abel_05_genomewide_793936.pdf.txt

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd"><html xmlns="http://www.w3.org/1999/xhtml">
<head>
<title>1471-2148-5-72.fm</title>
<meta name="Author" content="csproduction"/>
<meta name="Creator" content="FrameMaker 7.0"/>
<meta name="Producer" content="Acrobat Distiller 5.0.5 (Windows)"/>
<meta name="CreationDate" content=""/>
</head>
<body>
<pre>
BMC Evolutionary Biology

BioMed Central

Open Access

Research article

Genome-wide comparative analysis of the IQD gene families in
Arabidopsis thaliana and Oryza sativa
Steffen Abel*, Tatyana Savchenko and Maggie Levy
Address: Department of Plant Sciences, University of California, One Shields Avenue, Davis, CA 95616, USA
Email: Steffen Abel* - sabel@ucdavis.edu; Tatyana Savchenko - savchenko@ucdavis.edu; Maggie Levy - levym@agri.huji.ac.il
* Corresponding author

Published: 20 December 2005
BMC Evolutionary Biology 2005, 5:72

doi:10.1186/1471-2148-5-72

Received: 20 July 2005
Accepted: 20 December 2005

This article is available from: http://www.biomedcentral.com/1471-2148/5/72
© 2005 Abel et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract
Background: Calcium signaling plays a prominent role in plants for coordinating a wide range of
developmental processes and responses to environmental cues. Stimulus-specific generation of
intracellular calcium transients, decoding of calcium signatures, and transformation of the signal into
cellular responses are integral modules of the transduction process. Several hundred proteins with
functions in calcium signaling circuits have been identified, and the number of downstream targets of
calcium sensors is expected to increase. We previously identified a novel, calmodulin-binding nuclear
protein, IQD1, which stimulates glucosinolate accumulation and plant defense in Arabidopsis thaliana. Here,
we present a comparative genome-wide analysis of a new class of putative calmodulin target proteins in
Arabidopsis and rice.
Results: We identified and analyzed 33 and 29 IQD1-like genes in Arabidopsis thaliana and Oryza sativa,
respectively. The encoded IQD proteins contain a plant-specific domain of 67 conserved amino acid
residues, referred to as the IQ67 domain, which is characterized by a unique and repetitive arrangement
of three different calmodulin recruitment motifs, known as the IQ, 1-5-10, and 1-8-14 motifs. We
demonstrated calmodulin binding for IQD20, the smallest IQD protein in Arabidopsis, which consists of
a C-terminal IQ67 domain and a short N-terminal extension. A striking feature of IQD proteins is the high
isoelectric point (~10.3) and frequency of serine residues (~11%). We compared the Arabidopsis and rice
IQD gene families in terms of gene structure, chromosome location, predicted protein properties and
motifs, phylogenetic relationships, and evolutionary history. The existence of an IQD-like gene in
bryophytes suggests that IQD proteins are an ancient family of calmodulin-binding proteins and arose
during the early evolution of land plants.
Conclusion: Comparative phylogenetic analyses indicate that the major IQD gene lineages originated
before the monocot-eudicot divergence. The extant IQD loci in Arabidopsis primarily resulted from
segmental duplication and reflect preferential retention of paralogous genes, which is characteristic for
proteins with regulatory functions. Interaction of IQD1 and IQD20 with calmodulin and the presence of
predicted calmodulin binding sites in all IQD family members suggest that IQD proteins are a new class of
calmodulin targets. The basic isoelectric point of IQD proteins and their frequently predicted nuclear
localization suggest that IQD proteins link calcium signaling pathways to the regulation of gene expression.
Our comparative genomics analysis of IQD genes and encoded proteins in two model plant species
provides the first step towards the functional dissection of this emerging family of putative calmodulin
targets.

Page 1 of 25
(page number not for citation purposes)

BMC Evolutionary Biology 2005, 5:72

Background
The low solubility product constants of calcium phosphate salts provide a chemical rationale for the evolution
of Ca2+ as a universal second messenger. The necessity to
decrease cytosolic Ca2+ concentrations to submicromolar
levels by exporting the cation into extracellular spaces or
intracellular compartments that do not generate ATP,
such as the endoplasmic reticulum or vacuole, creates a
steep concentration gradient that allows for the controlled
and gated generation of rapid Ca2+ transients in response
to extracellular stimuli. Such intracellular Ca2+ signals are
not only characterized by their magnitudes but also by
their spatial and temporal resolution. The sum of these
parameters is often referred to as the 'Ca2+ signature' of a
primary stimulus [1-4]. Numerous environmental cues of
biotic and abiotic nature and endogenous physiological
and developmental conditions trigger specific Ca2+ signatures [2,5-8]. Stimulus-specific Ca2+ oscillations are generated by voltage- and ligand-gated Ca2+-permeable
channels (influx), and by Ca2+-ATPases and antiporters
(efflux) to regain resting Ca2+ levels [3,7]. Approximately
80 genes coding for potential Ca2+ channels, pumps and
antiporters have been identified in the Arabidopsis
genome, suggesting complex generation and regulation of
stimulus-specific Ca2+ signatures [8].
Calcium spikes are recognized by several Ca2+-binding
proteins and are decoded via Ca2+-dependent conformational changes in these sensor polypeptides and interacting target proteins [6,9-11]. Several classes of Ca2+ sensors
have been identified in plants that contain a Ca2+-binding
helix-loop-helix fold known as the EF-hand motif. Calmodulin is the archetypal Ca2+ sensor, which is exceptionally conserved in eukaryotes and contains four EF-hand
motifs. About 250 EF-hand motif-containing proteins
have been identified in Arabidopsis [12], including six
typical calmodulins and 50 calmodulin-like proteins that
differ significantly in sequence and number of EF-hand
motifs [13,14]. Members of a second, plant-specific family of Ca2+ sensors, which usually contain three EF-hand
motifs, have similarity to the regulatory B-subunit of calcineurin in animals and are referred to as calcineurin Blike (CBL) proteins [9,15-17]. While calmodulins and
CBL sensor proteins have no catalytic activity on their own
and therefore are sometimes referred to as 'Ca2+ sensor
relays', a third major class of Ca2+ sensors are bifunctional
proteins, known as Ca2+-dependent protein kinases
(CDPK), which contain a calmodulin-like domain with
four EF-hand motifs and a Ca2+-dependent, Ser/Thr protein kinase domain on a single polypeptide chain [18,19].
Because of their dual functions as Ca2+-binding proteins
and catalytic effectors the CDPK proteins are considered
'Ca2+ sensor responders'. In Arabidopsis, CDPK and CBL
proteins are encoded by multigene families of 34 and 10
members, respectively [16,19]. CDPKs play essential roles

http://www.biomedcentral.com/1471-2148/5/72

in hormone and stress signaling pathways as well as in
plant responses to pathogens [20,21].
To transmit the information of the second messenger,
Ca2+ sensor relays such as calmodulins and CBL proteins
interact with target proteins and regulate their biochemical activities. During the final phase of the transduction
process, the target proteins modulate diverse cellular
activities to establish the specific response to a given extracellular signal. The CBL sensor proteins interact specifically in a Ca2+-dependent fashion with a single family of
SNF1-like Ser/Thr protein kinases, known as CBL-interacting protein kinases or CIPKs, which are encoded by 25
genes in Arabidopsis [16,22-24]. Current data indicate
that CBL-CIPK interaction networks provide a signaling
module for integrating plant responses to an array of environmental stimuli [17,23,25,26]. In contrast to CBL sensor proteins, which regulate a select set of target protein
kinases, calmodulins interact with an astonishingly large
number of target proteins. These have been extensively
reviewed and include among other functional categories,
proteins implicated in generating Ca2+ signatures,
enzymes in signaling and metabolic pathways, and transcriptional regulators [6,8,11,27-29]. The calmodulininteracting domains of target proteins are not necessarily
related in structure and exhibit high sequence variability,
which may reflect the versatility of the calmodulin sensor
relay. Nonetheless, calmodulin-interacting domains usually consist of a short (16–35 residues) basic amphiphilic
helix, which is recognized by a flexible hydrophobic
pocket that forms upon Ca2+ binding to calmodulin
[9,10,30,31]. Three calmodulin recruitment motifs are
currently known although not all functionally characterized calmodulin-binding domains contain these specific
motifs: the IQ motif (IQxxxRGxxxR; Pfam 00612) is
thought to mediate calmodulin retention in a Ca2+-independent manner, whereas Ca2+-dependent interaction can
be achieved by two related motifs, termed 1-5-10 and 1-814, which are distinguished by their spacing of bulky
hydrophobic and basic amino acid residues [31-34].
Using various biochemical approaches, about 200 target
proteins have been identified in Arabidopsis, a number
that is expected to rise [8,11].
In a genetic screen for regulatory factors of the glucosinolate homeostasis in Arabidopsis thaliana [35], we have
recently identified a gene coding for a calmodulin-binding protein with similarity to SF16 from sunflower [36].
We termed this protein IQD1 for the presence of a plantspecific domain of 67 conserved amino acids (referred to
as IQ67 domain), which is characterized by a unique and
repetitive arrangement of IQ, 1-5-10 and 1-8-14 calmodulin recruitment motifs. We demonstrated by biochemical
and genetic studies that IQD1 is a nuclear calmodulinbinding protein that stimulates glucosinolate accumula-

Page 2 of 25
(page number not for citation purposes)

BMC Evolutionary Biology 2005, 5:72

http://www.biomedcentral.com/1471-2148/5/72

Table 1: The IQD gene family of Arabidopsis thaliana

Gene
Identifier

REFSEQ
Accession

Protein ID

cDNA Accessiona Protein ID

Expressionb

Protein
Namec

Size
(aa)

Mass
(kD)

IP

Predicted_Locationd
PSORT

At1g01110
At1g14380

NM_099993
NM_101305

NP_563618
NP_563950

At1g17480
At1g18840
At1g19870

NM_101610
NM_101741
NM_101842

NP_173191
NP_173318
NP_564097

At1g51960
At1g72670

NM_104077
NM_105926

NP_175608
NP_177411

At1g74690

NM_106127

NP_177607

At2g02790
At2g26180
At2g26410
At2g33990

NM_126334
NM_128176
NM_128198
NM_128950

NP_178382
NP_180187
NP_180209
NP_180946

At2g43680

NM_180068

NP_850399

At3g09710
At3g15050
At3g16490
At3g22190
At3g49260

NM_111805
NM_112367
NM_112520
NM_113116
NM_114785

NP_187582
NP_188123
NP_188270
NP_188858
NP_566917

At3g49380
At3g51380
At3g52290

NM_114798
NM_114997
NM_115089

NP_190507
NP_190706
NP_190797

At3g59690

NM_115831

NP_191528

At4g00820
At4g10640

NM_116308
NM_117132

NP_567191
NP_192802

At4g14750
At4g23060
At4g29150

NM_117560
NM_118435
NM_119059

NP_193211
NP_194037
NP_194644

At5g03040

NM_120382

NP_568110

At5g03960
At5g07240

NM_120478
NM_120806

NP_196016
NP_196341

At5g13460

NM_121349

NP_196850

At5g35670

NM_122958

NP_568529

At5g62070

NM_125600

NP_201013

AY085363*
BT005935A
AO64870
AY702665
AY702666
BT001081A
AN46862
BT010652A
AR07516
AY128860A
AM91260
BX818988
BX840898
AU237877
AV557487
BT008408A
AP37767
AY827468
BX825987
BX824788
BT000602A
AN18171
BX838271 (FL-EST)
BT005639A
AO64059
BT001176A
AN65063
BX826435
BT010145A
AQ22614
BX827601
AY702664
BT003896A
AO41944
AY143972A
AN28911
BX829656
BT006056A
AP04041
AY128736A
AM91136
AK128736B
AD43467
AY143917A
AN28856

TargetP

ACD
ABCD

IQD18
IQD28

527
664

59.2
72.8

10.3
9.7

N
N

?
?

ACD
ABCD
ABCD

IQD7
IQD30
IQD32

370
572
794

41.0
62.7
86.8

10.5
9.2
5.2

?
N
N

?
?
C 0.65/4

ACD

IQD27
IQD8

351
414

39.3
45.9

10.1
10.3

?
N

?
?

ACD

IQD31

587

65.2

9.6

?

?

AC
CD
A
AD

IQD29
IQD6
IQD4
IQD9

636
416
527
249

69.8
46.9
58.3
28.5

9.6
10.5
10.3
10.8

N
N
?
N

C 0.71/4
?
?
?

AB

IQD14

668

74.3

11.3

?

?

ACD
BCD
AD
A
ABD

IQD1
IQD10
IQD26
IQD5
IQD21

454
259
398
400
471

50.5
29.6
48.7
44.5
52.1

10.4
10.3
10.1
10.1
10.0

N
?
?
N
N

?
C 0.91/1
?
?

AD
ABCD

IQD15
IQD20
IQD3

352
103
430

40.8
11.8
48.1

10.2
12.4
10.6

N
M
?

?
M 0.80/2
?

AD

IQD13

517

58.5

10.9

?

?

ACD
AD

IQD17
IQD16

534
423

60.0
48.7

10.3
10.1

?
N

M 0.38/5
?

ACD
ABCD
AD

IQD19
IQD22
IQD25

387
543
383

43.9
60.3
41.4

9.7
10.2
10.7

?
?
?

?
M 0.50/4
M 0.78/3

ABCD

IQD2

461

50.5

10.6

N

C 0.55/3

ACD

IQD12
IQD24

403
401

46.0
45.3

10.6
10.3

?
?

M 0.76/2
M 0.54/4

CD

IQD11

443

50.8

10.0

N

?

CD

IQD33

442

49.5

8.5

?

