Molecular Characterization and Evolution of the Protein
Phosphatase 2A Bⴕ Regulatory Subunit Family in Plants1
Javier Terol2,3, Mónica Bargues2, Pedro Carrasco, Manuel Pérez-Alonso, and Nuria Paricio*
Departament de Genètica (J.T., M.B., M.P.-A., N.P.) and Departament de Bioquı́mica i Biologı́a Molecular
(P.C.), Universitat de València, Doctor Moliner 50, 46100 Burjassot, Spain
Type 2A serine/threonine protein phosphatases (PP2A) are important components in the reversible protein phosphorylation
events in plants and other organisms. PP2A proteins are oligomeric complexes constituted by a catalytic subunit and several
regulatory subunits that modulate the activity of these phosphatases. The analysis of the complete genome of Arabidopsis
allowed us to characterize four novel genes, AtB⬘⑀, AtB⬘␨, AtB⬘␩, and AtB⬘␪, belonging to the PP2A B⬘ regulatory subunit
family. Because four genes of this type had been described previously, this family is composed of eight members. Reverse
transcriptase-polymerase chain reaction experiments showed that AtB⬘⑀ mRNAs are present in all Arabidopsis tissues
analyzed, and their levels do not respond significantly to heat stress. Expressed sequence tags corresponding to AtB⬘␨, AtB⬘␩,
and AtB⬘␪ have been identified, indicating that the new genes are actively transcribed. The genomic organization of this
family of PP2A regulatory subunits is reported, as well as its chromosomal location. An extensive survey of the family has
been carried out in plants, characterizing B⬘ subunits in a number of different species, and performing a phylogenetic study
that included several B⬘ regulatory proteins from animals. Our results indicate that the animal and plant proteins have
evolved independently, that there is a relationship between the number of B⬘ isoforms and the complexity of the organism,
and that there are at least three main subfamilies of regulatory subunits in plants, which we have named ␣, ␩, and ␬.
Reversible protein phosphorylation is widely accepted as a major mechanism for the control of biological processes in eukaryotic cells. In plants, reversible protein phosphorylation is involved in processes
such as hormonal, pathogenic, or environmental
stress responses (Mumby and Walter, 1993; Smith
and Walker, 1993; Garbers et al., 1996; Schöntal, 1998;
Janssens and Goris, 2001). In this context, Ser/Thr
protein phosphatases (PPs) are important regulatory
components of many signal transduction pathways
(Ingebritsen and Cohen, 1983a; Schöntal, 1998). Several Ser/Thr phosphatases, grouped into different
categories, have been identified in a variety of plant
species. Specifically, homologs of the 1, 2A, and 2C
types of animal PPs have been described in plants
(Rodrı́guez, 1998; Lin et al., 1999; Meek et al., 1999).
All these types of PPs are distinguished by their
different sensitivity to inhibitors and their divalent
cation requirements, and are structurally different
(for review, see Mumby and Walter, 1993).
Type 2A phosphatases (PP2A) are oligomeric enzymes with no obvious requirements for ions or co1
This work was supported by the EC (grant no. BIO4 –CT98 –
0549) and by Dirección General de Enseñanza Superior e Investigación Cientı́fica, Spanish Ministerio de Educación y Cultura
(grant no. BIO99 –1320 –CE).
2
These authors contributed equally to the paper.
3
Present address: Sistemas Genómicos S.L., C/Benjamı́n Franklin 12, Parque Tecnológico the Valencia, Paterna 46980, Spain.
* Corresponding author; e-mail [email protected]; fax
34 –96 –398 –3029.
Article, publication date, and citation information can be found
at www.plantphysiol.org/cgi/doi/10.1104/pp.020004.
808
factors, and are implicated in a variety of cellular
processes (Mumby and Walter, 1993; Janssens and
Goris, 2001). In general, the native forms of PP2A
proteins exist as oligomeric complexes, constituted
by a catalytic subunit (PP2Ac), and one or more
regulatory subunits named A and B. Thus, PP2A
proteins can be heterodimers, consisting of a PP2Ac
catalytic subunit and a type A regulatory subunit, or
heterotrimers that contain an additional regulatory
subunit of the B type. PP2Ac subunits are highly
conserved in all organisms analyzed, and their activity, specificity, and subcellular localization depend
on the association of this subunit with different A
and B regulatory subunits (Hendrix et al., 1993b;
Strack et al., 1998). The A regulatory subunit has a
molecular mass of 65 kD, and consists of 15 imperfect
repeats of 38 to 43 amino acids, through which it
interacts with the PP2Ac catalytic subunit and the B
regulatory subunit (Groves et al., 1999). Type B regulatory subunits of PP2A are very diverse, and can be
clustered into at least three distinct groups including
the 55-kD B, the 52- to 74-kD B⬘, and the 72- to 130-kD
B⬘⬘ subunit families (Rundle et al., 1995; Corum et al.,
1996; Csortos et al., 1996; McCright et al., 1996a).
Each family is composed of several members with the
exception of the B⬘⬘ subunit family (Hendrix et al.,
1993a).
Homologs to all PP2A subunits have been described in plants. In Arabidopsis, the catalytic subunit of PP2 (PP2Ac) is encoded by at least five genes,
each of which appears to be expressed in all tissues
albeit at different levels (Ariño et al., 1993; Casamayor et al., 1994; Pérez-Callejón et al., 1998). Re-
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Copyright © 2002 American Society of Plant Biologists. All rights reserved.
Protein Phosphatase 2A B⬘ Regulatory Subunit Family in Plants
garding the regulatory subunits, three genes encoding the A 65-kD subunit (Slabas et al., 1994), two
genes encoding the B subunit (Rundle et al., 1995;
Corum et al., 1996), and one gene encoding the B⬘⬘
regulatory subunit (Sato et al., 1997) have been identified. In the last years, four isoforms of the B⬘ regulatory subunit of PP2A have been described in Arabidopsis, named AtB⬘␣, AtB⬘␤, AtB⬘␥ (Latorre et al.,
1997), and AtB⬘␦ (Haynes et al., 1999). However,
Southern-blot analyses of genomic DNA indicated
that at least another gene encoding a fifth B⬘ isoform
could be present in this plant (Haynes et al., 1999).
Five genes encoding B⬘ regulatory subunits (or PR56)
also have been described in humans (Homo sapiens)
that produce at least seven isoforms (McCright and
Virshup, 1995; McCright et al., 1996a; Tehrani et al.,
1996). Similarly, five genes encoding at least eight
isotypes of B⬘ exist in rabbits (Oryctologus cuniculus;
Csortos et al., 1996; Zolnierowicz et al., 1996).
All four AtB⬘ genes described so far are expressed
in all Arabidopsis organs and encode very similar
proteins, the central core of the B⬘ subunits being the
most conserved region (Latorre et al., 1997; Haynes et
al., 1999). It is interesting to mention that transcripts
from one of these genes, AtB⬘␥, seem to accumulate
in response to heat stress (Haynes et al., 1999), suggesting that PP2A heterotrimers containing this subunit could be involved in stress response mechanisms in plants. This has been proposed for the RTS1
(or SCS1) protein, the yeast (Saccharomyces cerevisiae)
homolog of the B⬘ subunit. RTS1 was isolated as
suppressor of mutant alleles of the ROX3 and Hsp60
genes. The ROX3 protein seems to be involved in the
global stress response pathway (Evangelista et al.,
1996) and Hsp60 is a mitochondrial heat shock protein (Shu and Hallberg, 1995). On the other hand, two
of the B⬘ subunits in Arabidopsis, AtB⬘␣ and AtB⬘␤,
contain putative nuclear targeting sequences, suggesting that the different isoforms could function to
target PP2A to unique subcellular locations (Latorre
et al., 1997). This idea has also been proposed for the
human B⬘ subunits, where different subcellular locations were found for ␣, ␤, and ⑀ subunits located in
the cytoplasm, and ␥ and ␦ in the nucleus (McCright
et al., 1996b; Zhao et al., 1997).
Our group has contributed to the European Union
Sequencing on Arabidopsis chromosome 3 (Salanoubat et al., 2000) as part of the worldwide Arabidopsis
Genome Initiative (AGI) that has sequenced the complete genome of Arabidopsis (AGI, 2000). In the
frame of this collaboration, we have characterized in
this work four additional members of the PP2A B⬘
regulatory subunit family in Arabidopsis (AtB⬘⑀,
AtB⬘␨, AtB⬘␩, and AtB⬘␪) and have analyzed their
genomic organization. We have also performed an
extensive study of this family in plants, describing
several new members coding for B⬘ subunits in rice
(Oryza sativa), tomato (Lycopersicon esculentum), corn
(Zea mays), medic barrel (Medicago truncatula), potato
Plant Physiol. Vol. 129, 2002
(Solanum tuberosum), soybean (Glycine max), etc. A
structural and phylogenetic analysis of the new proteins is also carried out.
