Data Mining the Arabidopsis Genome Reveals Fifteen
14-3-3 Genes. Expression Is Demonstrated for Two out of
Five Novel Genes1
Magnus Rosenquist*, Magnus Alsterfjord, Christer Larsson, and Marianne Sommarin
Department of Plant Biochemistry, Lund University, P.O. Box 117, SE–221 00 Lund, Sweden
In plants, 14-3-3 proteins are key regulators of primary metabolism and membrane transport. Although the current dogma
states that 14-3-3 isoforms are not very specific with regard to target proteins, recent data suggest that the specificity may
be high. Therefore, identification and characterization of all 14-3-3 (GF14) isoforms in the model plant Arabidopsis are
important. Using the information now available from The Arabidopsis Information Resource, we found three new GF14
genes. The potential expression of these three genes, and of two additional novel GF14 genes (Rosenquist et al., 2000), in
leaves, roots, and flowers was examined using reverse transcriptase-polymerase chain reaction and cDNA library polymerase chain reaction screening. Under normal growth conditions, two of these genes were found to be transcribed. These genes
were named grf11and grf12, and the corresponding new 14-3-3 isoforms were named GF14omicron and GF14iota, respectively. The gene coding for GF14omicron was expressed in leaves, roots, and flowers, whereas the gene coding for GF14iota
was only expressed in flowers. Gene structures and relationships between all members of the GF14 gene family were
deduced from data available through The Arabidopsis Information Resource. The data clearly support the theory that two
14-3-3 genes were present when eudicotyledons diverged from monocotyledons. In total, there are 15 14-3-3 genes (grfs 1–15)
in Arabidopsis, of which 12 (grfs 1–12) now have been shown to be expressed.
14-3-3 proteins were discovered by Moore and
Perez (1967) as major soluble proteins in brain tissue.
Since then it has become clear that 14-3-3 proteins are
present in all eukaryotic organisms (for review, see
Rosenquist et al., 2000) and have a wide range of
functions (for review, see Finnie et al., 1999; Fu et al.,
2000). 14-3-3s typically function as dimers and bind
to particular phosphorylated motifs in other proteins,
thereby regulating the activity of the respective target protein. In 1992, the first four plant 14-3-3 isoforms were reported: one in Arabidopsis (Lu et al.,
1992), one each in Spinacia oleracea and Oenothera
hookeri (Hirsch et al., 1992), and one in Hordeum vulgare (Brandt et al., 1992). The Arabidopsis isoform
was identified as part of a protein/G box complex
and therefore named “G box factor 14-3-3,” in short
“GF14.” This designation has been kept for the Arabidopsis isoforms, and the four additional 14-3-3 isoforms reported by Lu et al. (1994) were named
GF14chi, phi, psi, and upsilon, and the original isoform received the designation GF14omega. The same
year, Jarillo et al. (1994) reported two “rare coldinducible” 14-3-3s from Arabidopsis, RCI1 and RCI2.
The RCI1 was identical to GF14psi of Lu et al. (1994),
whereas RCI2 was a new isoform, very closely re1
This work was supported by the Swedish Foundation for
Strategic Research (to C.L. and M.S.), by the Swedish Natural
Science Research Council (to C.L. and M.S.), by the Kungliga
Fysiografiska Sällskapet (to M.R.), and by the Royal Swedish
Academy of Sciences (to M.R.).
* Corresponding author; e-mail [email protected];
fax 46 – 462224116.
142
lated to GF14lambda (Wu et al., 1997) and until now
regarded as identical. An additional four 14-3-3s,
GF14epsilon, kappa, mu, and nu, were identified in
1997 (Wu et al., 1997). After that, the members of the
14-3-3 gene family in Arabidopsis were regarded to
be 10.