M 0.47/5

ACD

IQD23

403

44.3

10.5

N

C 0.51/5

?

a Full-length

cDNAs (asterisk denotes a cDNA clone that is likely 5'-truncated).
evidence for IQD gene expression provided by (A) whole-genome array [105], (B) community microarray data [94], (C) Massively
Parallel Signature Sequencing (MPSS, [106]), (D) EST clones.
c Nomenclature of IQD genes is arbitrary. Levy et al. [37] cloned IQD1 and reported closely related genes IQD2-IQD6. The designation of IQD7IQD33 is based on the phylogenetic analysis presented in Figure 1a.
d PSORT predictions: N (nucleus), C (chloroplast), M (mitochondrion). TargetP predictions: values indicate score (0.00 – 1.00) and reliability class
(1–5; best class is 1).
b Additional

Page 3 of 25
(page number not for citation purposes)

BMC Evolutionary Biology 2005, 5:72

http://www.biomedcentral.com/1471-2148/5/72

(a)

(b)
I

At1g51960
1000
At3g16490

1000

At4g29150
990

a

At5g07240
1000
At5g62070

999
793

2

At4g23060

1

At3g49260
643

b

At3g51380

587

At4g14750

329

At1g01110
1000
At4g00820

1000

c

At4g10640

992

344

At3g49380
At2g43680

II

1000

*

At3g59690
668
At5g03960
946

*

At5g13460
691

*

III

At1g17480
1000
At1g72670

999

At2g26180
984

a

At2g33990
1000

*

At3g15050

587

At3g22190
610
At3g09710
1000
At5g03040

983

b

At3g52290

1000

At2g26410

1

IV

At1g14380
1000
At2g02790
998

1

At1g18840
1000

622

1

At1g74690

1

At1g19870
At5g35670

0.1

1000 nt

(c)

(d)
I

Os01m00895
1000
Os05m00863
990
Os01m05259
1000
Os05m04170
457

Os10m02409
1000
Os03m00584

1000
879

Os03m04199

298

Os04m04664
598

Os08m00125
Os02m01875

181

1000
Os06m02303

II

Os01m06663
Os05m04352

III

1000
Os01m04963

1000
296

Os01m00929

1

478

Os12m04168

a

1000
930

1

Os03m04309

1000

1
Os03m05627

514

Os06m00539

349

Os05m00240
1000

b

Os01m06082

922

Os05m03604
Os05m04307

1

IV

1000
602

1
Os01m05025

1
Os04m05532
Os03m00334
988

1

2

2

Os04m04570
Os06m03925

0.1

1000 nt

Figure 1
Phylogenetic analysis and exon-intron organization of IQD genes in Arabidopsis thaliana and Oryza sativa
Phylogenetic analysis and exon-intron organization of IQD genes in Arabidopsis thaliana and Oryza sativa. Neighbor-joining trees
of full-length amino acid sequences encoded by Arabidopsis (a) and rice (c) IQD genes are shown. The gene coding for the
protein containing a C-terminally truncated IQ67 domain in Arabidopsis, At5g35670, and in rice, Osm0603925, was used as
outgroup for each family. Bootstrap values (1,000 replicates) are placed at the nodes, and the scale bar corresponds to 0.1 estimated amino acid substitutions per site. Subfamilies and subgroups of IQD genes (I–IV) are highlighted by colored vertical bars
on the right of the trees. The exon-intron organization of the corresponding IQD genes is shown for the Arabidopsis (b) and
rice (d) gene family. Exons are depicted as boxes and introns as connecting thin lines. Protein-coding regions are colored in
red, and non-translated regions, when supported by full-length cDNA sequences, are shown in black. The gene structures are
drawn to scale and aligned along the left border (indicated by vertical dotted line) of the exon encoding amino acids 17–67 of
the IQ67 domain, with the exception of At5g03960, Os08m00126 and Os01m06663 that have lost the respective intron. Additional intron losses are indicated by asterisks between Arabidopsis gene pairs. The exon-intron organization of the Arabidopsis
IQD genes was taken from the TIGR Arabidopsis database, with the exception of At1g01110 for which the MIPS annotation
was used as template. The presentation of the exon-intron organization of rice IQD genes was adapted to match the TIGR format of Arabidopsis IQD genes. The length of the second and third intron of Os02m01875 and Os03m04309 is 3.8 kb and 2.1
kb, respectively. Most introns of IQD genes are in phase-0. Six Arabidopsis and seven rice IQD genes contain phase-1 and
phase-2 introns, which are labeled with the respective Arabic numeral. At2g02790, for which no full-length cDNA sequence is
available, may also contain a phase-1 intron on its 3'end.

Page 4 of 25
(page number not for citation purposes)

BMC Evolutionary Biology 2005, 5:72

http://www.biomedcentral.com/1471-2148/5/72

(a)
2

1

3

(b)
2

1

3

(c)

11

CONS.(50%)

15

EE#AA#+IQ.#FRGYLARRALRALKGLVRLQALVRG.#VR+QA##TL+CMQALVR#QA.VRARR#+#

Figure 2
Amino acid sequence conservation of the IQ67 domain
Amino acid sequence conservation of the IQ67 domain. Aligned are sequences of the IQ67 domain of 72 putative IQD proteins form Arabidopsis thaliana (a), Oryza sativa (b), Pinus spp. and Physcomitrella patens (c). Each protein is identified by its gene
identification (Arabidopsis and rice) or accession number (pine and moss). The numbers above the scheme (1–67) indicate the
position within the domain as defined in this study. The position of the conserved phase-0 intron that separates the coding
region of the IQ67 domain between codon 16 and 17 is marked by an arrow. The shading of the alignment presents residues
(white text) of the IQ motifs (red), the 1-5-10 motifs (blue) and the 1-8-14 motifs (green). If a residue is part of more than one
motif, the residue is shaded in the first assigned color as determined by the order of motifs listed above. In addition, acidic,
basic and hydrophobic amino acid residues that are conserved in at least 50% of the 72 sequences are shaded in grey, pink and
yellow, respectively. The scheme of connected triangles below panel C depicts the position and boundaries of the IQ (red), 15-10 (blue) and 1-8-14 (green) motifs. The consensus sequence at the bottom is based on the residues with greater than 50%
conservation among the 72 proteins shown (#, hydrophobic; +, basic). Black braces at right indicate the major subfamilies as
defined by the phylogenetic analysis of the 72 IQ67 domain sequences in Figure 7. Accession numbers of the putative pine and
moss IQD proteins are given the prefixes 'Ps' and 'Pp', respectively.

Page 5 of 25
(page number not for citation purposes)

BMC Evolutionary Biology 2005, 5:72

http://www.biomedcentral.com/1471-2148/5/72

Table 3: Average parameters of IQD genes and proteins from A. thaliana and O. sativa

Arabidopsis
No. of genes
Gene length (kb)
No. of translated exons
Protein length (residues)
Molecular mass (kD)
Isoelectric pointa
Frequency of Arg (%)a
Frequency of Lys (%)a
Frequency of Ser (%)
Frequency of Ala (%)
a Computation

Rice

33
2.4 ± 0.9
4.5 ± 1.2
454 ± 132
50.8 ± 14.3
10.3 ± 0.6
9.3 ± 2.4
8.3 ± 2.3
12.2 ± 2.2
8.6 ± 2.2

≥ 29
3.0 ± 1.6
4.4 ± 1.2
471± 106
51.4 ± 11.8
10.4 ± 0.6
10.6 ± 2.5
5.9 ± 2.5
10.2 ± 1.9
12.8 ± 3.4

does not include At1g19870 (pI of 5.2) and Os04m05532 (pI of 4.8).

tion and plant defense [37]. In this study, we present a
comparative genome-wide analysis of the entire IQD gene
families in Arabidopsis thaliana (33 loci) and Oryza sativa
(29 loci), which are predicted to encode proteins sharing
the IQ67 domain. Our genomics analysis provides the
framework for future studies to dissect the function of this
emerging family of novel calmodulin target proteins.

Results
Identification and structure of IQD genes in Arabidopsis
thaliana
In a previous study, we characterized IQD1 as a calciumdependent calmodulin-binding protein and identified six
closely related genes in Arabidopsis [37]. The encoded
proteins share a conserved central region of 67 amino acid
residues, referred to as the IQ67 domain, which is characterized by the occurrence of multiple calmodulin-binding
motifs [32,33] that are arranged in a unique repetitive pattern. The IQ67 domain contains 1–3 copies each of the IQ
motif (IQxxxRGxxxR or of its more relaxed version
[ILV]QxxxRxxxx
[R,
K]),
the
1-5-10
motif
([FILVW]x3[FILV]x4[FILVW]), and the 1-8-14 motif
([FILVW]x6[FAILVW]x5[FILVW]). In addition, several conserved basic and hydrophobic amino acid residues are
flanking these motifs, and the IQ67 domain is predicted
to fold into a basic amphiphilic helix ([37]; see Figure 2).

To uncover the entire family of genes coding for IQD proteins in the Arabidopsis genome, we searched available
Arabidopsis databases with multiple BLAST algorithms
using full-length IQD1 (454 amino acids) and its IQ67
domain as the query sequences, followed by additional
searches with related sequences (see Methods). In addition, we performed a pattern search with the IQ motif and
its degenerate versions as the query sequences and
inspected each hit for the presence of an IQ67 domain.
We subsequently performed pair-wise sequence comparisons to exclude redundant entries from the initial data set,
which is frequently caused by multiple identification
numbers of the same DNA or protein sequence in the

databases. A total of 33 non-redundant putative IQD
genes were extracted from these sources (Table 1 and Figure 1). Full-length cDNA or EST sequences were available
for 26 of those genes, and we attempted to clone by
reverse transcriptase-mediated PCR cDNA sequences for
the remaining seven genes. We succeeded to generate fulllength cDNAs for three additional genes, At1g17480,
At1g18840 and At4g23060, but were unable to amplify
cDNAs for At1g51960, At2g02790, At3g22190 and
At3g49380. To date, no evidence is available supporting
the expression of At1g51960 and At3g49380 (Table 1). A
comparison of the 29 genomic loci with their corresponding cDNA sequences revealed that most of the predicted
gene models are correct, with only three exceptions
(At4g10640, At2g26410, At1g01110). The full-length
cDNA of At4g10640 encodes a protein that is 16 amino
acid residues longer than the protein predicted by the
MIPS MATDB annotation. This discrepancy is caused by
the erroneous and superfluous annotation of a fifth intron
in the last coding exon. For At2g26410, the translational
start site and the 5' border of the first intron were misannotated for the MIPS MATDB entry when compared with
its full-length cDNA. The available cDNA for At1g01110,
annotated as a full-length cDNA (Arabidopsis TIGR db
Annotation Version 5.0), encodes only three exons but is
likely truncated at its 5'-end because (i) At1g01110 and
At4g00820 are paralogous genes that evolved by a segmental duplication event (see Figure 1a and Figure 5), and
(ii) the At4g00820 gene model of five coding exons is supported by a full-length cDNA sequence. We therefore consider the MIPS MATDB annotation of At1g01110 (five
coding exons) to be correct. The gene models of
At1g51960, At2g02790, At3g22190 and At3g49380
remain to be verified as no full-length cDNA sequences
are available. Structural examination of the 33 putative
IQD genes revealed the presence of 2–6 translated exons,
suggesting that IQD proteins are quite diverse. Almost
two-thirds of the gene family (20 members) contains
more than four protein-coding exons, and 12 genes
encode one or two non-translated exons in their 5'-region

Page 6 of 25
(page number not for citation purposes)

BMC Evolutionary Biology 2005, 5:72

(a)

2

3

http://www.biomedcentral.com/1471-2148/5/72

4

5

6

7

8

IQ66 Domain

At1g51960

At5g07240

12 13 14

b

*
*

At3g49380

*

At4g10640

c

*
*

At4g00820

*

At2g43680

*

At3g59690

II

*
*

At5g13460

*
*
*

At1g17480
At1g72670

At3g22190

III

*
*
*

At2g33990
At3g15050

*

At3g09710

*

At5g03040
At3g52290

b

*
*

At2g26410

*

At1g14380

*
*

At2g02790
At1g74690

IV

*

At1g18840

*

At1g19870

*

At5g35670

(b)

2

3

4

5

6

7

8

IQ66 Domain

9 10
19

11
20

12 13 14

15

16

17

18

*

Os05m00863

*
**

Os01m05259
Os05m04170

*

Os10m02409

*

I

Os03m00584

*

Os03m04199

*

Os04m04664

*

Os08m00125

*
*

Os02m01875
Os06m02303

II

Os01m06663

*
*
*
*

Os05m04352
Os01m04963
Os01m00929

*
*

III

Os12m04168
Os03m04309

*

Os03m05627

*
*

Os06m00539
Os05m00240
Os01m06082

*

b

*

Os05m03604

*

Os05m04307

*

IV

Os01m05025

*

Os04m05532

*

Os03m00334

Os06m03925

100 aa

*

Os01m00895

Os04m04570

18

*

At4g14750

At2g26180

17

*
*

At3g49260

At5g03960

16

*
*

At4g23060

At1g01110

15

**
I

At5g62070

At3g51380

11
20

*

At3g16490
At4g29150

9 10
19

*

*

*
*
100 aa

Figure 3
Motif patterns in IQD proteins of Arabidopsis thaliana and Oryza sativa
Motif patterns in IQD proteins of Arabidopsis thaliana and Oryza sativa. The schematic IQD proteins of Arabidopsis (a) and rice
(b) are aligned relative to the IQ67 domain (orange box). Total amino acid sequence length, boundaries of protein-coding
exons (vertical tick marks), and length and position of separate and distinct MEME motifs (shown as color-coded boxes) are
drawn to scale. Motifs shared by the primary structures of at least four Arabidopsis IQD proteins are depicted at the reference
bar on top of each alignment and numbered consecutively, beginning with motifs most N-terminal in the protein. Motif numbers are cross-indexed in Table 5 that lists the multilevel consensus sequence for each MEME motif. The position of putative
calmodulin-binding sites predicted by the Calmodulin Target Database [40] (see Table 4) is indicated by an asterisk above each
protein model. IQD proteins are aligned in the same order as they appear in the phylogenetic trees (see Figure 1). Subfamilies
and subgroups (I–IV) of IQD proteins are highlighted by colored vertical bars next to the gene identifiers.

Page 7 of 25
(page number not for citation purposes)

BMC Evolutionary Biology 2005, 5:72

Total

-

http://www.biomedcentral.com/1471-2148/5/72

Sup.

+
-

Pellet

+

+
-

Last Wash

+

+
-

+

Ca2+
EGTA

T7-IQD20

Figure 4
Interaction of Arabidopsis IQD20 and calmodulin in vitro
Interaction of Arabidopsis IQD20 and calmodulin in vitro. Calmodulin-agarose beads were incubated in the presence of Ca2+ or
absence of Ca2+ (+EGTA) with soluble proteins prepared from induced bacterial cultures expressing a T7-tagged IQD20 protein and treated as described in Methods. Proteins of the total bacterial extract, the supernatant fraction, the entire pellet
(beads) fraction, and of the last wash were resolved by SDS-PAGE, transferred to a membrane, and probed with a HRP conjugated T7-Tag monoclonal antibody.