RESULTS
Identification and Characterization of AtBⴕ⑀, a Gene
Encoding a New Isoform of the PP2A Bⴕ Regulatory
Subunit Family in Arabidopsis
As a result of our participation in the AGI, the
bacterial artificial chromosome (BAC) clone T15C9
was sequenced and analyzed to identify putative
coding regions. One of these regions was of particular interest due to its similarity to the B⬘ regulatory
subunits of PP2A. The open reading frame (ORF) was
composed of two exons of 1,188 and 306 nucleotides,
separated by an intron of 82 nucleotides. The predicted protein encoded by this gene was 497 amino
acids long, with a molecular mass of 57.5 kD. Similarity searches in protein databases showed that this
protein was highly homologous to the B⬘ regulatory
subunits of PP2A from Arabidopsis (Latorre et al.,
1997; Haynes et al., 1999) and other organisms. The
striking similarity with respect to the Arabidopsis B⬘
regulatory subunits (AtB⬘ ␣, ␤, ␥, and ␦), which
ranged from 53.1% to 77.9% at the protein level, led
us to consider this protein as a new member of the B⬘
family of regulatory subunits of PP2A. We named the
hypothetical gene AtB⬘⑀, following the previous nomenclature (Latorre et al., 1997; Haynes et al., 1999).
To know whether AtB⬘⑀ was normally expressed in
any stage of the Arabidopsis development, we used
the DNA sequence corresponding to the hypothetical
protein to perform a BLAST similarity search against
all the available Arabidopsis expressed sequence tag
(EST) databases. The searches yielded no cDNA
clones identical to AtB⬘⑀, so we decided to perform a
reverse transcriptase (RT)-PCR analysis to determine
the expression pattern of the gene (see “Materials
and Methods”). The primers were designed specifically for AtB⬘⑀, avoiding cross amplification of sequences from the other members of the B⬘ family. The
results show that the AtB⬘⑀ is an active gene and that
the transcripts accumulate in all organs analyzed:
leaves, seeds, stems, and flowers (Fig. 1A). This is in
accordance with the expression patterns reported for
other Arabidopsis PP2A B⬘ regulatory subunits (Latorre et al., 1997; Haynes et al., 1999). The experimental evidences obtained for AtB⬘⑀ confirm that it is
subject to transcription and, therefore, can be considered as an active gene.
It had been reported that the AtB⬘␥ isoform appears to be involved in the heat stress response because one of the mRNAs derived from this gene (1.5
kb) accumulates in Arabidopsis seedlings after 2 h of
heat shock at 37°C (Latorre et al., 1997). On the
contrary, transcripts from the AtB⬘␣, AtB⬘␤, and
AtB⬘␦ genes do not respond to such treatment (Latorre et al., 1997; Haynes et al., 1999). To determine
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809
Terol et al.
we performed the same RT-PCR experiments using a
set of specific primers to amplify AtB⬘␥ mRNAs. We
established that the PCR reactions were in a semiquantitative range performing a series of control reactions with ribosomal RNA with the presence of
competimers that inhibit amplification at different
ratios. As the ratio of competimer increases, the intensity of the band on the gel decreases (Fig. 1B,
lower), which indicates that, in the same conditions,
the variations observed in the experiment reactions
are not an artifact. This way, it can be observed that,
although there is a clear response to heat stress in
AtB⬘␥ expression, AtB⬘⑀ mRNA levels do not significantly vary under heat shock conditions (Fig. 1B,
upper).
Identification of Three Additional Members of the
PP2A Bⴕ Regulatory Subunit Family
Figure 1. RT-PCR experiments with AtB⬘⑀. A, Expression of the AtB⬘⑀
gene in different Arabidopsis organs. Total RNA was extracted from
leaves 4.5 weeks old (lane 1), leaves 7 weeks old (lane 2), seeds (lane
3), stems (lane 4), and flowers (lane 5). Lane M is the DNA size
marker, and numbers indicate size of the DNA fragments in nucleotides. Upper, Results of RT-PCR using oligo(dT)- and AtB⬘⑀-specific
primers to amplify AtB⬘⑀ transcripts. Lower, Results of the RT-PCR
using oligo(dT) and actin-1 primers as control. Equal amounts of both
RT-PCR reactions (with AtB⬘⑀-specific primers and actin-specific
primers) were loaded for each tissue. B, Expression of the AtB⬘⑀ gene
after heat shock conditions. Lower, rRNA amplification using 6:10,
7:10, 8:10, and 9:10 ratio competimers to amplify RNA from nonstressed plants (lanes 1–4) or heat-shocked plants (lanes 5–8). The
intensity of the bands on the gel decreases as the ratio of competimers increases, indicating that the PCR is in a quantitative range.
Upper, Expression of AtB⬘⑀ and AtB⬘␥ at 23°C (lanes 1 and 3,
respectively), and after heat shock, with 2 h at 37°C (lanes 2 and 4,
respectively). M is the DNA molecular mass marker VI (Roche
1062590); numbers indicate size of the DNA fragments in bp.
whether AtB⬘⑀ expression changes in response to
heat stress, we performed a semiquantitative RT-PCR
analysis using two different RNA samples, extracted
from adult Arabidopsis plants grown in standard
conditions and from plants subjected to heat stress at
37°C for 2 h. As a control for the heat shock response,
810
Besides the four previously described AtB⬘ isoforms, the BLAST searches performed to characterize
AtB⬘⑀, in the Arabidopsis databases, produced three
additional predicted proteins that showed a high
degree of conservation with respect to the B⬘PP2A
regulatory subunits and AtB⬘⑀ itself. Both the scores
and e values showed a clear cutoff, separating AtB⬘␣,
AtB⬘␤, AtB⬘␥, AtB⬘␦, AtB⬘⑀, and the three new proteins from the other ones produced by the BLAST
search. The three predicted proteins were the result
of the annotation of the complete genome of Arabidopsis performed by the AGI, and their accession
numbers were BAB02360, BAB01065, and AAG09562.
The high degree of similarity obtained allowed us to
consider the novel predicted proteins as members of
the B⬘PP2A regulatory subunit family and, following
the previous nomenclature, we named them AtB⬘␨,
AtB⬘␩, and AtB⬘␪, respectively.
In an attempt to identify additional members of
this family that had not been already described at the
protein level, we analyzed the whole Arabidopsis
genome, dynamically translated in all reading
frames, in a TBLASTN search, using the eight proteins as queries. The searches did not yield additional
new members, and no new DNA sequences, apart
from the previously characterized so far, produced
proteins with a significant similarity. The exhaustive
analyses performed, both at the protein and DNA
level, allowed us to discard the existence of additional genes producing PP2A B⬘ regulatory subunits
in the genome of Arabidopsis. Therefore, although
genomic Southern-blot analyses had suggested that
five genes encoding B⬘ isoforms were present in Arabidopsis (Latorre et al., 1997), the analysis of the
complete genome of the plant revealed that this family of regulatory proteins is composed of eight
members.
To know whether any of the three new members of
this Arabidopsis gene family are normally expressed
in any stage of the Arabidopsis development, we
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Copyright © 2002 American Society of Plant Biologists. All rights reserved.
Plant Physiol. Vol. 129, 2002
Protein Phosphatase 2A B⬘ Regulatory Subunit Family in Plants
used the DNA sequence corresponding to the hypothetical B⬘ proteins we had identified to perform a
BLAST similarity search against all the available Arabidopsis EST databases. cDNA clones corresponding
to AtB⬘␨, AtB⬘␩, and AtB⬘␪ were identified (Table I),
confirming that these hypothetical genes are subject
to transcription and, therefore, can be considered as
active genes.
Genomic Organization of the AtBⴕ Regulatory
Subunit Family
Table II. Location in the AtB⬘ genes
Gene
Chrom.a
G.C.b
Acc. No.c
AtB⬘␣
AtB⬘␤
AtB⬘␥
AtB⬘␦
AtB⬘⑀
AtB⬘␨
AtB⬘␩
AtB⬘␪
V
III
IV
III
III
III
III
I
F12E4
F8A24
ESSAI FCA
MPE11
T15C9
MIL23
MPE11
T6J4
AL162751
AC015985
Z97338
AB023041
AL132970
AB019232
AB023041
AC011810
a
b
Chromosome no.
Genomic BAC clone where the gene is
c
isolated.
GenBank accession no.
The sequencing of the complete genome of Arabidopsis allowed us to determine the genomic organization of the eight PP2A B⬘ regulatory subunit genes
as well as their chromosomal position. We did these
determinations for the previously described genes,
AtB⬘␣, AtB⬘␤, AtB⬘␥, and AtB⬘␦, only characterized at
the cDNA level, as well as for the ones we describe
here for the first time, to our knowledge, AtB⬘⑀,
AtB⬘␨, AtB⬘␩, and AtB⬘␪.