Although the first Arabidopsis 14-3-3 isoform,
GF14omega, was discovered as a constituent of a
protein/G box complex and implicated to be involved in regulation of gene transcription (Lu et al.,
1992), subsequent research also has shown plant 143-3s to have a broad range of functions (for review,
see Finnie et al., 1999). In particular, plant 14-3-3s
have been found to be important regulators of primary metabolism. For example, nitrate reductase and
Suc phosphate synthase, which are key enzymes in
nitrogen and carbon metabolism, respectively, are
both inhibited by binding of 14-3-3 (Bachmann et al.,
1996; Moorhead et al., 1996; Toroser et al., 1998),
whereas binding of 14-3-3 to the plasma membrane
H⫹ATPase activates H⫹ pumping (Jahn et al., 1997;
Oecking et al., 1997) and hence stimulates nutrient
uptake.
In a previous study, using surface plasmon resonance, we reported strong differences in binding affinity between nine GF14 isoforms and a known target, the phosphorylated C terminus of the Arabidopsis
plasma membrane H⫹ATPase isoform 2, AHA2
(Rosenquist et al., 2000). This suggests that one reason
for the large number of 14-3-3 isoforms in multicellular organisms, such as plants, is functional specificity.
Thus, the regulatory role of 14-3-3s in carbon and
nitrogen metabolism, in nutrient uptake, etc, may be
Plant Physiology, September
2001,from
Vol.on
127,
pp.18,
142–149,
www.plantphysiol.org
© 2001 American Society of Plant Biologists
Downloaded
June
2017 - Published
by www.plantphysiol.org
Copyright © 2001 American Society of Plant Biologists. All rights reserved.
The Arabidopsis 14-3-3 Gene Family
executed by different 14-3-3 isoforms. To determine
whether this is the case, it is important to first identify
and then have access to all isoforms expressed. The
completion of the sequencing of the Arabidopsis genome has enabled determination of gene numbers
within specific gene families. Using the information
now available via Internet, we recently identified two
novel GF14 genes (Rosenquist et al., 2000). After further data mining of the Arabidopsis genome, we have
now identified three additional GF14 genes, bringing
the total number of GF14 genes to 15. Using reverse
transcriptase (RT)-PCR and cDNA library PCR screening, we now demonstrate that two of the five novel
genes are expressed, bringing the total number of
expressed 14-3-3 isoforms in Arabidopsis to 12.
RESULTS AND DISCUSSION
Examining the Arabidopsis genome revealed three
additional novel GF14 genes, grf12, grf13, and grf14,
in addition to the two recently reported by Rosenquist et al. (2000), grf11 and grf15 (Fig. 1). One of the
two previously reported novel genes, grf15 (Table I),
only codes for approximately 30% of the length of a
GF14 protein (Fig. 1). Furthermore, the 82 amino
acids the gene encodes correspond to the C terminus
of a functional 14-3-3, and show only 44% identity
with its closest relatives, GF14psi and upsilon (data
not shown). Grf15 and its putative product were
therefore excluded from further analysis. To determine if the four other novel genes are expressed or
not, RT-PCR and cDNA library PCR screening were
performed. Thus, primers designed for the novel 143-3 genes, grfs 11 through 14, found in BAC clones
F21H2, T1K7, T16E15, and T11I11 (Table I), were
used to PCR screen a cDNA library made from developing flowers, as well as for RT-PCR on mRNA
prepared from leaves, roots, and flowers, respectively. Only two of these four novel GF14 genes, grf11
and grf12, were found to be expressed (Fig. 2). No
corresponding cDNAs or ESTs were found in the
database, which suggests that these mRNA species
are present in low abundancy. The two novel isoforms were named GF14omicron, encoded by grf11
(F21H2.3), and GF14iota, encoded by grf12 (T1K7.15;
Table I), in accordance with the designations used
earlier, using Greek letters for the proteins and Arabic numbers for the genes. Both GF14omicron and
Figure 1. Gene maps of all Arabidopsis 14-3-3 (GF14) genes. Exons are indicated as black boxes and introns as white boxes.
Exon and intron sizes are indicated with the number of bases within in each box. The names of the resulting proteins are
within parentheses. The genes can be divided into two groups based on exon patterns: grfs 1 through 8, and grfs 9 through
14, excluding the severely truncated grf15 that does not readily fit into either group. The five novel genes discussed in this
paper are grf11, grf12, grf13, grf14, and grf15, of which grf11 and grf15 were already reported by Rosenquist et al. (2000).