(Figure 1b). All introns of most IQD genes are phase-0
introns, separating exactly two triplet codons [38]. The
last intron of At1g23060 is in phase-2, which lies between
the second and third nucleotide of joining codons, and a
phase-1 intron is found in five other IQD genes (Figure
1b). The average size of IQD genes in Arabidopsis is 2.4 kb
(Table 3).
Predicted primary structure and properties of Arabidopsis
IQD proteins
Having identified non-redundant and verified potential
IQD protein coding sequences, we developed a set of criteria for the presence of the IQ67 domain in the 33 predicted Arabidopsis proteins. The IQ67 domain is
characterized by the precise spacing of three copies of the
11-amino acid IQ motif, which are separated by short
sequences of 11 and 15 amino acid residues (Figure 2a).
The first IQ motif is best conserved (present in 32 proteins), followed by the second (26 proteins) and third (12
proteins) IQ repeat. Although the third IQ motif shows
the highest degree of sequence degeneration, its initial
hydrophobic amino acid and following glutamine residue
are present in 31 proteins. Each IQ motif is congruent
with a 1-5-10 motif of hydrophobic amino acids, which
again is least conserved for the last IQ motif. A fourth 1-510 motif overlaps the first spacer sequence and second IQ
motif. Each IQ motif also partially overlaps with a 1-8-14
motif. Besides these repetitive motifs, the IQ67 domain is
characterized by the presence of additional conserved
hydrophobic and basic amino acid residues flanking each
IQ motif (Figure 2a). A hallmark of IQD genes is the pres-

ence of a phase-0 intron at an invariant position within
the coding region of the IQ67 domain that disrupts codon
16 and 17 (equivalent to codon 9 and 10 of the first IQ
motif). At5g03960 is the only exception to this rule,
which encodes the entire IQ67 domain on its second and
central exon (Figure 1b and Figure 3a). Given these criteria, 32 proteins contain at least two or three discernible IQ
motifs with the accompanying 1-5-10 and 1-8-14 motifs
in their IQ67 domain, which we therefore consider bona
fide IQD proteins. The protein encoded by At5g35670
does not meet these criteria because it only contains the
first, albeit truncated IQ motif provided by the N-terminal
exon of the IQ67 domain (exon 2 of At5g35670). The
exon coding for the remainder of the IQ67 domain (residues 17–67) is missing and replaced by an unrelated exon
in At5g35670 (Figure 2a and Figure 3a). However, the
At5g35670 protein shares five common amino acid
sequence motifs outside the IQ67 domain with a large set
of IQD proteins as detected by comparative MEME (Multiple Expectation Maximization for Motif Elicitation)
analysis [39] of the complete amino acid sequences of the
33 Arabidopsis proteins (Figure 3a). As most of these
motifs are unique to IQD proteins, we consider
At5g35670 a member of the IQD gene family in Arabidopsis. Since amino acids 17–67 of the IQ67 domain are
encoded by the second or third exon of IQD genes, the
IQ67 domain contributes to the core region of most IQD
proteins. An interesting exception is At3g51380, which is
the smallest member of the IQD protein family in Arabidopsis and consists of a C-terminal IQ67 domain and a
short N-terminal extension of 35 amino acid residues.

Page 8 of 25
(page number not for citation purposes)

BMC Evolutionary Biology 2005, 5:72

http://www.biomedcentral.com/1471-2148/5/72

Table 4: Predicted calmodulin-binding sites in Arabidopsis and rice IQD proteins

Groupa

Gene Identifier

Protein

Predicted calmodulin binding sequenceb

Ia

At1g51960
At3g16490
At4g29150
At5g07240
At5g62070
At4g23060
At3g49260
At3g51380
At4g14750
At3g49380
At4g10640
At1g01110
At4g00820
At2g43680
At3g59690
At5g03960
At5g13460
At1g17480
At1g72670
At2g26180
At3g22190
At2g33990
At3g15050
At3g09710
At5g03040
At3g52290
At2g26410
At1g14380
At2g02790
At1g74690
At1g18840
At1g19870
At5g35670
Os01m00895
Os05m00863
Os01m05259
Os05m04170

IQD27
IQD26
IQD25
IQD24
IQD23
IQD22
IQD21
IQD20
IQD19
IQD15
IQD16
IQD18
IQD17
IQD14
IQD13
IQD12
IQD11
IQD7
IQD8
IQD6
IQD5
IQD9
IQD10
IQD1
IQD2
IQD3
IQD4
IQD28
IQD29
IQD31
IQD30
IQD32
IQD33
OsIQD22
OsIQD21
OsIQD20
OsIQD19

Os10m02409

OsIQD18

Os03m00584
Os03m04199
Os04m04664
Os08m00125
Os02m01875
Os06m02303
Os01m06663
Os05m04352
Os01m04963
Os01m00929
Os12m04168
Os03m04309
Os03m05627
Os06m00539
Os05m00240
Os01m06082
Os05m03604
Os05m04307
Os01m05025
Os04m05532

OsIQD17
OsIQD16
OsIQD15
OsIQD14
OsIQD13
OsIQD12
OsIQD11
OsIQD10
OsIQD9
OsIQD5
OsIQD8
OsIQD3
OsIQD4
OsIQD7
OsIQD1
OsIQD2
OsIQD6
OsIQD23
OsIQD24
OsIQD25

(98) EERWAAVKIQKVFRGSL (114)
(137) ALVRGYLVRKRAAET (151)
(65) KERRTHAIAVA (75) (83) DAAVAAAKAAAA (94)
(105) EYKAAMKIQSAFRGYLA (121)
(115) QENIAAMKIQSAFRGYLAR (133)
(189) LKGLVRLQAIVRGHIERK (205)
(137) RALRALKGL (145)
(9) VVRRKLLRRSQSR (22)
(155) ALITLQAKAREQRIRMIG (172)
(140) ALVRGHNVRRRTSITLQRVQALVRI (164)
(235) EIAIKREKAQALALSNQI (252)
(146) LVKLQALVRGHNVRKQ (161)
(157) LVKLQALVRGHNVRKQA (172)
(1) MVKKGSWFSAI (11)
(1) MGKKGSWFSAI (11)
(8) FGWMKRLFICEAKARAEK (24)
(5) KGLFTVLKRIFISEVN (20)
(125) IFRGRQVRKQAAVTLRC (141)
(119) VRIQAIFRGRQVRK (132)
(116)VRGRQVRKQAAVTLRCMQALVRVQARVRARR (146)
(137) QALVRVQARVRARRV (151)
(59) AYKARKSLRRLKGIARAKLS (78)
(61) RAFKARKRLCS (71)
(103) GKSKEEAAAIL (113)
(141) VRLKLLMEGSVVKQAAN (158)
(213) MLNKQVATMRREKALAYAF (231)
(245) RSVNRKEASVRRERALAY (262)
(106) AHQARRAFRTL (116)
(159) VKVQALVRGKKARSS (173)
(149) LVRRQAVATLF (160)
(159) GIVRLQALARGREIRHSDIG (178)
(230) ARRELLRSKKVI (241)
(270) RERALAYAFSQQL (282)
(134) PRGRAAAVKIQTAFRGFL (151)
(434) NRVQEAFNFKTAVVGRLDR (453)
(94) MVIQKAYRGYLA (105)
(87) AVMIQKAFRGYLARRALRA (107)
(110) LKALVKIQALVRGYLVRKQAATT (129)
(48) KKRWSFRRSSASASAAAM (65)
(170) TLRRMQALLVAQARlRAQ (187)
(28) ALPGEAAKEKRWSFRRPVHG (47)
(138) KLQALVRGHLVRRQAS (153)
(121) KREEYAAVRIQAAFRG (136)
(269) TRKDAALKRERALSYA (284)
(127) ASREERAAVRIQ (138)
(120) AGREERAAVRIQA (132)
(1) MGKKGGWITA (11)
(114) RLVRRQLAVTLKCMNALLR (132)
(120) RGRRVRKQLAVTLKCMQALV (139)
(143) QVRKQAAVTLRCMQALVRVQARIRARRVRMST (176)
(147) AQARVRARRVRISL (160)
(161) ARVRARQVRVSLE (173)
(113) FLARRARRALKGL (125)
(174) VKRERAMAYAFNHQWRAR (191)
(219) AVRRERALAYAFSHQWK (235)
(1) MGKKGNWFSAV (11)
(132) RVYLGRRSQRARGLDRL (148)
(160) WLIVKFQALVRGRNVR (174)
(155) LVRGRNVRLSGASI (168)
(295) LVRRQAAESLQ (305)

Ib

Ic

II

IIIa

IIIb

IV

I

II
IIIa

IIIb

IV

Page 9 of 25
(page number not for citation purposes)

BMC Evolutionary Biology 2005, 5:72

http://www.biomedcentral.com/1471-2148/5/72

Table 4: Predicted calmodulin-binding sites in Arabidopsis and rice IQD proteins (Continued)

Os03m00334
Os04m04570

OsIQD26
OsIQD27

Os06m03925

OsIQD28

(154) GNAKLGRR (161)
(8) LEKKRVITVQGRDKAGRPI (26)
(132) GKLRYVSRLEYLWAHVRKG (150)
(252) LAYAFSQQLRSCGGGGGGTT (271)

a Roman

numerals correspond to subfamilies and subgroups of IQD proteins as used in Figure 1 and Figure 3.
calmodulin-binding sites predicted by the Calmodulin Target Database [40] are shown for strings of amino acid residues with a score of at
least "7". Residues with the highest score ("9") are highlighted in bold.

b Putative

Since At3g51380 is predicted to encode a 'minimal' IQD
protein (IQD20), we tested whether calmodulin interacts
with recombinant IQD20. We employed the same co-sedimentation assay that we recently used to demonstrate
Ca2+-dependent binding of IQD1 to bovine calmodulin
[37]. As shown in Figure 4, an epitope tagged T7-IQD20
fusion protein preferentially co-sedimented with calmodulin-agarose beads in the presence of Ca2+, whereas
noticeably less T7-IQD20 protein was bound to immobilized calmodulin when the incubation mix and wash
buffer were supplemented with EGTA. Thus, our data
indicate that the smallest member of the IQD protein
family in Arabidopsis interacts with calmodulin in a Ca2+independent manner but suggest that calmodulin binding
is possibly stimulated by the presence of Ca2+ ions. We
interrogated the web-based Calmodulin Target Database,
which computes various structural and biophysical
parameters of a given protein sequence to predict calmodulin binding sites [40]. This analysis predicted that IQD20
and all other IQD proteins of Arabidopsis contain, in
addition to multiple IQ motifs, strings of high-scoring
amino acid residues that indicate the location of putative
calmodulin interaction sites (Table 4). The predicted calmodulin binding sites overlap with the IQ67 domain in
23 of the 33 IQD protein sequences (see Figure 3a).
Although the predicted IQD proteins are quite diverse
with respect to size (103–794 residues) and computed
molecular mass (11.8–86.8 kD), they appear to be
remarkably uniform in terms of their relatively high theoretical isoelectric point (10.3 ± 0.6), the only exception
being At1g19870 (pI of 5.2), and with respect to the abundance of Ala (8.6 ± 2.2), Ser (12.2% ± 2.2%), and basic
amino acid residues (Arg/Lys, 17.6% ± 2.2%). To uncover
the possible subcellular localization of IQD proteins in
Arabidopsis, we searched for different signature motifs
specific to cellular compartments. Because of their high
content of basic residues, and as suggested by PSORT, at
least half of the IQD protein family (16 members) may be
localized in the cell nucleus (Table 1). This conjecture is
supported by the presence of several basic clusters in IQD
proteins that conform to the SV40-type, MATα2-type, and
bipartite type of nuclear localization signals [41], and by
the nuclear localization of an IQD1-GFP fusion protein
[37]. The remaining IQD proteins are predicted to be
localized in the mitochondria (7), chloroplasts (5), or
unknown compartments (Table 1).

Chromosomal distribution and homology of Arabidopsis
IQD genes
To infer clustering patterns that reflect IQD protein
sequence similarity and evolutionary ancestry, we constructed phylogenetic trees by the neighbor-joining
method [42] using IQD full-length sequences and the
amino acid sequence of At5g35670 as outgroup. The
At5g35670 gene encodes a C-terminally truncated IQ67
domain that lacks amino acid residues 17–67 (Figure 2a).
The phylogenetic analysis of the Arabidopsis IQD gene
family reveals four well-resolved subfamilies, two of
which can be further divided into subgroups supported by
the presence and position of introns, the occurrence of
common protein motifs outside the IQ67 domain, and
bootstrapping values (Figure 1a and 1b; Figure 3a). Large
segmental duplications of chromosomal regions during
evolution, followed by gene loss, small-scale duplications
and local rearrangements, have created the present complexities of the Arabidopsis genome [43-51]. These events
have likely shaped the size and structure of the current
IQD gene family. We therefore analyzed the evolutionary
history of IQD genes, which are relatively evenly distributed among all five Arabidopsis chromosomes (Figure 5
and Table 1). The topology of the phylogenetic tree (Figure 1a) suggests for several IQD genes in all subfamilies a
clear paralogous pattern of gene divergence by gene duplication. Using the Arabidopsis Redundancy Viewer
(MATDB), the Viewer of Segmental Genome Duplications
(TIGR) and the searchable supplementary material provided by Blanc et al. [45] and Simillion et al. [48], we
found that 26 of the 33 IQD genes are located in previously identified chromosomal duplications [45,47,48].
Eight pairs of duplicated IQD genes have been retained
during evolution, whereas the IQD sister gene has been
lost for each of the other 10 duplication events (Figure 5).
All 18 duplications involving IQD genes occurred during
the relatively recent genome-wide duplication event 75 ±
22 Myr ago, as estimated by Simillion et al. [48]. In most
cases, the paralogous relationships indicated by segmental duplication are supported by the exon-intron organization and the phylogeny of the IQD gene pairs (Figure 1a
and 1b). The following pairs of genes are therefore close
paralogous IQD genes in Arabidopsis, sharing 50–67%
amino acid sequence identity: At1g01110 and At4g00820;
At1g14380 and At2g02790; At1g17480 and At1g72670;
At1g18840 and At1g74690; At1g51960 and At3g16490;
At2g43680 and At3g59690; At3g09710 and At5g03040;

Page 10 of 25
(page number not for citation purposes)

BMC Evolutionary Biology 2005, 5:72

http://www.biomedcentral.com/1471-2148/5/72

Table 2: The IQD gene family of Oryza sativa
Gene
Identifiera

Clone
IDb

Positionc

Protein
ID Coded

cDNA
Accessione

Expressionf

Protein
Nameg

Size
(aa)

Mass
(kD)

IP

Predicted Locationh
PSORT

TargetP

Os01m00895

AP002743
02000445

70239–72382

NP_914546

AK119868

B

OsIQD22

465

49.7

10.5

N

M 0.69/3

Os01m00929

AP002746
02000453

152586–155207

NP_914588

AK073282

B

OsIQD5

442

48.9

10.3

?

?

Os01m04963

AP002901
02003727

7612–9986

NP_916574

AK102451

A

OsIQD9

441

48.2

11.0

?

M 0.55/5

Os01m05025

AP003288
02003743

38561–44222

9629.m05025i

AK062106

AB

OsIQD24

574

63.1

9.8

N

C 0.44/5

Os01m05259

AP003768
02003803

95943–99625

NP_916047j

-

AB

OsIQD20

378

42.4

10.7

?

?

Os01m06082

AP004366
02004199

106290–110032

BAD73780

AK072219

AB

OsIQD2

500

56.1

10.2

N

?

Os01m06368

AP003611
02004332

27187–28795

BAB63799k

AK120019*

-

n.d.

n.d.

n.d.

n.d.

n.d.

Os01m06663

AP003349
02004466

15479–17371

NP_915152

AK105622*

A

OsIQD11

563

61.7

11.5

?