For the first group of genes, we performed a
BLASTN search against the Arabidopsis genome database, using the cDNA sequences as a query, to find
the genomic clones in which the genes were located.
The chromosomal position of such clones was determined using the Map Viewer Tool at The Arabidopsis
Information Resource (http://www.Arabidopsis.org/
servlets/mapper). For the genes we describe here, this
information was available in the GenBank annotation
entries. The results are displayed in Table II, where
the chromosome and genomic clones of the genes are
shown. In summary, five of the genes (AtB⬘␤, AtB⬘␦,
AtB⬘␨, AtB⬘⑀, and AtB⬘␩) are located on chromosome
III, the three remaining ones, AtB⬘␣, AtB⬘␥, and
AtB⬘␪, being placed on chromosomes V, IV, and I,
respectively. Only chromosome II presents no genes
related to this family. It is noticeable that AtB⬘␦ and
AtB⬘␩ are adjacent genes, separated by less than 800
bp, which opens interesting questions about their
evolutionary origin that will be discussed below. The
chromosomal position of the genomic clones that
contain the eight genes is shown in Figure 2.
The comparison of the cDNA sequence from AtB⬘␣,
AtB⬘␤, AtB⬘␦, and AtB⬘␥ against the sequence of their
corresponding genomic clones allowed us to determine the intron-exon structure of the four genes,
which is described here for the first time, to our
knowledge. We also identified ESTs corresponding
to all the previously described genes (Table I), but the
partial sequences of the cDNA clones did not reveal
any new exon different from those described before,
although this cannot be completely discarded until
the sequences of the new cDNAs are completely determined. The structure of the four new genes presented in this work was determined using prediction
programs as GenScan, and, in some cases, partially
confirmed by the EST sequences. Table I summarizes
the data obtained, which are graphically depicted in
Figure 2.
The eight genes present ORFs of similar size, and
the differences are mostly produced at the variable 5⬘
and 3⬘ variable regions. All genes except AtB⬘␭ and
AtB⬘␩ present a simple exon-intron structure, with
Table I. Genomic organization of the Arabidopsis and rice genes
No. of
Exons
5⬘ URa
Lengthc
CR1b
Lengthc
Intron
Lengthc
AtB⬘␣, U73526
AtB⬘␤, U73527
AtB⬘␥, U73528
2
2
3
284
360
414
1,161
1,161
1,191
AtB⬘␦, AF0623396
2
393
AtB⬘⑀, AJ276037
AtB⬘␨, BAB02360
AtB⬘␩, BAB01065
AtB⬘␪, AAG09562
OsB⬘␨, AJ312314
OsB⬘␩, CAC09487
OsB⬘␪, AJ312315
OsB⬘␬, AJ312313
OsB␭, BAB07976
2d
2d
4
2d
2
2
2d
2
2d
NDe
ND
326
ND
ND
ND
ND
ND
ND
Gene Name and
Accession No.
a
Untranslated region.
b
CR2
Lengthc
3⬘ UR
Lengthc
ORF
Lengthc
592
530
73
324
336
375
447
664
587
1,485
1,497
1,566
1,128
95
303
588
1,431
1,188
1,254
1,227
1,164
1,194
1,173
1,206
1,164
1,125
82
94
690
200
1,644
1,389
1,228
404
1,238
306
384
303
312
360
357
345
369
387
ND
ND
ND
ND
427
303
ND
384
ND
1,494
1,638
1,530
1,476
1,664
1,530
1,551
1,533
1,512
Coding region.
Plant Physiol. Vol. 129, 2002
c
In bp.
d
ESTs Found
AV522466 and AW004404
AA395160, T43172, and AV561236
BE520334, AI999159, AA042311, BE520336, AV547536,
AA597610, and AA597613
AI993739, AV562733, BE521372, BE521374, BE521373,
AV558379, N97014, and N96717
Not found
BE524995
AV548590
BE524785 and AI996905
AU092036, AU092032, D22058, D22057, and D22055
AU032466, AU057748, AU057747, and AU032467
Not found
AJ272423, BE040415, AU085794, and C27982
Not found
Theoretically predicted.
e
Not determined.
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811
Terol et al.
Figure 2. Genomic organization of the AtB⬘ genes on the Arabidopsis chromosomes. Bars marked with roman numerals
represent Arabidopsis chromosomes (I and III–V), with several genetic markers indicated for each one. The centromere
regions are shown as black circles. Blowups of the regions between two genetic markers containing the genomic clones with
the AtB⬘ genes are shown, an arrow indicating the exact position of the clones. The intron-exon structure of the eight genes
is also illustrated, with each gene shown as a blowup of the genomic clones where they are located. Black boxes indicate
coding regions and white boxes represent untranslated ones.
two coding exons separated by an intron of variable
size. All the genes display exons of similar size, with
the first one being approximately one-half the size of
the second, whereas the intron length is more variable. The presence of an intron in the 5⬘-untranslated
region of AtB⬘␭ was reported by Haynes et al. (1999).
On the other hand, the sequence of the EST found for
AtB⬘␩ revealed the existence of two non-coding small
exons preceding the coding ones, indicating that this
gene is composed of at least four exons. The existence
of non-coding exons in the four newly predicted
genes will be determined when complete cDNAs are
812
sequenced and the 5⬘ region of the genes is analyzed.
The similar structure of the genes speaks about a
common origin.
The PP2A Bⴕ Subunit Family in Rice
We considered it of interest to study the family of
B⬘ regulatory subunits of PP2A in another plant genome, although the Arabidopsis genome is the only
one that has been completed so far. One of the most
advanced genome projects is The International Rice
Genome Sequencing Project, that, up to now, has
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Plant Physiol. Vol. 129, 2002
Protein Phosphatase 2A B⬘ Regulatory Subunit Family in Plants
released 78.8 Mb of DNA sequence to the DNA Data
Bank of Japan (http://www.ddbj.nig.ac.jp/). On the
other hand, the Monsanto Rice Genome Sequencing
project (http://www.rice-research.org/) has produced sequence from 3,391 BACs distributed across
the genome of rice cv Nipponbare (the same cultivar
used by The International Rice Genome Sequencing
Project) that constitute a “draft” dataset consisting of
52,202 contigs, corresponding to 259 Mb of assembled sequence data, which represents approximately
70% of the total genome size.
Thus, we decided to analyze the partially sequenced genome of rice, and we compared the eight
Arabidopsis proteins against the nonredundant DNA
Data Bank of Japan and Rice Research Organization
nucleotide sequence databases in a TBLASTN search.
This search yielded five genomic clones that contained
sequences similar to the B⬘ family two from GenBank,
and three from the Rice Research Organization (Table
I). The proteins we found in the GenBank clones had
been already annotated and correspond to entries
CAC09487 and BAB07976, respectively, although in
the first case the predicted polypeptide is much
shorter than the one we obtained, probably due to a
mistake in the automatic annotation process of the
clone. The genomic clones from the Rice Research
Organization were analyzed with the GenScan program, and three ORFs coding for three proteins of 510,
517, and 516 amino acids were predicted. Comparative analysis with respect to the Arabidopsis proteins
showed a high degree of conservation between them,
with similarities ranging from 58.6% to 77.5%.
To determine whether the five predicted genes
were transcriptionally active, a BLAST search was
performed on the EST databases, producing several
cDNA clones identical to three of the predicted genes.
The comparison of the genomic and cDNA sequences
allowed us to confirm the intron-exon structure of
these three genes, as well as to determine the length of
the untranslated regions. The structure determined
for the rice genes is similar to that of the Arabidopsis
ones, with ORFs about 1,200 bp long, composed of
two exons separated by a single intron. No EST
clones were found for the other two predicted genes.
All these data are summarized in Table I.
Based on the degree of conservation with respect to
Arabidopsis, we named the rice proteins OsB⬘␨ (accession no. AJ312314), OsB⬘␩ (accession no. CAC09487),
and OsB⬘␪ (accession no. AJ312315). For the two remaining proteins, it was not possible to determine an
ortholog with the Arabidopsis B⬘ isoforms, so we
decided to name them OsB⬘␬ and OsB⬘␭, following
the existing nomenclature.
