Copyright  2000 by the Genetics Society of America
Genetic Structure and Evolution of RAC-GTPases in Arabidopsis thaliana
Per Winge, Tore Brembu, Ralf Kristensen and Atle M. Bones
UNIGEN Center for Molecular Biology and Department of Botany, Norwegian University of Science
and Technology, N-7491 Trondheim, Norway
Manuscript received February 29, 2000
Accepted for publication August 4, 2000
ABSTRACT
Rho GTPases regulate a number of important cellular functions in eukaryotes, such as organization of
the cytoskeleton, stress-induced signal transduction, cell death, cell growth, and differentiation. We have
conducted an extensive screening, characterization, and analysis of genes belonging to the Ras superfamily
of GTPases in land plants (embryophyta) and found that the Rho family is composed mainly of proteins
with homology to RAC-like proteins in terrestrial plants. Here we present the genomic and cDNA sequences
of the RAC gene family from the plant Arabidopsis thaliana. On the basis of amino acid alignments and
genomic structure comparison of the corresponding genes, the 11 encoded AtRAC proteins can be divided
into two distinct groups of which one group apparently has evolved only in vascular plants. Our phylogenetic
analysis suggests that the plant RAC genes underwent a rapid evolution and diversification prior to the
emergence of the embryophyta, creating a group that is distinct from rac/cdc42 genes in other eukaryotes.
In embryophyta, RAC genes have later undergone an expansion through numerous large gene duplications.
Five of these RAC duplications in Arabidopsis thaliana are reported here. We also present an hypothesis
suggesting that the characteristic RAC proteins in higher plants have evolved to compensate the loss of
RAS proteins.
T
HE Rho family of GTPases, Rho, Rac, and Cdc42,
is a diverse group of proteins with an evolutionary
history dating back to the first unicellular eukaryotic
cells. Proteins of the Rho family are found in protists
as well as in fungi, plants, and animals (Madaule and
Axel 1985; Yang and Watson 1993; Lohia and Samuelson 1996). When activated by external (or internal)
signals the Rho family GTPases are converted to a GTPbinding form that interacts with cellular target proteins
or effectors and produces a variety of cellular responses
(Hall 1998). These include organization of the actin
cytoskeleton, regulating programmed cell death, stressinduced signal transduction, and cell growth and differentiation by mediating signals from membrane-bound
receptors. The Rac proteins are important for selection
of the location of actin polymerization during membrane ruffling and lamellipodia formation in mammalian cells (Ridley et al. 1992). In the budding yeast
Saccharomyces cerevisiae, which do not have Rac proteins,
the Cdc42 protein plays an important role coordinating
actin-dependent morphogenetic processes such as bud
emergence, the formation of mating projections, and
pseudohyphal growth (Johnson 1999). Recent reports
indicate that Rac proteins in plants have related functions to their counterparts in yeast and animals (Xia et
al. 1996; Lin and Yang 1997). In addition, plant RAC
proteins have been suggested to regulate pollen tube
Corresponding author: Atle M. Bones, UNIGEN Center for Molecular
Biology and Department of Botany, Norwegian University of Science
and Technology, N-7491Trondheim, Norway.
Genetics 156: 1959–1971 (December 2000)
tip growth through targeted secretions of vesicles (Kost
et al. 1999; Li et al. 1999). Another function of Rac
proteins is their ability to regulate the activation of a
multicomponent plasma membrane-bound NADPHdependent oxidase that triggers the “oxidative burst”
(Abo et al. 1991). There is now also compelling evidence
that RAC proteins in plants have similar regulatory function and that they have a key role in the production
of reactive oxygen species that is associated with plant
defense against pathogens (Kawasaki et al. 1999; Potikha et al. 1999).
In mammalian cells an extended family of 14 Rho
GTPases has been identified that can be further divided
into seven distinct subgroups (Nobes and Hall 1994;
Aspenstrom 1999). In contrast only two distinct groups
of the Rho family GTPase have been found in higher
plants. Only one of these proteins (GenBank accession
no. U88402; Newman et al. 1994) can be described as
being a RHO-like homologue while the others represent
RAC proteins (Yang and Watson 1993; Delmer et al.
1995; Winge et al. 1997). Little is known about the
genomic structure of the members of the Rho GTPase
family in plants and how they have evolved through
time. With the now almost complete sequence of Arabidopsis thaliana plus the genomes from Saccharomyces cerevisiae, Caenorhabditis elegans, and Drosophila melanogaster,
a large genetic resource is available to compare and
analyze the evolution of complete gene families.
In this article we determine and analyze the genomic
sequences of 11 RAC-like genes from A. thaliana (AtRAC1-11), including 1 gene sequenced by the genome
1960
P. Winge et al.
project. The RAC genes analyzed probably constitute
the complete set of RAC genes from A. thaliana. All
these genes have also recently been sequenced by the
Arabidopsis genome project, thereby providing information about their chromosome location. Nine of the
AtRAC gene sequences from A. thaliana (L.) Heynh. cv.
Landsberg erecta and cv. Columbia have been compared, providing a rare opportunity to study the shortterm evolution within a gene family. We also present
results that show that the RAC proteins found in angiosperms can be divided into two distinct groups, a division
that must have taken place ⬎200 million years ago.
Finally our analysis of the plant RAC proteins from
higher and lower plants suggests that the ancestral RAC
genes underwent a rapid evolution before land plants
appeared.
MATERIALS AND METHODS
Isolation of genomic DNA: DNA was isolated from four A.
thaliana ecotypes, Columbia, Landsberg erecta, Wassilewskaja,
and Cape Verde, with a modified minipreparation procedure
adapted from Dellaporta et al. (1983). After ethanol precipitation, the pellet was dissolved in TE-buffer with 10 ␮g/ml
RNase A (Sigma-Aldrich, St. Louis) and incubated 1 hr at 37⬚.
mRNA isolation and cDNA synthesis: mRNA was isolated
from 5-week-old A. thaliana (var. Columbia) plants using the
Dynabeads mRNA DIRECT kit according to the manufacturer’s instructions (Dynal, Oslo). Random-primed first-strand
cDNA was made from ⵑ50 ng mRNA with a first-strand cDNA
synthesis kit (Pharmacia Biotech, Piscataway, NJ). After firststrand cDNA synthesis the samples were heat inactivated at
85⬚ for 5 min and stored at ⫺20⬚ until used as a template for
PCR (see below).