?

Os02m01875

AP005534
02005830

59894–65564

XP_465098

AK105486

B

OsIQD13

485

52.0

10.4

?

?

Os03m00334

AC099399
02007792

57690–58691

XP_470188

-

B

OsIQD26

303

32.3

11.2

N

M 0.47/4

Os03m00584

AC105729
02029613

135566–136967

AAN06867

-

B

OsIQD17

417

44.3

10.2

?

M 0.61/4

Os03m04199

AC120505
02010452

144176–145684

XP_468989

-

-

OsIQD16

447

48.2

10.4

?

M 0.83/3

Os03m04309

AL731878
02014260

118442–126461

AK067192l

AAU89191

AB

OsIQD3

440

48.7

9.6

N

?

Os03m05627

AC084296
02011159

48853–52578

AAT75259

AK103438

AB

OsIQD4

422

47.0

9.8

?

?

Os04m04570

Chr.4m
02014535

27592940–27594955

9632.m04570

-

-

OsIQD27

368

41.6

11.4

N

?

Os04m04664

AL607001
02017716

151253–153796

XP_473550

AK100392

AB

OsIQD15

464

50.1

10.4

N

M 0.42/5

Os04m05532

AL606999
02015015

85710–91604

XP_474230

AK066310

AB

OsIQD25

893

98.5

4.8

N

?

Os05m00240

AC093089
02015233

81361–85365

AAV33309

AK065809

AB

OsIQD1

474

52.0

10.2

?

?

Os05m00863

AC093954
02015642

45436–47015

XP_476075

-

-

OsIQD21

497

52.6

10.4

?

M 0.57/4

Os05m03604

AC108500
02017442

23568–26042

AAU90174

-

-

OsIQD6

538

57.8

9.6

N

?

Os05m04170

Chr.5n
02017671

24971333–24973284

-

-

-

OsIQD19

367

40.5

10.8

N

?

Os05m04307

AC097112
02017716

54441–58756

XP_475770

AK101555

AB

OsIQD23

574

63.8

9.8

N

C 0.38/5

Os05m04352

AC104713
02017731

37570–40338

XP_475808

AK107193

B

OsIQD10

408

44.6

10.6

?

C 0.36/5

Os06m00539

AP004844
02018243

90441–94332

BAD69297

AK099462

AB

OsIQD7

353

39.4

10.4

N

?

Os06m02303

AP003572
02019223

18921–22322

BAD61625

-

-

OsIQD12

470

50.0

10.6

N

?

Os06m03925

AP0039440
2020217

329–2234

9634.m03925

AK109238*

-

OsIQD28

432

46.1

8.3

N

M 0.55/4

Page 11 of 25
(page number not for citation purposes)

BMC Evolutionary Biology 2005, 5:72

http://www.biomedcentral.com/1471-2148/5/72

Table 2: The IQD gene family of Oryza sativa (Continued)
Os08m00125

AP005657
02022817

88345–90355

XP_479772

AK100461

AB

OsIQD14

543

59.0

11.0

?

?

Os10m02409

AC027662
02029613

17834–19903

NP_921513

AK110922

B

OsIQD18

485

52.2

10.3

N

M 0.73/3

Os12m04168

AL732532
02035326

133867–139274

9640.m04168

AK102525

A

OsIQD8

442

48.2

10.1

N

?

a TIGR

V2 pseudo-molecules annotation.
line: nucleotide accession of a BAC clone coding for a rice IQD gene from O. sativa ssp. japonica [93]. Lower line: alternative rice BAC clone
from O. sativa ssp. indica (the prefix, AAAA, is omitted).
c Position of the IQD gene on the BAC clone from O. sativa ssp. japonica.
d For four IQD genes, protein identification numbers are only available from the TIGR Rice Genome Project database [74].
e cDNAs clones are full-length if not otherwise indicated. Asterisks denote cDNA sequences that are likely 5'-truncated by comparison with
predicted mRNAs and encoded OsIQD proteins.
f Additional evidence for expression provided by (A) EST clones and (B) Massively Parallel Signature Sequencing (MPSS, [106]).
g Nomenclature of OsIQD genes is arbitrary. Levy et al. [37] cloned AtIQD1 and reported closely related rice genes OsIQD1-OsIQD5. The
designation of OsIQD6-OsIQD29 is based on the phylogenetic analysis presented in Figure 1c.
h PSORT predictions: N (nucleus), C (chloroplast), M (mitochondrion). TargetP predictions: values indicate score (0.00 – 1.00) and reliability class
(1–5; best class is 1).
i Region of Os01m05025 is not annotated on BAC clone AP003288 as indicated for AK062106 full-length cDNA sequence on KOME website [98].
Therefore, no Protein ID is available and the TIGR gene model accession is given instead.
j Predicted gene model shows an N-terminal extension by 11 amino acids (possibly incorrect start codon), which was removed to meet consensus
of IQD protein N-termini for computational analysis of protein properties.
k Predicted protein of this gene locus is shorter for both O. sativa subspecies than the predicted polypeptide encoded by the partial cDNA clone
that is truncated in the coding region N-terminal to the predicted IQ67 domain. Therefore, theoretical physico-chemical parameters of the
predicted full-length protein could not be determined (n.d.).
l Protein coding region is misannotated when compared with the predicted protein encoded by the full-length cDNA sequence.
m BAC clone OJ1087C03 cannot be retrieved from GenBank.
n Incorrect hyperlink from gene locus to BAC clone on RiceGE website.
b Upper

At5g07240 and At5g62070. Two orphan genes contained
in opposite parts of a duplicated segment pair on chromosome III and IV, At3g22190 and At4g14750, group in different subfamilies of the phylogenetic tree and share
substantially lower primary structure identity (20%) as
well as less preservation of exon-intron organization (Figure 1a and 1b), suggesting reciprocal IQD sister gene loss
after duplication of a chromosomal segment that contained two ancestral IQD genes. The genes At2g33990 and
At3g15050 also appear to be closely related paralogs (Figure 1a, 43% identity); however they are positioned in different previously identified duplication segments, which
points to a more complex evolutionary history. As
expected, IQD genes of atypical structure (At5g03960, loss
of intron in IQ67 coding region) or encoding atypical proteins (At1g19870, acidic pI; At3g51380, C-terminal IQ67
domain; At5g35670, truncated IQ67 domain) are either
singleton genes (At5g35670, At3g51380), or orphan
genes (At1g19870, At5g03960) whose homologous sister
gene has been lost after duplication. Two pairs of closely
positioned singleton genes, one each on chromosome III
and IV, and two clustered genes in a duplicated segment
on chromosome IV (At4g49260, At4g49380), suggest
ancient tandem or local duplication events that have
already resulted in substantial gene diversification (<30%
identity for each gene pair). In summary, large-scale segmental duplication events appear to have exclusively contributed to the current complexity of the IQD gene family.

Identification and predicted properties of the IQD protein
complement in Oryza sativa
We next explored the occurrence and size of the IQD gene
family in the extensively sequenced genome of rice
[52,53]. BLAST searches in several databases of O. sativa
ssp. japonica and indica (see Materials and methods) using
several Arabidopsis full-length IQD protein sequences as
the queries identified 29 different loci that encode nonredundant putative IQD proteins in rice. The general features of rice IQD genes and proteins are summarized in
Table 2 and Table 3. Full-length cDNA sequences are
available for 16 genes and generally support the respective
gene model, with the exception of two loci
(Os01m05259, Os03m04309) that are incorrectly annotated (see Table 2). The putative full-length cDNA
sequences of two additional genes (Os01m06663,
Os06m3925) are likely truncated in their coding region
when compared with the conceptual translation products
of each corresponding locus. A gene model could not be
derived for the Os01m06368 locus in either O. sativa subspecies that covers the open reading frame of a corresponding partial cDNA sequence. To date, independent
evidence for gene expression has been obtained for six of
the remaining ten IQD family members for which a fulllength cDNA is currently not available, suggesting that
most IQD genes are functional in rice (Table 2). As for
Arabidopsis, rice IQD genes encode 2–6 translated exons;
however, less than half of the rice family members (13

Page 12 of 25
(page number not for citation purposes)

BMC Evolutionary Biology 2005, 5:72

http://www.biomedcentral.com/1471-2148/5/72

I

Mb

II

III

IV

0
01110

80.8

V
00820

02790

03040
03960
07240

70.1

09710

67.3

13460
14380

68.8

15050

17480
18840
19870

67.6

16490

2

69.4

3

10640
5

4

22190
3

14750

71.0

5

23060

69.9

2

29150

26180
26410
1

35670
62.2

1

33990

43680
70.1

51960

86.7

4

49260
49380
51380
52290

65.6

59690

62070

72670
74690

30

Figure 5
Chromosomal distribution and segmental duplication events for Arabidopsis IQD genes
Chromosomal distribution and segmental duplication events for Arabidopsis IQD genes. The five chromosomes are indicated
by Roman numerals and the centromeric regions by ellipses. Deduced chromosomal positions of the IQD genes are marked by
horizontal bars and gene identification numbers (last five digits only). The scale is in megabases (Mb) and is adapted from the
scale available on the TIGR database (see Materials and methods). Non-hidden duplicated chromosomal segments [48] that
contain at least one retained IQD gene pair are color-coded. In three such segments (blue, brown, light blue), one sister IQD
gene has been lost. Additional non-hidden duplicated segments that have lost sister IQD genes are shown in white and both
segments are labeled with the same Arabic numeral. The duplicated segments of one such event (number 3) have likely experienced reciprocal IQD gene losses as the remaining genes, At3g22190 and At4g14750, are only distantly related (see Figure 1a).
Numbers in italics at left indicate the estimated age (Myr) of the duplication event according to Simillion at al. [48]; the age estimates are given only once in the order of IQD gene location beginning with chromosome I.

Page 13 of 25
(page number not for citation purposes)

BMC Evolutionary Biology 2005, 5:72

genes) contain more than four exons (Figure 1d). Furthermore, all introns in most OsIQD genes are in phase-0;
only six genes contain a phase-1 intron in their 3'-region
and one gene (Os04m04570) is characterized by the presence of two phase-2 and one phase-1 intron in its 5'region (Figure 1d). Rice IQD genes are slightly larger than
Arabidopsis IQD genes, which is a result of increased
intron length (Figure 1b and 1d; Table 3).
Conceptual translation of full-length cDNA or predicted
mRNA sequences and computation of theoretical physico-chemical protein parameters reveal that the IQD protein complement in rice is remarkably similar to the IQD
protein family in Arabidopsis (Table 2 and Table 3). Comparative MEME analysis of the complete amino acid
sequences of the 28 rice IQD proteins identified a similar
set of conserved sequence motifs and their distribution
along the polypeptide chain as found for members of the
Arabidopsis IQD protein family (Figure 3b and Table 5).
The IQ67 domain is positioned close to the core region of
IQD polypeptides and is characterized by the same hallmarks as described for the Arabidopsis family, including
the location and spacing of the three calmodulin-binding
motifs (i.e., IQ, 1-5-10, 1-8-14), and the position of an
invariant phase-0 intron that separates codon 16 and 17
of the IQ67 domain (Figure 2b and Figure 3b). As predicted by interrogation of the Calmodulin Target Database [40], all rice IQD proteins contain additional
putative calmodulin binding sequences that often overlap
with the IQ67 domain (Figure 3b and Table 4). It is interesting to note that the rice IQD gene family contains
members with similar deviations from consensus properties as observed for the IQD gene family in Arabidopsis.
These exceptions include loss of the phase-0 intron
between the IQ67 domain-coding exons (Os01m06663,
Os08m00125), replacement of the second exon coding
for amino acids 17–67 of the IQ67 domain
(Os06m03925), C-terminal location of the IQ67 domain
(Os03m00334, Os04m04570), and an unusually large
and acidic protein (Os04m05532). Since the rice IQD
proteins display a similar range of structural and physicochemical characteristics as the IQD family in Arabidopsis,
it is very likely that we have identified most of the IQD
family members in rice. Again, the majority of the family
members (16 proteins) may be targeted to the cell
nucleus; the remaining IQD proteins are predicted to be
localized in the mitochondria (4), chloroplasts (1), or
unknown compartments (Table 2).
Chromosomal distribution of rice IQD genes
Unlike the Arabidopsis IQD gene family, which is evenly
distributed over all Arabidopsis chromosomes, the distribution of IQD genes in the rice genome is clearly biased
towards three chromosomes. Almost half of the rice IQD
gene family members (14 loci) are contained in chromo-

http://www.biomedcentral.com/1471-2148/5/72

somes I and V, and five genes are present on chromosome
III. Three IQD genes are each found on chromosomes IV
and VI, while seven of the twelve rice chromosomes contain either one or no IQD gene locus (Table 2). Such a heterogeneous distribution of IQD genes over the different
rice chromosomes is consistent with an ancient aneuploidy event, which has been proposed to have occurred
in rice about 70 Myr ago [51], and not with a wholegenome duplication or polyploidization event. Duplicated segments cover substantial regions of chromosome
V (16%) and chromosome I (11%), the second and third
largest fraction of segmental duplications after chromosome II (22%) [51]. The topology of the phylogenetic tree
of OsIQD genes suggests four pairs of paralogous genes
that evolved by segmental duplication (55–69% amino
acid sequence identity); interestingly, three such pairs
include IQD genes located on chromosome I and V (Figure 1c). Like the IQD protein family in Arabidopsis, the
phylogenetic analysis of the rice gene family reveals four
major subfamilies, and one can be divided into two subgroups. The two rice proteins containing the IQ67
domain at their C-terminus cluster as a separate subfamily
(Figure 1c and 1d, Figure 3b).
Comparative phylogenetic analyses
We further investigated the relationship between the Arabidopsis and rice IQD protein families by generating an
alignment of the 61 identified IQD amino acid sequences
followed by the generation of a neighbor-joining phylogenetic tree (Figure 6). The combined phylogeny between
the Arabidopsis and rice IQD sequences revealed six subfamilies of putative orthologous genes. Within each subfamily, the rice and Arabidopsis genes appear more
closely related to each other than to IQD genes of the
same species in a different subfamily, suggesting that an
ancestral set of IQD genes already existed before the
monocot-eudicot divergence. Four subfamilies of likely
orthologous genes (I–IV) are composed of nearly identical
sets of genes that constitute the respective subfamilies in
Arabidopsis and rice (compare Figure 6 with Figure 1a and
1c). The remaining two subfamilies contain the genes
encoding atypical IQD proteins in both species:
At3g51380, Os03m00334 and Os04m04570 (IQ67
domain on protein C-terminus) are members of subfamily V, whereas At5g35670 and Os06m03925 (truncated IQ67 domain) comprise subfamily VI (Figure 6).
The two genes coding for the acidic and unusually large
IQD proteins, At1g19870 and Os04m05532 (Table 1 and
Table 2), are members of subfamily IV and form a pair of
orthologous genes. These subgroups of orthologous genes
and other branches within the subfamilies are well-supported, which may be indicative for a relatively early
diversification of IQD gene structure and function during
plant evolution. The three genes that experienced loss of
the conserved intron separating the IQ67 domain-encod-