EST Analysis in Plants
The GenBank databases contain more than
1,000,000 plant ESTs, produced by large-scale cDNA
sequencing projects from many different species,
Plant Physiol. Vol. 129, 2002
from green algae to the angiosperms. To obtain a
broader view of the B⬘ regulatory subunit of PP2A
family in plants, we analyzed the plant EST sequences available in the databases in an attempt to
obtain cDNAs coding for proteins belonging to this
family. A TBLASTN search was carried out with the
eight Arabidopsis B⬘ isoforms as a query, and more
than 200 ESTs that produced protein fragments with
significant similarity were identified. The ESTs belonged to 20 plant species from five different classes,
including green algae, ferns, conifers, and mono- and
dicotyledons (Table III). The similarity with respect
to the Arabidopsis B⬘ regulatory subunits presented
by some protein fragments was quite striking: The
EST AI489160, from tomato, produced a 223-amino
acid fragment 95% similar to AtB⬘␣; the EST
BF006061, from medic barrel, produced a 197-amino
acid fragment 93% similar to AtB⬘␨, etc.
Despite the partial data, it was evident that the
protein fragments obtained for one species were not
identical, and showed different similarity scores with
respect to the Arabidopsis proteins, which suggested
the existence of several isoforms. As described in
“Materials and Methods,” we performed an extensive analysis of more than 100 EST sequences in an
effort to identify the minimum number of B⬘ isoforms
present in some of the species that showed related
EST sequences: Chlamydomonas reinhardtii, Ceratopteris richardii, corn, potato, soybean, medic barrel,
and tomato. All the ESTs available for one species
were assembled, and the resulting contigs that produced nonidentical, overlapping protein fragments
with significant similarity with respect to the B⬘ subunits were considered. Only identical EST sequences
were included in a contig to ensure that each consensus corresponded to a unique cDNA. This way, we
could establish that each contig corresponded to a
different cDNA coding for one member of the B⬘
regulatory family; thus, the number of contigs represented the minimum number of PP2A B⬘ genes existing in one species.
Table IV summarizes the results obtained for the
different species, showing the contigs from which the
consensus cDNA sequences were obtained, the best
similarity scores with respect to the Arabidopsis proteins, and the length of the compared fragments. It
can be appreciated that most of the species contain
between five and seven genes coding for these proteins in each plant species, and our hypothesis is that
the final number will be very similar to that of Arabidopsis. It is noteworthy that the eight EST sequences from the green algae C. reinhardtii, form a
single contig that produces a partial protein of 365
amino acids, suggesting that this species only possesses one representative of the B⬘ regulatory subunit
family, which we named CrPP2AB⬘. In the case of the
fern C. richardii, the three available ESTs might correspond to two different cDNAs, but it will be necessary to analyze more ESTs to confirm this result. In
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Terol et al.
Table III. ESTs coding for proteins similar to the Arabidopsis B⬘ regulatory subunits
Class
Chlorophyceae
Filicopsida
Conlferopsida
Dicotyledons
Species
C. reinhardtii
C. richardii
Pinus taeda
Soybean
Gossypium arboreum
Lotus japonicus
Medic barrel
Tomato
Potato
Monocotyledons
Hordeum vulgare
Sorghum bicolor
Triticum aestivum
Corn
EST Accession No.
BE860529, AV629664, AV627400, AV643417, and BF860529
BE642150, BE642127, and BE643333
BG275756 and AW461265
BE660710, BE555346, AW705726, BE023093, BE329729, BF596111,
AW705176, BG239516, BG650995, AW704844, AW132776, BE657883,
AW133398, BE658563, AW397699, BF424123, BF596789, BE347283,
BE330805, AI966136, BE802756, and AW306926
BF276574, BF270304, BG443130, BE055870, and BG445534
AU089016 and AV425928
BF006061, BF634957, BE248849, AL377837, BF003172, AW686915,
BF646322, AW693580, AW267888, BF004594, AL376694, BF003171,
BE325805, BE218374, BE998355, BF650340, and BG454035
AI489160, AW033985, BE344497, BG130386, AW220451, AI896639,
AW738120, AW931631, AI489441, BF113211, AI486727, AW650085,
AW093058, AI483511, AI486137, AW932061, AW932847, AW738117,
and BE353645
BG351390, BG598923, BE341841, BE923737, BG595709, BG595883, and
BE343144
BF258401, BF257656, and BF623532
AW563546, AW563590, AW677201, AW671157, BE594608, and BG356620
BE430742, BE400968, BE429666, BE430996, and BE412306
BG320458, AI621449, AI586835, AW165473, AW562969, AW052973, and
AW600513
general terms, the degree of conservation of all the
protein fragments obtained with respect to the Arabidopsis B⬘ isoforms is striking, with similarities
ranging from 64% to 97%.
The 25 ESTs from tomato assembled into eight
contigs, six of which were clearly identified as different cDNAs. Two of the cDNAs seemed to present
complete ORFs that coded for proteins of 511 and 506
amino acids, which is the average size of this type of
proteins. Based on the similarity data, we named
them LeB⬘␣, and LeB⬘␬. On the other hand, several
consensus cDNA sequences from C. reinhardtii, potato, soybean, medic barrel, and tomato produced
protein fragments larger than 300 amino acids, which
represent almost 60% of the average length of these
proteins, so we decided to use them in a phylogenetic
analysis. These partial proteins were also named
based on their similarity to the previously described
ones (CrPP2AB⬘, StB⬘␣, GmB⬘␣ and GmB⬘␩, MtB⬘␬,
and LeB⬘␩, respectively).
Comparative Analysis of the Proteins
A multiple alignment was performed with the
Clustal X program, including the Arabidopsis, rice,
and tomato B⬘ isoforms characterized so far, the partial proteins from potato, soybean, medic barrel, and
C. reinhardtii, and several B⬘ regulatory subunits from
animals: Caenorhabditis elegans (CePP2A-B⬘), Drosophila melanogaster (DmPP2A-B⬘), Xenopus laevis (XlB⬘⑀),
Oryctolagus cuniculus (␥), and humans (B56␣).
The multiple alignment of the protein sequences
shows the existence of a high degree of similarity
between them, with the central core regions being the
814
most conserved, and the amino- and carboxyterminal regions the most variable (Fig. 3). In this
central region, many amino acids are identical and
most of the substitutions are conservative ones. On
the contrary, the amino- and carboxy-terminal regions are highly variable and they accumulate most
of the size and amino acid identity changes.
The multiple alignment also shows differences between the proteins from plant and animal species.
This way, eight positions (indicated with circles in
Fig. 3) display a different conserved amino acid depending on the origin of the protein: All the animal
proteins display one residue, whereas the plant ones
present a different conserved amino acid.
Relationship Analysis
Because the Arabidopsis genome is completely
characterized and all the B⬘ regulatory subunits have
been identified in this species, we found it interesting
to perform the relationship analysis in this plant and,
posteriorly, to extend it to the remaining species. The
genetic distance between the AtB⬘ proteins was calculated using the poisson method (Table V) and the
distances obtained were of the same order between
all the proteins, confirming that the ones described in
this paper belong to the B⬘ subunit family. The presence of three pairs of very close proteins can be
observed that present the lowest distance values
(AtB⬘␣ and AtB⬘␤, AtB⬘␥ and AtB⬘␨, and AtB⬘␩ and
AtB⬘␪), suggesting that an event of gene duplication
might be involved in their origin. It is intriguing that
AtB⬘␦ and AtB⬘␩, which are located in tandem on
chromosome III, present a high genetic distance (d ⫽
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Plant Physiol. Vol. 129, 2002
Protein Phosphatase 2A B⬘ Regulatory Subunit Family in Plants
Table IV. Analysis of the EST sequences
Species
Consensusa
Similarityb
Lengthc
C. reinhardtii
(eight ESTs)
C. richardii
(three ESTs)
Corn
(eight ESTs)
Contig 1
CrPP2AB⬘
Contig 1
Contig 2
Contig 1
Contig 2
Contig 3
Contig 5
Contig 6
Contig 1
Contig 2
Contig 3
Contig 4
Contig 5
StB⬘␣
Contig 7
Contig 2
Contig 3
Contig 5
Contig 7
GmB⬘␣
Contig 9
Contig 11
GmB⬘␩
Contig 13
Contig 2
Contig 3
Contig 4
Contig 5
MtB⬘␬
Contig 6
Contig 9
Contig 2
LeB⬘␩
Contig 3
LeB⬘␣
Contig 5
LeB⬘␬
Contig 6
Contig 7
Contig 8
74
365
89
72
81
83
74
72
76
94
64
86
86
90
232
212
105
92
109
128
154
157
136
132
154
223
82
81
74
97
93
180
90
175
90
319
87
88
241
332
68
76
90
76
72
303
265
248
183
400
91
65
81
201
190
342
82
511
69
506
70
84
63
129
236
172
%
Potato
(10 ESTs)
Soybean
(31 ESTs)
Medic barrel
(31 ESTs)
Tomato
(25 ESTs)
a
b
Consensus sequence of the contig.