Amplification of AtRAC cDNA and genomic probes: Specific
AtRAC probes were amplified by PCR from both cDNA and
genomic DNA. PCR primers were designed from known AtRAC
cDNA sequences (Winge et al. 1997). The AtRAC1 probe was
amplified with the rac1f and rac1r primers (see below). The
AtRAC2 probe was amplified with the rac1f and Arac2C primers. The AtRAC9 probe was amplified with the A9f and A9r
primers. The probes from AtRAC7, AtRAC8, and AtRAC10 were
amplified with the rac2f and rac1R primers. The following
primers were used to amplify the AtRAC probes: Arac1F, 5⬘
TTG TTT CCT CAG GTT TTG TAG 3⬘; rac1f, 5⬘ AGG TTY
ATH AAG TGT GTS ACY GT 3⬘; rac1r, 5⬘ TCA AAI ACT SCY
TTC ACR TTC T 3⬘; Arac2C, 5⬘ TGA TCT CTT AGT CTT
CAA TGG T 3⬘; rac2f, 5⬘ GGK AAR ACI TGY ATG CTY ATY
TG 3⬘; A9f, 5⬘ GCA ACA TCA ACA TCA TCA GCA 3⬘; A9r,
5⬘ CTG GGA AGA TTG TGC AAG CA 3⬘ (Y, C/T; R, G/A; S,
G/C; K, G/T; H, not G; I, inosine). Buffers and reagents for
PCR were utilized according to standard procedures (Saiki et
al. 1988). AtRAC cDNA probes were amplified from ⵑ5 ng
first-strand cDNA, while genomic AtRAC probes were amplified from ⵑ50 ng genomic DNA. In general, the PCR was
performed at 94⬚ for 1 min, 50⬚ for 1 min, and 70⬚ for 1–2
min for a total of 40 cycles, using 1.5 units Amplitaq DNA
polymerase (Perkin-Elmer, Norwalk, CT). PCR products were
separated in low melt agarose gels (SeaPlaque GTG; FMC,
Rockland, ME), and excised DNA fragments were treated with
1 unit/50 ␮l agarase (Sigma) for 1 hr at 37⬚. The isolated
AtRAC PCR fragments, cDNA and genomic, were verified by
direct sequencing before they were used as probes.
Isolation of genomic AtRAC clones: A genomic lambda FIX
library from A. thaliana (L.) Heynh. cv. Landsberg erecta was
obtained from the Arabidopsis Biological Resource Center
(ABRC), Columbus, OH (Voytas et al. 1990). Plaque lifts and
phage work were conducted according to standard procedures
(Sambrook et al. 1989), using Hybond N membranes (Amersham, Uppsala, Sweden). The membranes were screened with
32
P-labeled AtRAC PCR probes, produced with the Megaprime
kit from Amersham/Pharmacia, Uppsala, Sweden. Approximately 300,000 plaque-forming units were screened with PCR
probes from AtRAC1, -2, and -7–10. Both cDNA and genomic
probes were used in four separate hybridizations, where one
or more probes were used in combination. Hybridizations
were done in 6⫻ SSC, 0.5% SDS at 56⬚ for 24 hr. Washes were
done with 1⫻ SSC, 0.1% SDS at 56⬚. Lambda DNA from
positive clones was prepared according to Sambrook et al.
(1989) and subcloned into pBluescript vectors (Stratagene,
La Jolla, CA).
DNA sequencing: DNA sequencing was performed manually
by dideoxy cycle sequencing (Murray 1989), with 33P-labeled
dideoxynucleotides and Thermo Sequenase (Amersham).
The AtRAC genomic clones were sequenced from derived
subclones and from PCR fragments amplified with degenerate
PCR primers matching conserved DNA motifs in the exons
(primers are not shown). Both DNA strands were sequenced
and gaps in the DNA sequences closed by primer walking.
Phylogenetic analyses: A RAC protein alignment was made
with the Clustal X program (Thompson et al. 1994, 1997) that
in addition to the plant RAC proteins also included sequences
of RAC proteins from various eukaryotes. This alignment was
manually refined with the GeneDoc program version 2.4.017
(http://www.cris.com/ⵑketchup/current.html) and the multiple sequence file was reimported to the Clustal X program.
Protein weight matrices of the PAM series were used to calculate the distances and an unrooted neighbor-joining (N-J) tree
was created using the neighbor-joining algorithm (Saitou
and Nei 1987). Bootstrapping of the N-J tree was done with
1000 bootstrap trials and the resulting tree file was imported
into the Treeview program (Page 1996).
An alignment of plant RAC cDNA sequence (including 17
ESTs) was produced with the GeneDoc program and a N-J
tree was produced from the multiple sequence file with the
Clustal X program. For comparison a parsimony analysis of
the DNA alignment was performed with Seqboot, Dnapars,
and Consense programs from the Phylip package version 3.5c
(Felsenstein 1984).