The abbreviation grf stands for G box regulating factor.
Plant Physiol. Vol. 127, 2001
Downloaded from on June 18, 2017 - Published by www.plantphysiol.org
Copyright © 2001 American Society of Plant Biologists. All rights reserved.
143
Rosenquist et al.
Table I. Summary of available database information on Arabidopsis 14-3-3 (GF14) genes and proteins
The chromosomes and bacteria artificial chromosome (BAC) clones the different genes are found on are indicated, as well as the accession
no. of the BAC clone, the gene no. in the BAC, and the accession no. of the mRNA. Accession nos. to putative and expressed gene products are
also indicated.
GF14 Isoform
Chromosome 1
Putative 14-3-3
Similar to epsilon
Iota
Omicron
Epsilon
Omega
Phi
Chromosome 2
Mu
14-3-3 Like
Chromosome 3
Nu
Chromosome 4
Chi
Chromosome 5
Upsilon
Psif
Kappa
Lambda (or AFT1)
14-3-3-Like protein AFT1g
RCI2h
BAC
Gene
mRNA
Accession No.
BAC Accession
No.
Protein
Accession No.
T11I11
T16E15
T1K7
F21H2
T16E15
F3F9
T32G9
grf13a (T11I11.16)
grf14 (T16E15.9c)
grf12 (T1K7.15e)
grf11 (F21H2.3)
grf10 (T16E15.8)
grf2 (F3F9.16)
grf4 (T32G9.30)
–
–
AF335544
AF323920
U36446
M96855
L09111
AC012680
AC068562
AC013427
AC007894
AC068562
AC013430
AC079605
AAG52105b
AAF87262d
AAF98570
AAG47840
P48347
Q01525
P46077
F14N22
F12P23
grf9 (F14N22.14)
grf15 (F12P23.4)
U60444
–
AC007087
AC007264
AAD51784
AAD28654
F16B3
grf7 (F16B3.15)
U60445
AC021640
AAD51782
Contig. 25 (F23J3)
grf1
L09112
AL161513
P42643
F1N13
P1 Clone MXI10 ⫹
MBB18
P1 Clone MNA5
F12B17
F12B17
–
grf5 (F1N13㛭190)
grf3 (MXI10.21)
L09109
L09110
AAB62225
P42644
grf8 (MNA5.16)
grf6
grf6 (F12B17㛭200)
RCIIB
U36447
U68545
–
X74141
AL391145
AB005248 ⫹
AB005231
AB011479
AL353995
AL353995
–
AAD51783
P48349
CAB89398
CAA52238
a
b
The abbreviation grf stands for G-box regulating factor.
Incorrect annotation resulted in a shortened N terminus (first exon and 5⬘ part
c
d
of second exon were not included).
T16E15.9 is located 1,329 bp from grf10 (T16E15.8) in the same BAC clone.
A single nucleotide
e
f
was counted as a fifth intron when sequence was annotated in the original entry.
Splicing suggested in database is incorrect.
The
g
h
isoform GF14psi is identical to the entry RCI1 (protein accession no. S47969).
Putative product due to alternative splicing.
An
additional thymine at the end of exon 3 of grf6 causes a frame shift resulting in this putative isoform, which is probably the result of erroneous
sequence analysis.
GF14iota contain the expected conserved regions
found in all other 14-3-3s (Rosenquist et al., 2000),
and most divergence is found in the N and C termini
(Fig. 3). The annotation of grf12 (T1K7.15) in the
Arabidopsis sequencing project did not correspond
to the sequence we obtained for the cDNA, suggesting that the predicted splicing in the database is
incorrect.
