c

f

d

g a be

c

f g

d

h

i

a bd

c a

b

ec

e f g f g hd

ae

c

f g

ad

b

h

b

e

a

c dd e

b

f

c

Fig. 4. Expression profile of proBPM:GUS and proRAP2.4:GUS in Arabidopsis thaliana. (A) proBPM1:GUS: hydathodes and stipules of 5-day-

old seedlings showed staining (a–c), as did fully developed siliques (d; base and stigma region) and vascular tissue of rosette leaves (f). In

flowers, expression was restricted to pollen and anthers (e). The primary root (g) was stained throughout, but the strongest expression was

detectable at the points of emerging lateral roots, indicated by the arrows. (B) proBPM2:GUS showed the strongest expression of all pro-

moter:GUS lines, detected in all tested tissues. Although cotyledons, hypocotyls and rosette leaves were strongly stained (a–c), GUS expres-

sion in cauline leaves was restricted to the base and apex of the lamina (c). Siliques showed staining at their bases and tips (stigma region)

(d). In flowers, the petals, stamens, receptacle and upper pistil were stained (e). Close-up of stigma with strong staining of the stigma’s

papillae (f). proBPM2:GUS plants showed GUS expression in the primary root (g, i) and lateral root primordia (h), but not in developed lateral

roots (g). (C) proBPM3:GUS lines showed altogether very weak expression. In seedlings, staining was only detectable in the stipules (a).

Rosette and cauline leaves showed good GUS expression in the central vein and petioles (b, c), as well as the anthers in flowers (d). proB-

PM3:GUS plants also showed expression in the root tips (e–g). (D) GUS expression under the BPM4 promoter was also weak, but with clear

expression in the stipules (a), midrib of rosette leaves (b), mature anthers and stigmata (c, d). In roots, faint expression was detected along

the primary root and its tip (g, h), whereas the lateral root primordia and base of the developed lateral roots showed stronger staining (e–g).

(E) GUS staining for proBPM5:GUS lines was strong in the vascular tissue of the cotyledons (a) and in mature leaves (b, rosette; c, caulin),

but also in the hypocotyl (a), young siliques (d) and flowers (g). (F) For BPM6, strong expression was observed in 3-day-old seedlings (a), as

well as in the entire lamina and petiole of rosette leaves, including vascular veins (b). In flowers, GUS expression started in the early stages

of the receptacles and stigmata (c) In older flowers, mature anthers and, later, filaments were also stained. In roots, GUS was expressed

only in the tips of primary roots (d), and at the base of differentiated siliques (e). (G) proRAP2.4:GUS lines showed GUS expression in 3-day-

old seedlings in cotyledons and in the central cylinder of the root, but not in the lower parts of the hypocotyl (a). Rosette leaves were

stained in the vascular tissue of the leaf blade, whereas the petiole and older parts of the midrib remained unstained (b). However, the cau-

line leaf blade was stained very evenly (c). In flowers, staining was only detectable in the pollen (d). Expression levels in siliques were very

low, with staining mainly present at the base and at the tip (e). A close-up of a root section showed that the central cylinder was stained (f),

whereas the lateral root primordia (marked by arrows) did not show any GUS expression.



localization for the different BPM proteins, which

might reflect their diverse biological roles in the cell. In

addition, they indicate that, except for BPM4, all other

BPM proteins are potentially able to interact with

RAP2.4 in planta, and also that CUL3a can assemble

with other proteins either in the nucleus or the cyto-

plasm.

Discussion

This work provides novel information, including the

first description linking ERF ⁄AP2 proteins to the

BPM family, but also a detailed report of BPM

expression and subcellular localization.

Our intensive interaction studies have identified the

domains required for RAP2.4 ⁄BPM assembly to the

BPM MATH domain and a region of RAP2.4 that

stretches from amino acid residues 125 to 251, which

encompasses the AP2 domain. Although the point

mutation G179S in the AP2 domain of RAP2.4 does

not affect assembly with BPMs, this does not rule

out a possible role of the domain for this type of

interaction. However, to date, there is no evidence

that the AP2 domain mediates protein–protein inter-

action and, because of this, we consider it probable

that it is also not involved in assembly with the BPM

proteins.

Both RAP2.4 and RAP2.13 belong to a small sub-

group within the ERF ⁄AP2 superfamily that comprises

eight members and is called the A-6 subfamily [32,36].