RESULTS
Cloning of AtRAC genes from A. thaliana: In a previous screen for cDNAs encoding RAC- and RHO-like
proteins, 10 A. thaliana cDNAs with high homologies to
human rac genes were found (Winge et al. 1997). To
find the corresponding genomic clones, an A. thaliana
Landsberg erecta genomic library was screened with
probes derived from AtRAC1, -2, and -7–10. Both cDNA
and genomic probes were used. After four separate library screens, a total of 63 genomic clones were isolated
and further characterized. All of the lambda clones were
verified by sequencing to contain AtRAC genes. Ten
different AtRAC genes were isolated in this process and
9 of them did contain full-length AtRAC genes. The
deduced protein sequences of the AtRAC genes are
shown in Figure 1. The AtRAC9 gene, which we had
found previously as a cDNA clone, was not found in
The Arabidopsis RAC Gene Family
1961
Figure 1.—A protein alignment of the AtRAC proteins. The two closest related pairs of AtRAC proteins, AtRAC1 and AtRAC6
and AtRAC4 and AtRAC5, are 98 and 97% identical, respectively. AtRAC9, the most divergent protein, is maximum 71% identical
with the other AtRAC proteins. GenBank accession nos. AtRAC1, AF115466; AtRAC2, AF115469; AtRAC3, AF115470; AtRAC4,
AF115471; AtRAC5, AF115472; AtRAC6, AF115473; AtRAC7, AF115474; AtRAC8, AF115475; AtRAC9, AF156896; AtRAC10,
AF115467; AtRAC11, AF085480.
these screens. The AtRAC9 gene was, however, recently
sequenced by the Arabidopsis genome initiative, GenBank accession no. AC003672. As the AtRAC7 from
Landsberg was partial, the full-length sequence was determined from several overlapping PCR fragments amplified from A. thaliana ecotype Columbia genomic DNA.
The AtRAC gene structure: The size of the AtRAC
coding regions ranged from 585 (AtRAC4) to 645 bp
(AtRAC10). The coding regions were interrupted by five
to seven introns and the first six splice sites were 100%
conserved for all but one gene, AtRAC11, which has lost
intron 5. Three of the genes, AtRAC7, -8, and -10, have
an extra exon at the 3⬘ end, which is probably the result
of the insertion of an intron in exon 7 of an ancestral
plant RAC gene (Figure 2, a and b). The intron sizes
are generally small, ranging from 71 to 474 bp, with an
average size of 140 bp. The first intron is usually the
largest, but in AtRAC1 and -6, intron 4 is the longest
(474 and 428 bp, respectively). The introns show little
or no sequence homology, except close to the splice
site junctions. One exception is the first intron of the
AtRAC8 and AtRAC10 genes, which contain a cryptic
exon of unknown function, GenBank accession no.
AF115468. The majority of the AtRAC introns do not
contain repetitive DNA, but some introns have small
stretches of AT repeats. A study of the splice sites shows
that most introns have common splice donor and acceptor sites. The exceptions are intron 2 in AtRAC3 and
intron 6 in AtRAC1, which both have a 5⬘ GC splice
donor. The 5⬘ GC splice donor sites are found only in
ⵑ1% of the A. thaliana introns (Brown and Simpson
1998). In most introns it is possible to identify motifs
1962
P. Winge et al.
Figure 2.—(a) Splice sites between exons 7 and 8 in AtRAC7, -8, and -10 genes.
The position of the splice donor site is identical for AtRAC8 and AtRAC10 but is shifted
3 bp for AtRAC7. Just a part of intron 7,
which lies in phase two, is shown. As discussed in the text, we think the RAC genes
in group II evolved after insertion of an
intron in the 3⬘ end of an ancient RAC-like
gene. (b) Alignment of the C-terminal part
of the AtRAC proteins with homologues
from Oryza sativa (OsRAC1, 2 and 3) and
Zea Mays (ZmRACA and RACC). The arrow
indicates the position in the CaaL box (a,
aliphatic amino acid) where the corresponding AtRAC7, -8, and -10 genes have
an intron inserted.
with similarity to branch point sequences that are located 15–60 bp upstream of the 3⬘ splice sites, (results
not shown). Together with AT-rich introns the branch
point sequences are important guide signals that ensure
the proper splicing of plant introns (Brown et al. 1996).
Comparison of AtRAC genes from Columbia and
Landsberg: Comparison of the AtRAC1 promoter from
the Columbia and Landsberg erecta ecotypes revealed
a 155-bp insertion/deletion (indel) 590–745 bp upstream of the start codon. This 155-bp indel, which
contains regions with repeated DNA, probably represents an insertion in the AtRAC1 Landsberg erecta promoter. To verify the indel, PCR primers flanking the
indel were used to amplify this promoter region from
four ecotypes, Columbia, Landsberg erecta, Wassilewskaja, and Cape Verde. The 155-bp insert was found in
both Landsberg erecta and Cape Verde while Columbia
and Wassilewskaja lacked the insert (results not shown).
The AtRAC8 gene has a repeated element inserted
ⵑ2.4 kb upstream of the start codon in Landsberg erecta
that is missing in the AtRAC8 promoter of Columbia
(results not shown). Repeated elements of this type are
found throughout the Arabidopsis genome (P. Winge,
personal observations). The AtRAC8 promoter has a
unique large repeat element ⵑ1290 bp in size that begins 331 bp upstream of the start codon. This repeat
element, registered in GenBank (accession no. AC003952),
has been found in several intergenic regions on all five
chromosomes and it is found in the AtRAC8 promoter
of both Landsberg erecta and Columbia.
To analyze the short-term evolution of the AtRAC
genes, pairwise comparisons of nine AtRAC genes from
Columbia and Landsberg erecta, AtRAC1–6, -8, -10, and
-11, were conducted. Seven of the genes, AtRAC3, -4, -5,
-6, -8, -10, and -11, were highly conserved between these
two ecotypes and few single nucleotide polymorphisms
(SNPs) and indels were found. The AtRAC3 gene had,
for instance, just one SNP in the 1792 bp analyzed, and
no polymorphisms were found in 1564 bp from the
AtRAC5 gene. In contrast, a 2091-bp sequence of the
AtRAC2 gene revealed 41 SNPs; 7 of these were located
in coding regions, but none resulted in amino acid
changes (Figure 3). Just 5 SNPs in AtRAC2 were located
upstream of intron 3. Assuming a Poisson distribution
The Arabidopsis RAC Gene Family
1963
Figure 3.—A graphical description of the SNPs found in the AtRAC2 gene when comparing the Landsberg erecta and the
Columbia ecotypes. The boxes indicate the exons and the vertical lines mark the positions of the SNPs. There appears to be a
biased distribution of SNPs with the majority localized in the 3⬘ end of the gene.