Page 14 of 25
(page number not for citation purposes)

BMC Evolutionary Biology 2005, 5:72

http://www.biomedcentral.com/1471-2148/5/72

Table 5: Major motifs in Arabidopsis and rice IQD proteins

Motifa

Multilevel consensus sequenceb

1

EEWAAIKIQTAFRGYLARRALRALKGLVRLQALVRGHLVRKQAAMTL
RCMQALVRVQAQVRR
MGKKGKWFKSLFGGF
SWFTAVKRIFISPTK
NKKWKLWRTSSED
EKRRWSFRKSS
PPCPPPPPPHH
KHAIAVAIATAAAAEAAVAAA
QAAAEVVRLTS
SEENQALQKQLHQKHHHE
GEDWDDSILSK
EEIEAKLQMRQEAAIKRERAMAYAFSHQW
WKNSSKTGNPTFMDP
DNPNWGWNWLERWMA
ARPWENRLMDD
YEENPKIVEMDTGKPYY
GSMNDDESFTSCPDF
PNYMANTESAKAKVRCQSAPR
SAKKRLSFPN
DHVKEIEEGWCDSIG
WMEKLTNNAFADKLLASSPTTLPLH

2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
a Numbers

(1–20) correspond to the motifs schematically presented in the reference bars of Figure 3. Motif 1 corresponds to the IQ67 consensus
sequence. The remaining motifs are listed in the order as they occur in the primary structures of IQD proteins, continuing with motifs most Nterminal.
b Sequences were obtained from the MEME analysis of the 61 Arabidopsis and rice IQD full length proteins. Only consensus sequences that are
shared by at least four Arabidopsis IQD proteins are listed.

ing exons, At5g03960, Os01m06663 and Os08m00125,
are members of different subfamilies (Figure 6), which
suggests that intron loss occurred after the divergence of
both evolutionary lineages. The phylogeny of Arabidopsis
and rice IQD genes supports the occurrence of species-specific IQD gene duplications events. For example the two
closely related IQD gene pairs in subfamily I
(Os05m00863/Os01m00895
and
At3g16490/
At1g51960)
or
subfamily
IV
(Os05m04307/
Os01m05025 and At1g18840/At1g74690) result from
duplication events that occurred independently in both
species.
To explore the evolutionary history of the IQD gene family in greater detail, we searched publicly available
genomic and EST databases for homologous sequences in
other plant species. We identified ESTs corresponding to
IQD proteins for all angiosperm species represented in the
TIGR Plant Gene Indices as well as for the gymnosperm
Pinus ssp. (three putative full-length cDNA and six additional EST sequences). As expected, the putative fulllength IQD proteins of pine (TIGR Pinus Gene Index
entries TC41979, TC52213, and TC52519) are very similar to the Arabidopsis and rice IQD proteins with respect
to calculated molecular masses (38.9–56.8 kD), isoelectric points (pI of 10.1–10.3) and frequencies of Ala, Ser,

Arg, and Lys residues. A combined phylogenetic analysis
of the Arabidopsis, rice and pine full-length IQD protein
sequences reveals that the IQD proteins from Pinus cluster
with different subfamilies (see Figure 6), suggesting that
IQD proteins predated the evolution of vascular plants.
We also performed a BLAST search of the moss database
(see Materials and methods) and identified one contig
EST sequence from Physcomitrella patens that encodes an
IQD-like protein (contig5180). Although the deduced
amino acid sequence appears to be truncated at the C-terminus (20 amino acid residues downstream of the IQ67
domain), an appreciable similarity with the protein
encoded by At1g01110 is evident (33% identity), which
includes the presence of MEME motif 3 at its N-terminus
(data not shown). Interestingly, alignment of the deduced
IQ67 domain of the moss polypeptide reveals a deletion
of six residues that correspond to the N-terminus of the
second IQ67 domain-encoding exon of most Arabidopsis
and rice IQD proteins (Figure 2c). As the IQ67 intron is in
phase-0 (see above) and since A. thaliana and O. sativa
both express an IQD-like gene in which the second IQ67
domain-encoding exon is replaced by an unrelated exon,
it is unlikely that the contig5180 DNA sequence is an artifact and probably represents either a novel variant of IQDlike genes or an ancestral gene of the IQD genes found in
vascular plants.

Page 15 of 25
(page number not for citation purposes)

BMC Evolutionary Biology 2005, 5:72

http://www.biomedcentral.com/1471-2148/5/72

Os01m06082
At1g17480
At5g03040 Os05m00240
At1g72670
At2g26410 At3g09710 At3g52290
At2g26180
Os05m03604
Os01m00929
Os05m04352
At5g13460
Os01m04963
At5g03960

At3g22190
Os03m05627

At3g59690

Os12m04168

At2g43680

Os03m04309

Os01m06663

At2g33990

Os03m00334
At3g15050
Os06m00539
At3g51380

*

Os06m03925

**

At5g35670

Os04m04570

At1g14380
Os06m02303

***

Os02m01875

At2g02790

At3g49260

Os05m04307

At4g00820

Os01m05025

At1g18840

At1g01110
Os08m00125

At1g74690

At4g10640
At1g19870
At3g49380
At4g29150
At1g51960
At3g16490

Os05m04170
Os01m05259

Os04m05532

Os03m00584

Os01m00895
Os05m00863
At5g62070
Os03m04199
At5g07240
At4g23060
At4g14750
Os04m04664

Os10m02409

0.1

Figure 6
Phylogenetic relationships of Arabidopsis thaliana and Oryza sativa IQD proteins
Phylogenetic relationships of Arabidopsis thaliana and Oryza sativa IQD proteins. The unrooted tree, constructed using ClustalX
(1.81), summarizes the evolutionary relationship among the 61 members of both IQD protein families. The neighbor-joining
tree was constructed using aligned full-length amino acid sequences. The scale bar corresponds to 0.1 estimated amino acid
substitutions per site. Nodes supported by high bootstrap results (>75%) are indicated by dots. The same color code was used
as in Figures 1 and 3 to highlight the different subfamilies (red, I; yellow, II; blue, III; green, IV; black, V [proteins with IQ67
domain on C-terminus]; brown, VI [proteins with truncated IQ67 domain]). The asterisks indicate the approximate position of
branches corresponding to putative IQD proteins from pine (*TC522213, **TC41979, ***TC52519; Tentative Consensus of
TIGR Unique Gene Indices).

We finally examined the relationships between the IQ67
domains of the four plant species by constructing a neighbor-joining phylogenetic tree using the PAUP*4.0 program and the amino acid sequence alignment shown in
Figure 2. Three major subfamilies of IQ67 domain
sequences can be observed, which each contain members
of the Arabidopsis, rice and pine IQD families. In addition, two small subfamilies and two single branches originate deeply in the unrooted tree and are only distantly

related to the three major subfamilies, which can be further divided into subgroups (Figure 7). Bootstrap analyses
indicated that the deep nodes of the tree have low statistical support, which may be attributed to the small size of
the IQ67 domain. Low bootstrap support has also been
observed for the phylogeny of the similarly sized DNAbinding domains of bHLH [54], Dof [55], or GATA [56]
transcription factor families. Nevertheless, the IQ67 tree
has better resolution in the outer clades. The short

Page 16 of 25
(page number not for citation purposes)

BMC Evolutionary Biology 2005, 5:72

http://www.biomedcentral.com/1471-2148/5/72

57

100
100
90

53
70
100
95

100

99

96
51
98

88
57
79
100
100
76
100
53
77
85
58
52
74
59

51
86
100
92

58
99

At3g22190
Os12m04168
PsCO163170
Os03m05627
Os03m04309
At4g23060
At5g07240
At5g62070
At1g01110
At4g00820
Os08m00125
At4g10640
At3g49380
PsTC40942
PsTC52519
At2g26180
Os01m00929
At1g72670
At1g17480
Os01m04963
Os05m04352
PsTC52213
PsTC43617
Os02m01875
Os06m02303
At3g49260
Os04m04664
PpContig5180
At3g16490
At1g51960
PsCN785626
Os01m00895
Os05m00863
Os01m06368*
Os01m05259
Os05m04170
At4g29150
Os03m00334
Os04m04570
At3g51380
PsCF403032
At1g18840
At1g74690
At1g14380
At2g02790
Os05m04307
Os01m05025
PsCO368832
At1g19870
Os04m05532
Os10m02409
Os03m00584
At4g14750
Os03m04199
At3g52290
At2g26410
Os05m00240
Os01m06082
Os05m03604
At3g09710
At5g03040
PsTC41979
At3g59690
At2g43680
Os01m06663
At5g13460
At5g03960
At5g35670
Os06m03925
At3g15050
Os06m00539
At2g33990

1

2

3

Phylogenetic relationships of the IQ67 domains encoded by IQD genes from Arabidopsis thaliana, Oryza sativa, Pinus ssp. and
Figure 7
Physcomitrella patens
Phylogenetic relationships of the IQ67 domains encoded by IQD genes from Arabidopsis thaliana, Oryza sativa, Pinus ssp. and
Physcomitrella patens. The unrooted tree was constructed from the alignment shown in Figure 2 using PAUP* 4.0 and the neighbor-joining method. Numbers on branches indicate the percentage of 1000 bootstrap replicates that support the adjacent
node; low bootstrap support (<50%) was not reported. Black braces and Arabic numerals at right indicate the three major subfamilies as defined by the phylogenetic analysis of the 72 IQ67 domain sequences. Gene identification and accession numbers
are colored using the same code as in Figure 6 to denote the different subfamilies of the parental IQD proteins. Accession
numbers of the putative pine and moss IQD proteins are given the prefixes 'Ps' and 'Pp', respectively. The asterisk denotes the
putative rice IQD protein for which a full-length amino acid sequence could not be predicted (see Table 2).

Page 17 of 25
(page number not for citation purposes)

BMC Evolutionary Biology 2005, 5:72

http://www.biomedcentral.com/1471-2148/5/72

0

0

IQD

IQ-a

0
11

2

15

IQ-b

IQ-c

0

CAMTA

IQ-a

12

0

IQ-b

7

1

0

Myosin

IQ-a

12

IQ-b

0
14

IQ-c

12

IQ-d

14

0

IQ-e

2

CNGC

11

IQ-a

C-terminus

Figure 8
Organization of IQ motifs in major families of calmodulin-binding proteins
Organization of IQ motifs in major families of calmodulin-binding proteins. The scheme depicts the arrangement of the multiple
IQ motifs present in proteins of the IQD family (this study; [37]), the CAMTA family of calmodulin-binding transcriptional activators [59-61], the myosin family [58], and the CNGC family of cyclic nucleotide gated channels [57, 104]. The IQ motifs are
shown as light-blue boxes. Predicted and experimentally verified calmodulin-interacting peptide sequences are shown in
orange. The numbers in the white spacers equal the number of separating amino acid residues. The triangles and numbers
above each protein family model indicate the position and the phase of conserved introns, respectively. The positions of the
left and right most introns are not drawn to scale.

branches at the tips of the tree indicate high sequence conservation and strong evolutionary relationships among
subfamily members. Interestingly, although the major
subfamilies of IQ67 domain sequences (1–3) and of IQD
full-length protein sequences (I–IV) overlap only partially
(compare color code in Figure 6 and Figure 7), subgroups
of IQ67 domain sequences largely correspond to subgroups of full-length IQD protein sequences as identified
in Figure 6, which is suggestive of exon shuffling during
the evolution of IQD proteins. We also investigated the
effect of different programs and methods on IQ67
domain tree topology. Using ClustalX and the neighborjoining algorithm or the PAUP*4.0 program and maximum parsimony analysis resulted in a similar tree topology (data not shown), which indicates that the neighborjoining tree presented in Figure 7 is robust and reflective
of likely phylogenetic relationships between IQ67
domains within subfamilies.

Discussion
The IQ67 domain – a plant-specific arrangement of
putative calmodulin-interacting motifs
In this study we characterized a possibly complete set of
IQ67 domain-encoding genes in the current version of the
Arabidopsis thaliana and Oryza sativa genomes. The defining features of the IQ67 domain are the invariant arrangement of three IQ motifs [32] separated by 11 and 15
intervening amino acid residues, and the conserved exon-

intron organization (Figure 2). A pattern search of the Arabidopsis proteome with the conventional IQ motif
(IQxxxRGxxxR) and its more generalized versions
([ILV]QxxxRxxxx[R,K]) as the queries confirmed a set of
33 IQD genes identified by reiterative BLAST searches. As
expected from previous reports, our pattern search evidenced three additional major families and numerous
miscellaneous proteins that contain at least one IQ motif:
the CNGC family of cyclic nucleotide gated channels (20
members; [57]), the myosin family (17 members; [58]),
and the CAMTA family of calmodulin-binding transcriptional activators (6 members; [59-61]). For each of these
families, the spacing of IQ motifs and the exon-intron
organization of the respective regions are unique and distinctive from the IQD family, which establishes the IQD
proteins as a separate class of putative calmodulin targets
of unknown biochemical functions (see Figure 8). The
IQD proteins possibly constitute the largest class of putative calmodulin targets in plants. The size of the IQD family in Arabidopsis (33 proteins) and rice (29 proteins)
clearly exceeds the size of other families of calmodulinbinding proteins [8] and is only comparable with the
CIPK family (25–30 proteins) that interact with CBL Ca2+
sensors in Arabidopsis and rice [16]. In addition to the IQ
motif, the IQ67 domain contains multiple copies the 1-510 and 1-8-14 motifs, which are related and typified by
their spacing of hydrophobic and basic amino acid residues. While the IQ motif is thought to mediate calmodu-

Page 18 of 25
(page number not for citation purposes)