We tested more members of this subgroup, At1g36060,

At4g39780 and At4g13620, in pulldown assays with

GST:BPM1. Although At4g39780 and At4g13620 were

both able to assemble with GST:BPM1, At1g36060

was not (Fig. S1), indicating that assembly with

ERF ⁄AP2 proteins is restricted to the A-6 subfamily

and, in this case, even to a subset of eight members.

The finding that, within the A-6 subfamily, not all of

its members interact with BPM1 indicates that BPM

proteins assemble only with a very limited set of

ERF ⁄AP2 proteins, which potentially does not extend

beyond the A-6 subgroup. However, we experienced a

high degree of redundancy from the BPM site, as all

BPMs were able to interact with RAP2.4. It will be of

interest to determine whether this redundancy is also

present for all other ERF ⁄AP2 proteins that bind to

BPM1.

Studies on gene expression showed that BPMs have

a widely overlapping pattern of expression. This was

further corroborated by the promoter:GUS lines,

GFP:BPM1 GFP:BPM2

GFP:BPM3 GFP:BPM4

GFP:BPM5 GFP:BPM6

GFP:RAP2.4 GFP:CUL3a

100 µm 100 µm100 µm 100 µm

50 µm50 µm50 µm50 µm

50 µm 50 µm 50 µm 50 µm

100 µm100 µm200 µm200 µm

Fig. 5. Localization of transiently expressed

GFP fusion proteins of BPM, RAP2.4 and

CUL3A in Nicotiana benthamiana leaves

was analyzed by confocal laser scanning

microscopy. GFP signals (green) and

merged images of GFP against the red chlo-

rophyll autofluorescence background signal

are shown. Like GFP:RAP2.4, GFP:BPM1

and GFP:BPM2 showed a predominant

localization to the nucleus, whereas

GFP:BPM4 showed an opposite pattern and

was detectable only outside the nucleus.

GFP:BPM3, GFP:BPM5, GFP:BPM6 and

GFP:CUL3A accumulated inside and outside

the nucleus (nuclei of single cells are indi-

cated by arrows).



which showed that most BPMs were expressed in

anthers, root tips and rosette leaves. Although some

patterns were highly specific, such as, for example,

expression of BPM1 and BPM4 at the junction of

primary to lateral roots, or BPM3 expression specifi-

cally in root tips, overall our results indicated that

BPM proteins were functionally redundant. Conse-

quently, one would expect no obvious developmental

defects in plants affected in single BPM genes, which

is the case for available T-DNA insertion mutants (H.

Weber and H. Hellmann, unpublished work). How-

ever, on the basis of the expression patterns

described, it is predictable that mutants affected in

multiple BPMs will show aberrant flower, leaf and

root development. Likewise, the inducible expression

of BPM1, BPM4 and BPM5 on abiotic stress treat-

ment suggests that corresponding single or multiple

mutants will display an altered tolerance when

exposed to these stressors.

The widely overlapping expression patterns of BPMs

with RAP2.4 also suggest that the transcription factor

can potentially assemble with most BPM proteins. This

was further supported by our subcellular localization

studies, in which all of the BPMs, except BPM4, were

present in the nucleus. However, the specific nuclear

localization of BPM1 and BPM2 currently makes both

proteins the most favorable candidates for in planta

assembly with the transcription factor RAP2.4. As

BPM3, BPM5 and BPM6 were also present in the

cytosol, it is probable that they assemble with addi-

tional, yet unknown proteins in this cellular compart-

ment, and this is especially likely for BPM4, which

was never found in the nucleus. In this case, it is of

interest that CUL3a was also localized to the nucleus

and the cytosol. Because a proposed role of BPMs is

to function as substrate adaptors to a CUL3-based

E3-ligase, the current findings suggest that such an

assembly can occur within the nucleus and the cyto-

plasm and that, in both compartments, proteins can be

ubiquitinated and potentially marked for degradation

via the 26S proteasome.