of the SNPs, the chance of having ⱕ5 SNPs located
upstream of intron 3 is small [P(X ⱕ 5) ⫽ 0.0014 (X ⫽
number of SNPs; ␭ (the mean) ⫽ 16)]. A similar biased
distribution of SNPs was found in AtRAC1, but there
the majority of SNPs were located in the 5⬘ end of the
gene and no polymorphisms were identified downstream of exon 4. For the other AtRAC genes the SNPs
are more evenly distributed throughout the genes. In
noncoding regions a SNP was found every ⵑ211 bp
while in coding regions a SNP was found every ⵑ416
bp. Coding regions represent 29% of the analyzed gene
sequences. When both coding and noncoding regions
are included, the SNP frequency is one change every
ⵑ246 bp. This is in good agreement with previous published data of polymorphism levels between Landsberg
erecta and Columbia (Konieczny and Ausubel 1993).
The results from these analyses are summarized in Table
1. The SNPs had an almost equal proportion of transitions and transversions. In addition to the SNPs the
AtRAC1 and AtRAC2 genes have several indels, four of
them ⬎34 bp. Intron 4 of AtRAC2 is the most variable
intron. This is partly due to a number of indels, two of
which are relatively large, 67 and 342 bp in size. The
AtRAC2 gene also has a TA repeat in intron 6 that
was found in Columbia and Wassilewskaja but not in
Landsberg erecta and Cape Verde (results not shown).
Thus, the indel in the AtRAC1 promoter and the TA
repeat in AtRAC2 intron 6 show that there is a close
genetic relationship between the Landsberg erecta and
the Cape Verde ecotypes.
Genomic gene structure and evolution of rac genes
in higher plants: To clarify the evolution and ancestry
of RAC genes in plants, the AtRAC gene structure was
compared to other genes of the Rho family. Until now
few complete genomic Rho family genes have been characterized, but in recent years sequences from Schizosaccharomyces pombe, D. melanogaster, C. elegans, and Homo
sapiens have appeared in databases. Several of these rho
genes, especially from unicellular organisms such as
yeasts and amoebas, are without introns. The 11 AtRAC
genes reported in this study are the first complete set
of genomic RAC genes characterized from a plant. From
other organisms ⬎30 Rho family genomic genes are
known, but so far only 17 have introns. These 17 genes
are shown in Table 2.
The rac and cdc42 genes from yeast and animals differ
in gene structure when compared with the AtRAC genes.
In general, the rac and cdc42 genes have fewer splice
sites and the splice junction between exons 3 and 4 is
often the only splice site conserved with the AtRAC
genes. The amino acids flanking this splice site, Lys and
Trp, are nearly 100% conserved among the Rho family
members. Among the rac and cdc42 genes the human
cdc42 gene has the highest structural similarity to the
AtRAC genes and two of the splice sites, between exons
2 and 3 and 3 and 4, are 100% conserved.
The human rho7 gene and a RHO-like gene from S.
pombe (GenBank accession no. Z97185) have a genomic
structure that is surprisingly similar to the AtRAC genes.
Four of the AtRAC introns, introns 1, 3, 5, and 6, lie in
phase 0 (the splice site does not split a codon), while
introns 2 and 4 lie in phase 1 (the splicing occurs between nucleotides 1 and 2 in a codon). In both the
human rho7 and the S. pombe RHO-like gene, their introns are 100% conserved and lie in the same phases
as the corresponding AtRAC introns. The chance that
the AtRAC, human rho7, and the S. pombe rho gene have
had their introns inserted at exactly the same position
independent of each other at a later stage in evolution is
unlikely. These introns may therefore represent ancient
introns inherited from a primordial rac/rho gene. Comparisons of AtRAC genes with rho genes, such as rhoA
from C. elegans (EMBL accession no. AL031823), show
that they have a clearly different gene structure (Figure
4) and they are also lacking the conserved splice site
found between exons 3 and 4 in most rac and cdc42
genes.
Recently we have also characterized a partial RAC
gene from the moss Physcomitrella patens, GenBank accession no. AF146341, which had an almost identical exon/
intron structure as the AtRAC genes (P. Winge, R. Kristensen and A. M. Bones, unpublished results). This
demonstrates that the genomic structure of the plant
RAC genes has remained more or less unchanged since
the divergence of vascular and nonvascular plants ⬎400
million years ago.
Phylogenetic analysis: The plant RAC proteins were
aligned with Rac and Cdc42 proteins from S. pombe, H.
sapiens, C. elegans, D. melanogaster, Dictyostelium discoideum,
and Entamoeba histolytica. Figure 5 shows a bootstrapped
neighbor-joining tree created from this alignment,
where an Arabidopsis Rho-like protein was selected as
an outgroup. The phylogram revealed a major division
between the Rac and Cdc42 proteins and it appears that
the plant RAC proteins are a sister group to the Rac/
Cdc42 proteins. The plant RAC proteins have the highest identity with human Rac1, up to 61% identity, and
slightly lower identity with the Cdc42 proteins, up to
The SNP frequency for the eight genes compared pairwise varies from 0 (AtRAC5) to 1.96% (AtRAC2). There is no bias in the number of transitions vs. transversions
and all SNPs in the coding regions are silent.