The GF14omicron gene grf11 was expressed in
leaves, roots, and flowers, whereas the GF14iota gene
grf12 was only expressed in flowers (Fig. 2). The
expression of these isoforms in flowers, and particularly the exclusive expression of GF14iota in this
tissue, is interesting because when examining binding of the 10 previously identified GF14s to the phosphorylated C terminus of the flower-specific Arabidopsis plasma membrane H⫹ ATPase isoform 9,
AHA9, using surface plasmon resonance, all 10
GF14s were classified as nonbinding (data not
shown). The C terminus of AHA9 differs from the one
in AHA2 (which showed different degrees of binding
to the nine GF14 isoforms tested; Rosenquist et al.,
2000) on three out of six amino acid positions, and the
14-3-3 binding motifs of AHA9 and AHA2 thus are
sufficiently different to suggest that they are activated
144
by different GF14s. Therefore, GF14omicron and particularly GF14iota are good candidates as regulators of
AHA9.
The 14-3-3 genes grf13 (T11I11.16; Table I) and grf14
(T16E15.9; Table I) were not expressed in any of the
examined tissues. The coding sequence of gene grf14
contains a single nucleotide insertion in exon 4
(which we have confirmed by additional sequencing
[data not shown]) causing a frame shift, which creates a stop codon and loss of the last 45 amino acids
(Fig. 1). Given the fact that seven of the 17 conserved
target-binding amino acid residues, as well as the
nuclear export signal (Rittinger et al., 1999), are located within the missing area, grf14 does not likely
produce a functional 14-3-3 isoform, although possible expression cannot be ruled out. The fact that no
expression of the gene grf13 was observed is intriguing. The amino acid sequence it encodes is the most
divergent of all GF14s, suggesting that if it is expressed it might have a very specific function. Therefore, our failure to detect transcription may be due to
a rather specific expression of this isoform, either
regarding developmental stage, tissue, or stress condition. We have refrained from naming the gene
Downloaded from on June 18, 2017 - Published by www.plantphysiol.org
Copyright © 2001 American Society of Plant Biologists. All rights reserved.
Plant Physiol. Vol. 127, 2001
The Arabidopsis 14-3-3 Gene Family
products of grf13 and grf14 as no expression has been
shown so far.
When examining the 10 previously characterized
grfs, we found that the gene coding for GF14psi, grf3
(MXI10.21), is divided between P1clone MXI10 and
MBB18 (Table I). A striking discovery was that the
annotation in the Arabidopsis genome project of BAC
clone F12B17 corresponds to a putative protein, “143-3-like protein AFT1,” and not to the expressed
GF14lambda of Wu et al. (1997). Thus, an alternative
splicing of the pre-mRNA of the GF14lambda coding
gene, grf6, is suggested, resulting in the putative
GF14 protein “14-3-3-like protein AFT1” (CAB89398)
with a completely different C terminus from the previously reported GF14lambda (P48349). The C terminus of the putative “14-3-3-like protein AFT1” is 25
amino acids longer than the C terminus of
GF14lambda. Our attempts to detect mRNA corresponding to “14-3-3-like protein AFT1” have been
negative, suggesting that the gene grf6 codes for no
other product than GF14lambda, and that the predicted splicing in the database is incorrect. There is,
in fact, no experimental evidence to date that alternative splicing of 14-3-3 genes exists. Thus, the annotation in the database of the grf6 gene (F12B17_200)
in the BAC F12B17 should be changed to correspond
to GF14lambda. The RCIIB gene reported by Jarillo et
al. (1994) results in a protein, RCI2 (CAA52238), that
only differs from GF14lambda in the last eight amino
acids. The RCIIB gene cannot be found among the
data from the Arabidopsis genome project, but is due
to an additional thymine at the end of exon 3 in grf6
(Fig. 1). Because this is most likely the result of
erroneous sequence analysis, RCIIB has been excluded from the present analysis.