Conclusion

In future work, it will be critical to verify our findings

on BPM–ERF ⁄AP2 assembly in planta and to identify

the motif in RAP2.4 that mediates this type of interac-

tion, as this has the potential to predict possible pro-

tein binding to the BPMs. It will also be of importance

to define the functional impact of the BPM family on

the activity of ERF ⁄AP2 transcription factors. For

example, do they affect the stability of these proteins

and does this require CUL3s? In this case, it is note-

worthy that we observed the instability of in planta-

expressed myc-tagged RAP2.4 in a 26S proteasome-

dependent manner (Fig. S4). We currently favor a

working model in which they bind to ERF ⁄AP2s,

potentially interfering with their DNA-binding ability,

but which ultimately results in the degradation of

ERF ⁄AP2 proteins (Fig. 6). Independent of this hypo-

thetical role, the work described here opens up a novel

and important connection between two plant protein

families, and provides a forecast on a new regulatory

mechanism controlling ERF ⁄AP2 transcription factor

activities.

Materials and methods

Plant growth conditions and transformation

Arabidopsis thaliana (ecotype Col0) and tobacco (Nicoti-

ana benthamiana) plants were grown under standard condi-

tions with 16 h : 8 h light : dark cycles in either soil or

sterile culture, using ATS medium [37] without supple-

mented sucrose. Arabidopsis floral dip transformation was

performed as described in [38].

Fig. 6. Schematic model for assembly and

functional impact of BPM–ERF ⁄ AP2 assem-

bly. BPM proteins function as substrate

adaptors to CUL3-based E3-ligases. They

also assemble with ERF ⁄ AP2 transcription

factors, and this interaction serves to bring

bound ERF ⁄ AP2 proteins to the core

E3-ligase. Docking of the BPM–ERF ⁄ AP2

complex to the E3-ligase results in ubiquiti-

nation and subsequent degradation of bound

transcription factor.



Molecular cloning and mutagenesis

Full-length cDNAs of BPM genes were amplified from a

seedling-specific cDNA library [39]. The promoters of the

different BPM genes and RAP2.4 (for sizes, see Table S1),

as well as AP2 ⁄ERF transcription factors, were amplified

directly from Col0 genomic DNA. In all cases, Pfu poly-

merase (Promega, Mannheim, Germany) was used and the

PCR products were controlled for correct sequences. The

cDNAs obtained were subcloned into pDONR221 (Invitro-

gen, Carlsbad, CA, USA) and shuffled into different desti-

nation vectors using GATEWAY technology (Invitrogen):

pACT2 and pBTM116 [2] for Y2H studies, pDEST15 (Invi-

trogen) for Escherichia coli expression and the binary vector

pK7FWG2 [40] for subcellular localization analysis. The

amplified promoters were subcloned into pCR2.1 by TOPO

TA reaction (Invitrogen). Afterwards, using the BamHI and

XbaI restriction sites, the promoters were fused to the GUS

gene in the binary vector pCB308 [41]. The primers used in

this and other sections are given in Table S2. Mutagenesis

was performed using a mutagenesis kit from Stratagene

(La Jolla, CA, USA) as described previously [2].

Expression analysis

Expression was studied by RT-PCR with gene-specific

primers, and histochemically using promoter-GUS fusions.

RT-PCR was performed on 100 ng of total RNA isolated

from different tissues of mature Arabidopsis ecotype Col0

plants grown on soil, or on total RNA extracted from

7-day-old seedlings grown on plates, respectively. For histo-

chemical analysis, promoters in the binary vector pCB308

were introduced into Arabidopsis plants. Transgenic plants

were selected by BASTA herbicide (Aventis Crop Science,

Leverkusen, Germany). GUS staining was carried out by

vacuum infiltration of plant material with staining solution

[2] and subsequent incubation at room temperature for up

to 24 h. For stress treatment, 7-day-old sterile-grown seed-

lings were transferred for 6 h into liquid ATS medium sup-

plemented with either 200 mm NaCl or sorbitol. To impose

drought stress on 7-day-old seedlings, the lids from culture

dishes were removed for 6 h before the samples were har-

vested.

Y2H assay

Screening for BPM-interacting clones was performed using

a root-specific suspension cell cDNA library in the prey

vector pACT2-GW [42]. The MATH domain of BPM1 and

full-length BPM3 were cloned into the bait vector

pBTM116-D9-GW [42]. Yeast transformation and testing

for interaction were performed as described in [2]. Clones

were transformed into yeast with an efficiency of 1.5 million

clones per transformation. All BPM-interacting clones were

tested for auto-activation and sequenced for the correct

open reading frame in pACT2.