2976
1
0
1
0
0
2
4
0.13
1762
2
2
1
1
0
0
6
0.34
2804
2
0
6
2
1
0
11
0.39
Base pairs analyzed
C ↔ T transition
G ↔ A transition
T ↔ A transversion
G ↔ T transversion
G ↔ C transversion
A ↔ C transversion
Total
Frequency (%)
2091
12
13
5
4
3
4
41
1.96
1792
0
0
0
1
0
0
1
0.06
1794
3
0
0
1
1
1
6
0.33
1564
0
0
0
0
0
0
0
0
2459
2
1
0
0
1
0
4
0.16
AtRAC10
AtRAC8
AtRAC6
AtRAC5
AtRAC4
AtRAC3
AtRAC2
AtRAC1
Single nucleotide
polymorphisms
Single nucleotide polymorphisms (SNPs) found in nine AtRAC genes by comparison
of A. thaliana Landsberg erecta and Columbia ecotypes
TABLE 1
1439
1
0
0
1
0
1
3
0.21
P. Winge et al.
AtRAC11
1964
TABLE 2
An overview of genomic sequences of the Rho GTPase gene
family registered in GenBank
Organism
Animals
Yeast
Plants
Slime molds
amoeba
rac
Ce rac1
Ce rac2
Ce MIG2
Hs rac2
Yl rac1
cdc42
rho
Hs cdc42
Dm cdc42
Ce cdc42
Hs rho7
Mm rhoB a
Ce rhoA
Dm rho1
Sp rhy
Sp rhob
Sp rhoc
At RHO d
Sp cdc42
Gc cdc42
AtRAC1 → 11
Ph rac3 e
Not found
Dd rac1Ad
Eh racAa
Not found
Dd racE
Eh rho1a
Abbreviations: At, Arabidopsis thaliana; Ce, Caenorhabditis elegans; Dd, Dictyostelium discoideum; Dm, Drosophila melanogaster;
Eh, Entamoeba histolytica; Gc, Glomerella cingulata; Hs, Homo
sapiens; Mm, Mus musculus; Ph, Physcomitrella patens; Sp, Schizosaccharomyces pombe; Yl, Yarrowia lipolytica.
a
Gene without introns.
b
GenBank no. Z97185.
c
GenBank no. Z99753.
d
mRNA sequence.
e
P. Winge, R. Kristensen, R. Reski and A. M. Bones, unpublished results. GenBank no. AF146341.
53% identical with S. pombe CDC42. Due to several
unique amino acid substitutions and an N-terminal extension not found in other plant Rac proteins (Figure
1), the AtRAC9 protein is singled out into a separate
group. A parsimony analysis of the same protein alignment using the Protpars program (Felsenstein 1984)
produces a tree with a slightly different topology, but
the major divisions are the same and they were also
supported by significant bootstrap values (results not
shown). Thus, the topology of the tree, whatever
method used, suggests that the AtRAC proteins can be
divided into distinct groups and that they have a deep
branch linking them to the Rac/Cdc42 proteins. The
placement of RAC group II near the base of the plant
RAC branch is probably an artifact due to the rapid
evolution of this group.
A DNA alignment was made, where the coding regions of 17 plant RAC expressed sequence tags (ESTs)
were included together with 34 full-length plant RAC
cDNAs registered in GenBank. All ESTs were ⬎230 bp
with an average size of 488 bp. Regions with low quality
sequences were excluded and in a few sequences some
minor changes had to be made to avoid gaps. The DNA
alignment was analyzed with both parsimony and distance matrix methods. Figure 6 shows an unrooted
neighbor-joining tree where the divisions of plant RAC
genes into defined groups are evident. In this tree the
RAC genes from mosses, conifers, and genes related to
AtRAC2 appeared to form a clade that was more closely
related to AtRAC9 than to the RAC genes within group
The Arabidopsis RAC Gene Family
1965
Figure 4.—Gene structure of rho GTPases. The exon and intron structure of the AtRAC group I and II genes is compared
with the gene structure of various rho, rac, and cdc42 genes (Hs, Homo sapiens; Sp, Schizosaccharomyces pombe; Dd, Dictyostelium
discoideum; Ce, Caenorhabditis elegans; Yl, Yarrowia lipolytica). The numbers between the exons indicate the splice phase of the
intron (in phase zero the splicing occurs between codons). The sizes of introns are not drawn to scale. All genes are registered
in GenBank under the given name in the figure. Sp rho*, GenBank accession no. Z97185.
II. This is in slight contrast to the results from the analysis of the plant RAC proteins shown in Figure 5, where
AtRAC9 appeared to be more closely related to the RAC
proteins from group II. A parsimony analysis of the same
DNA alignment produced a tree with an almost identical
topology, even though the bootstrap values for some
clades are slightly lower (results not shown).
Chromosomal location of the AtRAC genes: All the
11 AtRAC genes identified and characterized here have
also recently been sequenced by the Arabidopsis Genome Initiative. The AtRAC genes are scattered throughout the A. thaliana genome and are located on all five
chromosomes. There is no obvious clustering of the
genes, but the AtRAC3 and AtRAC6 genes are separated
by just 250 kb on chromosome IV. In Table 3, the genomic locations of the AtRAC genes are shown together
with their nearest genetic markers. A comparison of the
AtRAC gene neighbors revealed the dynamic nature of
the Arabidopsis genome and showed that duplications,
insertions, and deletions have been shaping the genome
through evolution. Figure 7 shows the nearest gene
neighbors to AtRAC1, -4–6, and -11, and clearly shows
that these AtRAC genes were created through a number
of large duplications. One of these duplications, encompassing 4.6 Mb of the middle part of chromosome II
and the tip of the large arm of chromosome IV (Lin et
al. 1999; Mayer et al. 1999), resulted in the creation of
AtRAC1 and AtRAC6. The high similarity between the
AtRAC1 and AtRAC6 proteins suggests that this is a
relatively recent duplication, but comparison of the noncoding regions of the AtRAC1 and AtRAC6 genes and
the detection of several gene rearrangements suggest
that the duplication must have occurred tens of millions
of years ago. This is further supported by our studies
of RAC genes from other plants within Brassicaceae
(Cheiranthus cheiri, GenBank accession no. AF161017;
Lepidium sativum, and others). These results indicated
that all duplications leading to the AtRAC genes detected in Arabidopsis today must have taken place before the split between Arabidopsis and the other members of the mustard family, which is estimated to have
occurred 10–35 million years ago (Lagercrantz 1998).