BMC Evolutionary Biology 2005, 5:72

lin retention in a Ca2+-independent manner, the 1-5-10
and 1-8-14 motifs are involved in Ca2+-dependent association of calmodulin with its target [33,34]. However, it
should be noted that not all characterized calmodulinbinding domains contain these features [31,32].
We previously demonstrated that Arabidopsis IQD1
binds to bovine calmodulin in a Ca2+-dependent fashion
[37]. In this study, we tested calmodulin binding for
IQD20, the smallest member of the Arabidopsis IQD protein family (103 residues), which consists only of the
IQ67 domain at its C-terminus and a short N-terminal
extension of 35 amino acid residues. Interestingly, we
observed interaction of recombinant IQD20 with calmodulin in the absence of Ca2+, which is possibly augmented
when the metal ion is present (Figure 4). This observation
and the prediction of putative calmodulin binding sites in
IQD20 and all IQD proteins in Arabidopsis and rice,
using the algorithm provided by the Calmodulin Target
Database [40], strongly suggest that all IQD proteins have
the potential to interact with calmodulin (Figure 3 and
Table 4). Given our results with Arabidopsis IQD1 and
IQD20, the prospect arises that different IQD proteins
may interact with calmodulin in different modes, which
could be Ca2+-independent, Ca2+-dependent, or more
complex. The precise mechanism for each IQD protein is
likely determined by the number and specific composition of the IQ, 1-5-10 and 1-8-14 motifs in the IQ67
domain, by the predicted calmodulin binding site adjacent to or overlapping with the IQ67 domain, and by the
overall tertiary structure of the IQD protein. These structural features differ substantially between IQD1 and
IQD20 (Figure 2, Table 1, Table 4), which are likely
responsible for the observed differences in calmodulin
interaction with respect to Ca2+ dependency. The identification of interacting calmodulin or calmodulin-like proteins [14] and the biochemical characterization of
calmodulin binding sites for each IQD protein are important tasks for future research.
It is interesting to note that the Calmodulin Target Database successfully predicts experimentally verified calmodulin-interacting peptides in CNGC [57] and CAMTA [5961] proteins, which are located at conserved positions
adjacent to the IQ motifs (see Figure 8). Although the IQ
motif is likely as widely distributed as calmodulin and calmodulin-like proteins, the IQ67-specific arrangement of
the three calmodulin retention motifs is confined to plant
proteins and not found outside the plant kingdom, suggesting that this calmodulin-interaction module arose
early in plant evolution.
Evolution of IQD proteins
The presence of at least one putative IQD-like gene in
Physcomitrella patens indicates that the IQD gene family

http://www.biomedcentral.com/1471-2148/5/72

originated during the early evolution of land plants, possibly before the divergence of bryophyte and vascular
plant lineages 450–700 Myr ago [62], but not later than
the split of gymnosperms and angiosperms about 300
Myr ago [63] as evidenced by EST and full-length cDNA
sequences coding for at least nine IQD genes in pine.
Molecular and phylogenetic analysis of IQD and IQD-like
genes from ferns, bryophytes and green algae will be necessary to resolve the evolutionary origin of the IQD gene
family.
To explore how the IQD gene family has evolved since the
monocot-eudicot divergence 170–235 Myr ago [64], we
performed a genome-wide comparative analysis of the
IQD gene complement between Arabidopsis and rice. The
phylogenetic trees of the 33 Arabidopsis and 28 rice IQD
genes showed relatively long branches and closely clustered nodes, reflecting a high degree of sequence divergence, which is further indicated by the large variation in
the number of protein-coding exons (2–6) and computed
molecular masses of the predicted IQD proteins (Figure 1
and Tables 1, 2, 3). Based on their phylogenetic relationships, up to six different subfamilies of IQD genes can be
defined for both species. This classification is supported
by conserved exon-intron organization and protein motif
patterns within each subfamily. The combined phylogenetic analysis revealed that members of all six subfamilies
are present in the Arabidopsis and rice genome, indicating
a relatively early diversification of the IQD gene family
before the monocot-eudicot split (Figure 6). In those subfamilies, seven members of both IQD gene families are
clearly recognizable as distinct orthologous pairs (e.g.
genes coding for atypical IQD proteins), suggesting that
the encoded proteins exert similar functions in both species. On the other hand, it is currently impossible to
assign potential functions to IQD genes that are the result
of recent species-specific duplication events leading to
independent functional diversification.
The topology of the phylogenetic trees at the outer
branches suggests that gene duplication played a prominent role in the evolution of both gene families, which is
supported by the analysis of duplicated segments in the
Arabidopsis genome (Figure 5). More than 80% of all
genes in the annotated Arabidopsis genome reside in
duplicated segments, and systematic analyses indicate
that the Arabidopsis genome experienced a large-scale or
even complete genome duplication event 30–90 Myr ago,
sometime between the Arabidopsis-Gossypium and Arabidopsis-Brassica splits [48,49,51,65,66]. Evidence for older
(>100 Mya) large scale-duplications exist, however, the
frequency and precise timing of polyploidizations
remains to be resolved and is a focus of current research
[45,47-50,65,66]. The location of IQD genes in the Arabidopsis genome is clearly reflective of the recent large-scale

Page 19 of 25
(page number not for citation purposes)

BMC Evolutionary Biology 2005, 5:72

duplication event. The IQD gene family is uniformly distributed among the five chromosomes, and 26 (or 79%)
of the 33 IQD loci are found in duplicated segments of the
recent age class (Figure 5). It is important to point out that
16 of those 26 genes in duplicated loci correspond to 8
IQD sister gene pairs, which represents an unusually high
fraction of paralogous genes (44.5%) that have been
retained from the extra gene set since the duplication
event. Nonfunctionalization and subsequent gene loss is
the most likely fate of a gene duplicate, and less than 27%
of the entire paralogous gene set originating from polyploidy have been retained in Arabidopsis [45,48]. Preferential retention of duplicated genes has been observed for
gene families in Arabidopsis with functions in signal
transduction and transcriptional regulation [44]. Specific
examples include the gene families encoding Aux/IAA
(71.5% [67]), GATA (39% [56]) and GRAS (40% [68])
transcription factors, or genes coding for 20S proteasome
subunits (64% [69]); the given percentages equal fractions of retained gene duplicates that we calculated from
published data. Empirical evidence indicates that regulatory processes in metazoa such as signal transduction or
gene transcription are dependent on gene dosage and stoichiometric protein-protein interactions [70]. As pointed
out by Blanc and Wolfe [44], retention of a near-complete
set or subset of duplicated genes coding for regulatory
components such as transcription factors, kinases, phosphatases or Ca2+-binding proteins would minimize disturbances in sensitive stoichiometric and concentrationdependent relationships.
The evolutionary history of the rice genome is less understood. The view of an ancient polyploidy event has
recently been questioned by evidence suggesting that rice
experienced a partial or entire duplication of one chromosome about 70 Myr ago and can thus be considered an
ancient aneuploid [43,51,52,71-73]. The observed nonuniform distribution of the 29-member IQD gene family
in the rice genome, 50% of all IQD loci and three of the
four paralogous IQD gene pairs are present on chromosomes I and V (Table 2), is more consistent with an aneuploidy than whole-genome duplication event. If
polyploidization had occurred, it would be expected that
IQD genes are randomly distributed over the whole rice
genome, as observed for the IQD gene family in Arabidopsis. Given the significant differences in genome size and
estimated gene count between rice (420 Mb, 57,900 genes
[52,53,74]) and Arabidopsis (119 Mb, 27,500 genes
[75]), the slightly larger size of the IQD gene family in Arabidopsis (33 members) versus rice (29 genes) is in agreement with a whole-genome duplication event in the
evolutionary history of the Arabidopsis genome. A similar
difference in membership has been reported for the Arabidopsis and rice gene families encoding Dof and GRAS
transcription factors [55,68]. Nonetheless, IQD genes

http://www.biomedcentral.com/1471-2148/5/72

tend to be larger in rice than in Arabidopsis, which is
mainly due to an increased intron length (Figure 1 and
Table 3). In addition to polyploidization and segmental
duplication events, tandem duplication is another important mechanism in the evolution of gene families [76] and
plays a significant role in Arabidopsis as 17% of all genes
are arranged in tandem arrays [48,77]. However, there is
no evidence for tandem proliferation of the IQD gene
families in the recent history of Arabidopsis and rice
genomes.
Our analysis further suggests that exon shuffling played a
major role during the evolution of IQD genes. Exon insertions and duplications, the major mechanisms of exon
shuffling, contributed significantly to the complexities of
eukaryotic proteomes [38,78,79]. A striking correlation
between functional domains in protein and exons flanked
by introns of matching phases, referred to as symmetrical
exons, has been observed [38,80]. As stated by the phasecompatibility rules of exon shuffling [81], symmetrical
exons and their flanking introns can be deleted, duplicated and inserted into introns of the same phase class
without causing frame shifts. Thus, symmetrical exons
flanked by introns of a single phase class tend to predominate in genes that largely evolved by exon shuffling and
their nonrandom usage may be indicative of gene assembly by exon recruitment [38,78]. An intriguing feature of
IQD gene organization in Arabidopsis and rice is the
almost exclusive presence of symmetrical exons flanked
by phase-0 introns (Figure 1). The strong bias for one
intron phase class and the variation in the number of
exons (2–6), and consequently size of the encoded proteins, is consistent with exon shuffling during the evolution of IQD genes. Exon shuffling is also suggested by the
comparisons of patterns of protein motifs (Figure 3) and
by the phylogenetic analysis of IQD full-length proteins
and IQ67 domains, which indicate that phylogenetic relationships based on the IQ67 domain do not necessarily
recapitulate patterns of protein and gene structure (Figures 5 and 6). Putative exon shuffling events may be recognized in some of the IQD gene structures. For example,
At5g35670 and Os06m03925 encode a partial IQ67
domain and may have experienced exon swapping, or
At4g10640 may have acquired its penultimate exon when
compared with At3g49380 of the same subgroup (Figure
1). Exon shuffling may have played a prominent role in
the diversification of IQD genes and their hitherto
unknown functions. The above-mentioned gene families
of transcription factors [55,56,67] contain introns of
mixed phase classes, suggesting that exon shuffling played
only a minor role during the evolution of these proteins
with relatively defined functions. On the other hand, for
example, all introns of genes coding for CIPKs are in
phase-0 [16]. The exclusive usage of one phase class may
indicate exon shuffling to generate the domain diversity

Page 20 of 25
(page number not for citation purposes)

BMC Evolutionary Biology 2005, 5:72

necessary for kinase regulation and the ability to recognize a wide spectrum of protein substrates.
Potential roles for IQD proteins
We have recently identified At3g09710 (IQD1) in a screen
for Arabidopsis mutants with altered glucosinolate accumulation [37]. Glucosinolates are synthesized mainly by
cruciferous species and constitute a class of secondary
metabolites with roles in plant defense against pathogens
and herbivores [35]. Characterization of gain- and loss-offunction alleles of IQD1 demonstrated that the encoded
protein functions as a modulator of glucosinolate pathway-related gene expression. Tissue-specific expression of
IQD1 is consistent with glucosinolate accumulation and
mainly confined to the vascular tissues. We further demonstrated that an IQD1-GFP fusion protein is targeted to
the cell nucleus and that recombinant IQD1 interacts with
calmodulin in a Ca2+-dependent fashion [37]. It is therefore intriguing to hypothesize that IQD1 integrates intracellular Ca2+ signals elicited by environmental cues such
as herbivorous attack to fine-tune glucosinolate synthesis
and accumulation. It should be pointed out that the rice
genome does not contain an ortholog of At3g09710 (Figure 6), which is consistent with the absence of the glucosinolate pathway in this species and with functional
diversification of the Arabidopsis and rice IQD gene families.

We are left to speculate on the biochemical and cellular
functions of IQD proteins. One of the most intriguing features of IQD proteins is their high isoelectric point
(~10.3), which has been maintained irrespective of protein size variation and domain composition, except for
one family member each in Arabidopsis and rice. This
observation suggests that the basic nature of IQD proteins
is important for their biochemical functions. Although
IQD proteins do not contain currently known DNA- or
RNA-binding motifs, the basic isoelectric point and high
frequency of serine residues, which are reminiscent of certain splicing factors [82], suggest that IQD proteins may
associate with nucleic acids and regulate gene expression
at the transcriptional or post-transcriptional level. Interestingly, we have recently observed that Arabidopsis IQD1
binds to nucleic acids (T. Savchenko, B. Zipp and S. Abel,
unpublished results). A regulatory role for IQD proteins is
also suggested by the relatively high fraction of retained
duplicated IQD genes in the Arabidopsis genome. Preferential retention of paralogous gene pairs is thought to
counteract disturbances in gene dosage and stoichiometric ratios of regulatory protein complexes after large-scale
segmental duplication events and the onset of gene inactivation and loss of gene duplicates [44]. In this context, it
is interesting to point out that the multiple Ca2+-dependent and Ca2+-independent calmodulin recruitment motifs
of the IQ67 domains are likely involved in specific and

http://www.biomedcentral.com/1471-2148/5/72

cooperative interactions with calmodulins or calmodulinlike proteins. These interactions may dramatically alter
the dynamic range of Ca2+-binding kinetics and, in turn,
modulate interactions of the oligomeric protein complex
with additional target proteins [31,83]. Many, if not most,
members of the Arabidopsis and rice IQD protein families
are likely to function in the cell nucleus (Tables 1 and 2).
There is increasing evidence for the generation of nucleusspecific Ca2+-signatures in plant cells [1,84-86] and for a
potential regulatory role of calmodulin and related Ca2+
sensor proteins in nuclear processes such as transcription
or gene silencing [9,60,61,87-90].

Conclusion
We have systematically identified and characterized by
bioinformatics a novel family of putative calmodulin target proteins in two model plant species, Arabidopsis thaliana and Oryza sativa. Our phylogenetic analyses indicate
that the major IQD gene lineages originated before the
monocot-eudicot divergence and that the expansion of
the IQD gene family in the genomes of Arabidopsis and
rice is consistent with a recent polyploidization and aneuploidization event, respectively. The extant IQD loci in
Arabidopsis primarily resulted from segmental duplication and reflect preferential retention of paralogous genes,
which is characteristic for proteins with regulatory functions. The almost exclusive usage of phase-0 introns and
variable number of exons suggests a role for exon shuffling during the diversification of IQD proteins, which is
also supported by phylogenetic relationships between the
IQ67 domain and full-length IQD proteins. The unusually basic isoelectric point of IQD proteins and their frequently predicted nuclear localization suggest that IQD
proteins link calcium signaling pathways to the regulation
of gene expression. Our study provides a framework for
the functional dissections of this emerging family of putative calmodulin target proteins.