Subcellular localization analysis

Fluorescent fusion proteins of the six BPM proteins, CUL3a

and RAP2.4 were transiently expressed in tobacco epidermal

cells using the method of Agrobacterium infiltration as

described in [43]. The bacterial attenuance (D) at 600 nm was

0.01–0.03 for all constructs. In addition, BPM4 localization

was also analyzed in stable transgenic Arabidopsis plants

expressing GFP:BPM4 fusion protein. In all cases, binary

GFP expression vectors obtained from [40] were used.

Transfected leaf sections were imaged using a Zeiss (Jena,

Germany) LSM 510 Meta confocal microscope.

In vitro transcription ⁄ translation assays

For interaction studies, full-length BPM proteins, fragments

of BPM1 and selected ERF ⁄AP2 proteins were expressed in

the TNT-reticulocyte lysate system (Promega) as described

previously [2]. In vitro-translated proteins were labeled with

either [35S]methionine (Amersham, Chalfont St Giles, UK)

or greenLysine (Promega).

Acknowledgements

We thank Sutton Mooney for critical reading of the

manuscript. Financial support for this project was pro-

vided by the Deutsche Forschungsgemeinschaft (DFG)

grants HE3224 ⁄ 5-1 and 5-2 and Washington State

University to HH.

Accession numbers

BPM1 (At5g19000 ⁄Q8L765); BPM2 (At3g06190 ⁄Q9-

M8J9); BPM3 (At2g39760 ⁄O22286); BPM4 (At3g-

03740 ⁄Q9SRV1); BPM5 (At5g21010 ⁄Q1EBV6); BPM6

(At3g43700 ⁄A1L4W5); RAP2.4 (At1g78080 ⁄Q8H1E4);

RAP2.13 (At1g22190 ⁄Q9LM15).

References

1 Stogios PJ, Downs GS, Jauhal JJ, Nandra SK & Prive

GG (2005) Sequence and structural analysis of BTB

domain proteins. Genome Biol 6, R82.

2 Weber H, Bernhardt A, Dieterle M, Hano P, Mutlu A,

Estelle M, Genschik P & Hellmann H (2005) Arabidop-

sis AtCUL3a and AtCUL3b form complexes with

members of the BTB ⁄POZ-MATH protein family.

Plant Physiol 137, 83–93.

3 Figueroa P, Gusmaroli G, Serino G, Habashi J, Ma L,

Shen Y, Feng S, Bostick M, Callis J, Hellmann H et al.



(2005) Arabidopsis has two redundant Cullin3 proteins

that are essential for embryo development and that

interact with RBX1 and BTB proteins to form multi-

subunit E3 ubiquitin ligase complexes in vivo. Plant

Cell 17, 1180–1195.

4 Gingerich DJ, Gagne JM, Salter DW, Hellmann H,

Estelle M, Ma L & Vierstra RD (2005) Cullins 3a and

3b assemble with members of the broad complex ⁄ tram-

track ⁄ bric-a-brac (BTB) protein family to form essential

ubiquitin–protein ligases (E3s) in Arabidopsis. J Biol

Chem 280, 18810–18821.

5 Gingerich DJ, Hanada K, Shiu SH & Vierstra RD

(2007) Large-scale, lineage-specific expansion of a bric-

a-brac ⁄ tramtrack ⁄ broad complex ubiquitin-ligase gene

family in rice. Plant Cell 19, 2329–2348.

6 Zollman S, Godt D, Prive GG, Couderc JL & Laski

FA (1994) The BTB domain, found primarily in zinc

finger proteins, defines an evolutionarily conserved fam-

ily that includes several developmentally regulated genes

in Drosophila. Proc Natl Acad Sci USA 91, 10717–

10721.

7 Bardwell VJ & Treisman R (1994) The POZ domain: a

conserved protein–protein interaction motif. Genes Dev

8, 1664–1677.

8 Ahmad KF, Engel CK & Prive GG (1998) Crystal

structure of the BTB domain from PLZF. Proc Natl

Acad Sci USA 95, 12123–12128.

9 Ahmad KF, Melnick A, Lax S, Bouchard D, Liu J,

Kiang CL, Mayer S, Takahashi S, Licht JD & Prive

GG (2003) Mechanism of SMRT corepressor recruit-

ment by the BCL6 BTB domain. Mol Cell 12, 1551–

1564.

10 Csankovszki G, Nagy A & Jaenisch R (2001) Synergism

of Xist RNA, DNA methylation, and histone hypoacet-

ylation in maintaining X chromosome inactivation.

J Cell Biol 153, 773–784.