Other interesting observations seen from Figure 7 are
the recent inversion of the AtRAC1 gene and the loss
of the VPS35-like genes next to AtRAC4 and AtRAC6. In
addition, an ascorbate peroxidase and a histidine kinase
have been inserted upstream and downstream of AtRAC6
and AtRAC1, respectively. Several genes encoding proteins involved in the regulation of the cytoskeleton and
plant defense responses are found in close proximity to
the AtRAC genes, but this could be a mere coincidence.
The two closely related genes, AtRAC4 and AtRAC5,
are located in two large duplicated regions on chromosome I. A computer-based search shows that the duplicated regions extend from ⵑ24–39 cM [bacterial artificial chromosome (BAC) clone F9L1 → F21J9] to 114–
131 cM (BAC clone F20P5 → F23A5). The low sequence
homology of the noncoding regions in AtRAC4 and
AtRAC5 and the many gene rearrangements show that
this is an old duplication. As far as we know this is the
first time this large duplication has been reported.
Even though the AtRAC3 gene is located close to
AtRAC6 and the region flanking AtRAC3 is found duplicated on chromosome II, there is no indication that it has
been duplicated together with the AtRAC1 and AtRAC6
genes, as no close homologues to AtRAC3 exist. This is
also supported by results that show the genes flanking
AtRAC3 have no homologous genes located near other
1966
P. Winge et al.
Figure 5.—A neighbor-joining tree of Rac proteins from
various eukaryotes was created
with the Clustal X program. An
Arabidopsis Rho-like protein,
AtRHO (GenBank accession
no. U88402) was selected as an
outgroup. To evaluate the confidence limits of the internal
branches of the tree a bootstrap analysis with 1000 replications was performed on the
data set. Bootstrap values ⬎500
are shown to the right of each
branch point. The scale bar indicates the number of amino
acid substitutions per site. Three
full-length EST clones are included. EST1, AI759954; EST2,
AW039993; and EST3, AI937960.
Abbreviations: Br, Brassica rapa;
Bv, Beta vulgaris; Ca, Cicer arietinum; Ce, Caenorhabditis elegans;
Dd, Dictyostelium discoideum; Dm,
Drosophila melanogaster; Eh, Entamoeba histolytica; Gh, Gossypium hirsutum; Gm, Glycine max;
Hs, Homo sapiens; Le, Lycopersicon esculentum; Lj, Lotus japonicus; Ms, Medicago sativa; Nt,
Nicotiana tabacum; Os, Oryza sativa; Ph, Physcomitrella patens;
Pm, Picea mariana; Ps, Pisum
sativum; Sp, Schizosaccharomyces
pombe; Zm, Zea mays.
AtRAC genes. This lack of synteny suggests that the AtRAC3
gene has been transposed to its current position after the
large duplication, but a deletion of an allele on chromosome II cannot be excluded.
The duplicated region that includes the AtRAC11 gene
appears to be more confined and extends from ⵑ74 to
76 cM on chromosome III (BAC clone T3A5 → F26O13).
This region, which spans ⵑ200 kb, is found duplicated
on chromosomes II and IV together with the AtRAC1 and
AtRAC6 genes. The AtRAC11 gene and the AtRAC5 gene
on chromosome I represent duplication events prior to
the AtRAC1 and AtRAC6 duplication. A computer-based
search indicates that these old duplications probably span
several hundred kilobases.
The relicts of an ancient duplication can also be found
when scrutinizing the BAC clones with the AtRAC11
and AtRAC2 genes. Both these genes share a common
neighbor, a homologue of the D. melanogaster crooked
neck gene (Crn), which is located ⵑ60 kb upstream of
AtRAC11 and 6 kb downstream of AtRAC2. So far these
are the only Crn homologues found in Arabidopsis and
they probably arose from the same duplication event.
Located next to AtRAC2 (BAC clone K15I22) is a cluster
of genes encoding anther-specific proline-rich proteins
(APGs). A similar cluster of APG genes is found next to
AtRAC4 and AtRAC5 (Figure 7), providing more support
for this ancient gene duplication.
A study of the BAC clones flanking AtRAC8 and
AtRAC10 genes reveals a duplication that spans ⬎1 Mb
on the long arms of chromosomes III and V. Currently
available data indicate that the duplication covers the regions 65–73 cM from chromosome III (BAC F14D17 →
The Arabidopsis RAC Gene Family
1967
Figure 6.—Phylogram of
RAC genes from embryophytes.
An unrooted neighbor-joining
tree of RAC genes from various
embryophytes was created with
the Clustal X program. Expressed sequence tags from 17
genes were included in the
data set; four of them are fulllength genes. Some of the sequences were compiled from
two or more ESTs and sequences of low quality were excluded. Branches with bootstrap values ⬎500 are indicated
as thick lines (1000 replications
were analyzed). The scale bar
indicates the number of nucleotide substitutions per site. Abbreviations: As, Populus tremuloides (Aspen); Br, Brassica rapa;
Bv, Beta vulgaris; Ca, Cicer arietinum; Gh, Gossypium hirsutum;
Gm, Glycine max; Le, Lycopersicon esculentum; Lj, Lotus japonicus; Ms, Medicago sativa; Nt, Nicotiana tabacum; Os, Oryza sativa;
Ph, Physcomitrella patens; Pm,
Picea mariana; Ps, Pisum sativum; Pt, Pinus taeda; Zm, Zea
mays. EST1, AI730323; EST2,
AI727570; EST3, AI731040;
EST4, AI775563; EST5, D41104 and C26233; EST6, AW039993; EST7, AI937960; EST8, AW218480; EST9, AI759954; EST10,
AW102025 and AI900160; EST11, AI162198; EST12, AU029919 and C73805; EST13, AI164960 and AI161509; EST14, AI901151;
EST15, AW225989; EST16, AW056772; and EST17, AI812534.
T8P19) and 121–128 cM from chromosome V (BAC
K19M22 → MBK5). As far as we know this apparently
old duplication has not been reported previously. More
than 40 genes are found duplicated in these regions
and the actin genes ACT4 and ACT12 are among the
duplicated genes.