Methods
Identification of IQD genes
To identify members of the Arabidopsis thaliana IQD protein family, multiple database searches were performed
using the Basic Local Alignment Search Tool (BLAST
[91,92]) algorithms BLASTP and TBLASTN available on
the National Center of Biotechnology Information
(NCBI) and The Arabidopsis Information Resource
(TAIR) databases [93-95]. We used the amino acid
sequence of IQD1 and of its IQ67 domain as initial query
sequences, followed by the amino acid sequences of other
IQD family members. Amino acid sequence pattern
searches were performed on the TAIR website using Patmatch. Arabidopsis nucleotide and protein sequences as
well as information regarding the gene structure were
obtained from the Munich Information Center for Protein
Sequences (MIPS) Arabidopsis thaliana Database (MATDB)

Page 21 of 25
(page number not for citation purposes)

BMC Evolutionary Biology 2005, 5:72

[96], The Institute for Genomic Research (TIGR) Arabidopsis thaliana Database [74], and the Arabidopsis thaliana
Plant Genome Database (AtPGD) [97]. To identify members of the rice (Oryza sativa) IQD protein family
(OsIQD), we searched four different databases using the
same BLAST algorithms. Sequences for O. sativa
ssp.japonica were retrieved from the database at the TIGR
Rice Genome Project [74]. Genomic sequences for ssp.
japonica and ssp. indica were also obtained from the GenBank database containing the results of the International
Rice Genome Sequencing Project and the draft rice
genome sequence of the Chinese Academy of Sciences
[53,93]. Rice full-length cDNA and EST sequences were
searched in the Knowledge-based Oryza Molecular biological Encyclopedia (KOME) at the National Institute of
Agrobiological Sciences [98] and in the TIGR Gene Indices
[74]. Nucleotide and amino acid sequences as well as gene
structure and chromosomal duplications were obtained
from the same databases mentioned above. Genomic
sequences that appeared to be misannotated by comparison with available cDNA sequences (full-length cDNAs,
ESTs) were corrected for subsequent analysis. Sequences
encoding putative IQD proteins in Pinus ssp. and Physcomitrella patens were identified by BLAST searches of the
TIGR Gene Indices [74] and of the moss database NIBB
PHYSCObase [99].
Chromosomal duplication in the Arabidopsis genome
For the detection of large segmental duplications, we used
the redundancy viewer at the MATDB [96], the duplicated
blocks map provided by TIGR [74], the interactive supplementary material by Simillion et al. [48], and the interactive maps of duplicated blocks in Arabidopsis by Blanc et
al. [45].
Computational analysis of IQD proteins
The amino acid sequences of all IQD proteins were analyzed for physico-chemical parameters (ProtParam) and
predicted subcellular localization (PSORT, TargetP) on
the ExPASy Proteomics Server [100]. MEME (Multiple
Expectation Maximization for Motif Elicitation) was used
to identify conserved motif structures among IQD protein
sequences [39]. Putative calmodulin-binding sites in IQD
protein sequences were predicted by the Calmodulin Target Database [40].
Alignment and phylogenetic analysis of IQD sequences
Multiple alignments of amino acid sequences were performed using ClustalW [101] or ClustalX [102] and were
manually corrected. For generating the phylogenetic trees
of full-length IQD protein sequences reported in Figures
1, 2 and 5, we used ClustalX (1.81) and the neighbor-joining algorithm [42]. Bootstrap analysis with 1,000 replicates was used to evaluate the significance of the nodes.
The trees of the Arabidopsis and rice IQD protein families

http://www.biomedcentral.com/1471-2148/5/72

were rooted using each atypical protein containing a truncated IQ67 domain as an outgroup; an unrooted tree is
shown for the combined analysis of all Arabidopsis and
rice IQD proteins (Figure 6). For the creation of the
unrooted phylogenetic tree of IQ67 domain sequences in
Figure 7, we used in addition the PAUP*4.0 (b10) program to perform distance and parsimony analyses [103].
The same program was used for subsequent bootstrap
analysis with 1,000 replicates to evaluate tree topology.
cDNA cloning
The identification and cloning of a full-length cDNA for
At3g09710 has been described previously [37]. Using
similar conditions for reverse transcriptase-mediated PCR,
we amplified predicted full-length cDNA sequences for

At1g17480
GGATT-3';

(forward:

5'-ATGGGTGGGTCAGGAAATT-

reverse: 5'-TTAGCTTCGCTGGCTCTTGG-3'),
At1g18840 (forward: 5'-ATGGGAAAGCCTGCAAGGTG3';
reverse: 5'-TAACCGTTTCCTTCTCGGGACGA-3'), and
At4g23060 (forward: 5'-ATGGGAAAAGCGTCCCGGTGGTT-3';
reverse: 5'-TCAGTACCTATACCCAATTGGCATCC-3').
The resulting PCR products were subcloned into the vector pGEMT (Promega, Madison, WI) by TA cloning followed by DNA sequencing of the insert with T7 and SP6
primers.
Expression of AtIQD20 and calmodulin binding assay
A full-length cDNA fragment encoding the predicted
IQD20 protein of Arabidopsis was generated by RT-PCR
using gene-specific primers

At3g51380 (forward: 5'-CGCGGATCCATGGCCAACTCCAAACGTTTG-3') and At3g51380 (reverse: 5'-GAGGAATTCTTAATGAGAGAG-3'). The PCR fragment was
subcloned into the BamHI and EcoRI sites of vector
pET21a (Novagen, Madison, WI, USA), which provides an
N-terminal T7-epitope tag. Expression of recombinant T7IQD20 and calmodulin-binding assays using calmodulinagarose beads (phosphodiesterase-3':5'-cyclic nucleotide
activator from bovine brain; Sigma-Aldrich, St. Louis,
MO, USA) were performed as previously described [37].

Authors' contributions
SA carried out most of the bioinformatics analyses and
wrote the entire manuscript. TS demonstrated calmodulin

Page 22 of 25
(page number not for citation purposes)

BMC Evolutionary Biology 2005, 5:72

binding of IQD20. TS and ML contributed to data collection and IQD sequence analysis.

Acknowledgements
We thank Carla Ticconi and Raymond Kwong for critical reading of the
manuscript. This work was supported by the National Research Initiative of
the United States Department of Agriculture Cooperative State Research,
Education and Extension Service to S.A. (grant number 2005-02507).

References
1.
2.
3.
4.
5.
6.
7.
8.
9.

10.
11.
12.
13.
14.
15.

16.

17.
18.
19.

20.
21.
22.

23.

Rudd JJ, Franklin-Tong VE: Unravelling response-specificity in
Ca2+-signaling pathways in plant cells. New Phytologist 2001,
151:7-33.
Evans NH, McAinsh MR, Hetherington AM: Calcium oscillations in
higher plants. Curr Opin Plant Biol 2001, 4(5):415-420.
Harper JF: Dissecting calcium oscillators in plant cells. Trends
Plant Sci 2001, 6(9):395-397.
Scrase-Field SA, Knight MR: Calcium: just a chemical switch?
Curr Opin Plant Biol 2003, 6(5):500-506.
Knight H, Knight MR: Abiotic stress signalling pathways: specificity and cross-talk. Trends Plant Sci 2001, 6(6):262-267.
Snedden WA, Fromm H: Calmodulin as a versatile calcium signal transducer in plants. New Phytol 2001, 151:35-66.
Sanders D, Pelloux J, Brownlee C, Harper JF: Calcium at the crossroads of signaling. Plant Cell 2002, 14 Suppl:S401-17.
Reddy VS, Reddy AS: Proteomics of calcium-signaling components in plants. Phytochemistry 2004, 65(12):1745-1776.
Luan S, Kudla J, Rodriguez-Concepcion M, Yalovsky S, Gruissem W:
Calmodulins and calcineurin B-like proteins: calcium sensors
for specific signal response coupling in plants. Plant Cell 2002,
14 Suppl:S389-400.
Yang T, Poovaiah BW: Calcium/calmodulin-mediated signal
network in plants. Trends Plant Sci 2003, 8(10):505-512.
Bouche N, Yellin A, Snedden WA, Fromm H: Plant-Specific Calmodulin-Binding Proteins. Annu Rev Plant Biol 2005, 56:435-466.
Day IS, Reddy VS, Shad Ali G, Reddy AS: Analysis of EF-hand-containing proteins in Arabidopsis.
Genome Biol 2002,
3(10):RESEARCH0056.
McCormack E, Braam J: Calmodulin and related potential calcium sensors of Arabidopsis. New Phytol 2003, 159:585-598.
McCormack E, Tsai YC, Braam J: Handling calcium signaling:
Arabidopsis CaMs and CMLs.
Trends Plant Sci 2005,
10(8):383-389.
Kudla J, Xu Q, Harter K, Gruissem W, Luan S: Genes for calcineurin B-like proteins in Arabidopsis are differentially regulated by stress signals. Proc Natl Acad Sci U S A 1999,
96(8):4718-4723.
Kolukisaoglu U, Weinl S, Blazevic D, Batistic O, Kudla J: Calcium
sensors and their interacting protein kinases: genomics of
the Arabidopsis and rice CBL-CIPK signaling networks. Plant
Physiol 2004, 134(1):43-58.
Batistic O, Kudla J: Integration and channeling of calcium signaling through the CBL calcium sensor/CIPK protein kinase
network. Planta 2004, 219(6):915-924.
Harmon AC, Gribskov M, Harper JF: CDPKs - a kinase for every
Ca2+ signal? Trends Plant Sci 2000, 5(4):154-159.
Hrabak EM, Chan CW, Gribskov M, Harper JF, Choi JH, Halford N,
Kudla J, Luan S, Nimmo HG, Sussman MR, Thomas M, Walker-Simmons K, Zhu JK, Harmon AC: The Arabidopsis CDPK-SnRK
superfamily of protein kinases.
Plant Physiol 2003,
132(2):666-680.
Sheen J: Ca2+-dependent protein kinases and stress signal
transduction in plants. Science 1996, 274(5294):1900-1902.
Romeis T, Ludwig AA, Martin R, Jones JD: Calcium-dependent
protein kinases play an essential role in a plant defence
response. Embo J 2001, 20(20):5556-5567.
Shi J, Kim KN, Ritz O, Albrecht V, Gupta R, Harter K, Luan S, Kudla
J: Novel protein kinases associated with calcineurin B-like
calcium sensors in Arabidopsis.
Plant Cell 1999,
11(12):2393-2405.
Halfter U, Ishitani M, Zhu JK: The Arabidopsis SOS2 protein
kinase physically interacts with and is activated by the cal-

http://www.biomedcentral.com/1471-2148/5/72

24.
25.
26.

27.
28.
29.

30.

31.
32.
33.

34.
35.
36.

37.

38.
39.
40.
41.
42.
43.
44.
45.
46.
47.
48.

cium-binding protein SOS3. Proc Natl Acad Sci U S A 2000,
97(7):3735-3740.
Kim KN, Cheong YH, Gupta R, Luan S: Interaction specificity of
Arabidopsis calcineurin B-like calcium sensors and their target kinases. Plant Physiol 2000, 124(4):1844-1853.
Zhu JK: Regulation of ion homeostasis under salt stress. Curr
Opin Plant Biol 2003, 6(5):441-445.
Pandey GK, Cheong YH, Kim KN, Grant JJ, Li L, Hung W, D'Angelo
C, Weinl S, Kudla J, Luan S: The calcium sensor calcineurin Blike 9 modulates abscisic acid sensitivity and biosynthesis in
Arabidopsis. Plant Cell 2004, 16(7):1912-1924.
Zielinski RE: Calmodulin And Calmodulin-Binding Proteins In
Plants. Annu Rev Plant Physiol Plant Mol Biol 1998, 49:697-725.
Zhang L, Lu YT: Calmodulin-binding protein kinases in plants.
Trends Plant Sci 2003, 8(3):123-127.
Reddy AS, Day IS, Narasimhulu SB, Safadi F, Reddy VS, Golovkin M,
Harnly MJ: Isolation and characterization of a novel calmodulin-binding protein from potato.
J Biol Chem 2002,
277(6):4206-4214.
Osawa M, Swindells MB, Tanikawa J, Tanaka T, Mase T, Furuya T,
Ikura M: Solution structure of calmodulin-W-7 complex: the
basis of diversity in molecular recognition. J Mol Biol 1998,
276(1):165-176.
Hoeflich KP, Ikura M: Calmodulin in action: diversity in target
recognition and activation mechanisms.
Cell 2002,
108(6):739-742.
Bahler M, Rhoads A: Calmodulin signaling via the IQ motif.
FEBS Lett 2002, 513(1):107-113.
Choi JY, Lee SH, Park CY, Heo WD, Kim JC, Kim MC, Chung WS,
Moon BC, Cheong YH, Kim CY, Yoo JH, Koo JC, Ok HM, Chi SW,
Ryu SE, Lee SY, Lim CO, Cho MJ: Identification of calmodulin isoform-specific binding peptides from a phage-displayed random 22-mer peptide library.
J Biol Chem 2002,
277(24):21630-21638.
Rhoads AR, Friedberg F: Sequence motifs for calmodulin recognition. Faseb J 1997, 11(5):331-340.
Wittstock U, Halkier BA: Glucosinolate research in the Arabidopsis era. Trends Plant Sci 2002, 7(6):263-270.
Dudareva N, Evrard JL, Pillay DT, Steinmetz A: Nucleotide
sequence of a pollen-specific cDNA from Helianthus annuus
L. encoding a highly basic protein.
Plant Physiol 1994,
106(1):403-404.
Levy M, Wang Q, Kaspi R, Parrella MP, Abel S: Arabidopsis IQD1,
a novel calmodulin-binding nuclear protein, stimulates glucosinolate accumulation and plant defense. Plant J 2005,
43(1):79-96.
Liu M, Grigoriev A: Protein domains correlate strongly with
exons in multiple eukaryotic genomes--evidence of exon
shuffling? Trends Genet 2004, 20(9):399-403.
Bailey TL, Elkan C: The value of prior knowledge in discovering
motifs with MEME. Proc Int Conf Intell Syst Mol Biol 1995, 3:21-29.
Yap KL, Kim J, Truong K, Sherman M, Yuan T, Ikura M: Calmodulin
target database. J Struct Funct Genomics 2000, 1(1):8-14.
Abel S, Theologis A: A polymorphic bipartite motif signals
nuclear targeting of early auxin-inducible proteins related to
PS-IAA4 from pea (Pisum sativum). Plant J 1995, 8(1):87-96.
Saitou N, Nei M: The neighbor-joining method: a new method
for reconstructing phylogenetic trees. Mol Biol Evol 1987,
4(4):406-425.
Blanc G, Wolfe KH: Widespread paleopolyploidy in model
plant species inferred from age distributions of duplicate
genes. Plant Cell 2004, 16(7):1667-1678.
Blanc G, Wolfe KH: Functional divergence of duplicated genes
formed by polyploidy during Arabidopsis evolution. Plant Cell
2004, 16(7):1679-1691.
Blanc G, Hokamp K, Wolfe KH: A recent polyploidy superimposed on older large-scale duplications in the Arabidopsis
genome. Genome Res 2003, 13(2):137-144.
Blanc G, Barakat A, Guyot R, Cooke R, Delseny M: Extensive duplication and reshuffling in the Arabidopsis genome. Plant Cell
2000, 12(7):1093-1101.
Vision TJ, Brown DG, Tanksley SD: The origins of genomic duplications in Arabidopsis. Science 2000, 290(5499):2114-2117.
Simillion C, Vandepoele K, Van Montagu MC, Zabeau M, Van de Peer
Y: The hidden duplication past of Arabidopsis thaliana. Proc
Natl Acad Sci U S A 2002, 99(21):13627-13632.

Page 23 of 25
(page number not for citation purposes)

BMC Evolutionary Biology 2005, 5:72

49.

50.

51.
52.

53.

54.
55.
56.
57.

58.
59.

60.
61.
62.
63.

64.

65.