Best similarity score
obtained in the BLAST search respect the Arabidopsis B⬘ subunit
c
proteins.
Length, in amino acids, of the compared sequences.
0, 244), which indicates that, if there was any, the
duplication event happened long ago. AtB⬘␦, as well
as AtB⬘⑀, are the most distant proteins, with similar
distance values with respect to the remaining members of the family, which might indicate that they
diverged very early during the evolution of the
family.
A phylogenetic tree was constructed using the
neighbor joining method, based on the distance matrix previously calculated (Fig. 4). It can be appreciated that the eight proteins cluster into two groups,
one formed by the pairs AtB⬘␥ and AtB⬘␨, and AtB⬘␩
and AtB⬘␪, plus AtB⬘␦, and the second composed of
the cluster AtB⬘␣ and AtB⬘␤, plus AtB⬘⑀. The topolPlant Physiol. Vol. 129, 2002
ogy of the tree reflects the relationships between the
proteins established by the genetic distances, presenting three clearly differentiated clusters (composed of the B⬘ isoforms ␣ and ␤, ␥ and ␨, and ␩ and
␪), confirmed by bootstrap values of 100.
A similar analysis was performed for all the plant
protein sequences used in the alignment: The genetic
distances were calculated with the poisson correction
distance and a phylogenetic tree was constructed
with the unweighted pair group method analysis
(UPGMA) method (Fig. 5), using the bootstrap test
with 1,000 iterations. It can be observed that the
animal and plant proteins form two separated clusters, supported by a bootstrap value of 100. On the
other hand, all the plant sequences group in three
separated clusters, which are supported by the bootstrap values obtained and by the fact that trees constructed with the neighbor joining and maximum
parsimony methods present an identical topology
(data not shown). This clustering suggests the existence of three main subfamilies of B⬘ regulatory subunits, which we have designated as ␣, ␩, and ␬. The
protein from the green algae, CrPP2AB⬘, as well as
AtB⬘␦ and AtB⬘⑀, appear in separated branches,
which, in the first case, can reflect the evolutionary
distance between the unicellular green algae and the
complex higher plants. In the case of AtB⬘␦ and
AtB⬘⑀, it could not be discarded that close relationships can be found in other species when more sequence data are available.
DISCUSSION
The PP2A Bⴕ Regulatory Subunit Family in Arabidopsis
In this work, we report the identification and molecular characterization of four novel Arabidopsis
genes, AtB⬘⑀, AtB⬘␨, AtB⬘␩, and AtB⬘␪, which encode
four new isoforms of the PP2A B⬘ regulatory subunit
family in this species. Previous studies revealed the
presence of four different AtB⬘ isoforms in Arabidopsis: ␣, ␤, ␥, and ␦, but also suggested the existence of
an additional B⬘ gene (Latorre et al., 1997; Haynes et
al., 1999). The analysis of the complete genome of
Arabidopsis has allowed us to identify the ⑀, ␨, ␩, and
␪ isoforms, and to ensure that there are no more
additional members of the family. The finding of
cDNAs identical to AtB⬘␨, AtB⬘␩, and AtB⬘␪, and the
experimental evidences obtained for AtB⬘⑀, confirm
that the four predicted genes are actively transcribed
and, therefore, should be considered functional
genes. This way, the description of PP2A B⬘ regulatory subunit family in Arabidopsis is now completed,
and it has been established that it is composed of
eight genes that code for eight different B⬘ isoforms.
RT-PCR experiments show that AtB⬘⑀ mRNAs are
present in all the Arabidopsis tissues analyzed. Similar results were obtained when the expression pattern of the genes encoding the ␣, ␤, ␥, and ␦ AtB⬘
isoforms was analyzed (Latorre et al., 1997; Haynes et
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815
Terol et al.
Figure 3. A, Alignment profile obtained with the Clustal X program of the B⬘ proteins from the species mentioned below. The
height of the bars indicates the number of identical residues per position. The arrowheads indicate the region shown in the
multiple alignment. B, Sequence alignment of the core region of the B⬘ regulatory subunits from various organisms, performed
with the Clustal X software. The protein sequences included are from Arabidopsis (AtB⬘␣, AtB⬘␤, AtB⬘␥, AtB⬘␦, AtB⬘⑀, AtB⬘␨,
AtB⬘␥, AtB⬘␩, and AtB⬘␪), rice (OsB⬘␨, OsB⬘␩, OsB⬘␪, OsB⬘␬, and OsB⬘␭), tomato (LeB⬘␣, LeB⬘␩, and LeB⬘␬), soybean (GmB⬘␣
and GmB⬘␩), medic barrel (MbB⬘k), potato (StB⬘a), C. reinhardtii (CrPP2AB⬘), C. elegans (CePP2A-B⬘, accession no.
CAA98422), D. melanogaster (DmPP2A-B⬘, accession no. CAB86364), X. laevis (XlB⬘⑀, accession no. AAG22076), O.
cuniculus (OcB⬘␥, accession no. Q28651), and human (HsB56␣, accession no. NM 006243). Black boxes indicate amino
acid identity and gray boxes indicate conservative changes. Dashes represent gaps to maximize amino acid alignment. Circles
indicate residues that are differently conserved between animals and plants.
816
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Plant Physiol. Vol. 129, 2002
Protein Phosphatase 2A B⬘ Regulatory Subunit Family in Plants
Table V. Genetic distances between the AtB⬘ isoforms estimated with the poisson correction
AtB⬘␣
AtB⬘␤
AtB⬘␥
AtB⬘␦
AtB⬘⑀
AtB⬘␨
AtB⬘␩
AtB⬘␪
HsB56␣
AtB⬘␣
AtB⬘␤
AtB⬘␥
AtB⬘␦
AtB⬘⑀
AtB⬘␨
AtB⬘␩
AtB⬘␪
–
0.157
0.461
0.544
0.444
0.486
0.482
0.496
0.718
–
0.403
0.518
0.427
0.430
0.437
0.464
0.682
–
0.454
0.548
0.152
0.324
0.358
0.740
–
0.656
0.444
0.420
0.447
0.750
–
0.575
0.548
0.544
0.754
–
0.352
0.365
0.754
–
0.124
0.695
–
0.736
al., 1999). All of them are ubiquitously expressed,
suggesting that the PP2A B⬘ regulatory subunits are
required for basic housekeeping function in plant
cells. It is interesting to mention that in some cases
different levels of expression were observed in different organs (Latorre et al., 1997; Haynes et al.,
1999).
It had been reported that several B⬘ regulatory
subunits were differentially expressed in response to
heat shock stress. In Arabidopsis, only mRNAs derived from the AtB⬘␥ gene accumulate differentially
after heat shock conditions. In this work, we demonstrate that AtB⬘⑀ expression levels, as previously reported for AtB⬘␣, ␤, and ␦ genes (Latorre et al., 1997;
Haynes et al., 1999), do not fluctuate in response to
heat shock. Similar experiments will have to be carried out with AtB⬘␨, AtB⬘␩, and AtB⬘␪.
The PP2A Bⴕ Regulatory Subunit Family in Plants
In an effort to characterize the PP2A B⬘ regulatory
subunit family in other plants, we have analyzed the
genomic information available for rice. We could
identify five B⬘ isoforms, which presented a high
Figure 4. Unrooted phylogeny of the eight isoforms of the Arabidopsis B⬘ regulatory subunit family. The neighbor-joining tree was obtained from an alignment using Clustal X and Molecular Evolutionary
Genetic Analysis programs. The HsR5␣ protein from humans was
used as an outgroup. The bootstrap values, shown at the nodes, are
percentages for 1,000 replications. Tree branches are proportional to
genetic distances.
Plant Physiol. Vol. 129, 2002
degree of conservation in sequence and structure
with respect to the Arabidopsis B⬘ subunit genes and
proteins. We have also performed an extensive analysis of the EST databases, which produced a large
number of cDNAs coding for proteins similar to the
Arabidopsis B⬘ regulatory subunits. The cDNAs corresponded to species from at least five different orders, including green algae (Chloroficeae), ferns (Filicopsida), conifers (Coniferopsida), monocotyledons,
and dicotyledons. This fact suggests that the family
of PP2A B⬘ regulatory subunits is present throughout
the whole plant kingdom. The EST analysis also produced two complete proteins from tomato (LeB⬘␣ and
LeB⬘␩), as well as a number of large protein fragments from potato, soybean, and medic barrel. Comparison of all these protein sequences suggest a high
degree of conservation of these proteins in plants, a
fact that is not surprising if we consider the striking
similarity found between the B⬘ proteins from organisms as different as Arabidopsis, yeast, insects, and
vertebrates.