Examining the gene maps of all GF14 genes, one
can observe two major groups based on exon patterns; grfs 1 through 8 form one group, where all
genes are expressed, and grfs 9 through 14 form a
second group (Fig. 1). Unrooted phylogenetic trees
based on either the resulting mRNA or amino acid
sequences (including the putative products of grf13
and grf14) show that the gene products of grfs 1
through 8 form three branches in one part of the tree,
and that the products of grfs 9 through 14 form the
other, more divergent, part of the tree (Fig. 4; mRNA
data not shown). It is intriguing that a branch-wise
specificity was found when the affinity between nine
of the GF14s and the phosphorylated C terminus of
AHA2 was measured (Rosenquist et al., 2000).
In Figure 5, the locations of the different BAC
clones containing GF14 genes are indicated on the
Figure 2. Detection of novel 14-3-3 gene transcripts in flower (A, B,
and F), leaf (C and E), and root (D) tissues by RT-PCR and cDNA
library PCR. Primary RT-PCR reactions were performed with primers
corresponding to exon 2 (see Fig. 1) of grf11 (omicron; A and C),
grf12 (iota; A and C), grf13 (A, C, and D), and grf14 (B, C, and D).
Subsequent RT-PCR reactions were performed with full-length primers for grf11 on leaf (E) and root (D) cDNA, and for grf12 on flower
(F) and root (D) cDNA. Products were run on 1.5% (w/v) agarose gel
Plant Physiol. Vol. 127, 2001
(inverted image) and bands of interest are indicated with black
arrowheads. The identities of these bands were confirmed by sequencing. Sizes of standard (std) markers are indicated to the left.
Note that the band grf12 (iota) exon2 (A) only corresponds to 290 bp
of the exon due to optimization of primer positions, and therefore is
17 bp shorter than grf11 (omicron) exon2.
Downloaded from on June 18, 2017 - Published by www.plantphysiol.org
Copyright © 2001 American Society of Plant Biologists. All rights reserved.
145
Rosenquist et al.
Figure 3. DNA sequences of the two expressed novel Arabidopsis 14-3-3 (GF14) genes, grf11 and grf12, and corresponding
amino acid sequences for the proteins, GF14omicron and GF14iota, respectively. Conserved identity between all expressed
GF14s is indicated with gray boxes, and conserved homology is indicated with white boxes.
chromosomes, and two major duplication events 170
and 50 million years ago (Vision et al., 2000), which
may be responsible for the formation of some of the
isoforms, are indicated. The genes grf5 (upsilon) and
grf7 (nu) are located in regions of chromosome 5 and
3, respectively, that were duplicated 170 million
years ago. However, considering the very close relation between GF14 upsilon and nu (Fig. 4), it is more
likely that the duplication leading to the formation of
grf5 (upsilon) and grf7 (nu) was a much more recent
event. The gene grf2 (omega) and grf13 are situated
close together on chromosome 1, and this is also the
case for grf4 (phi) and grf11 (omicron). These two
146
gene pairs, as well as grf1 (chi) on chromosome 4, are
located within areas that were duplicated 50 million
years ago. It is notable that no other GF14 gene is
located close to grf1 (chi), suggesting that a gene
related to grf11 (omicron) and grf13 has been lost. The
fact that GF14phi, chi, and omega are much more
closely related than GF14Grf13 (the putative product
of grf13) is to omicron (Fig. 4) is noteworthy because
they all seem to originate from the major duplication
event 50 million years ago (Fig. 5). Considering their
common evolutionary history, one would expect
GF14Grf13 and omicron to show about the same high
homology as GF14phi, chi, and omega (Fig. 4). As
Downloaded from on June 18, 2017 - Published by www.plantphysiol.org
Copyright © 2001 American Society of Plant Biologists. All rights reserved.
Plant Physiol. Vol. 127, 2001
The Arabidopsis 14-3-3 Gene Family
The completion of the sequencing of the Arabidopsis genome finally enabled the determination of the
total number of 14-3-3 genes to be 15. The challenge
now is to examine the expression pattern of all GF14
isoforms, to identify new target proteins, and to determine the functional specificity of all GF14 isoforms, in order to reveal all the cellular processes
involving regulatory 14-3-3 proteins in the model
plant Arabidopsis.