Bowers JE, Chapman BA, Rong J, Paterson AH: Unravelling
angiosperm genome evolution by phylogenetic analysis of
chromosomal
duplication
events.
Nature
2003,
422(6930):433-438.
Ziolkowski PA, Blanc G, Sadowski J: Structural divergence of
chromosomal segments that arose from successive duplication events in the Arabidopsis genome. Nucleic Acids Res 2003,
31(4):1339-1350.
Vandepoele K, Simillion C, Van de Peer Y: Evidence that rice and
other cereals are ancient aneuploids.
Plant Cell 2003,
15(9):2192-2202.
Goff SA, Ricke D, Lan TH, Presting G, Wang R, Dunn M, Glazebrook
J, Sessions A, Oeller P, Varma H, Hadley D, Hutchison D, Martin C,
Katagiri F, Lange BM, Moughamer T, Xia Y, Budworth P, Zhong J,
Miguel T, Paszkowski U, Zhang S, Colbert M, Sun WL, Chen L,
Cooper B, Park S, Wood TC, Mao L, Quail P, Wing R, Dean R, Yu Y,
Zharkikh A, Shen R, Sahasrabudhe S, Thomas A, Cannings R, Gutin A,
Pruss D, Reid J, Tavtigian S, Mitchell J, Eldredge G, Scholl T, Miller RM,
Bhatnagar S, Adey N, Rubano T, Tusneem N, Robinson R, Feldhaus J,
Macalma T, Oliphant A, Briggs S: A draft sequence of the rice
genome (Oryza sativa L. ssp. japonica). Science 2002,
296(5565):92-100.
Yu J, Hu S, Wang J, Wong GK, Li S, Liu B, Deng Y, Dai L, Zhou Y,
Zhang X, Cao M, Liu J, Sun J, Tang J, Chen Y, Huang X, Lin W, Ye C,
Tong W, Cong L, Geng J, Han Y, Li L, Li W, Hu G, Huang X, Li W, Li
J, Liu Z, Li L, Liu J, Qi Q, Liu J, Li L, Li T, Wang X, Lu H, Wu T, Zhu
M, Ni P, Han H, Dong W, Ren X, Feng X, Cui P, Li X, Wang H, Xu X,
Zhai W, Xu Z, Zhang J, He S, Zhang J, Xu J, Zhang K, Zheng X, Dong
J, Zeng W, Tao L, Ye J, Tan J, Ren X, Chen X, He J, Liu D, Tian W,
Tian C, Xia H, Bao Q, Li G, Gao H, Cao T, Wang J, Zhao W, Li P,
Chen W, Wang X, Zhang Y, Hu J, Wang J, Liu S, Yang J, Zhang G,
Xiong Y, Li Z, Mao L, Zhou C, Zhu Z, Chen R, Hao B, Zheng W, Chen
S, Guo W, Li G, Liu S, Tao M, Wang J, Zhu L, Yuan L, Yang H: A draft
sequence of the rice genome (Oryza sativa L. ssp. indica). Science 2002, 296(5565):79-92.
Toledo-Ortiz G, Huq E, Quail PH: The Arabidopsis basic/helixloop-helix transcription factor family.
Plant Cell 2003,
15(8):1749-1770.
Lijavetzky D, Carbonero P, Vicente-Carbajosa J: Genome-wide
comparative phylogenetic analysis of the rice and Arabidopsis Dof gene families. BMC Evol Biol 2003, 3(1):17.
Reyes JC, Muro-Pastor MI, Florencio FJ: The GATA family of transcription factors in Arabidopsis and rice. Plant Physiol 2004,
134(4):1718-1732.
Kohler C, Merkle T, Neuhaus G: Characterisation of a novel
gene family of putative cyclic nucleotide- and calmodulinregulated ion channels in Arabidopsis thaliana. Plant J 1999,
18(1):97-104.
Reddy AS, Day IS: Analysis of the myosins encoded in the
recently completed Arabidopsis thaliana genome sequence.
Genome Biol 2001, 2(7):RESEARCH0024.
Reddy AS, Reddy VS, Golovkin M: A calmodulin binding protein
from Arabidopsis is induced by ethylene and contains a
DNA-binding motif.
Biochem Biophys Res Commun 2000,
279(3):762-769.
Yang T, Poovaiah BW: A calmodulin-binding/CGCG box DNAbinding protein family involved in multiple signaling pathways in plants. J Biol Chem 2002, 277(47):45049-45058.
Bouche N, Scharlat A, Snedden W, Bouchez D, Fromm H: A novel
family of calmodulin-binding transcription activators in multicellular organisms. J Biol Chem 2002, 277(24):21851-21861.
Hedges SB: The origin and evolution of model organisms. Nat
Rev Genet 2002, 3(11):838-849.
Bowe LM, Coat G, dePamphilis CW: Phylogeny of seed plants
based on all three genomic compartments: extant gymnosperms are monophyletic and Gnetales' closest relatives are
conifers. Proc Natl Acad Sci U S A 2000, 97(8):4092-4097.
Yang YW, Lai KN, Tai PY, Li WH: Rates of nucleotide substitution in angiosperm mitochondrial DNA sequences and dates
of divergence between Brassica and other angiosperm lineages. J Mol Evol 1999, 48(5):597-604.
Ermolaeva MD, Wu M, Eisen JA, Salzberg SL: The age of the Arabidopsis thaliana genome duplication. Plant Mol Biol 2003,
51(6):859-866.

http://www.biomedcentral.com/1471-2148/5/72

66.
67.
68.
69.
70.

71.
72.
73.
74.
75.

76.
77.
78.
79.
80.

81.
82.

83.
84.
85.
86.
87.

88.

89.

90.

Raes J, Vandepoele K, Simillion C, Saeys Y, Van de Peer Y: Investigating ancient duplication events in the Arabidopsis
genome. J Struct Funct Genomics 2003, 3(1-4):117-129.
Remington DL, Vision TJ, Guilfoyle TJ, Reed JW: Contrasting
modes of diversification in the Aux/IAA and ARF gene families. Plant Physiol 2004, 135(3):1738-1752.
Tian C, Wan P, Sun S, Li J, Chen M: Genome-wide analysis of the
GRAS gene family in rice and Arabidopsis. Plant Mol Biol 2004,
54(4):519-532.
Cannon SB, Young ND: OrthoParaMap: distinguishing
orthologs from paralogs by integrating comparative genome
data and gene phylogenies. BMC Bioinformatics 2003, 4(1):35.
Birchler JA, Bhadra U, Bhadra MP, Auger DL: Dosage-dependent
gene regulation in multicellular eukaryotes: implications for
dosage compensation, aneuploid syndromes, and quantitative traits. Dev Biol 2001, 234(2):275-288.
Bancroft I: Insights into cereal genomes from two draft
genome sequences of rice.
Genome Biol 2002,
3(6):REVIEWS1015.
Paterson AH, Bowers JE, Peterson DG, Estill JC, Chapman BA:
Structure and evolution of cereal genomes. Curr Opin Genet
Dev 2003, 13(6):644-650.
Simillion C, Vandepoele K, Saeys Y, Van de Peer Y: Building
genomic profiles for uncovering segmental homology in the
twilight zone. Genome Res 2004, 14(6):1095-1106.
The Institute for Genomic Research (TIGR)
[http://
www.tigr.org]
Wortman JR, Haas BJ, Hannick LI, Smith RK Jr., Maiti R, Ronning CM,
Chan AP, Yu C, Ayele M, Whitelaw CA, White OR, Town CD:
Annotation of the Arabidopsis genome. Plant Physiol 2003,
132(2):461-468.
Meyers BC, Kozik A, Griego A, Kuang H, Michelmore RW:
Genome-wide analysis of NBS-LRR-encoding genes in Arabidopsis. Plant Cell 2003, 15(4):809-834.
AGI: Analysis of the genome sequence of the flowering plant
Arabidopsis thaliana. Nature 2000, 408(6814):796-815.
Patthy L: Genome evolution and the evolution of exon-shuffling--a review. Gene 1999, 238(1):103-114.
Long M: Evolution of novel genes. Curr Opin Genet Dev 2001,
11(6):673-680.
de Souza SJ, Long M, Klein RJ, Roy S, Lin S, Gilbert W: Toward a resolution of the introns early/late debate: only phase zero
introns are correlated with the structure of ancient proteins.
Proc Natl Acad Sci U S A 1998, 95(9):5094-5099.
Patthy L: Intron-dependent evolution: preferred types of
exons and introns. FEBS Lett 1987, 214(1):1-7.
Chaudhary N, McMahon C, Blobel G: Primary structure of a
human arginine-rich nuclear protein that colocalizes with
spliceosome components. Proc Natl Acad Sci U S A 1991,
88(18):8189-8193.
Putkey JA, Kleerekoper Q, Gaertner TR, Waxham MN: A new role
for IQ motif proteins in regulating calmodulin function. J Biol
Chem 2003, 278(50):49667-49670.
van Der Luit AH, Olivari C, Haley A, Knight MR, Trewavas AJ: Distinct calcium signaling pathways regulate calmodulin gene
expression in tobacco. Plant Physiol 1999, 121(3):705-714.
Pauly N, Knight MR, Thuleau P, van der Luit AH, Moreau M, Trewavas
AJ, Ranjeva R, Mazars C: Control of free calcium in plant cell
nuclei. Nature 2000, 405(6788):754-755.
Xiong TC, Jauneau A, Ranjeva R, Mazars C: Isolated plant nuclei as
mechanical and thermal sensors involved in calcium signalling. Plant J 2004, 40(1):12-21.
Anandalakshmi R, Marathe R, Ge X, Herr JM Jr., Mau C, Mallory A,
Pruss G, Bowman L, Vance VB: A calmodulin-related protein
that suppresses posttranscriptional gene silencing in plants.
Science 2000, 290(5489):142-144.
Du L, Poovaiah BW: A novel family of Ca2+/calmodulin-binding
proteins involved in transcriptional regulation: interaction
with fsh/Ring3 class transcription activators. Plant Mol Biol
2004, 54(4):549-569.
Perruc E, Charpenteau M, Ramirez BC, Jauneau A, Galaud JP, Ranjeva
R, Ranty B: A novel calmodulin-binding protein functions as a
negative regulator of osmotic stress tolerance in Arabidopsis thaliana seedlings. Plant J 2004, 38(3):410-420.
Yoo JH, Park CY, Kim JC, Heo WD, Cheong MS, Park HC, Kim MC,
Moon BC, Choi MS, Kang YH, Lee JH, Kim HS, Lee SM, Yoon HW,

Page 24 of 25
(page number not for citation purposes)

BMC Evolutionary Biology 2005, 5:72

91.
92.

93.
94.
95.

96.
97.
98.
99.
100.
101.

102.

103.
104.
105.

106.

Lim CO, Yun DJ, Lee SY, Chung WS, Cho MJ: Direct interaction
of a divergent CaM isoform and the transcription factor,
MYB2, enhances salt tolerance in arabidopsis. J Biol Chem
2005, 280(5):3697-3706.
Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ: Basic local
alignment search tool. J Mol Biol 1990, 215(3):403-410.
Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ: Gapped BLAST and PSI-BLAST: a new generation of
protein database search programs. Nucleic Acids Res 1997,
25(17):3389-3402.
National Center of Biotechnology Information (NCBI)
[http://www.ncbi.nlm.nih.gov]
The Arabidopsis Information Resource (TAIR)
[http://
www.arabidopsis.org]
Rhee SY, Beavis W, Berardini TZ, Chen G, Dixon D, Doyle A, GarciaHernandez M, Huala E, Lander G, Montoya M, Miller N, Mueller LA,
Mundodi S, Reiser L, Tacklind J, Weems DC, Wu Y, Xu I, Yoo D,
Yoon J, Zhang P: The Arabidopsis Information Resource
(TAIR): a model organism database providing a centralized,
curated gateway to Arabidopsis biology, research materials
and community. Nucleic Acids Res 2003, 31(1):224-228.
Munich Information Center for Protein Sequences (MIPS)
Arabidopsis thaliana Database (MATDB) [http://mips.gsf.de/
proj/thal/db/]
Arabidopsis thaliana Plant Genome Database (AtPGD)
[http://www.plantgdb.org]
Knowledge-based Oryza Molecular biological Encyclopedia
(KOME) [http://cdna01.dna.affrc.go.jp/cDNA/]
PHYSCObase [http://moss.nibb.ac.jp]
ExPASy Proteomics Server [http://us.expasy.org/]
Thompson JD, Higgins DG, Gibson TJ: CLUSTAL W: improving
the sensitivity of progressive multiple sequence alignment
through sequence weighting, position-specific gap penalties
and weight matrix choice.
Nucleic Acids Res 1994,
22(22):4673-4680.
Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG: The
CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools.
Nucleic Acids Res 1997, 25(24):4876-4882.
Swofford D: PAUP*: Phylogenetic analysis using parsimony.
Sunderland, MA , Sinauer; 2000.
Talke IN, Blaudez D, Maathuis FJ, Sanders D: CNGCs: prime targets of plant cyclic nucleotide signalling? Trends Plant Sci 2003,
8(6):286-293.
Yamada K, Lim J, Dale JM, Chen H, Shinn P, Palm CJ, Southwick AM,
Wu HC, Kim C, Nguyen M, Pham P, Cheuk R, Karlin-Newmann G,
Liu SX, Lam B, Sakano H, Wu T, Yu G, Miranda M, Quach HL, Tripp
M, Chang CH, Lee JM, Toriumi M, Chan MM, Tang CC, Onodera CS,
Deng JM, Akiyama K, Ansari Y, Arakawa T, Banh J, Banno F, Bowser
L, Brooks S, Carninci P, Chao Q, Choy N, Enju A, Goldsmith AD,
Gurjal M, Hansen NF, Hayashizaki Y, Johnson-Hopson C, Hsuan VW,
Iida K, Karnes M, Khan S, Koesema E, Ishida J, Jiang PX, Jones T, Kawai
J, Kamiya A, Meyers C, Nakajima M, Narusaka M, Seki M, Sakurai T,
Satou M, Tamse R, Vaysberg M, Wallender EK, Wong C, Yamamura
Y, Yuan S, Shinozaki K, Davis RW, Theologis A, Ecker JR: Empirical
analysis of transcriptional activity in the Arabidopsis
genome. Science 2003, 302(5646):842-846.
Meyers BC, Vu TH, Tej SS, Ghazal H, Matvienko M, Agrawal V, Ning
J, Haudenschild CD: Analysis of the transcriptional complexity
of Arabidopsis thaliana by massively parallel signature
sequencing. Nat Biotechnol 2004, 22(8):1006-1011.

http://www.biomedcentral.com/1471-2148/5/72

Publish with Bio Med Central and every
scientist can read your work free of charge
"BioMed Central will be the most significant development for
disseminating the results of biomedical researc h in our lifetime."
Sir Paul Nurse, Cancer Research UK

Your research papers will be:
available free of charge to the entire biomedical community
peer reviewed and published immediately upon acceptance
cited in PubMed and archived on PubMed Central
yours — you keep the copyright

BioMedcentral

Submit your manuscript here:
http://www.biomedcentral.com/info/publishing_adv.asp

Page 25 of 25
(page number not for citation purposes)

</pre>
</body>
</html>