Analysis of the Structure of the Bⴕ Regulatory Subunits
Sequence comparison of the plant B⬘ subunits
shows that they contain a highly conserved central
domain, and very diverged amino- and carboxyterminal regions. The conservation of the central region is strikingly high, even when we compare the
plant B⬘ subunits and their homologs in other organisms like D. melanogaster, C. elegans, X. laevis, rabbits,
and humans. The high similarity found in the central
region suggests that it could be the putative functional core of the protein that could be essential for
the assembly of the B⬘ regulatory subunits with the
other components of the PP2A complex, which are
also highly conserved proteins (Depaoli-Roach et al.,
1994).
On the other hand, different studies suggest that
the highly variable amino- and carboxy-terminal regions of these proteins may play an important role in
defining properties such as the substrate specificity
and/or the cellular localization of the Ser/Thr PPs
(Janssens and Goris, 2001). In fact, in some mammal
isoforms of B and B⬘ regulatory subunits, several
regions in their carboxy termini have been identified
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817
Terol et al.
Figure 5. Phylogenetic tree based on the multiple alignment in Figure 1, constructed with the
UPGMA method, with the genetic distances calculated by the poisson correction for proteins.
The gray boxes indicate the main clusters obtained. The plant and animal protein sequences
separate into two clusters. Most of the plant
protein sequences group in three clusters, considered B⬘ subfamilies named ␣, ␩, and ␬.
as being responsible for PP2A subcellular targeting,
and also for being the subject of phosphorylation,
which supports the notion that the variable regions
of B⬘ regulatory subunits control PP2A function
(Zhao et al., 1997). On the other hand, considering
that reversible protein phosphorylation, which is the
mechanism by which many biological functions are
regulated, requires the coordinated action of protein
kinases and PPs, and that only a limited number of
PP catalytic subunits are present in the cell, the existence of B and B-related regulatory subunits, present
in multiple isoforms, raises the possibility that a combinatorial association could generate enough PPs to
counterbalance the action of the more numerous protein kinases (Depaoli-Roach et al., 1994). In the case
of PP2A, heterotrimer complexes are formed by a
catalytic subunit and two regulatory subunits (A and
B), each with different isoforms, which can give rise
to a number of different combinations that could
explain how the PP2A phosphatases are involved in
a variety of processes such as the initiation and termination of translation, apoptosis, or stress responses, in multiple types of eukaryotic cells (for
review, see Janssens and Goris, 2001). The elucidation of the specific function of these complexes will
818
be very important to understand the regulation of
protein phosphorylation in plants.
The Number of Bⴕ Isoforms and the
Organisms’ Complexity
Our analysis of the Arabidopsis genome has shown
that there are eight isoforms of the B⬘ family. In rice,
we have found that the partially sequenced genome
contains five regulatory subunits and, considering
that about 30% the genome remains to be sequenced,
we estimate that the number of B⬘ isoforms will be
very similar to that of Arabidopsis. The study performed on the ESTs has allowed us to identify the
minimum number of genes coding for B⬘ regulatory
subunits in the genome of the analyzed species. This
way, we have found that there are at least five genes
in corn, six in potato, medic barrel, and tomato and
seven in soybean. Our data indicate that the number
of isoforms in this species would also be on the same
order than in Arabidopsis. On the contrary, the unicellular green algae, C. reinhardtii, seems to have only
one B⬘ regulatory subunit, as is suggested by the fact
that all the ESTs found are identical and form a single
contig representing a unique cDNA. The difference
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Plant Physiol. Vol. 129, 2002
Protein Phosphatase 2A B⬘ Regulatory Subunit Family in Plants
in the number of regulatory proteins between the
higher plants and C. reinhardtii is significant, and it is
not biased by the number of ESTs available in the
databases, which is in the same range: There are
83,453 ESTs from C. reinhardtii, 42,602 from potato,
and 96,793 from corn (National Center for Biotechnology Information, dbEST release 070601).
In the animal kingdom, we find a similar situation:
In humans and rabbits, five genes encoding PP2A B⬘
regulatory subunits have been described (McCright
and Virshup, 1995; McCright et al., 1996a; Csortos et
al., 1996; Tehrani et al., 1996; Zolnierowicz et al.,
1996), whereas the lower eukaryote yeast possesses a
single member of the family, RTS1 (Shu et al., 1997),
Schizosaccharomyces pombe contains two genes, par1⫹
and par2⫹ (Jiang and Hallberg, 2000), there is only a
single B⬘ protein in D. melanogaster (Berry and Gehring, 2000), and we have identified only two members of the family in C. elegans (C13G3.3b and
W08g11.4). It appears that in plant and animal kingdoms, there is a correlation between the complexity
of the organism and the number of existing B⬘ isoforms, so that the more complex the organism is, the
larger the number of PP2A B⬘ isoforms.
Interestingly, plants and animals would have developed different strategies to increase the number of
these regulatory subunits. The five B⬘ genes of human and rabbit produce seven and eight isoforms,
respectively, due to alternative splicing of one of
them (Csortos et al., 1996). On the contrary, there is
no evidence of this mechanism in Arabidopsis: Only
one transcript is derived from the AtB⬘␣, ␤, and ␦
genes, as detected by northern blot (Latorre et al.,
1997; Haynes et al., 1999), and, although three
mRNAs with different sizes are derived from the
AtB⬘␥ gene, it has been suggested that the three
produced identical proteins (Haynes et al., 1999). We
cannot rule out the possibility of alternative splicing
in all the other B⬘ isoforms analyzed in Arabidopsis
and the other plants, although the extensive EST
analysis we have performed does not provide any
evidence suggesting it. This way, the eight genes
found in Arabidopsis, and probably in the higher
plants, would produce the same number of proteins
as the five genes described in rabbits or humans.
These data suggest that plants chose to increase the
number of genes to obtain the necessary number of B⬘
regulatory subunits, whereas vertebrates, with a limited number of genes, produce a similar number of
different isoforms by means of alternative splicing.
However, given the level of current information regarding this hypothesis, more information will be
necessary to strongly support these conclusions.
Evolutionary History
Two main conclusions can be drawn from the phylogenetic analysis (Fig. 5): First, despite the high degree of conservation, the plant and animal B⬘ proteins
Plant Physiol. Vol. 129, 2002
have evolved independently; and second, the B⬘ isoforms from plants can be subdivided into three subfamilies, which we have named ␣, ␩, and ␬. The
phylogenetic trees constructed with different methods (neighbor joining, UPGMA, and maximum parsimony) and significant bootstrap values support
both ideas. First, all the plant proteins cluster into a
group clearly separated from the animal sequences,
indicating an independent evolutionary history. This
separation is illustrated by the comparison of the B⬘
subunit core region from plants and animals, with
several positions presenting a conserved amino acid
in plants and a different one in animals (Fig. 3B).
Second, most of the plant proteins group in three
clusters, indicating that isoforms from different species are more similar between them than to the other
B⬘ subunits from the same species. The grouping of
the proteins has been shown to be consistent in all the
trees constructed with different methods, suggesting
the existence of at least three subfamilies, which we
have named ␣, ␩, and ␬. The number of subfamilies
could increase if close relationships are found for
AtB⬘␦ and AtB⬘⑀, which are left outside the main
clusters, or new isoforms from other plant species are
characterized. On the other hand, the position of the
C. reinhardtii B⬘ protein may be a reflection of its
primitive evolutionary position with respect to the
higher plants.
Our results suggest that the family of PP2A B⬘
regulatory subunits appeared very early during the
evolution of the eukaryotes, even before the separation of plants and animals, probably as a single-copy
gene. The evolutionary process produced increasingly complex organisms, with new functions that
required new regulatory subunits for PP2A; thus,
new genes appeared by successive duplications of
the original ancestor. This hypothesis would explain
why the more complex the organism, the larger the
number of genes encoding for PP2A B⬘ subunits.
Plants and animals would have adopted different
strategies to increase the number of isoforms, which
in the case of animals implies alternative splicing.
The evolution of the family of B⬘ regulatory subunits
occurred in parallel in both kingdoms, starting from
a common ancestor, which produced, by duplication
and subsequent divergence, the different subfamilies
and isoforms present nowadays in all the organisms.
MATERIALS AND METHODS
DNA Sequencing
Within the frame of an international consortium for the
Arabidopsis genome sequencing, we determined the nucleotide sequence of the P1 and BAC genomic clones
T15C9, F11C1, F3A4, T20E23, and T12C14, corresponding
to the short arm of chromosome III (Salanoubat et al., 2000).
The accession numbers for these sequences are AL132970,
AL132976, AL132970, AL133363, and AL162507. Information about performance of analysis and a detailed annota-
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Copyright © 2002 American Society of Plant Biologists. All rights reserved.
819
Terol et al.
tion of these database entries can be viewed at the Munich
Information Center for Protein Sequences (http://mips.gsf.
de/). Our contribution to the sequencing of chromosome
III has been the determination of the nucleotide sequence of
the inserts of these five clones, which amounts to a total of
540,430 bp.