MATERIALS AND METHODS
Examination of Database Entries
Figure 4. A phylogenetic tree with topology representative for the
Arabidopsis 14-3-3 (GF14) protein family. The putative gene products of grf13 and grf14 are designated Grf13 and Grf14, respectively.
A heuristic search using the maximum parsimony method was done
on the alignment of GF14 protein sequences using the PAUP 4.0b4
software (Sinauer Associates, Inc. Publishers, Sunderland, MA), with
gaps treated as missing data. Bootstrap values for 12,000 replicates
are indicated on each branch. The hypothetical 14-3-3-like protein
coded by grf15 (Table I; Fig. 1) was excluded from the analysis due
to strong deviance in both sequence and length. The 82 amino acids
encoded by grf15 show highest homology (44% identity) to GF14psi
and upsilon, and correspond to the C terminus of a functional 14-3-3
(data not shown).
proteins involved in complex formation, 14-3-3s are
subject to a high selection pressure, which acts to
conserve these proteins. Thus, if GF14phi, chi, and
omega have retained their functional specificity this
will have limited their evolution. In a converse manner, if GF14omicron and the putative gene product of
grf13 (GF14Grf13) have obtained different functions
this may explain their low homology, or if grf13 is not
expressed at all, it has of course evolved without any
constraints.
Previous phylogenetic analyses of plant 14-3-3 isoforms indicate that at the time of the split into eudicotyledons and monocotyledons, about 200 million
years ago, two 14-3-3 isoforms were already present
(Wang and Shakes, 1996; Rosenquist et al., 2000). This
is in accordance with both the gene map in Figure 1
and the phylogenetic tree in Figure 4. The gene map
(Fig. 1) divides the Arabidopsis 14-3-3 genes into two
major groups; grfs 1 through 8, and grfs 9 through 14,
respectively. The phylogenetic tree (Fig. 4) similarly
suggests two major branches: the GF14 upsilon/nu/
psi, phi/chi/omega, and lambda/kappa branches
forming a major branch in the lower part of the tree,
and the GF14 mu, iota, omicron, epsilon, Grf14, and
Grf13 forming an upper major branch. One of the
gene pairs, grf2 (omega)/grf13, grf4 (phi)/grf11 (omicron), or grf1 (chi) and its putative aborted neighbor,
is probably the direct descendant of the two ancestral
14-3-3 genes (Fig. 5).
Plant Physiol. Vol. 127, 2001
GF14 database entries were obtained by a simple keyword search at the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov). A blast search
of the Arabidopsis BAC clones at The Arabidopsis Information Resource (http://www.Arabidopsis.org) revealed
additional isoforms, and allowed mapping of the genes.
Computational Analysis of GF14 Gene Sequences
DNA sequences corresponding to GF14 genes were obtained from the GenBank database via the National Center
for Biotechnology Information. Clustal W multiple alignments of sequences were performed using the MacVector
7.0 software (Oxford Molecular Group Plc, Oxford). The
alignments were carried out using the Blossum series matrix, with an open gap penalty of 10 and an extend-gap
penalty of 0.05. Alignments were examined and adjusted
manually. A heuristic search using the maximum parsimony method was done on the alignment using the PAUP
4.0b4a software (Sinauer Associates, Inc. Publishers), with
gaps treated as missing data. The tree bisection reconnection branch-swapping algorithm was used. Tree building
and calculation of Bootstrap values, with 12,000 replicates,
were also carried out using the PAUP 4.0b4a software.
Gene maps were drawn from splicing patterns indicated in
the respective BAC entries. The splicing patterns of grf13
(T11I11.16) and grf14 (T16E15.9) suggested in the database
were incorrect, and hence adjusted.