DISCUSSION
After an extensive screen for RAC- and RHO-like genes
in A. thaliana, we have identified a large family of RAC
genes, AtRAC1 → AtRAC11. Given the large number of
clones analyzed in this screen and that a previous cDNA
screen identified 10 of the corresponding cDNAs (Winge
et al. 1997), it is likely that all the AtRAC genes in A.
thaliana have now been found. Probes and hybridization
conditions used in these screens were also carefully selected to include the more divergent AtRAC genes such
as AtRAC2, -7, -9, and -10. Results from various EST
projects shows that all RAC genes found in higher plants
are highly similar to the Arabidopis RAC genes (Figures
5 and 6). Even though distinct subgroups of RAC genes
exist in mosses and monocotyledons, there are no indications that other distantly related groups of RAC genes
exist in embryophyta.
A new class of RAC genes identified in vascular plants:
On the basis of their genomic structure and a number
of distinct amino acid differences (Winge et al. 1997),
the 11 AtRAC genes can be divided into two distinct
groups. The encoded proteins of group I AtRACs
(AtRAC1–6, -9, and -11) have a C-terminal motif, CaaL
(a, aliphatic amino acid), which indicates that they are
geranylgeranylated (Trainin et al. 1996). AtRAC genes
belonging to group II have an additional exon at the
3⬘ end, which is most likely the result of the insertion
of an intron in the extreme 3⬘ end of an ancestral RAC
gene. One effect of the insertion of this intron is that
the group II-encoded proteins lack the typical C-terminal CaaL motif but they have nevertheless retained a
cysteine-containing motif, suggesting that they have a
different C-terminal modification. The AtRAC7 protein,
for instance, has a C-terminal motif (CTAA), which indicates it is farnesylated (Nambara and McCourt 1999).
Comparison of the C-terminal parts of the AtRAC proteins suggests that an AtRAC3-like gene may have been
the ancestor to the AtRAC genes belonging to RAC
group II.
Phylogenetic analysis shows that the members of the
RAC group II can be divided into two defined subgroups, which are present in both monocotyledons and
dicotyledons. This suggests that the creation of group
II and the following duplication of this ancestral gene
must have happened before the split between monocotyledonous and dicotyledonous plants that occurred
1968
P. Winge et al.
Figure 7.—A physical map of a 50-kb region flanking the AtRAC1, -4–6, and -11 genes indicating numerous duplications,
deletions, and rearrangements. The labels above the genes flanking AtRAC6 are registered BAC gene numbers. An inversion of
the AtRAC1 gene can be observed. Three of the genes, AtRAC1, -5, and -11, have a VPS35 homologue as their closest neighbor,
while in AtRAC4 and AtRAC6 it has been deleted. The structural similarities of the genes flanking AtRAC1 and AtRAC6 indicate
that this is a recent gene duplication. The complete sequence of the BAC clones can be found in GenBank: AtRAC1, AF024504
and AC003952; AtRAC4, AC022472; AtRAC5, AC007396; AtRAC6, AL022373; and AtRAC11, AL132980.
ⵑ200 million years ago (Yang et al. 1999). Our analyses
of the RAC genes from the moss P. patens and other
bryophyta suggest that the group II RAC genes have
evolved only in vascular plants (P. Winge, R. Kristensen and A. M. Bones, unpublished results). This
indicates that the insertion of the intron, which resulted
in the creation of group II RAC genes, must have occurred 200–400 million years ago.
The involvement of RAC proteins in the regulation of
the actin cytoskeleton in eukaryotes is firmly established
(Hall 1998). It is therefore interesting to note that the
evolution and diversity of the AtRAC genes show many
similarities with the Arabidopsis actin gene family
(McDowell et al. 1996; An et al. 1999; Meagher et al.
1999). They are both multigene families that diversified
and split into two distinct groups approximately at the
same time and both encode proteins that have distinct
spatial and temporal expression patterns in vegetative
and reproductive tissues (Delmer et al. 1995; Li et al.
1998; Kost et al. 1999; P. Winge, unpublished results).
Four of the actin genes, ACT3, ACT4, ACT12, and
T6D20.1 (an actin-like gene), have a chromosomal localization close to AtRAC genes. Whether there has been
a coevolution of these two gene families remains an
open question.
Organization and evolution of RAC genes in higher
plants: Our results show that most of the AtRAC genes
were created through large duplications encompassing
several genes instead of being created through tandem
duplications as is often seen in Arabidopsis. One of
these recent duplications involving ⬎4 Mb from chromosomes II and IV (Lin et al. 1999; Mayer et al. 1999)
resulted in the generation of the AtRAC1 and AtRAC6
genes. The AtRAC1 and AtRAC6 homologues may be
restricted to the Brassicaceae family or possibly the Capparales order, but genes related to the AtRAC1, -6, and
-11 subgroup are present in both rosids and asterids.
This shows that the gene duplication, which resulted in
the creation of this subgroup, happened before the split
between asterids and rosids ⵑ90 million years ago.
We also report the findings of two other large duplications that have created new AtRAC homologues and
report evidence for three additional ancient duplications. The duplicated regions on chromosome I, which
includes the AtRAC4 and AtRAC5 genes, probably span
3 Mb or more. Our detection of an AtRAC4 homologue
in L. sativum also shows that this duplication predates
the split between Arabidopsis and other members of
the Brassicaceae. The number and sizes of these ancient
duplicated regions also raise the question of whether
The Arabidopsis RAC Gene Family
TABLE 3
The genomic location of A. thaliana AtRAC1–11
Gene
AtRAC1
AtRAC2
AtRAC3
AtRAC4
AtRAC5
AtRAC6
AtRAC7
AtRAC8
ATRAC9
AtRAC10
AtRAC11
Chromosome
II
V
IV
I
I
IV
IV
III
II
V
III
cM
Nearest
genetic
marker
BAC
clone
40
101
109
32
122
111
92
70
90
127
75
CPK6
mi61
pCITd99
mi203
ATHATPAS
POL2LS
RLK5
m249
m336
m211A
MUR_1
T17A5
MCL19
M4E13
T20H2
T4O12
T19K4
F25O24
T17F15
F16B22
MQB2
F24M12
Approximate gene positions are indicated in centimorgans
(cM). The physical and genetic map information was gathered
from The Arabidopsis Information Resource (TAIR).
they are due to real duplication events or if the early
ancestor was a tetraploid, as has also been suggested by
others (Grant et al. 2000).