The BAC insert DNAs were sequenced using a shotgun
library approach, which produced a library containing
about 2,000 clones with inserts in the range of 0.5 to 1.5 kb
long for each BAC clone. Plasmid DNA extractions were
made with the High Pure Plasmid Isolation Kit (Boehringer
Mannheim/Roche, Basel). DNA sequences were obtained
with an ABI PRISM 377 Automated DNA Sequencer
(Perkin-Elmer Applied Biosystems, Foster City, CA), using
the ABI PRISM Big Dye Terminator Cycle Sequencing
Ready Reaction Kit and the ABI PRISM dRhodamine Terminator Cycle Sequencing Ready Reaction Kit (PerkinElmer Applied Biosystems). Specific primers were designed when necessary to fill gaps in the sequence. Editing
and assembling of the DNA sequences were made using
the STADEN software package (Staden, 1996). A total of
84,233 bp were analyzed, with an average redundancy of
7.62.
Sequence Analysis
The GenScan program (Burge and Karlin, 1997) was used
to predict the presence of putative ORFs in the complete
DNA sequence of the BAC insert (http://genes.mit.edu/
GENSCAN.html). Databases searches looking for sequence
similarities were performed using the BLAST program (Altschul et al., 1997) at the National Center for Biotechnology
Information (http://www.ncbi.nlm.nih.gov:80/BLAST), or
in specific Arabidopsis databases such as The Arabidopsis
Information Resource (http://godot.ncgr.org/Blast/
index.html) or the Arabidopsis Database (http://genomewww.stanford.edu/Arabidopsis/index.old.html). Multiple
sequence alignments were made using the Clustal X program (Thompson et al., 1997). Analyses of the putative
protein sequences deduced from the DNA were performed
using different programs such as EMOTIF SEARCH
(http://dna.stanford.edu/identify/), to look for functional
domains in the sequences, or PSORT (http://psort.nibb.
ac.jp/form.html), to identify putative signals for subcellular localization of the proteins. Genetic distances were calculated with the Poisson correction method (Nei and
Chakraborty, 1976) for amino acid sequences; the phylogenetic trees were constructed with the neighbor joining (Saitou and Nei, 1987), UPGMA (Swofford and Selander, 1981),
and maximum parsimony (Fitch and Farrish, 1974) methods; and the bootstrap test was carried out with 1,000
iterations. These analyses were performed with the Molecular Evolutionary Genetic Analysis platform (Sudhir et al.,
1993).
Analysis of AtBⴕ⑀ Expression
Arabidopsis ecotype Columbia was used in all experiments. Plants were grown under a 16-h-light/8-h-dark il820
lumination regime in a growth cabinet at 23°C. Plants
grown in standard conditions were used as control. Plant
material was harvested and immediately frozen in liquid
N2 at ⫺80°C. Total RNA was extracted from leaves 4.5
weeks old, leaves 7 weeks old, seeds, stems, and flowers
following a standard procedure (Prescott and Martin, 1987)
and purified by LiCl precipitation. cDNAs were obtained
using SuperScript II RT (Life Technologies/Gibco-BRL,
Cleveland) and oligo(dT) primers.
PCR reactions were carried out using the following conditions: initial denaturation at 94°C for 5 min, followed by
40 cycles of 30 s at 94°C, 30 s at 55°C, and 30 s at 72°C. Upon
completion of the last cycle, samples were incubated for 4
min at 68°C. Primers BE1 and BE2 were used to amplify
AtB⬘⑀, BG1, and BG2 for AtB⬘␥ (Latorre et al., 1997), and
AC1 and AC2 for actin-1. The sizes of the amplified fragments were 500, 1,032, and 1,131 bp, respectively. Actin
transcripts were amplified to check RNA amounts in all
samples.
Heat Shock Experiments
Adult plants were incubated at 37°C for 2 h for heat
shock experiments. Plants grown in standard conditions
were used as control. Total RNA extraction and PCR reaction were performed as indicated above. AtB⬘⑀ and AtB⬘␥
were amplified by RT-PCR from heat-shocked and control
plants’ RNA.
As a control for quantitation of RNA levels in all samples, ribosomal RNA amplification with primers 18S1 and
18S2 and competimers was carried out. 3⬘-Modified oligonucleotides were used as competimers to amplify an approximately 500-bp fragment of the 18S rRNA. Ratios from
6:10 to 9:10 modified:unmodified primers were used.
The sequences of the oligonucleotides used in the PCR
reactionsare:rRNA,18S1,5⬘-TGGGATATCCTGCCAGTAGTCAT-3⬘ and 18S2, 5⬘-CTGGATCCAATTACCAGACTCAA-3⬘; AtB⬘⑀, BE1, 5⬘-TTCTGGTAAAGTCAATGAGACG-3⬘
and BE2, 5⬘-ATCAGCCTATGCTCCTCTCTC-3⬘; AtB⬘␥,
BG1, 5⬘-TCCTTCTGCGAATCACGAGAG-3⬘ and BG2, 5⬘GACGAGCACTGCCTCGTTGC-3⬘; and actin-1, AC1, 5⬘ATGATGCACCTAGAGCTG-3⬘ and AC2, 5⬘-TTCCAGGGAACATTGTGG-3⬘.
EST Analysis
The search for ESTs in Arabidopsis and other plant
species was performed by comparing the Arabidopsis protein sequences against different EST databases dynamically
translated in all reading frames in a TBLASTN search. For
rice (Oryza sativa), tomato (Lycopersicon esculentum), soybean (Glycine max), medic barrel (Medicago truncatula), potato (Solanum tuberosum), corn (Zea mays), Chlamydomonas
reinhardtii, and Ceratopteris richardii, the searches were limited to one species at a time to detect all the existing ESTs
that presented significant similarity. The selected ESTs
were assembled with the STADEN software package (Staden, 1996), with the assembling parameters such that only
identical sequences were included in one contig. The re-
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Copyright © 2002 American Society of Plant Biologists. All rights reserved.
Plant Physiol. Vol. 129, 2002
Protein Phosphatase 2A B⬘ Regulatory Subunit Family in Plants
sulting contigs were translated in all reading frames, and
the amino acid sequences were tested in a BLASTP search,
selecting those that presented a significant similarity with
respect to the PP2A B⬘ regulatory subunits existing in the
databases. Finally, the amino acid sequences were aligned
with the Clustal X program (Thompson et al., 1997), and
only the contigs that produced overlapping nonidentical
protein fragments were considered to estimate the number
of genes coding for B⬘ subunits.
ACKNOWLEDGMENTS
We thank Francisco Marco for his technical support and
good advice about the RT-PCR experiments. We are most
grateful to Ana Martinez for providing the actin
oligonucleotides.
Received January 23, 2002; accepted February 9, 2002.
LITERATURE CITED
AGI (2000) Analysis of the genome sequence of the flowering plant Arabidopsis thaliana. Nature 408: 796–815
Altschul SF, Madden TL, Schäffer AA, Zhang Z, Miller
W, Lipman DJ (1997) Gapped BLAST and PSI-BLAST: a
new generation of protein database search programs.