Plant Material
Arabidopsis ecotype Columbia-0 was grown on soil at
22°C with a 9-h-light/15-h-dark photoperiod (170 ␮E) and
70% relative humidity. Root cultures were grown in darkness in flasks with 100 mL of Murashige and Skoog medium, pH 5.7, supplemented with 3% (w/v) Suc (Malamy
and Benfey, 1997). Plants for flower RNA preparation were
grown under a long-day light regime (16 h light).
mRNA Isolation and RT-PCR
Leaves were harvested after 4 weeks and total RNA was
prepared with a conventional phenol/chloroform extraction. Subsequent mRNA isolation was carried out according to the manufacturer’s protocol using the QIAGEN Oligotex mRNA Purification Kit (Merck KGaA, Darmstadt,
Downloaded from on June 18, 2017 - Published by www.plantphysiol.org
Copyright © 2001 American Society of Plant Biologists. All rights reserved.
147
Rosenquist et al.
Figure 5. Arabidopsis chromosome map. BAC clones harboring 14-3-3 (GF14) genes are indicated with black lines. Gene
names, with corresponding gene products written in parentheses, are indicated by each BAC. Centromeres are indicated
with white circles. The gray dashed arrow indicates a major duplication event that took place 170 million years ago, and
the two gray arrows indicate another major duplication event that took place 50 million years ago (Vision et al., 2000). Thin
dashed and dotted lines connect relatively closely related genes and hence suggest other duplication events. The gene grf14
(T16E15.9) is located 1,329 bases from grf10 (T16E15.8) within the same BAC clone. Given the low homology of the severely
truncated 14-3-3 gene grf15 in F12P23, no connection to other genes is indicated.
Germany). First-strand synthesis was performed with the
First Strand cDNA Synthesis Kit (Boehringer Mannheim,
Basel). The same procedures were used on roots harvested
after 10 weeks of cultivation. Total RNA was extracted
according to the method developed by Chang et al. (1993)
from a mix of flowers in all stages of anthesis.
Primary RT-PCR was carried out with Taq polymerase
(Roche, Basel) and primers designed against the largest
exon of the newly identified GF14 genes (see Fig. 1), as well
as for an alternatively spliced grf6. Fragments obtained
were run on a 1.5% (w/v) agarose gel and sequenced for
identification. RT-PCR subsequently was done with primers corresponding to the full-length genes. Full-length fragments were cloned into the TA vector pGEM (Promega
Corp., Madison, WI), and sequenced. Restriction sites were
added to full-length primers to facilitate cloning into suitable vectors.
cDNA Library PCR Screening
A cDNA library from Arabidopsis ecotype Landsberg
erecta developing flowers (Weigel et al., 1992) was obtained
from the Arabidopsis Biological Resource Center. Conventional in vivo excision on the entire library was performed
on infected Escherichia coli XL1-Blue with E. coli SOLR (AH
Diagnostics, Stockholm). PCR with exon-specific primers
was performed on the excised library, for detection purpose only. Full-length clones were amplified from cDNA
148
synthesized from the flower mRNA preparation from Arabidopsis (Columbia-0) described above.
ACKNOWLEDGMENT
We thank Dr. Ove Nilsson (Swedish Agricultural University, Umeå, Sweden) for preparation of total RNA from
Arabidopsis flowers.
Received April 6, 2001; returned for revision April 30, 2001;
accepted May 18, 2001.
LITERATURE CITED
Bachmann M, Huber JL, Athwal GS, Wu K, Ferl RJ,
Huber SC (1996) 14-3-3 proteins associate with the regulatory phosphorylation site of spinach leaf nitrate reductase in an isoform-specific manner and reduce dephosphorylation of Ser-543 by endogenous protein
phosphatases. FEBS Lett 398: 26–30
Brandt J, Thordal-Christensen H, Vad K, Gregersen PL,
Collinge DB (1992) A pathogen-induced gene of barley
encodes a protein showing high similarity to a protein
kinase regulator. Plant J 2: 815–820
Chang S, Puryear J, Cairney J (1993) A simple and efficient
method for isolating RNA from pine trees. Plant Mol Biol
Rep 11: 113–116
Downloaded from on June 18, 2017 - Published by www.plantphysiol.org
Copyright © 2001 American Society of Plant Biologists. All rights reserved.
Plant Physiol. Vol. 127, 2001
The Arabidopsis 14-3-3 Gene Family
Finnie C, Borch J, Collinge DB (1999) 14-3-3 proteins:
eukaryotic regulatory proteins with many functions.