11 Thomas JH (2006) Adaptive evolution in two large

families of ubiquitin-ligase adapters in nematodes and

plants. Genome Res 16, 1017–1030.

12 Tsukiyama T, Becker PB & Wu C (1994) ATP-depen-

dent nucleosome disruption at a heat-shock promoter

mediated by binding of GAGA transcription factor.

Nature 367, 525–532.

13 Huynh KD & Bardwell VJ (1998) The BCL-6 POZ

domain and other POZ domains interact with the co-

repressors N-CoR and SMRT. Oncogene 17, 2473–

2484.

14 Wang KL, Yoshida H, Lurin C & Ecker JR (2004)

Regulation of ethylene gas biosynthesis by the Arabid-

opsis ETO1 protein. Nature 428, 945–950.

15 Pintard L, Willems A & Peter M (2004) Cullin-based

ubiquitin ligases: Cul3–BTB complexes join the family.

EMBO J 23, 1681–1687.

16 Weber H, Hano P & Hellmann H (2007) The charming

complexity of CUL3. Int J Plant Dev Biol 1, 178–184.

17 Hellmann H & Estelle M (2002) Plant development:

regulation by protein degradation. Science (NY) 297,

793–797.

18 Pedmale UV & Liscum E (2007) Regulation of photo-

tropic signaling in Arabidopsis via phosphorylation

state changes in the phototropin 1-interacting protein

NPH3. J Biol Chem 282, 19992–20001.

19 Ha CM, Jun JH, Nam HG & Fletcher JC (2004)

BLADE-ON-PETIOLE1 encodes a BTB ⁄POZ domain

protein required for leaf morphogenesis in Arabidop-

sis thaliana. Plant Cell Physiol 45, 1361–1370.

20 Kim S, Choi HI, Ryu HJ, Park JH, Kim MD &

Kim SY (2004) ARIA, an Arabidopsis arm repeat

protein interacting with a transcriptional regulator of

abscisic acid-responsive gene expression, is a novel

abscisic acid signaling component. Plant Physiol 136,

3639–3648.

21 Cheng Y, Qin G, Dai X & Zhao Y (2007) NPY1, a

BTB-NPH3-like protein, plays a critical role in auxin-

regulated organogenesis in Arabidopsis. Proc Natl Acad

Sci USA 104, 18825–18829.

22 Dong X, Li X, Zhang Y, Fan W, Kinkema M & Clarke

J (2001) Regulation of systemic acquired resistance by

NPR1 and its partners. Novartis Found Symp 236, 165–

173.

23 Sunnerhagen M, Pursglove S & Fladvad M (2002) The

new MATH: homology suggests shared binding surfaces

in meprin tetramers and TRAF trimers. FEBS Lett 530,

1–3.

24 Uren AG & Vaux DL (1996) TRAF proteins and mep-

rins share a conserved domain. Trends Biochem Sci 21,

244–245.

25 Bond JS & Beynon RJ (1995) The astacin family of

metalloendopeptidases. Protein Sci 4, 1247–1261.

26 Bertenshaw GP, Turk BE, Hubbard SJ, Matters GL,

Bylander JE, Crisman JM, Cantley LC & Bond JS

(2001) Marked differences between metalloproteases

meprin A and B in substrate and peptide bond specific-

ity. J Biol Chem 276, 13248–13255.

27 Bauvois B (2001) Transmembrane proteases in focus:

diversity and redundancy? J Leukoc Biol 70, 11–17.

28 Baker SJ & Reddy EP (1996) Transducers of life and

death: TNF receptor superfamily and associated

proteins. Oncogene 12, 1–9.

29 Arch RH & Thompson CB (1998) 4-1BB and Ox40 are

members of a tumor necrosis factor (TNF)-nerve

growth factor receptor subfamily that bind TNF

receptor-associated factors and activate nuclear factor

kappaB. Mol Cell Biol 18, 558–565.

30 Oelmuller R, Peskan-Berghofer T, Shahollaria B,

Trebickab A, Sherametia I & Varmac A (2005) MATH

domain proteins represent a novel protein family in

Arabidopsis thaliana, and at least one member is

modified in roots during the course of a plant–microbe

interaction. Physiol Plant 124, 152–166.



31 Lin R-C, Park H-J & Wang H-Y (2008) Role of

Arabidopsis RAP2.4 in regulating light- and

ethylene-mediated developmental processes and drought

stress tolerance. Mol Plant 1, 42–57.