Nucleic Acids Res 25: 3389–3402
Ariño J, Pérez-Callejón E, Cunillera N, Camps M, Posas F,
Ferrer A (1993) Protein phosphatases in higher plants:
multiplicity of type 2A phosphatases in Arabidopsis thaliana. Plant Mol Biol 21: 475–485
Berry M, Gehring W (2000) Phosphorylation of the SCR
homeodomain determines its functional activity: essential role for protein phosphatase 2AB⬘. EMBO J 19:
2946–2957
Burge C, Karlin S (1997) Prediction of complete gene structures in human genomic DNA. J Mol Biol 268: 78–94
Casamayor A, Pérez-Callejón E, Pujol G, Ariño J, Ferrer A
(1994) Molecular characterization of a fourth isoform of
the catalytic subunit of protein phosphatase 2A from
Arabidopsis thaliana. Plant Mol Biol 26: 523–528
Corum JW, Hartung AJ, Stamey RT, Rundle SJ (1996)
Characterization of DNA sequences encoding a novel
isoform of the 55 kDa B regulatory subunit of the type 2A
protein serine/threonine phosphatase of Arabidopsis
thaliana. Plant Mol Biol 31: 419–427
Csortos C, Zolnierowicz S, Bako E, Durbin SD, DePaoliRoach AA (1996) High complexity in the expression of
the B⬘ subunit of protein phosphatase 2A. Evidence for
the existence of at least seven novel isoforms. J Biol
Chem 271: 2578–2588
Depaoli-Roach AA, Park IK, Cerovsky V, Csortos C,
Durbin SD, Kuntz MJ, Sitikov A, Tang PM, Verin A,
Zolnierowicz S (1994) Serine/threonine protein phosphatases in the control of cell function. Adv Enzyme
Regul 34: 199–224
Evangelista CCJ, Rodriguez TA, Limbach MP, Zitomer RS
(1996) Rox3 and Rts1 function in the global stress response
pathway in baker’s yeast. Genetics 142: 1083–1093
Plant Physiol. Vol. 129, 2002
Fitch WM, Farris JS (1974) Evolutionary trees with minimum nucleotide replacements from amino acid sequences. J Mol Evol 3: 263–278
Garbers C, DeLong A, Deruere J, Bernasconi P, Soll D
(1996) A mutation in protein phosphatase 2A regulatory
subunit A affects auxin transport in Arabidopsis. EMBO J
15: 2115–2124
Groves MR, Hanlon N, Turowski P, Hemmings BA, Barford D (1999) The structure of the protein phosphatase
2A PR65/A subunit reveals the conformation of its 15
tandemly repeated HEAT motifs. Cell 96: 99–110
Haynes JG, Hartung AJ, Hendershot JD, Passingham RS,
Rundle SJ (1999) Molecular characterization of the B⬘
regulatory subunit gene family of Arabidopsis protein
phosphatase 2A. Eur J Biochem 260: 127–136
Hendrix P, Mayer-Jackel RE, Cron P, Goris J, Hofsteenge
J, Merlevede W, Hemmings BA (1993a) Structure and
expression of a 72 kDa regulatory subunit of protein
phosphatase 2A. Evidence for different size forms produced by alternative splicing. J Biol Chem 268:
15267–15276
Hendrix P, Turowski P, Mayer-Jaekel RE, Goris J, Hofsteenge J, Merlevede W, Hemmings BA (1993b) Analysis of
subunit isoforms in protein phosphatase 2A holoenzymes
from rabbit and Xenopus. J Biol Chem 268: 7330–7337
Ingebritsen TS, Cohen P (1983a) Protein phosphatases:
properties and role in cellular regulation. Science 221:
331–338
Janssens V, Goris J (2001) Protein phosphatase 2A: a
highly regulated family of serine/threonine phosphatases implicated in cell growth and signalling. Biochem J
353: 417–439
Jiang W, Hallberg RL (2000) Isolation and characterization
of par1⫹ and par2⫹. Two Schizosaccharomyces pombe genes
encoding B⬘ subunits of protein phosphatase 2A. Genetics 154: 1025–1038
Latorre KA, Harris DM, Rundle SJ (1997) Differential
expression of three Arabidopsis genes encoding the B⬘
regulatory subunit of protein phosphatase 2A. Eur J Biochem 245: 156–163
Lin Q, Buckler ES, Muse SV, Walker JC (1999) Molecular
evolution of type 1 serine/threonine protein phosphatases. Mol Phylogenet Evol 12: 57–66
McCright B, Brothman AR, Virshup DM (1996a) Assignment of human protein phosphatase 2A regulatory subunit genes b56alpha, b56beta, b56gamma, b56delta, and
b56epsilon (PPP2R5A-PPP2R5E), highly expressed in
muscle and brain, to chromosome regions 1q41, 11q12,
3p21, 6p211, and 7p1123p12. Genomics 36: 168–170
McCright B, Rivers AM, Audlin S, Virshup DM (1996b)
The B56 family of protein phosphatase 2A (PP2A) regulatory subunits encodes differentiation-induced phosphoproteins that target PP2A to both nucleus and cytoplasm. J Biol Chem 271: 22081–22089
McCright B, Virshup DM (1995) Identification of a new
family of protein phosphatase 2A regulatory subunits.
J Biol Chem 270: 26123–26128
Meek S, Morrice N, MacKintosh C (1999) Microcystin
affinity purification of plant protein phosphatases: PP1C,
Downloaded from on June 17, 2017 - Published by www.plantphysiol.org
Copyright © 2002 American Society of Plant Biologists. All rights reserved.
821
Terol et al.
PP5 and a regulatory A-subunit of PP2A. FEBS Lett 457:
494–498
Mumby MC, Walter G (1993) Protein serine/threonine
phosphatases: structure, regulation, and functions in cell
growth. Physiol Rev 73: 673–699
Nei M, Chakraborty R (1976) Empirical relationship between the number of nucleotide substitutions and interspecific identity of amino acid sequences in some proteins. J Mol Evol 26: 313–323
Pérez-Callejón E, Casamayor A, Pujol G, Camps M, Ferrer
A, Ariño J (1998) Molecular cloning and characterization
of two phosphatase 2A catalytic subunit genes from Arabidopsis thaliana. Gene 209: 105–112
Prescott A, Martin C (1987) A rapid method for the quantitative assessment of levels of specific mRNAs in plants.
Plant Mol Biol Rep 4: 219–224
Rodrı́guez PL (1998) Protein phosphatase 2C (PP2C) function in higher plants. Plant Mol Biol 38: 919–927
Rundle SJ, Hartung AJ, Corum JW, O’Neill M (1995)
Characterization of a cDNA encoding the 55 kDa B regulatory subunit of Arabidopsis protein phosphatase 2A.
Plant Mol Biol 28: 257–266
Saitou N, Nei M (1987) The neighbour-joining method: a
new method for reconstructing phylogenetic trees. Mol
Biol Evol 4: 406–425
Salanoubat M, Lemcke K, Rieger M, Ansorge W, Unseld
M, Fartmann B, Valle G, Blocker H, Perez-Alonso M,
Obermaier B et al. (2000) Sequence and analysis of chromosome 3 of the plant Arabidopsis thaliana. Nature 408:
820–822
Sato S, Kotani H, Nakamura Y, Kaneko T, Asamizu E,
Fukami M, Miyajima N, Tabata S (1997) Structural analysis of Arabidopsis thaliana chromosome 5 I Sequence
features of the 16 Mb regions covered by twenty physically assigned P1 clones. DNA Res 4: 215–230
Schöntal AH (1998) Role of PP2A in intracellular signal
transduction pathways. Front Biosci 3: D1262–D1273
Shu Y, Hallberg RL (1995) SCS1, a multicopy suppressor of
hsp60-ts mutant alleles, does not encode a mitochondrially targeted protein. Mol Cell Biol 15: 5618–5626
Shu Y, Yang H, Hallberg E, Hallberg RL (1997) Molecular
genetic analysis of RTS1p, a B⬘ regulatory subunit of
822
Saccharomyces cerevisiae protein phosphatase 2A. Mol Cell
Biol 17: 3242–3253
Slabas AR, Fordham-Skelton AP, Fletcher D, MartinezRivas JM, Swinhoe R, Croy RR, Evans IM (1994) Characterization of cDNA and genomic clones encoding homologues of the 65 kDa regulatory subunit of protein
phosphatase 2A in Arabidopsis thaliana. Plant Mol Biol 26:
1125–1138
Smith RD, Walker JC (1993) Expression of multiple type 1
phosphoprotein phosphatases in Arabidopsis thaliana.
Plant Mol Biol 21: 307–316
Staden R (1996) The Staden sequence analysis package.
Mol Biotechnol 5: 233–241
Strack S, Zaucha JA, Ebner FF, Colbran RJ, Wadzinski BE
(1998) Brain protein phosphatase 2A: developmental regulation and distinct cellular and subcellular localization
by B subunits. J Comp Neurol 392: 515–527
Sudhir K, Koichir T, Masatosahi N (1993) Molecular Evolutionary Genetic Analysis, Version 10. Pennsylvania
State, University Park
Swofford DL, Selander RR (1981) BIOSYS-1: A Computer
Program for the Analysis of Allelic Variation in Genetics.
University of Illinois, Urbana
Tehrani MA, Mumby MC, Kamibayashi C (1996) Identification of a novel protein phosphatase 2A regulatory
subunit highly expressed in muscle. J Biol Chem 271:
5164–5170
Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG (1997) The ClustalX windows interface: flexible
strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 24: 4876–4882
Zhao Y, Boguslawski G, Zitomer RS, DePaoli-Roach AA
(1997) Saccharomyces cerevisiae homologs of mammalian B
and B⬘ subunits of protein phosphatase 2A direct the
enzyme to distinct cellular functions. J Biol Chem 272:
8256–8262
Zolnierowicz S, Van Hoof C, Andjelkovic N, Cron P,
Stevens I, Merlevede W, Goris J, Hemmings BA (1996)
The variable subunit associated with protein phosphatase 2A defines a novel multimember family of regulatory subunits. Biochem J 317: 187–194
Downloaded from on June 17, 2017 - Published by www.plantphysiol.org
Copyright © 2002 American Society of Plant Biologists. All rights reserved.
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