Phylogenetic analyses: The phylogenetic trees show
that the AtRAC proteins/genes can be divided into several distinct groups that can be partly explained by the
ancient gene duplications we report here. One interesting observation is that a large group of RAC homologues
within group I that also include the genes AtRAC1,
-3–6, and -11 appear to be restricted to dicotyledons
(see Figure 6). This suggests that a number of RAC gene
duplications have occurred in dicotyledons after the
split with the monocotyledons. There is no indication
that a similar gene expansion within this group has
taken place in monocotyledons. On the other hand,
additional RAC gene duplications within group II appear to have occurred in monocotyledons and the majority of RAC genes from monocotyledons and magnoliids registered in GenBank at this date belong to RAC
group II.
The phylogenetic study also shows that the AtRAC2
gene is one of the most divergent members within RAC
group I, and the existence of AtRAC2-like genes in conifers suggests that a similar gene existed well before the
development of flowering plants. This is also supported
by our studies of AtRAC gene duplications, which show
that an AtRAC2-like gene probably was a progenitor
to the AtRAC5 and AtRAC11 genes. Furthermore, our
expression analysis of AtRAC2 shows that it is primarily
expressed in roots and vascular tissues (Winge et al.
1997), an expression pattern it may have inherited from
a functionally developed AtRAC2 gene that existed in
early nonflowering plants.
AtRAC polymorphisms: Comparison of the AtRAC
genes sequenced from the Columbia and Landsberg
erecta ecotypes revealed large differences in the number of polymorphisms and indels. For instance, AtRAC2
1969
had approximately one SNP every 50 bp, which is five
times higher than the average. Several factors are known
to influence the mutation rates of individual genes.
From both eukaryotic and prokaryotic organisms it has
been shown that expression levels influence DNA repair
rates, through so-called transcription-coupled DNA repair (Hanawalt 1989). Compared with the other
AtRAC genes, AtRAC2 has a distinct expression pattern
and has a relatively low expression (Winge et al. 1997).
The high number of polymorphisms found in AtRAC2
can therefore be due to its low expression and thereby
lack of transcription-coupled DNA repair, which in turn
results in the observed accumulation of SNPs.
Evolutionary considerations: Phylogenetic comparison of the AtRAC proteins with Rho family members
from other eukaryotes suggests that the RAC proteins
in plants developed into a distinct group at an early
stage during eukaryotic evolution. An alternative explanation is that the RAC proteins in higher plants have
gone through a rapid evolution at a later stage. Support
for this latter view comes from studies of algae within
the paraphyletic Prasinophyte lineage, which show that
their RAC proteins are more similar to RAC proteins
found in animals and amoebas (P. Winge, T. Brembu
and A. M. Bones, unpublished results). Furthermore,
the studies of RAC proteins from bryophyta show that
they have remained virtually unchanged in land plants.
This suggests that RAC proteins in early plants underwent a rapid evolution before terrestrial plants appeared
480 million years ago (Kenrick and Crane 1997).
Whether this occurred when the first multicellular
plants evolved or if the transition took place even earlier
is still unresolved.
The existence of Rac and Rho proteins in eukaryotes
is universal. In vertebrates and invertebrates all subgroups of the Rho family (Rac, Rho, and Cdc42) are
present. In contrast some of the lower eukaryotes seem
to lack certain subgroups of the Rho family. For instance, “true” Rac proteins do not exist in S. cerevisiae
and S. pombe, but are found in the dimorphic yeast
Yarrowia lipolytica (Hurtado et al. 2000), while the
amoebas and slime molds appear to lack the Cdc42
proteins. The absence of Cdc42-like proteins in amoebas, slime molds, and higher plants could therefore
indicate that the Cdc42 group evolved later during evolution, but it cannot be ruled out that they have been
selectively lost in some of these kingdoms at a later
stage. Because RAC proteins in embryophyta appear to
have diverged and evolved faster than most Rac/Rho
proteins from other organisms, the phylogenetic analysis may give the impression that they have evolved early
and even predate the Rac/Cdc42 proteins. However,
the most likely scenario is that the RAC proteins in
embryophyta have evolved from a RAC-like ancestor.
Conservation of gene structure and specifically the
conservation of splice site positions are additional factors that can be used to deduce the phylogenetic relationship between genes (Kloek et al. 1993; de Souza et
1970
P. Winge et al.
al. 1998). Comparison of the AtRAC gene structure with
rac and rho genes from other eukaryotes shows that
AtRAC genes are more closely related to rac/cdc42 genes
than to rho genes, the exception being human rho7 and
a rho-like gene from S. pombe (GenBank accession no.
Z97185).
The evolution of the RAC proteins in higher plants
leaves some tantalizing questions. Why did the multicellular plants evolve such a distinct group of RAC proteins
in the first place and what type of selection pressure
could have been involved? One clue to these questions
may come from our and others’ observations that higher
plants do not have true Ras proteins (Winge et al. 1997;
Meyerowitz 1999). Ras and Rap-like proteins have
been found in various lower eukaryotes, including Trypanosoma brucei, E. histolytica, Dictyostelium discoideum, and
various fungi (Reymond et al. 1984; Lohia and Samuelson 1996; Sowa et al. 1999). The evolution of specific types of RAC proteins in higher plants may have
been an adaptation to the loss of Ras proteins, such that
the RAC proteins in plants now have dual functions.
This may also explain why there has been a selection
pressure to evolve a RAC multigene family in higher
plants. If the RAC proteins in embryophyta have both
Rac and Ras-like functions, then these proteins may play
the role of master regulators in plants.
This work was supported by The Norwegian Research Council grant
100370/410.
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Communicating editor: C. S. Gasser