32 Nakano T, Suzuki K, Fujimura T & Shinshi H (2006)

Genome-wide analysis of the ERF gene family in

Arabidopsis and rice. Plant Physiol 140, 411–432.

33 Okamuro JK, Caster B, Villarroel R, Van Montagu M

& Jofuku KD (1997) The AP2 domain of APETALA2

defines a large new family of DNA binding proteins in

Arabidopsis. Proc Natl Acad Sci USA 94, 7076–7081.

34 Jofuku KD, den Boer BG, Van Montagu M &

Okamuro JK (1994) Control of Arabidopsis flower and

seed development by the homeotic gene APETALA2.

Plant Cell 6, 1211–1225.

35 Magnani E, Sjolander K & Hake S (2004) From endo-

nucleases to transcription factors: evolution of the AP2

DNA binding domain in plants. Plant Cell 16, 2265–

2277.

36 Sakuma Y, Liu Q, Dubouzet JG, Abe H, Shinozaki K

& Yamaguchi-Shinozaki K (2002) DNA-binding speci-

ficity of the ERF ⁄AP2 domain of Arabidopsis DREBs,

transcription factors involved in dehydration- and cold-

inducible gene expression. Biochem Biophys Res

Commun 290, 998–1009.

37 Estelle MA & Somerville C (1987) Auxin-resistant

mutants of Arabidopsis thaliana with an altered mor-

phology. Mol Gen Genet 206, 200–206.

38 Clough SJ & Bent AF (1998) Floral dip: a simplified

method for Agrobacterium-mediated transformation of

Arabidopsis thaliana. Plant J 16, 735–743.

39 Minet M, Dufour ME & Lacroute F (1992) Comple-

mentation of Saccharomyces cerevisiae auxotrophic

mutants by Arabidopsis thaliana cDNAs. Plant J 2,

417–422.

40 Karimi M, Inze D & Depicker A (2002) GATEWAY

vectors for Agrobacterium-mediated plant transforma-

tion. Trends Plant Sci 7, 193–195.

41 Xiang C, Han P, Lutziger I, Wang K & Oliver DJ

(1999) A mini binary vector series for plant transforma-

tion. Plant Mol Biol 40, 711–717.

42 Dortay H, Gruhn N, Pfeifer A, Schwerdtner M,

Schmulling T & Heyl A (2008) Toward an interaction

map of the two-component signaling pathway of

Arabidopsis thaliana. J Proteome Res 7, 3649–3660.

43 Sparkes IA, Runions J, Kearns A & Hawes C (2006)

Rapid, transient expression of fluorescent fusion

proteins in tobacco plants and generation of stably

transformed plants. Nat Protoc 1, 2019–2025.

44 Campanella JJ, Bitincka L & Smalley J (2003)

MatGAT: an application that generates similarity ⁄identity matrices using protein or DNA sequences.

BMC Bioinformatics 4, 29.

Supporting information

The following supplementary material is available:

Fig. S1. In vitro-translated A-6 ERF ⁄AP2 proteins

At4g39780 and At4g13620 coprecipitate with

GST:BPM1, but not At1g36060.

Fig. S2. Verification of nuclear localization for

GFP:BPM1 and GFP:RAP2.4 in transient expression

experiments using Nicotiana benthamiana leaves.

Fig. S3. Generation of stable transgenic Arabidopsis

P35S:GFP:BPM4 plants.

Fig. S4. Instability of RAP2.4:myc in plants overex-

pressing myc-epitope-tagged RAP2.4 under the control

of a 35S promoter (pro35S:RAP2.4:myc).

Table S1. Comparison of whole BPM proteins (A) and

the BPM MATH domains only (B). Similarities and

identities between the amino acid sequences were deter-

mined by matgat 2.02 software using the default set-

tings (BLOSUM62, first gap 12, extending gap 2) [44].

Table S2. Overview of primers used in this work and

expected PCR products.

This supplementary material can be found in the

online version of this article article.

Please note: As a service to our authors and readers,

this journal provides supporting information supplied

by the authors. Such materials are peer-reviewed and

may be re-organized for online delivery, but are not

copy-edited or typeset. Technical support issues arising

from supporting information (other than missing files)

should be addressed to the authors.



Arabidopsis thaliana BTB POZ-MATH proteins interact...

Documents

Transcript of Arabidopsis thaliana BTB POZ-MATH proteins interact...