Plant Mol Biol 40: 545–554
Fu H, Subramanian RR, Masters SC (2000) 14-3-3 proteins:
structure, function, and regulation. Annu Rev Pharmacol
Toxicol 40: 617–647
Hirsch S, Aitken A, Bertsch U, Soll J (1992) A plant
homologue to mammalian brain 14-3-3 protein and protein kinase C inhibitor. FEBS Lett 296: 222–224
Jahn T, Fuglsang AT, Olsson A, Brüntrup IM, Collinge
DB, Volkmann D, Sommarin M, Palmgren MG, Larsson C (1997) The 14-3-3 protein interacts directly with the
C-terminal region of the plant plasma membrane H⫹ATPase. Plant Cell 9: 1805–1814
Jarillo JA, Capel J, Leyva A, Martinez-Zapater JM, Salinas
J (1994) Two related low-temperature-inducible genes of
Arabidopsis encode proteins showing high homology to
14-3-3 proteins, a family of putative kinase regulators.
Plant Mol Biol 25: 693–704
Lu G, DeLisle AJ, de Vetten NC, Ferl RJ (1992) Brain
proteins in plants: an Arabidopsis homolog to neurotransmitter pathway activators is part of a DNA binding
complex. Proc Natl Acad Sci USA 89: 11490–11494
Lu G, Rooney MF, Wu K, Ferl RJ (1994) Five cDNAs
encoding Arabidopsis GF14 proteins. Plant Physiol 105:
1459–1460
Malamy JE, Benfey PN (1997) Organization and cell differentiation in lateral roots of Arabidopsis thaliana. Development 124: 33–44
Moore B, Perez V (1967) Specific acidic proteins of the
nervous system. In FD Carlson, ed, Physiological and
Plant Physiol. Vol. 127, 2001
Biochemical Aspects of Nervous Integration. Prentice
Hall, Englewood Cliffs, NJ, pp 343–359
Moorhead G, Douglas P, Morrice N, Scarabel M, Aitken
A, MacKintosh C (1996) Phosphorylated nitrate reductase from spinach leaves is inhibited by 14-3-3 proteins
and activated by fusicoccin. Curr Biol 1996 6: 1104–1113
Oecking C, Piotrowski M, Hagemeier J, Hagemann K
(1997) Topology and target interaction of the fusicoccinbinding 14-3-3 homologs of Commelina communis. Plant J
12: 441–453
Rittinger K, Budman J, Xu J, Volinia S, Cantley LC,
Smerdon SJ, Gamblin SJ, Yaffe MB (1999) Structural
analysis of 14-3-3 phosphopeptide complexes identifies a
dual role for the nuclear export signal of 14-3-3 in ligand
binding. Mol Cell 4: 153–166
Rosenquist M, Sehnke P, Ferl RJ, Sommarin M, Larsson C
(2000) Evolution of the 14-3-3 protein family: does the
large number of isoforms in multicellular organisms reflect functional specificity? J Mol Evol 51: 446–458
Toroser D, Athwal GS, Huber SC (1998) Site-specific regulatory interaction between spinach leaf sucrose-phosphate
synthase and 14-3-3 proteins. FEBS Lett 435: 110–114
Vision TJ, Brown DG, Tanksley SD (2000) The origins of
genomic duplications in Arabidopsis. Science 290:
2114–2117
Wang W, Shakes DC (1996) Molecular evolution of the
14-3-3 protein family. J Mol Evol 43: 384–398
Weigel D, Alvarez J, Smyth DR, Yanofsky MF, Meyerowitz EM (1992) Leafy controls floral meristem identity in
Arabidopsis. Cell 69: 843–859
Wu K, Rooney MF, Ferl RJ (1997) The Arabidopsis 14-3-3
multigene family. Plant Physiol 114: 1421–1431
Downloaded from on June 18, 2017 - Published by www.plantphysiol.org
Copyright © 2001 American Society of Plant Biologists. All rights reserved.
149